The InteractiveFly: Drosophila as a Model for Human Diseases


Miscellaneous Drosophila Disease Models

Miscellaneous Drosophila Disease Models

Adams-Oliver syndrome

Adenoid cystic carcinoma (ACC)

Adrenoleukodystrophy

Adult-onset inherited myopathy

Alcoholism

alpha-Synucleinopathies

Amyloidosis

Ataxia

Azoospermia/Cryptozoospermia
Berardinelli-Seip congenital lipodystrophy type 2

Birt-Hogg-Dubé syndrome

Cachexia

Cardiac Lipotoxicity

Cerebellar hypoplasia and dysplasia
CerTra Syndrome
Charcot-Marie-Tooth Disease

Chediak-Higashi syndrome

Chronic kidney disease

Chronic Obstructive Pulmonary Disease

Ciliopathy

Cocaine Use Disorder
Colorectal cancer

Congenital disorders of glycosylation

Covid
Creutzfeldt-Jakob Disease and Prion Disease
Cystic fibrosis

Epilepsy and seizure disorders

Fanconi Syndrome

Fatty acid hydroxylase-associated neurodegeneration (FAHN)

Floating-Harbor syndrome

Freeman Sheldon Syndrome, a skeletal muscle contracture syndromes

Galactosemia

Galloway-Mowat syndrome - a severe, inherited kidney-neurological disease

Glioma and Glioblastoma

Heart Disease

Hereditary neuropathy

Human papillomavirus E6-induced malignancy

Hypertrophic cardiomyopathy
Hyperuricemia
Hypotonia
Inclusion body myopathy type 3

Inflammatory Gut Disease

Insomnia

Intellectual Disability associated with WAC

Jacobsen syndrome, an 11q terminal deletion developmental disorder

Kohlschutter-Tonz syndrome

Laminopathy

Leigh Syndrome

Limb-girdle muscular dystrophy D2

Lysosomal storage disease

Maple syrup urine disease

Meier-Gorlin syndrome

Microcephly

Mitochondrial disease

Mucopolysaccharidosis

Myeloproliferative neoplasms

Muscular Dystrophies

Myopathy

Nephrotic Syndrome

Neurofibromatosis - an autosomal dominant condition
Neurological disorder with regression, abnormal movements, loss of speech, and seizures (NEDAMSS)
Neuronal ceroid lipofuscinoses

Diabetic Neuropathy

N-Glycanase 1 deficiency

Nonalcoholic fatty liver disease

Nonsyndromic X-linked mental retardation

Noonan syndrome<

Oculopharyngeal muscular dystrophy
Obesity

Peripheral neuropathy

Peroxisome biogenesis disorders

Perry syndrome

Polycystic kidney disease

Polyglutamine (polyQ) disorders

Pontocerebellar Hypoplasia

Primary Ciliary Dyskinesia

Robinow syndrome

Renal Fibrosis

Retinitis pigmentosa

Ribosomopathies

Sarcopenia

Schizophrenia

Shwachman-Diamond syndrome

16p12.1 deletion

Skin cancer and nucleotide excision repair

Snyder-Robinson Syndrome

Spinal muscular atrophy

Spliceosomopathy


Spondyloarthritis

Squamous cell carcinoma

Stenosis

TANGO2 deficiency disease

Tauopathy

Traumatic Brain Injury

Trichohepatoenteric syndrome

Triosephosphate isomerase deficiency

Tuberous Sclerosis Complex

Ultraviolet Damage

Warburg micro syndrome a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2

Werner syndrome

Wilms' tumour

Williams-Beuren Syndrome

Wolfram syndrome

Zinc Deficiency

Adams-Oliver syndrome

Gagliani, E. K., Gutzwiller, L. M., Kuang, Y., Odaka, Y., Hoffmeister, P., Hauff, S., Turkiewicz, A., Harding-Theobald, E., Dolph, P. J., Borggrefe, T., Oswald, F., Gebelein, B. and Kovall, R. A. (2022). A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects. PLoS Genet 18(8): e1010335. PubMed ID: 35951645

Abstract
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. This study found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

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Adenoid cystic carcinoma

Bangi, E., Smibert, P., Uzilov, A. V., Teague, A. G., Gopinath, S., Antipin, Y., Chen, R., Hecht, C., Gruszczynski, N., Yon, W. J., Malyshev, D., Laspina, D., Selkridge, I., Wang, H., Gomez, J., Mascarenhas, J., Moe, A. S., Lau, C. Y., Taik, P., Pandya, C., Sung, M., Kim, S., Yum, K., Sebra, R., Donovan, M., Misiukiewicz, K., Ang, C., Schadt, E. E., Posner, M. R. and Cagan, R. L. (2021). A Drosophila platform identifies a novel, personalized therapy for a patient with adenoid cystic carcinoma. iScience 24(3): 102212. PubMed ID:
33733072

Abstract

Adenoid cystic carcinoma (ACC) is a rare cancer type that originates in the salivary glands. Tumors commonly invade along nerve tracks in the head and neck, making surgery challenging. Follow-up treatments for recurrence or metastasis including chemotherapy and targeted therapies have shown limited efficacy, emphasizing the need for new therapies. This study reports a Drosophila-based therapeutic approach for a patient with advanced ACC disease. A patient-specific Drosophila transgenic line was developed to model the five major variants associated with the patient's disease. Robotics-based screening identified a three-drug cocktail-vorinostat, pindolol, tofacitinib-that rescued transgene-mediated lethality in the Drosophila patient-specific line. Patient treatment led to a sustained stabilization and a partial metabolic response of 12 months. Subsequent resistance was associated with new genomic amplifications and deletions. Given the lack of options for patients with ACC, these data suggest that this approach may prove useful for identifying novel therapeutic candidates (Bangi, 2021).

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Adrenoleukodystrophy

Sivachenko, A., Gordon, H. B., Kimball, S. S., Gavin, E. J., Bonkowsky, J. L. and Letsou, A. (2016). Neurodegeneration in a Drosophila model of Adrenoleukodystrophy: the roles of the bubblegum and double bubble acyl-CoA synthetases. Dis Model Mech 9(4): 377-87. PubMed ID:
26893370

Abstract

Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease- modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. This study presents the first analysis of a very long chain acyl-CoA synthetase double mutant. The Drosophila bubblegum (bgm) and double bubble (dbb) genes have overlapping functions, and the consequences of bubblegum double bubble double knockout in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of Adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very long chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, this study shows that dysregulation of fatty acid metabolism via disruption of ACS function in vivo is causal of neurodegenerative pathologies evident in both neuronal cells and their support cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of the model system to the study of human disease, identification of a leukodystrophy patient who harbors a rare mutation in a human homologue of Bgm and Dbb was found: the SLC27a6-encoded very-long-chain acyl-CoA synthetase.

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Adult-onset inherited myopathy

Li, S., Zhang, P., Freibaum, B.D., Kim, N.C., Kolaitis, R.M., Molliex, A., Kanagaraj, A.P., Yabe, I., Tanino, M., Tanaka, S., Sasaki, H., Ross, E.D., Taylor, J.P. and Kim, H.J. (2016). Genetic interaction of hnRNPA2B1 and DNAJB6 in a Drosophila model of multisystem proteinopathy. Hum Mol Genet 25(5): 936-50. PubMed ID:
26744327

Abstract

This study sought to establish a mechanistic link between diseases caused by mutations in two genes associated with adult-onset inherited myopathies, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila homologs of human hnRNPA2B1 and DNAJB6, respectively. Disease-homologous mutations were introduced to Hrb98DE. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins, suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover wild-type, but not disease-associated mutant forms of MRJ, interacted with RNA-binding proteins after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interaction between disease-linked RNA-binding proteins and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.

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Alcoholism

Lange, A. P. and Wolf, F. W. (2023). Alcohol tolerance encoding in sleep regulatory circadian neurons in Drosophila. bioRxiv. PubMed ID:
36778487

Alcohol tolerance is a simple form of behavioral and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. A forward neuroanatomical screen was used to identify three new neural sites for rapid tolerance encoding. One of these was comprised of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. Rapid tolerance was localized to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link (Lang, 2023).

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Tremblay, S., Zeng, Y., Yue, A., Chabot, K., Mynahan, A., Desrochers, S., Bridges, S. and Ahmad, S. T. (2022). Caffeine Delays Ethanol-Induced Sedation in Drosophila. Biology (Basel) 12(1). PubMed ID: 36671755

Abstract

Caffeine and ethanol are among the most widely available and commonly consumed psychoactive substances. Both interact with adenosine receptor-mediated signaling which regulates numerous neurological processes including sleep and waking behaviors. In mammals, caffeine is an adenosine receptor antagonist and thus acts as a stimulant. Conversely, ethanol is a sedative because it promotes GABAergic neurotransmission, inhibits glutamatergic neurotransmission, and increases the amount of adenosine in the brain. Despite seemingly overlapping interactions, not much is known about the effect of caffeine on ethanol-induced sedation in Drosophila. In this study, using Drosophila melanogaster as a model, it was shown that caffeine supplementation in food delays the onset of ethanol-induced sedation in males and females of different strains. The resistance to sedation reverses upon caffeine withdrawal. Heterozygous adenosine receptor mutant flies are resistant to sedation. These findings suggest that caffeine and adenosine receptors modulate the sedative effects of ethanol in Drosophila (Tremblay, 2022).

Drosophila is an excellent model to study the effects of caffeine, adenosine receptor signaling, and alcohol on a variety of behaviors such as circadian rhythm, locomotion, and cognition. This study is the first to examine interplay among caffeine, Drosophila adenosine receptor, and ethanol-induced sedation in Drosophila. The results demonstrate that exposure to caffeine prolongs the onset of sedation in both male and female w1118 and OR-R flies, commonly used control and wild type Drosophila strains, respectively. Sex differences in ethanol-induced sedation time are observed in both vertebrates and invertebrates including Drosophila. Female flies are reported to have a shorter sedation time than males. However, in w1118 sexual dimorphism of sedation does not resolve when exposed to 100% ethanol. It was observed that w1118 females showed a trend (not statistically significant) for a higher ST50 than males in control at all concentrations of caffeine tested (Tremblay, 2022).

The effect of caffeine on sedation time when sedated with 100% ethanol was most robust and statistically significant at 0.5 mg/mL dose and did not hold at 1 mg/mL dose. The effect of caffeine on sedation time was more prominent with 50% and 75% ethanol possibly because the effect of caffeine manifests more effectively when the rate of onset of sedation is less drastic at lower doses of ethanol. Other studies in flies have examined the effects of caffeine, at a similar dose range, on adenosine receptor expression, fecundity, egg laying, and life span. It is possible that at higher doses, flies are generally weaker or less viable, as caffeine appears to begin to confer mortality in flies at 1 mg/mL concentration. Significant widespread mortality was observed at 3 mg/mL and 5 mg/mL dose after one-week exposure. At higher caffeine doses, a weakened state is possible either because flies are avoiding consuming highly caffeinated food due to its bitter taste and/or due to the direct physiological effects of caffeine. There is support for a direct effect of caffeine on physiological functions leading to mortality in flies as shown by the correlation between caffeine-induced mortality and reduced transcript levels of neuromodulators and adenosine receptors. The effect of caffeine on sedation is reversible and wears off within three days of caffeine withdrawal, likely due to its metabolic clearance. Caffeine is metabolized by the Cytochrome P-450 enzymes in both Drosophila and humans. Caffeine metabolites (theophylline, theobromine, paraxanthine) are also detected within 3 h of caffeine ingestion in flies. The half-life of caffeine in humans is 3-7 h (Tremblay, 2022).

Caffeine primarily acts as a stimulant in mammals because of its antagonism of adenosine receptor signaling, which promotes sleep. This study addressed the relationship between adenosine receptor gene dosage and sedation in flies. Heterozygous adenosine receptor mutant flies were found to be resistant to sedation. It was not possible to conduct sedation assays on homozygous mutant flies for the AdoRMB04401 allele because they generally do not survive to adulthood. This finding is in accordance with a previous study in which adenosine A2A receptor knockout mice were shown to be resistant to ethanol-induced hypnotic effects. Mammalian adenosine A2A receptor has the highest homology to the only adenosine receptor isoform in Drosophila. However, in Drosophila, caffeine may also act through additional pathways, such as the dopamine receptor-mediated signaling. The fly dopamine/ecdysteroid receptor (DopEcR) mediates ethanol-induced sedation. The Drosophila dopamine receptor (dDA1) and dopamine signaling have been independently shown to modulate the wake-promoting effect of caffeine, suggesting a potential adenosine receptor-independent mechanism of action for caffeine in flies (Tremblay, 2022).

Additionally, the effects of caffeine on sleep does not depend on adenosine activity. Further, caffeine was unable to inhibit adenosine receptor-mediated signaling in Drosophila neuroblast cell line in vitro. Therefore, it appears that caffeine and adenosine receptor either through coordination and/or independently play a role in ethanol-induced sedation (Tremblay, 2022).

The data suggests that pharmacological (caffeine exposure) and genetic (adenosine receptor mutation) disruption of adenosine receptor function delays ethanol sedation. Therefore, it can be hypothesized that agonists of the adenosine receptor, especially the endogenous ligand, adenosine, will promote sedation. Ethanol elevates extracellular adenosine levels which in turn activate the adenosine receptors. In mammalian systems, the ethanol-mediated elevation of adenosine modulates ethanol-induced behaviors primarily through adenosine A1 and A2 receptors. In future studies, it will be interesting to determine effects of adenosine receptor agonists on sedation and the cellular and molecular characteristics of signaling pathways downstream of adenosine receptors that mediate ethanol-induced sedation in flies (Tremblay, 2022).

Broadly, this study further supports the use of Drosophila as a model to study complex human behavior and to examine the colloquial notion of the negative implications of mixing caffeine with alcohol. Human correlational studies have found that individuals concurrently consuming caffeinated energy drinks and alcohol are more likely to consume more alcohol and have more severe negative consequences due to alcohol consumption [41]. When both substances are consumed in conjunction, caffeine reduces perceived inebriation, which leads to further consumption, thereby promoting binge drinking and risky behavior such as driving under the influence of alcohol. Further studies on the interaction between caffeine and ethanol will improve understanding of the biochemical and behavioral consequences of their consumption and aid in creating awareness of this public health crisis, especially for adolescents and young adults (Tremblay, 2022).

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Aleyakpo, B., Umukoro, O., Kavlie, R., Ranson, D. C., Thompsett, A., Corcoran, O. and Casalotti, S. O. (2019). G-protein alphaq gene expression plays a role in alcohol tolerance in Drosophila melanogaster. Brain Neurosci Adv 3: 2398212819883081. PubMed ID: 32166184

Abstract

Ethanol is a psychoactive substance causing both short- and long-term behavioural changes in humans and animal models. This study used the fruit fly Drosophila melanogaster to investigate the effect of ethanol exposure on the expression of the Galphaq protein subunit. Repetitive exposure to ethanol causes a reduction in sensitivity (tolerance) to ethanol, which was measured as the time for 50% of a set of flies to become sedated after exposure to ethanol (ST50). It was demonstrated that the same treatment that induces an increase in ST50 over consecutive days (tolerance) also causes a decrease in Galphaq protein subunit expression at both the messenger RNA and protein level. To identify whether there may be a causal relationship between these two outcomes, strains of flies were developed in which Galphaq messenger RNA expression is suppressed in a time- and tissue-specific manner. In these flies, the sensitivity to ethanol and the development of tolerance are altered. This work further supports the value of Drosophila as a model to dissect the molecular mechanisms of the behavioural response to alcohol and identifies G proteins as potentially important regulatory targets for alcohol use disorders (Aleyakpo, 2010).

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Mokashi, S. S., Shankar, V., MacPherson, R. A., Hannah, R. C., Mackay, T. F. C. and Anholt, R. R. H. (2021). Developmental Alcohol Exposure in Drosophila: Effects on Adult Phenotypes and Gene Expression in the Brain. Front Psychiatry 12: 699033. PubMed ID: 34366927

Abstract

Fetal alcohol exposure can lead to developmental abnormalities, intellectual disability, and behavioral changes, collectively termed fetal alcohol spectrum disorder (FASD). In 2015, the Centers for Disease Control found that 1 in 10 pregnant women report alcohol use and more than 3 million women in the USA are at risk of exposing their developing fetus to alcohol. Drosophila exposed to alcohol undergo physiological and behavioral changes that resemble human alcohol-related phenotypes. This study shows that adult flies that developed on ethanol-supplemented medium have decreased viability, reduced sensitivity to ethanol, and disrupted sleep and activity patterns. To assess the effects of exposure to alcohol during development on brain gene expression, single cell RNA sequencing was performed, and cell clusters were resolved with differentially expressed genes which represent distinct neuronal and glial populations. Differential gene expression showed extensive sexual dimorphism with little overlap between males and females. Gene expression differences following developmental alcohol exposure were similar to previously reported differential gene expression following cocaine consumption, suggesting that common neural substrates respond to both drugs. Genes associated with glutathione metabolism, lipid transport, glutamate and GABA metabolism, and vision feature in sexually dimorphic global multi-cluster interaction networks. These results provide a blueprint for translational studies on alcohol-induced effects on gene expression in the brain that may contribute to or result from FASD in human populations (Mokashi, 2021).

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Belhorma, K., Darwish, N., Benn-Hirsch, E., Duenas, A., Gates, H., Sanghera, N., Wu, J. and French, R. L. (2021). Developmental ethanol exposure causes central nervous system dysfunction and may slow the aging process in a Drosophila model of fetal alcohol spectrum disorder. Alcohol. PubMed ID: 33961967

Abstract

Alcohol is a known teratogen, and developmental exposure to ethanol results in Fetal Alcohol Spectrum Disorder (FASD). Children born with FASD can exhibit a range of symptoms including low birth weight, microcephaly, and neurobehavioral problems. Treatment of patients with FASD is estimated to cost 4 billion dollars per year in the United States alone, and 2 million dollars per affected individual's lifetime. This study has established Drosophila melanogaster as a model organism for the study of FASD. This study reports that mutations in Dementin (Dmtn), the Drosophila ortholog of the Alzheimer Disease associated protein TMCC2, convey sensitivity to developmental ethanol exposure, and provide evidence that Dmtn expression is disrupted by ethanol. In addition, it was found that flies reared on ethanol exhibit mild climbing defects suggestive of neurodegeneration. Surprisingly, the data also suggest that flies reared on ethanol age more slowly than control animals, and a number of slow-aging mutants were found to be sensitive to developmental ethanol exposure. Finally, this study found that flies reared on ethanol showed a persistent upregulation of genes encoding antioxidant enzymes, which may contribute to a reduced rate of central nervous system aging. Thus, in addition to the well-documented negative effects of developmental alcohol exposure on the nervous system, there may be a previously-unsuspected neuroprotective effect in adult animals (Belhorma, 2021).

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Weston, R. M., Schmitt, R. E., Grotewiel, M. and Miles, M. F. (2021). Transcriptome analysis of chloride intracellular channel knockdown in Drosophila identifies oxidation-reduction function as possible mechanism of altered sensitivity to ethanol sedation. PLoS One 16(7): e0246224. PubMed ID: 34228751

Abstract
Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. This study targeted for RNAi knockdown the sole Drosophila melanogaster ortholog, Clic, to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, the effect of hyperoxia on ethanol sedation was evaluated in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly (Weston, 2021).

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Bonilla, M., McPherson, M., Coreas, J., Boulos, M., Chavol, P., Alrabadi, R. I. and Loza-Coll, M. (2021). Repeated ethanol intoxications of Drosophila melanogaster adults increases the resistance to ethanol of their progeny. Alcohol Clin Exp Res 45(7): 1370-1382. PubMed ID: 34120365

Abstract
For decades, Drosophila has been used as a model organism to understand the genetics and neurobiology of ethanol intoxication and tolerance. Previous research has shown that acute and chronic pre-exposures to ethanol can trigger the development of functional ethanol tolerance in flies and has unveiled some of the genetic pathways involved in the process. No previous work has systematically explored whether repeated intoxications of adult flies can affect the ethanol tolerance of their progeny. Adult flies were intoxicated several times (once daily, over several days), and their F1 and F2 progeny were subjected to a functional tolerance test in which flies are exposed to ethanol and video recorded twice within 5 hr. Their behavior was subsequently analyzed to determine how long it took them to become sedated during the first and second exposures. One- and 2-way ANOVAs were used to determine whether parental treatment had an effect on their progeny's baseline resistance and/or acquired functional tolerance to ethanol. Parental flies that were intoxicated several times produced F1 and F2 progeny with a significantly higher resistance to ethanol than progeny from unexposed controls. Further, parental intoxications inconsistently increased the progeny's capacity to develop rapid functional tolerance upon re-exposure to ethanol. The transmission of increased ethanol resistance to progeny lasted several days after the last parental intoxication. This is the first demonstration that repeated parental daily intoxications affect the progeny's response to ethanol in fruit flies. These findings support the use of D. melanogaster to explore conserved pathways underlying the transmission of ethanol tolerance and can help in the identification of novel strategies for managing alcohol use disorder (Bonilla, 2021).

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Gambini, J., Gimeno-Mallench, L., Olaso-Gonzalez, G., Mastaloudis, A., Traber, M. G., Monleon, D., Borras, C. and Vina, J. (2021). Moderate Red Wine Consumption Increases the Expression of Longevity-Associated Genes in Controlled Human Populations and Extends Lifespan in Drosophila melanogaster. Antioxidants (Basel) 10(2). PubMed ID: 33669360

Abstract
The beneficial effects of moderate red wine consumption on cardiovascular health are well known. The composition of red wine includes several compounds, such as the phytoestrogen resveratrol, that exert these beneficial effects, although not all the mechanisms by which they act are known. The aim of this work was to study the effect of red wine consumption on longevity-related genes in controlled human populations, such as cloistered nuns. The expression of catalase, manganese-superoxide dismutase, Sirt1, and p53 was increased in peripheral blood mononuclear cells after 14 days of moderate red wine consumption. This increase was accompanied by an enhanced metabolic wellness: fatty acids, cholesterol, branched chain amino acids (isoleucine and leucine), ketone bodies (acetoacetate), bacterial co-metabolites (trimethylamine), and cellular antioxidants (taurine) contributed to a change in metabolic profile after moderate red wine consumption by the nuns. No serious unwanted side effects were observed. Finally, the effect of moderate red wine consumption was tested on longevity in a controlled animal population, such as D. melanogaster, and found that it increased average life span by 7%. In conclusion, moderate red wine consumption increases the expression of key longevity-related genes and improves metabolic health in humans and increases longevity in flies (Gambini, 2021).

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Lee, K. M., Talikoti, A., Shelton, K. and Grotewiel, M. (2021). Tyramine synthesis, vesicular packaging, and the SNARE complex function coordinately in astrocytes to regulate Drosophila alcohol sedation. Addict Biol: e13019. PubMed ID: 33538092

Abstract
Identifying mechanisms underlying alcohol-related behaviors could provide important insights regarding the etiology of alcohol use disorder. To date, most genetic studies on alcohol-related behavior in model organisms have focused on neurons, leaving the causal roles of glial mechanisms less comprehensively investigated. This paper reports studies on the role of Tyrosine decarboxylase 2 (Tdc2), which converts tyrosine to the catecholamine tyramine, in glial cells in Drosophila alcohol sedation. Using genetic approaches that drove transgene expression constitutively in all glia, constitutively in astrocytes and conditionally in glia during adulthood, this study found that knockdown and overexpression of Tdc2, respectively, increased and decreased the sensitivity to alcohol sedation in flies. Manipulation of the genes tyramine β-hydroxylase and tyrosine hydroxylase, which respectively synthesize octopamine and dopamine from tyramine and tyrosine, had no discernable effect on alcohol sedation, suggesting that Tdc2 affects alcohol sedation by regulating tyramine production. It was also found that knockdown of the vesicular monoamine transporter (VMAT) and disruption of the SNARE complex in all glia or selectively in astrocytes increased sensitivity to alcohol sedation and that both VMAT and the SNARE complex functioned downstream of Tdc2. These studies support a model in which the synthesis of tyramine and vesicle-mediated release of tyramine from adult astrocytes regulates alcohol sedation in Drosophila. Considering that tyramine is functionally orthologous to norepinephrine in mammals, these results raise the possibility that gliotransmitter synthesis release could be a conserved mechanism influencing behavioral responses to alcohol as well as alcohol use disorder (Lee, 2021).

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Wilson, A., Periandri, E. M., Sievers, M. and Petruccelli, E. (2023). Absolute ethanol intake predicts ethanol preference in Drosophila melanogaster. Drosophila Stat92E Signaling Following Pre-exposure to Ethanol. Neurosci Insights 18: 26331055221146755. PubMed ID: 36643884

Abstract
Repeated exposure to alcohol alters neuromolecular signaling that influences acute and long-lasting behaviors underlying Alcohol Use Disorder (AUD). Recent animal model research has implicated changes in the conserved JAK/STAT pathway, a signaling pathway classically associated with development and the innate immune system. How ethanol exposure impacts STAT signaling within neural cells is currently unclear. This study investigated the role of Drosophila Stat92E in ethanol-induced locomotion, signaling activity, and downstream transcriptional responses. Findings suggest that expressing Stat92E-RNAi causes enhanced ethanol-induced hyperactivity in flies previously exposed to ethanol. Furthermore, alternative splicing of Stat92E itself was detected after repeated ethanol exposure, although no changes were found in downstream transcriptional activity. This work adds to our growing understanding of altered neuromolecular signaling following ethanol exposure and suggests that STAT signaling may be a relevant target to consider for AUD treatment.

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Park, S. J. and Ja, W. W. (2020). Absolute ethanol intake predicts ethanol preference in Drosophila melanogaster. J Exp Biol 223(Pt 11). PubMed ID: 32366685

Abstract
Factors that mediate ethanol preference in Drosophila melanogaster are not well understood. A major confound has been the use of diverse methods to estimate ethanol consumption. This study measured fly consumptive ethanol preference on base diets varying in nutrients, taste and ethanol concentration. Both sexes showed an ethanol preference that was abolished on high nutrient concentration diets. Additionally, manipulating total food intake without altering the nutritive value of the base diet or the ethanol concentration was sufficient to evoke or eliminate ethanol preference. Absolute ethanol intake and food volume consumed were stronger predictors of ethanol preference than caloric intake or the dietary caloric content. These findings suggest that the effect of the base diet on ethanol preference is largely mediated by total consumption associated with the delivery medium, which ultimately determines the level of ethanol intake. It is speculated that a physiologically relevant threshold for ethanol intake is essential for preferential ethanol consumption (Park, 2020).

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Kanno, M., Hiramatsu, S., Kondo, S., Tanimoto, H. and Ichinose, T. (2021). Voluntary intake of psychoactive substances is regulated by the dopamine receptor Dop1R1 in Drosophila. Sci Rep 11(1): 3432. PubMed ID: 33564023

Abstract
Dysregulated motivation to consume psychoactive substances leads to addictive behaviors that often result in serious health consequences. Understanding the neuronal mechanisms that drive drug consumption is crucial for developing new therapeutic strategies. The fruit fly Drosophila melanogaster offers a unique opportunity to approach this problem with a battery of sophisticated neurogenetic tools available, but how they consume these drugs remains largely unknown. This study examined drug self-administration behavior of Drosophila and the underlying neuronal mechanisms. The preference of flies for five different psychoactive substances was measured using a two-choice feeding assay and its long-term changes were monitored. Flies were found to show acute preference for ethanol and methamphetamine, but not for cocaine, caffeine or morphine. Repeated intake of ethanol, but not methamphetamine, increased over time. Preference for methamphetamine and the long-term escalation of ethanol preference required the dopamine receptor Dop1R1 in the mushroom body. The protein level of Dop1R1 increased after repeated intake of ethanol, but not methamphetamine, which correlates with the acquired preference. Genetic overexpression of Dop1R1 enhanced ethanol preference. These results reveal a striking diversity of response to individual drugs in the fly and the role of dopamine signaling and its plastic changes in controlling voluntary intake of drugs (Kanno, 2021).

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Park, A., Tran, T., Scheuermann, E. A., Smith, D. P. and Atkinson, N. S.(2020). Alcohol potentiates a pheromone signal in flies. Elife 9. PubMed ID: 33141025

Abstract
For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. This study finds that when male flies inhabit ethanol-containing food substrates they become more aggressive. A possible mechanism was identified for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, it was observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors (Park, 2020).

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Nunez, K. M., Azanchi, R. and Kaun, K. R. (2018). Cue-induced ethanol seeking in Drosophila melanogaster is dose-dependent. Front Physiol 9: 438. Pubmed ID: 29740347

Abstract
Alcohol use disorder generates devastating social, medical and economic burdens, making it a major global health issue. The persistent nature of memories associated with intoxication experiences often induces cravings and triggers relapse in recovering individuals. Despite recent advances, the neural and molecular mechanisms underlying these memories are complex and not well understood. This makes finding effective pharmacological targets challenging. The investigation of persistent alcohol-associated memories in the fruit fly, Drosophila melanogaster, presents a unique opportunity to gain a comprehensive understanding of the memories for ethanol reward at the level of genes, molecules, neurons and circuits. This study characterizes the dose-dependent nature of ethanol on the expression of memory for an intoxication experience. The concentration of ethanol, number of ethanol exposures, length of ethanol exposures, and timing between ethanol exposures are critical in determining whether ethanol is perceived as aversive or appetitive, and in how long the memory for the intoxicating properties of ethanol last. This study highlights that fruit flies display both acute and persistent memories for ethanol-conditioned odor cues, and that a combination of parameters that determine the intoxication state of the fly influence the seemingly complex retention and expression of memories associated with intoxication. This thorough behavioral characterization provides the opportunity to interrogate the biological underpinnings of these observed preference differences in future studies (Nunez, 2018).

Lee, K. M., Mathies, L. D. and Grotewiel, M. (2019). Alcohol sedation in adult Drosophila is regulated by Cysteine proteinase-1 in cortex glia. Commun Biol 2: 252. PubMed ID: 31286069

Abstract
Although numerous studies have demonstrated that neuronal mechanisms regulate alcohol-related behaviors, very few have investigated the direct role of glia in behavioral responses to alcohol. The results described in this study begin to fill this gap in the alcohol behavior and gliobiology fields. Since Drosophila exhibit conserved behavioral responses to alcohol and their CNS glia are similar to mammalian CNS glia, this study used Drosophila to begin exploring the role of glia in alcohol behavior. Knockdown of Cysteine proteinase-1 (Cp1) in glia increased Drosophila alcohol sedation and that this effect was specific to cortex glia and adulthood. These data implicate Cp1 and cortex glia in alcohol-related behaviors. Cortex glia are functionally homologous to mammalian astrocytes and Cp1 is orthologous to mammalian Cathepsin L. These studies raise the possibility that cathepsins may influence behavioral responses to alcohol in mammals via roles in astrocytes (Lee, 2019).

Xu, S., Pany, S., Benny, K., Tarique, K., Al-Hatem, O., Gajewski, K., Leasure, J. L., Das, J. and Roman, G. (2018). Ethanol regulates presynaptic activity and sedation through presynaptic Unc13 proteins in Drosophila. eNeuro 5(3). PubMed ID: 29911175

Abstract
One possible presynaptic effector for ethanol is the Munc13-1 protein. This study shows that ethanol binding to the rat Munc13-1 C1 domain, at concentrations consistent with binge exposure, reduces diacylglycerol (DAG) binding. The inhibition of DAG binding is predicted to reduce the activity of Munc13-1 and presynaptic release. In Drosophila, this study shows that sedating concentrations of ethanol significantly reduce synaptic vesicle release in olfactory sensory neurons (OSNs), while having no significant impact on membrane depolarization and Ca(2+) influx into the presynaptic compartment. These data indicate that ethanol targets the active zone in reducing synaptic vesicle exocytosis. Drosophila, haploinsufficent for the Munc13-1 ortholog Dunc13, are more resistant to the effect of ethanol on presynaptic inhibition. Genetically reducing the activity of Dunc13 through mutation or expression of RNAi transgenes also leads to a significant resistance to the sedative effects of ethanol. The neuronal expression of Munc13-1 in heterozygotes for a Dunc13 loss-of-function mutation can largely rescue the ethanol sedation resistance phenotype, indicating a conservation of function between Munc13-1 and Dunc13 in ethanol sedation. Hence, reducing Dunc13 activity leads to naive physiological and behavioral resistance to sedating concentrations of ethanol. It is propose that reducing Dunc13 activity, genetically or pharmacologically by ethanol binding to the C1 domain of Munc13-1/Dunc13, promotes a homeostatic response that leads to ethanol tolerance (Xu, 2018).

Choi, H. J., Cha, S. J. and Kim, K. (2018). Ethanol regulates presynaptic activity and sedation through presynaptic Unc13 proteins in Drosophila. Glutathione transferase modulates acute ethanol-induced sedation in Drosophila neuron. Insect Mol Biol. PubMed ID: 30347459

Abstract
Heavy alcohol consumption leads to neuropathological damage and alcohol use disorder (AUD), which affects the health of people and results in a cost burden. However, the genes modulating sensitivity to ethanol remains largely unknown. This study identified a novel gene, Drosophila glutathione transferase omega 1 (GstO1), which plays a critical role in regulating sensitivity to ethanol sedation. The GstO1 mutant flies showed highly increased ethanol sensitivity. Furthermore, the expression level of GstO1 regulates the behavioral response to ethanol, because decreasing and increasing GstO1 affects sedation sensitivity in a contrasting manner. In addition, the RNAi-mediated knockdown of GstO1 expression reveals that GstO1 mediates sensitivity to ethanol sedation in neurons, including dopaminergic and serotonergic neurons. Altogether, our findings provide the first evidence for the involvement of glutathione transferase in the response to alcohol in Drosophila and provide a novel mechanistic insight into the toxicity and sensitivity of ethanol exposure (Choi, 2018).

Petruccelli, E., Feyder, M., Ledru, N., Jaques, Y., Anderson, E. and Kaun, K. R. (2018). Alcohol activates Scabrous-Notch to influence associated memories. Neuron 100(5): 1209-1223 PubMed ID: 30482693

Abstract
Drugs of abuse, like alcohol, modulate gene expression in reward circuits and consequently alter behavior. However, the in vivo cellular mechanisms through which alcohol induces lasting transcriptional changes are unclear. This study shows that Drosophila Notch/Su(H) signaling and the secreted fibrinogen-related protein Scabrous in mushroom body (MB) memory circuitry are important for the enduring preference of cues associated with alcohol's rewarding properties. Alcohol exposure affects Notch responsivity in the adult MB and alters Su(H) targeting at the dopamine-2-like receptor (Dop2R). Alcohol cue training also caused lasting changes to the MB nuclear transcriptome, including changes in the alternative splicing of Dop2R and newly implicated transcripts like Stat92E. Together, these data suggest that alcohol-induced activation of the highly conserved Notch pathway and accompanying transcriptional responses in memory circuitry contribute to addiction. Ultimately, this provides mechanistic insight into the etiology and pathophysiology of alcohol use disorder (Petruccelli, 2018).

Scepanovic, G. and Stewart, B. A. (2019). Analysis of Drosophila nervous system development following an early, brief exposure to ethanol. Dev Neurobiol. PubMed ID: 31472090

Abstract
The effects of ethanol on neural function and development have been studied extensively, motivated in part by the addictive properties of alcohol and the neurodevelopmental deficits that arise in children with Fetal Alcohol Spectrum Disorder (FASD). Absent from this research area is a genetically tractable system to study the effects of early ethanol exposure on later neurodevelopmental and behavioural phenotypes. Embryos of the fruit fly, Drosophila melanogaster have been used as a model system to investigate the neuronal defects that arise after an early exposure to ethanol. Several disruptions of neural development and morphology were found following a brief ethanol exposure during embryogenesis along with subsequent changes in larval behaviour. Altogether, this study establishes a new system to examine the effects of alcohol exposure in embryos and the potential to conduct large scale genetics screens to uncover novel factors that sensitize or protect neurons to the effects of alcohol (Scepanovic, 2019).

Choi, H. J., Cha, S. J. and Kim, K. (2019). Glutathione transferase modulates acute ethanol-induced sedation in Drosophila neurones. Insect Mol Biol 28(2): 246-252. PubMed ID: 30347459

Abstract
Heavy alcohol consumption leads to neuropathological damage and alcohol use disorder, which affects the health of people and results in a cost burden. However, the genes modulating sensitivity to ethanol remain largely unknown. This study identified a novel gene, Drosophila glutathione transferase omega 1 (GstO1), which plays a critical role in regulating sensitivity to ethanol sedation. GstO1 mutant flies showed highly increased ethanol sensitivity. Furthermore, the expression level of GstO1 regulates the behavioural response to ethanol, because decreasing and increasing GstO1 affects sedation sensitivity in a contrasting manner. In addition, the RNA interference-mediated knockdown of GstO1 expression reveals that GstO1 mediates sensitivity to ethanol sedation in neurones, including dopaminergic and serotonergic neurones. Altogether, these findings provide the first evidence for the involvement of glutathione transferase in the response to alcohol in Drosophila and provide a novel mechanistic insight into the toxicity and sensitivity of ethanol exposure (Choi, 2019).

Ranson, D. C., Ayoub, S. S., Corcoran, O. and Casalotti, S. O. (2019). Pharmacological targeting of the GABAB receptor alters Drosophila's behavioural responses to alcohol. Addict Biol. PubMed ID: 30761704

Abstract
When exposed to ethanol, Drosophila melanogaster display a variety of addiction-like behaviours similar to those observed in mammals. Sensitivity to ethanol can be quantified by measuring the time at which 50% of the flies are sedated by ethanol exposure (ST50); an increase of ST50 following multiple ethanol exposures is widely interpreted as development of tolerance to ethanol. Sensitivity and tolerance to ethanol were measured after administration of the gamma-aminobutyric acid receptor B (GABAB) agonist (SKF 97541) and antagonist (CGP 54626), when compared with flies treated with ethanol alone. Dose-dependent increases and decreases in sensitivity to ethanol were observed for both the agonist and antagonist respectively. Tolerance was recorded in the presence of GABAB drugs, but the rate of tolerance development was increased by SKF 97451 and unaltered in presence of CGP 54626. This indicates that the GABAB receptor contributes to both the sensitivity to ethanol and mechanisms by which tolerance develops. The data also reinforce the usefulness of Drosophila as a model for identifying the molecular components of addictive behaviours and for testing drugs that could potentially be used for the treatment of alcohol use disorder (Ranson, 2019).

Schmitt, R. E., Messick, M. R., Shell, B. C., Dunbar, E. K., Fang, H. F., Shelton, K. L., Venton, B. J., Pletcher, S. D. and Grotewiel, M. (2019). Pharmacological targeting of the GABAB receptor alters Drosophila's behavioural responses to alcohol. Dietary yeast influences ethanol sedation in Drosophila via serotonergic neuron function. Addict Biol: e12779. PubMed ID: 31169340

Abstract
Abuse of alcohol is a major clinical problem with far-reaching health consequences. Understanding the environmental and genetic factors that contribute to alcohol-related behaviors is a potential gateway for developing novel therapeutic approaches for patients that abuse the drug. To this end, Drosophila melanogaster was used as a model to investigate the effect of diet, an environmental factor, on ethanol sedation. Providing flies with diets high in yeast, a routinely used component of fly media, increased their resistance to ethanol sedation. The yeast-induced resistance to ethanol sedation occurred in several different genetic backgrounds, was observed in males and females, was elicited by yeast from different sources, was readily reversible, and was associated with increased nutrient intake as well as decreased internal ethanol levels. Inhibition of serotonergic neuron function using multiple independent genetic manipulations blocked the effect of yeast supplementation on ethanol sedation, nutrient intake, and internal ethanol levels. These results demonstrate that yeast is a critical dietary component that influences ethanol sedation in flies and that serotonergic signaling is required for the effect of dietary yeast on nutrient intake, ethanol uptake/elimination, and ethanol sedation. These studies establish the fly as a model for diet-induced changes in ethanol sedation and raise the possibility that serotonin might mediate the effect of diet on alcohol-related behavior in other species (Schmitt, 2019).

Butts, A. R., Ojelade, S. A., Pronovost, E. D., Seguin, A., Merrill, C. B., Rodan, A. R. and Rothenfluh, A. (2019). Altered actin filament dynamics in the Drosophila mushroom bodies lead to fast acquisition of alcohol consumption preference. J Neurosci. PubMed ID: 31558618

Abstract
Alcohol use is highly prevalent in the United States and across the world, and every year millions of people suffer from alcohol use disorders (AUDs). While the genetic contribution to developing AUDs is estimated to be 50-60%, many of the underlying molecular mechanisms remain unclear. Previous studies have revealed that Drosophila melanogaster lacking RhoGAP18B and Ras Suppressor 1 (Rsu1) display reduced sensitivity to ethanol-induced sedation. Both Rsu1 and RhoGAP18B are negative regulators of the small Rho-family GTPase, Rac1, a modulator of actin dynamics. This study investigated the role of Rac1 and its downstream target, the actin-severing protein cofilin, in alcohol consumption preference. These two regulators of actin dynamics can alter male experience-dependent alcohol preference in a bidirectional manner: expressing either activated Rac1 or dominant-negative cofilin in the mushroom bodies (MB) abolishes experience-dependent alcohol preference. Conversely, dominant-negative Rac1 or activated cofilin MB expression lead to faster acquisition of alcohol preference. These data show that Rac1 and cofilin activity are key to determining the rate of acquisition of alcohol preference, revealing a critical role of actin dynamics regulation in the development of voluntary self-administration in Drosophila

Petruccelli, E., Brown, T., Waterman, A., Ledru, N. and Kaun, K. R. (2020). Alcohol Causes Lasting Differential Transcription in Drosophila Mushroom Body Neurons. Genetics. PubMed ID: 32132098

Abstract
Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become long-lasting and inflexible remain unclear. New methods to analyze gene expression within precise neuronal cell-types can provide further insight towards AUD prevention and treatment. This study used genetic tools in Drosophila melanogaster to investigate the lasting consequences of ethanol on transcription in memory-encoding neurons. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Differential expression analyses revealed that distinct transcripts, but not genes, in the MB were associated with experiencing ethanol alone compared to forming a memory of an odor cue associated with ethanol. Adult MB specific knockdown of spliceosome-associated proteins demonstrated the necessity of RNA processing in ethanol memory formation. These findings highlight the dynamic, context-specific regulation of transcription in cue-encoding neurons, and the lasting impact of ethanol on transcript usage during memory formation (Petruccelli, 2020).

Kohlmeier, P., Zhang, Y., Gorter, J. A., Su, C. Y. and Billeter, J. C. (2021). Mating increases Drosophila melanogaster females' choosiness by reducing olfactory sensitivity to a male pheromone. Nat Ecol Evol. PubMed ID: 34155384

Abstract
Females that are highly selective when choosing a mate run the risk of remaining unmated or delaying commencing reproduction. Therefore, low female choosiness would be beneficial when males are rare but it would be maladaptive if males become more frequent. How can females resolve this issue? Polyandry would allow mating-status-dependent choosiness, with virgin females selecting their first mate with little selectivity and becoming choosier thereafter. This plasticity in choosiness would ensure timely acquisition of sperm and enable females to increase offspring quality during later mating. This study shows that Drosophila melanogaster females display such mating-status-dependent choosiness by becoming more selective once mated and identifies the underlying neurohormonal mechanism. Mating releases juvenile hormone, which desensitizes Or47b olfactory neurons to a pheromone produced by males, resulting in increased preference for pheromone-rich males. Besides providing a mechanism to a long-standing evolutionary prediction, these data suggest that intersexual selection in D. melanogaster, and possibly in all polyandrous, sperm-storing species, is mainly the domain of mated females since virgin females are less selective. Juvenile hormone influences behaviour by changing cue responsiveness across insects; the neurohormonal modulation of olfactory neurons uncovered in D. melanogaster provides an explicit mechanism for how this hormone modulates behavioural plasticity (Kohlmeier, 2021).

Lange, A. P. and Wolf, F. W. (2023). Alcohol tolerance encoding in sleep regulatory circadian neurons in Drosophila. bioRxiv. PubMed ID: 36778487

Abstract
Alcohol tolerance is a simple form of behavioral and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. A forward neuroanatomical screen was used to identify three new neural sites for rapid tolerance encoding. One of these was comprised of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. Rapid tolerance was localized to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link.

Lange, A. P. and Wolf, F. W. (2023). Alcohol tolerance encoding in sleep regulatory circadian neurons in Drosophila. bioRxiv. PubMed ID: 36778487

Abstract

Abstract:
Alcohol tolerance is a simple form of behavioral and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. A forward neuroanatomical screen was used to identify three new neural sites for rapid tolerance encoding. One of these was comprised of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. Rapid tolerance was localized to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link. Larnerd, C., Adhikari, P., Valdez, A., Del Toro, A. and Wolf, F. W. (2023). Rapid and Chronic Ethanol Tolerance Are Composed of Distinct Memory-Like States in Drosophila. J Neurosci 43(12): 2210-2220. PubMed ID: 36750369
Summary:
Ethanol tolerance is the first type of behavioral plasticity and neural plasticity that is induced by ethanol intake, and yet its molecular and circuit bases remain largely unexplored. This study characterize the following three distinct forms of ethanol tolerance in male Drosophila: rapid, chronic, and repeated. Rapid tolerance is composed of two short-lived memory-like states, one that is labile and one that is consolidated. Chronic tolerance, induced by continuous exposure, lasts for 2 d, induces ethanol preference, and hinders the development of rapid tolerance through the activity of histone deacetylases (HDACs). Unlike rapid tolerance, chronic tolerance is independent of the immediate early gene Hr38/Nr4a Chronic tolerance is suppressed by the sirtuin HDAC Sirt1, whereas rapid tolerance is enhanced by Sirt1. Moreover, rapid and chronic tolerance map to anatomically distinct regions of the mushroom body learning and memory centers. Chronic tolerance, like long-term memory, is dependent on new protein synthesis and it induces the kayak/c-fos immediate early gene, but it depends on CREB signaling outside the mushroom bodies, and it does not require the Radish GTPase. Thus, chronic ethanol exposure creates an ethanol-specific memory-like state that is molecularly and anatomically different from other forms of ethanol tolerance.

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alpha-Synucleinopathies

Olsen, A. L. and Feany, M. B. (2019). Glial alpha-synuclein promotes neurodegeneration characterized by a distinct transcriptional program in vivo. Glia. PubMed ID:
31267577

Abstract
alpha-Synucleinopathies are neurodegenerative diseases that are characterized pathologically by alpha-synuclein inclusions in neurons and glia. The pathologic contribution of glial alpha-synuclein in these diseases is not well understood. Glial alpha-synuclein may be of particular importance in multiple system atrophy (MSA), which is defined pathologically by glial cytoplasmic alpha-synuclein inclusions. Previous work has described Drosophila models of neuronal alpha-synucleinopathy, which recapitulate key features of the human disorders. This study now expands this model to express human alpha-synuclein in glia. Expression of alpha-synuclein in glia alone results in alpha-synuclein aggregation, death of dopaminergic neurons, impaired locomotor function, and autonomic dysfunction. Furthermore, co-expression of alpha-synuclein in both neurons and glia worsens these phenotypes as compared to expression of alpha-synuclein in neurons alone. Unique transcriptomic signatures induced by glial as opposed to neuronal alpha-synuclein were identified. These results suggest that glial alpha-synuclein may contribute to the burden of pathology in the alpha-synucleinopathies through a cell type-specific transcriptional program. This new Drosophila model system enables further mechanistic studies dissecting the contribution of glial and neuronal alpha-synuclein in vivo, potentially shedding light on mechanisms of disease that are especially relevant in MSA but also the alpha-synucleinopathies more broadly (Olsen, 2019).

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Amyloidosis

Ibrahim, R. B., Yeh, S. Y., Lin, K. P., Ricardo, F., Yu, T. Y., Chan, C. C., Tsai, J. W. and Liu, Y. T. (2019). Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration. Cell Mol Life Sci. PubMed ID:
31728576

Transthyretin amyloidosis (ATTR) is a progressive life-threatening disease characterized by the deposition of transthyretin (TTR) amyloid fibrils. Several pathogenic variants have been shown to destabilize TTR tetramers, leading to aggregation of misfolded TTR fibrils. However, factors that underlie the differential age of disease onset amongst amyloidogenic TTR variants remain elusive. This study examined the biological properties of various TTR mutations and found that the cellular secretory pattern of the wild-type (WT) TTR was similar to those of the late-onset mutant (Ala97Ser, p. Ala117Ser), stable mutant (Thr119Met, p. Thr139Met), early-onset mutant (Val30Met, p. Val50Met), but not in the unstable mutant (Asp18Gly, p. Asp38Gly). Cytotoxicity assays revealed their toxicities in the order of Val30Met > Ala97Ser > WT > Thr119Met in neuroblastoma cells. Surprisingly, while early-onset amyloidogenic TTR monomers (M-TTRs) are retained by the endoplasmic reticulum quality control (ERQC), late-onset amyloidogenic M-TTRs can be secreted extracellularly. Treatment of thapsigargin (Tg) to activate the unfolded protein response (UPR) alleviates Ala97Ser M-TTR secretion. Interestingly, Ala97Ser TTR overexpression in Drosophila causes late-onset fast neurodegeneration and a relatively short lifespan, recapitulating human disease progression. This study demonstrates that the escape of TTR monomers from the ERQC may underlie late-onset amyloidogenesis in patients and suggests that targeting ERQC could mitigate late-onset ATTR (Ibrahim, 2019).

Bergkvist, L., Richards, D. R., Bernardo-Gancedo, A., Kumita, J. R., Nilsson, P. R. and Brorsson, A. C. (2020). Serum amyloid P component promotes formation of distinct aggregated lysozyme morphologies and reduces toxicity in Drosophila flies expressing F57I lysozyme. PLoS One 15(1): e0227227. PubMed ID: 31978114

Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using a Drosophila model of systemic lysozyme amyloidosis, SAP has been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. It was found that SAP can counteract the toxicity induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway (Bergkvist, 2019)

Ibrahim, R. B., Yeh, S. Y., Lin, K. P., Ricardo, F., Yu, T. Y., Chan, C. C., Tsai, J. W. and Liu, Y. T. (2019). Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration. Cell Mol Life Sci. PubMed ID: 31728576

Transthyretin amyloidosis (ATTR) is a progressive life-threatening disease characterized by the deposition of transthyretin (TTR) amyloid fibrils. Several pathogenic variants have been shown to destabilize TTR tetramers, leading to aggregation of misfolded TTR fibrils. However, factors that underlie the differential age of disease onset amongst amyloidogenic TTR variants remain elusive. This study examined the biological properties of various TTR mutations and found that the cellular secretory pattern of the wild-type (WT) TTR was similar to those of the late-onset mutant (Ala97Ser, p. Ala117Ser), stable mutant (Thr119Met, p. Thr139Met), early-onset mutant (Val30Met, p. Val50Met), but not in the unstable mutant (Asp18Gly, p. Asp38Gly). Cytotoxicity assays revealed their toxicities in the order of Val30Met > Ala97Ser > WT v Thr119Met in neuroblastoma cells. Surprisingly, while early-onset amyloidogenic TTR monomers (M-TTRs) are retained by the endoplasmic reticulum quality control (ERQC), late-onset amyloidogenic M-TTRs can be secreted extracellularly. Treatment of thapsigargin (Tg) to activate the unfolded protein response (UPR) alleviates Ala97Ser M-TTR secretion. Interestingly, Ala97Ser TTR overexpression in Drosophila causes late-onset fast neurodegeneration and a relatively short lifespan, recapitulating human disease progression. This study demonstrates that the escape of TTR monomers from the ERQC may underlie late-onset amyloidogenesis in patients and suggests that targeting ERQC could mitigate late-onset ATTR (Ibrahim, 2019). Oliveira da Silva, M. I., Lopes, C. S. and Liz, M. A. (2020). Transthyretin interacts with actin regulators in a Drosophila model of familial amyloid polyneuropathy. Sci Rep 10(1): 13596. PubMed ID: 32788615

Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder whose major hallmark is the deposition of mutated transthyretin (TTR) in the form of amyloid fibrils in the peripheral nervous system (PNS). The exposure of PNS axons to extracellular TTR deposits leads to an axonopathy that culminates in neuronal death. However, the molecular mechanisms underlying TTR-induced neurodegeneration are still unclear, despite the extensive studies in vertebrate models. This work used a Drosophila FAP model, based on the expression of the amyloidogenic TTR (V30M) in the fly retina, to uncover genetic interactions with cytoskeleton regulators. TTR interacts with actin regulators and induces cytoskeleton alterations, leading to axonal defects. Moreover, this study pinpoints an interaction between TTRV30M and members of Rho GTPase signaling pathways, the major actin regulators. Based on these findings it is proposed that actin cytoskeleton alterations may mediate the axonopathy observed in FAP patients, and highlight a molecular pathway, mediated by Rho GTPases, underlying TTR-induced neurodegeneration. This work should prompt novel studies and approaches towards FAP therapy (Lopes, 2020).

Abstract

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Ataxia

Olmos, V., Thompson, E. N., Gogia, N., Luttik, K., Veeranki, V., Ni, L., Sim, S., Chen, K., Krause, D. S., Lim, J. (2024). Dysregulation of alternative splicing in spinocerebellar ataxia type 1. Hum Mol Genet, 33(2):138-149 PubMed ID:
37802886

Abstract

Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. W RNA sequencing was performed in mouse models of spinocerebellar ataxia type 1 and mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. The diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing identification of overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. Evidence is provided that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1 (Olmos, 2024). Blount, J. R., Patel, N. C., Libohova, K., Harris, A. L., Tsou, W. L., Sujkowski, A., Todi, S. V. (2023). Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3. Journal of the neurological sciences, 454:120828 PubMed ID: 37865002

Abstract

Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when the enzyme is itself ubiquitinated at lysine (K) at position 117: in vitro, K117-ubiqutinated Atxn3 cleaves poly-ubiquitin markedly more rapidly compared to its unmodified counterpart. How polyQ expansion causes SCA3 remains unclear. To gather insights into the biology of disease of SCA3, the following question was posited: is K117 important for toxicity caused by pathogenic Atxn3? To answer this question, transgenic Drosophila lines were generated that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. It was found that mutating K117 mildly enhances the toxicity and aggregation of pathogenic Atxn3. An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest that Atxn3 ubiquitination is a regulatory step of SCA3, in part by modulating its aggregation (Blount, 2023). Servettini, I., Talani, G., Megaro, A., Setzu, M. D., Biggio, F., Briffa, M., Guglielmi, L., Savalli, N., Binda, F., Delicata, F., Bru-Mercier, G., Vassallo, N., Maglione, V., Cauchi, R. J., Di Pardo, A., Collu, M., Imbrici, P., Catacuzzeno, L., D'Adamo, M. C., Olcese, R. and Pessia, M. (2023). An activator of voltage-gated K(+) channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1. Proc Natl Acad Sci U S A 120(31): e2207978120. PubMed ID: 37487086

Abstract

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. This study uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. Through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5)s. By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery (Servettini, 2023).

Sousa, A., Rocha, S., Vieira, J., Reboiro-Jato, M., Lopez-Fernandez, H. and Vieira, C. P. (2023). On the identification of potential novel therapeutic targets for ataxia type 1 (SCA1) neurodegenerative disease using EvoPPI3. J Integr Bioinform. PubMed ID: 36848492

Abstract
EvoPPI, a meta-database for protein-protein interactions (PPI), has been upgraded (EvoPPI3) to accept new types of data, namely, PPI from patients, cell lines, and animal models, as well as data from gene modifier experiments, for nine neurodegenerative polyglutamine (polyQ) diseases caused by an abnormal expansion of the polyQ tract. The integration of the different types of data allows users to easily compare them, as here shown for Ataxin-1, the polyQ protein involved in spinocerebellar ataxia type 1 (SCA1) disease. Using all available datasets and the data here obtained for Drosophila melanogaster wt and exp Ataxin-1 mutants (also available at EvoPPI3), this study showed that, in humans, the Ataxin-1 network is much larger than previously thought (380 interactors), with at least 909 interactors. The functional profiling of the newly identified interactors is similar to the ones already reported in the main PPI databases. 16 out of 909 interactors are putative novel SCA1 therapeutic targets, and all but one are already being studied in the context of this disease. The 16 proteins are mainly involved in binding and catalytic activity (mainly kinase activity), functional features already thought to be important in the SCA1 disease.

Patel, N., Alam, N., Libohova, K., Dulay, R., Todi, S. V. and Sujkowski, A. (2023). Phenotypic defects from the expression of wild-type and pathogenic TATA-Binding Proteins in new Drosophila models of Spinocerebellar Ataxia Type 17. G3 (Bethesda). PubMed ID: 37551423

Abstract
Spinocerebellar Ataxia Type 17 (SCA17) is the most recently identified member of the polyglutamine (polyQ) family of disorders, resulting from abnormal CAG/CAA expansion in the TATA box binding protein (TBP), an initiation factor essential for of all eukaryotic transcription. A largely autosomal dominant inherited disease, SCA17 is unique in both its heterogeneous clinical presentation and low incidence of genetic anticipation, the phenomenon in which subsequent generations inherit longer polyQ expansions that yield earlier and more severe symptom onset. Like other polyQ disease family members, SCA17 patients experience progressive ataxia and dementia, and treatments are limited to preventing symptoms and increasing quality of life. This study reports two new Drosophila models that express human TBP with polyQ repeats in either wild-type or SCA17 patient range. TBP expression was found to have age- and tissue-specific effects on neurodegeneration, with polyQ-expanded SCA17 protein expression generally having more severe effects. In addition, SCA17 model flies accumulate more aggregation-prone TBP, with a greater proportion localizing to the nucleus. These new lines provide a new resource for the biochemical characterization of SCA17 pathology and the future identification of therapeutic targets (Patel, 2023).

Sousa, E. S. R., Sousa, A. D., Vieira, J. and Vieira, C. P. (2023). The Josephin domain (JD) containing proteins are predicted to bind to the same interactors: Implications for spinocerebellar ataxia type 3 (SCA3) studies using Drosophila melanogaster mutants. Front Mol Neurosci 16: 1140719. PubMed ID: 37008788

Abstract
Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/ MJD), is the most frequent polyglutamine (polyQ) neurodegenerative disorder. It is caused by a pathogenic expansion of the polyQ tract, located at the C-terminal region of the protein encoded by the ATXN3 gene. This gene codes for a deubiquitinating enzyme (DUB) that belongs to a gene family, that in humans is composed by three more genes (ATXN3L, JOSD1, and JOSD2), that define two gene lineages (the ATXN3 and the Josephins). These proteins have in common the N-terminal catalytic domain (Josephin domain, JD), that in Josephins is the only domain present. In ATXN3 knock-out mouse and nematode models, the SCA3 neurodegeneration phenotype is not, however, reproduced, suggesting that in the genome of these species there are other genes that are able to compensate for the lack of ATXN3. Moreover, in mutant Drosophila melanogaster, where the only JD protein is coded by a Josephin-like gene, expression of the expanded human ATXN3 gene reproduces multiple aspects of the SCA3 phenotype, in contrast with the results of the expression of the wild type human form. In order to explain these findings, phylogenetic, as well as, protein-protein docking inferences are here performed. This study showed multiple losses of JD containing genes across the animal kingdom, suggesting partial functional redundancy of these genes. Accordingly, it is predicted that the JD is essential for binding with ataxin-3 and proteins of the Josephin lineages, and that D. melanogaster mutants are a good model of SCA3 despite the absence of a gene from the ATXN3 lineage. The molecular recognition regions of the ataxin-3 binding and those predicted for the Josephins are, however, different. This study also reports different binding regions between the two ataxin-3 forms (wild-type (wt) and expanded (exp)). The interactors that show an increase in the interaction strength with exp ataxin-3, are enriched in extrinsic components of mitochondrial outer membrane and endoplasmatic reticulum membrane. On the other hand, the group of interactors that show a decrease in the interaction strength with exp ataxin-3 is significantly enriched in extrinsic component of cytoplasm (Sousa, 2023).

Blount, J. R., Patel, N. C., Libohova, K., Harris, A. L., Tsou, W. L., Sujkowski, A. and Todi, S. V. (2023). Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3. bioRxiv. PubMed ID: 37398109

Abstract
Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when it is ubiquitinated at lysine (K) at position 117. K117-ubiqutinated Atxn3 cleaves poly-ubiquitin more rapidly in vitro compared to its unmodified counterpart and this residue is also important for Atxn3 roles in cell culture and in Drosophila melanogaster. How polyQ expansion causes SCA3 remains unclear. To gather insight into the biology of disease of SCA3, the following question was posited: is K117 important for toxicity caused by Atxn3? Transgenic Drosophila lines were generated that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. K117 mutation mildly enhances the toxicity and aggregation of pathogenic Atxn3 in Drosophila. An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest Atxn3 ubiquitination as a regulatory step of SCA3, in part by modulating its aggregation (Blount, 202).

Prifti, M. V., Libohova, K., Harris, A. L., Tsou, W. L. and Todi, S. V. (2022). Ubiquitin-binding site 1 of pathogenic ataxin-3 regulates its toxicity in Drosophila models of Spinocerebellar Ataxia Type 3. Front Neurosci 16: 1112688. PubMed ID: 36733922

Abstract
Spinocerebellar Ataxia Type 3 (SCA3) is a member of the family of polyglutamine (polyQ) diseases that are caused by anomalous CAG triplet repeat expansions in several genes. SCA3 results from abnormal polyQ expansion in the deubiquitinase (DUB), ataxin-3 (Atxn3). To understand the role of the different domains of mutant Atxn3 on its pathogenicity, with the hope that they can be explored for therapeutic interventions, their individual and collective effects on its toxicity was systematically studied. One such domain is ubiquitin-binding site 1 (UbS1) on the catalytic domain of Atxn3; UbS1 is necessary for the enzymatic activity of Atxn3. This study investigated the importance of UbS1 on the toxicity of pathogenic Atxn3 was investigated. Transgenic Drosophila melanogaster lines that express polyQ-expanded Atxn3 were investigated with and without a functional UbS1. Mutating UbS1 markedly exacerbates the toxicity of pathogenic Atxn3. Additional studies indicated that UbS1 regulates the toxicity of Atxn3 not by affecting its aggregation or sub-cellular localization, but by impacting its role in ubiquitin processing. These findings provide additional insights into the role of Atxn3's domains in the pathogenicity of SCA3.

Johnson, S. L., Prifti, M. V., Sujkowski, A., Libohova, K., Blount, J. R., Hong, L., Tsou, W. L. and Todi, S. V. (2022). Drosophila as a Model of Unconventional Translation in Spinocerebellar Ataxia Type 3. Cells 11(7). PubMed ID: 35406787

Abstract
RNA toxicity contributes to diseases caused by anomalous nucleotide repeat expansions. Recent work demonstrated RNA-based toxicity from repeat-associated, non-AUG-initiated translation (RAN translation). RAN translation occurs around long nucleotide repeats that form hairpin loops, allowing for translation initiation in the absence of a start codon that results in potentially toxic, poly-amino acid repeat-containing proteins. Discovered in Spinocerebellar Ataxia Type (SCA) 8, RAN translation has been documented in several repeat-expansion diseases, including in the CAG repeat-dependent polyglutamine (polyQ) disorders. The ATXN3 gene, which causes SCA3, also known as Machado-Joseph Disease (MJD), contains a CAG repeat that is expanded in disease. ATXN3 mRNA possesses features linked to RAN translation. The potential contribution of RAN translation to SCA3/MJD was tested in Drosophila by using isogenic lines that contain homomeric or interrupted CAG repeats. No unconventional translation was observed in fly neurons or glia. However, these investigations indicate differential toxicity from ATXN3 protein-encoding mRNA that contains pure versus interrupted CAG repeats. Additional work suggests that this difference may be due in part to toxicity from homomeric CAG mRNA. It is concluded that Drosophila is not suitable to model RAN translation for SCA3/MJD, but offers clues into the potential pathogenesis stemming from CAG repeat-containing mRNA in this disorder.

Nath, S., Caron, N. S., May, L., Gluscencova, O. B., Kolesar, J., Brady, L., Kaufman, B. A., Boulianne, G. L., Rodriguez, A. R., Tarnopolsky, M. A. and Truant, R. (2022). Functional characterization of variants of unknown significance in a spinocerebellar ataxia patient using an unsupervised machine learning pipeline. Hum Genome Var 9(1): 10. PubMed ID: 35422034

Abstract

CAG-expanded ATXN7 has been previously defined in the pathogenesis of spinocerebellar ataxia type 7 (SCA7), a polyglutamine expansion autosomal dominant cerebellar ataxia. Pathology in SCA7 occurs as a result of a CAG triplet repeat expansion in excess of 37 in the first exon of ATXN7, which encodes ataxin-7 (see Drosophila Ataxin 7). SCA7 presents clinically with spinocerebellar ataxia and cone-rod dystrophy. This study presents a novel spinocerebellar ataxia variant occurring in a patient with mutations in both ATXN7 and TOP1MT, which encodes mitochondrial topoisomerase I (top1mt). Using machine-guided, unbiased microscopy image analysis, alterations were demonstrated in ataxin-7 subcellular localization, and through high-fidelity measurements of cellular respiration, bioenergetic defects in association with top1mt mutations. Ataxin-7 Q35P and top1mt R111W were identified as deleterious mutations, potentially contributing to disease states. These mutations were recapitulated through Drosophila genetic models. This work provides important insight into the cellular biology of ataxin-7 and top1mt and offers insight into the pathogenesis of spinocerebellar ataxia applicable to multiple subtypes of the illness. Moreover, this study demonstrates an effective pipeline for the characterization of previously unreported genetic variants at the level of cell biology.

Wu, Q., Akhter, A., Pant, S., Cho, E., Zhu, J. X., Garner, A., Ohyama, T., Tajkhorshid, E., van Meyel, D. J. and Ryan, R. M. (2022). Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function. J Clin Invest 132(7). PubMed ID: 35167492

Abstract
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1; see Drosophila Eaat1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. This study investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. The results indicate that mutations resulting in decreased hEAAT1 Cl- channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. This study also identified what is believed to be a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Denha, S. A., Atang, A. E., Hays, T. S. and Avery, A. W. (2022). beta-III-spectrin N-terminus is required for high-affinity actin binding and SCA5 neurotoxicity. Sci Rep 12(1): 1726. PubMed ID: 35110634

Abstract
Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). This study investigated in Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). Pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P substitution. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.

Johnson, S. L., Libohova, K., Blount, J. R., Sujkowski, A. L., Prifti, M. V., Tsou, W. L. and Todi, S. V. (2021). Targeting the VCP-binding motif of ataxin-3 improves phenotypes in Drosophila models of Spinocerebellar Ataxia Type 3. Neurobiol Dis 160: 105516. PubMed ID: 34563642

Abstract
Of the family of polyglutamine (polyQ) neurodegenerative diseases, Spinocerebellar Ataxia Type 3 (SCA3) is the most common. Like other polyQ diseases, SCA3 stems from abnormal expansions in the CAG triplet repeat of its disease gene resulting in elongated polyQ repeats within its protein, ataxin-3. Various ataxin-3 protein domains contribute to its toxicity, including the valosin-containing protein (VCP)-binding motif (VBM). A previous study reported that VCP, a homo-hexameric protein, enhances pathogenic ataxin-3 aggregation and exacerbates its toxicity. These findings led to an exploration of the impact of targeting the SCA3 protein by utilizing a decoy protein comprising the N-terminus of VCP (N-VCP) that binds ataxin-3's VBM. The notion was that N-VCP would reduce binding of ataxin-3 to VCP, decreasing its aggregation and toxicity. This study found that expression of N-VCP in Drosophila melanogaster models of SCA3 ameliorated various phenotypes, coincident with reduced ataxin-3 (see Drosophila Ataxin-2)aggregation. This protective effect was specific to pathogenic ataxin-3 and depended on its VBM. Increasing the amount of N-VCP resulted in further phenotype improvement. This work highlights the protective potential of targeting the VCP-ataxin-3 interaction in SCA3, a key finding in the search for therapeutic opportunities for this incurable disorder (Johnson, 2021).

Bhat, S. A., Yousuf, A., Mushtaq, Z., Kumar, V. and Qurashi, A. (2021). Hum Mol Genet. PubMed ID: 33772546

Abstract
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. This study used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. Presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by rCGG repeats mediates presynaptically during larval NMJ development. It is also suggested that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats (Bhat, 2021).

Ghosh, B., Karmakar, S., Prasad, M. and Mandal, A. K. (2021). Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity. Mol Biol Cell: mbcE20110747. PubMed ID: 34161122

Abstract
A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington's disease (HD), this study aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, these findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates (Ghosh, 2021).

Shibata, T., Nagano, K., Ueyama, M., Ninomiya, K., Hirose, T., Nagai, Y., Ishikawa, K., Kawai, G. and Nakatani, K. (2021). Small molecule targeting r(UGGAA)(n) disrupts RNA foci and alleviates disease phenotype in Drosophila model. Nat Commun 12(1): 236. PubMed ID: 33431896

Abstract
Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. This paper reports the discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)(n) repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarifies the mode of binding that recognizes four guanines in the UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)(n) repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSB-mediated splicing events. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates the disease phenotype induced by toxic r(UGGAA)(n) repeat RNA. These studies demonstrate that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases (Shibata, 2021).

Nan, Y., Lin, J., Cui, Y., Yao, J., Yang, Y. and Li, Q. (2021). Protective role of vitamin B6 against mitochondria damage in Drosophila models of SCA3. Neurochem Int 144: 104979. PubMed ID: 33535071

Abstract
Polyglutamine (polyQ)-mediated mitochondria damage is one of the prime causes of polyQ toxicity, which leads to the loss of neurons and the injury of non-neuronal cells. With the discovery of the crucial role of the gut-brain axis and gut microbes in neurological diseases, the relationship between visceral damage and neurological disorders has also received extensive attention. This study successfully simulated the polyQ mitochondrial damage model by expressing 78 or 84 polyglutamine-containing Ataxin3 proteins in Drosophila intestinal enterocytes. In vivo, polyQ expression can reduce mitochondrial membrane potential, mitochondrial DNA damage, abnormal mitochondrial morphology, and loose mitochondrial cristae. Expression profiles evaluated by RNA-seq showed that mitochondrial structural genes and functional genes (oxidative phosphorylation and tricarboxylic acid cycle-related) were significantly down-regulated. More importantly, bioinformatic analyses demonstrated that pathological polyQ expression induced vitamin B6 metabolic pathways abnormality. Active vitamin B6 participates in hundreds of enzymatic reactions and is very important for maintaining mitochondria's activities. In the Spinocerebellar ataxia type 3 Drosophila model, Vitamin B6 supplementation significantly suppressed ECs mitochondria damage in guts and inhibited cellular polyQ aggregates in fat bodies, indicating a promising therapeutic strategy for the treatment of polyQ. Taken together, these results reveal a crucial role for the Vitamin B6-mediated mitochondrial protection in polyQ-induced cellular toxicity, which provides strong evidence for this process as a drug target in polyQ diseases treatment (Nan, 2021).

Singh, A., Hulsmeier, J., Kandi, A. R., Pothapragada, S. S., Hillebrand, J., Petrauskas, A., Agrawal, K., Rt, K., Thiagarajan, D., Jayaprakashappa, D., VijayRaghavan, K., Ramaswami, M. and Bakthavachalu, B. (2021). Antagonistic roles for Ataxin-2 structured and disordered domains in RNP condensation. Elife 10. PubMed ID: 33689682

Abstract

Ataxin-2 (Atx2) is a translational control molecule mutated in spinocerebellar ataxia type II and Als. While intrinsically disordered domains (IDRs) of Atx2 facilitate mRNP condensation into granules, how IDRs work with structured domains to enable positive and negative regulation of target mRNAs remains unclear. Using the Targets of RNA-Binding Proteins Identified by Editing technology, this study identified an extensive data set of Atx2-target mRNAs in the Drosophila brain and S2 cells. Atx2 interactions with AU-rich elements in 3'UTRs appear to modulate stability/turnover of a large fraction of these target mRNAs. Further genomic and cell biological analyses of Atx2 domain deletions demonstrate that Atx2 (1) interacts closely with target mRNAs within mRNP granules, (2) contains distinct protein domains that drive or oppose RNP-granule assembly, and (3) has additional essential roles outside of mRNP granules. These findings increase the understanding of neuronal translational control mechanisms and inform strategies for Atx2-based interventions under development for neurodegenerative disease (Singh, 2021).

Russi, M., Martin, E., D'Autreaux, B., Tixier, L., Tricoire, H. and Monnier, V. (2020). A Drosophila model of Friedreich Ataxia with CRISPR/Cas9 insertion of GAA repeats in the frataxin gene reveals in vivo protection by N-acetyl cysteine. Hum Mol Genet. PubMed ID: 32744307

Abstract
Friedreich Ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, this study generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. It was possible to by-pass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, a progressive increase was observed of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in the fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, it was observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, this model provides the opportunity to elucidate in vivo the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance (Russi, 2020).

Rodriguez, L. R., Calap-Quintana, P., Lapena-Luzon, T., Pallardo, F. V., Schneuwly, S., Navarro, J. A. and Gonzalez-Cabo, P. (2020). Oxidative stress modulates rearrangement of endoplasmic reticulum-mitochondria contacts and calcium dysregulation in a Friedreich's ataxia model. Redox Biol 37: 101762. PubMed ID: 33128998

Abstract
Friedreich ataxia (FRDA) is a neurodegenerative disorder characterized by neuromuscular and neurological manifestations. It is caused by mutations in the FXN gene, which results in loss of the mitochondrial protein frataxin. Endoplasmic Reticulum-mitochondria associated membranes (MAMs) are inter-organelle structures involved in the regulation of essential cellular processes, including lipid metabolism and calcium signaling. This study has analyzed in both, unicellular and multicellular models of FRDA, calcium management and integrity of MAMs. Function of MAMs was observed to be compromised in the cellular model of FRDA, which was improved upon treatment with antioxidants. In agreement, promoting mitochondrial calcium uptake was sufficient to restore several defects caused by frataxin deficiency in Drosophila melanogaster. Remarkably, the findings describe for the first time frataxin as a member of the protein network of MAMs, where interacts with two of the main proteins implicated in endoplasmic reticulum-mitochondria communication. These results suggest a new role of frataxin, indicate that FRDA goes beyond mitochondrial defects and highlight MAMs as novel therapeutic candidates to improve patient's conditions (Rodriguez, 2020).
Suart, C. E., Perez, A. M., Al-Ramahi, I., Maiuri, T., Botas, J. and Truant, R. (2021). Spinocerebellar Ataxia Type 1 protein Ataxin-1 is signalled to DNA damage by Ataxia Telangiectasia Mutated kinase. Hum Mol Genet. PubMed ID: 33772540

Abstract
Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the ataxin-1 protein. Recent genetic correlational studies have implicated DNA damage repair pathways in modifying the age at onset of disease symptoms in SCA1 and Huntington's Disease, another polyglutamine expansion disease. This study demonstrates that both endogenous and transfected ataxin-1 localizes to sites of DNA damage, which is impaired by polyglutamine expansion. This response is dependent on ataxia telangiectasia mutated (ATM) kinase activity. Further, an ATM phosphorylation motif within ataxin-1 at serine 188 was characterized. Reduction of the Drosophila ATM homolog levels in a ATXN1[82Q] Drosophila model through shRNA or genetic cross ameliorates motor symptoms. These findings offer a possible explanation as to why DNA repair was implicated in SCA1 pathogenesis by past studies. The similarities between the ataxin-1 and the huntingtin responses to DNA damage provide further support for a shared pathogenic mechanism for polyglutamine expansion diseases (Suart, 2021).

Johnson, S. L., Ranxhi, B., Libohova, K., Tsou, W. L. and Todi, S. V. (2020). Ubiquitin-interacting motifs of ataxin-3 regulate its polyglutamine toxicity through Hsc70-4-dependent aggregation. Elife 9. PubMed ID: 32955441

Abstract
Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3's non-polyglutamine domains in disease, a new, allelic series of Drosophila melanogaster was utilized. Ataxin-3 pathogenicity was found to be saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. These studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration (Johnson, 2020).

Sujkowski, A., Richardson, K., Prifti, M. V., Wessells, R. J. and Todi, S. V. (2020). (2022). Endurance exercise ameliorates phenotypes in Drosophila models of spinocerebellar ataxias. Elife 11. PubMed ID: 35170431

Abstract
Endurance exercise is a potent intervention with widespread benefits proven to reduce disease incidence and impact across species. While endurance exercise supports neural plasticity, enhanced memory, and reduced neurodegeneration, less is known about the effect of chronic exercise on the progression of movement disorders such as ataxias. This study focused on three different types of ataxias, spinocerebellar ataxias type (SCAs) 2, 3, and 6, belonging to the polyglutamine (polyQ) family of neurodegenerative disorders. In Drosophila models of these SCAs, flies progressively lose motor function. This study observe marked protection of speed and endurance in exercised SCA2 flies and modest protection in exercised SCA6 models, with no benefit to SCA3 flies. Causative protein levels are reduced in SCA2 flies after chronic exercise, but not in SCA3 models, linking protein levels to exercise-based benefits. Further mechanistic investigation indicates that the exercise-inducible protein, Sestrin (Sesn), suppresses mobility decline and improves early death in SCA2 flies, even without exercise, coincident with disease protein level reduction and increased autophagic flux. These improvements partially depend on previously established functions of Sesn that reduce oxidative damage and modulate mTOR activity. This study suggests differential responses of polyQ SCAs to exercise, highlighting the potential for more extensive application of exercise-based therapies in the prevention of polyQ neurodegeneration. Defining the mechanisms by which endurance exercise suppresses polyQ SCAs will open the door for more effective treatment for these diseases.

Zhao, J., Petitjean, D., Haddad, G. A., Batulan, Z. and Blunck, R. (2020). A Common Kinetic Property of Mutations Linked to Episodic Ataxia Type 1 Studied in the Shaker Kv Channel. Int J Mol Sci 21(20). PubMed ID: 33066705

Abstract
Episodic ataxia type 1 is caused by mutations in the KCNA1 gene encoding for the voltage-gated potassium channel Kv1.1. There have been many mutations in Kv1.1 linked to episodic ataxia reported and typically investigated by themselves or in small groups. The aim of this article is to determine whether it is possible to define a functional parameter common to all Kv1.1 mutants that have been linked to episodic ataxia. The disease mutations linked to episodic ataxia were introduced in the drosophila analog of Kv1.1, the Shaker Kv channel, and the channels were expressed in Xenopus oocytes. Using the cut-open oocyte technique, the gating and ionic currents were characterized. The episodic ataxia mutations were found to variably alter the different gating mechanisms described for Kv channels. The common characteristic was a conductance voltage relationship and inactivation shifted to less polarized potentials. It is suggested that a combination of a prolonged action potential and slowed and incomplete inactivation leads to development of ataxia when Kv channels cannot follow or adapt to high firing rates (Zhao, 2020).

Das, S., Kumar, P., Verma, A., Maiti, T. K. and Mathew, S. J. (2019). Myosin heavy chain mutations that cause Freeman-Sheldon syndrome lead to muscle structural and functional defects in Drosophila. Dev Biol. PubMed ID: 30826400

Abstract
Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. Transgenic Drosophila were generated expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and the effect of their expression on Drosophila muscle structure and function was characterized. The results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. Flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, these findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins (Das, 2019).

Ishikawa, K. and Nagai, Y. (2019). Molecular Mechanisms and Future Therapeutics for Spinocerebellar Ataxia Type 31 (SCA31). Neurotherapeutics. PubMed ID: 31755042

Abstract
Spinocerebellar ataxia type 31 (SCA31) is one of the autosomal-dominant neurodegenerative disorders that shows progressive cerebellar ataxia as a cardinal symptom. This disease is caused by a 2.5- to 3.8-kb-long complex pentanucleotide repeat containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n in an intron of the gene called BEAN1 (brain expressed, associated with Nedd4). By comparing various pentanucleotide repeats in this particular locus among control Japanese and Caucasian populations, it was found that (TGGAA)n was the only sequence segregating with SCA31, strongly suggesting the pathogenicity of (TGGAA)n. The complex repeat also lies in an intron of another gene, TK2 (thymidine kinase 2), which is transcribed in the opposite direction, indicating that the complex repeat is bi-directionally transcribed as noncoding repeats. In SCA31 human brains, (UGGAA)n, the BEAN1 transcript of SCA31 mutation was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. Subsequent RNA pulldown analysis disclosed that (UGGAA)n binds to RNA-binding proteins TDP-43, FUS, and hnRNP A2/B1. In fact, TDP-43 was found to co-localize with RNA foci in human SCA31 Purkinje cells. To dissect the pathogenesis of (UGGAA)n in SCA31, transgenic fly models of SCA31 were generated by overexpressing SCA31 complex pentanucleotide repeats in Drosophila. The toxicity of (UGGAA)n was found to be length- and expression level-dependent, and it was dampened by co-expressing TDP-43, FUS, and hnRNP A2/B1. Further investigation revealed that TDP-43 ameliorates (UGGAA)n toxicity by directly fixing the abnormal structure of (UGGAA)n. This led to a proposal that TDP-43 acts as an RNA chaperone against toxic (UGGAA)n. Further research on the role of RNA-binding proteins as RNA chaperones may provide a novel therapeutic strategy for SCA31 (Ishikawa, 2019).

Johnson, S. L., Blount, J. R., Libohova, K., Ranxhi, B., Paulson, H. L., Tsou, W. L. and Todi, S. V. (2019). Differential toxicity of ataxin-3 isoforms in Drosophila models of Spinocerebellar Ataxia Type 3. Neurobiol Dis: 104535. PubMed ID: 31310802

Abstract
\The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein ataxin-3, a deubiquitinase. Two major full-length isoforms of ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. This study took advantage of the fruit fly Drosophila melanogaster to model SCA3 and to examine the toxicity of each ataxin-3 isoform. The assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to these results, although both full-length ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. These findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein (Johnson, 2019).

Ristic, G., Sutton, J. R., Libohova, K. and Todi, S. V. (2018). Toxicity and aggregation of the polyglutamine disease protein, ataxin-3 is regulated by its binding to VCP/p97 in Drosophila melanogaster. Neurobiol Dis [Epub ahead of print]. PubMed ID: 29704548

Abstract
Among the nine dominantly inherited, age-dependent neurodegenerative diseases caused by abnormal expansion in the polyglutamine (polyQ) repeat of otherwise unrelated proteins is Spinocerebellar Ataxia Type 3 (SCA3). SCA3 is caused by polyQ expansion in the deubiquitinase (DUB), ataxin-3. Molecular sequelae related to SCA3 remain unclear. This study sought to understand the role of protein context in SCA3 by focusing on the interaction between this DUB and Valosin-Containing Protein (VCP). VCP is bound directly by ataxin-3 through an arginine-rich area preceding the polyQ repeat. The importance of this interaction was examined in ataxin-3-dependent degeneration in Drosophila melanogaster. Assays with new isogenic fly lines expressing pathogenic ataxin-3 with an intact or mutated VCP-binding site show that disrupting the ataxin-3-VCP interaction delays the aggregation of the toxic protein in vivo. Importantly, early on flies that express pathogenic ataxin-3 with a mutated VCP-binding site are indistinguishable from flies that do not express any SCA3 protein. Also, reducing levels of VCP through RNA-interference has a similar, protective effect to mutating the VCP-binding site of pathogenic ataxin-3. Based on in vivo pulse-chases, aggregated species of ataxin-3 are highly stable, in a manner independent of VCP-binding. Collectively, these results highlight an important role for the ataxin-3-VCP interaction in SCA3, based on a model that posits a seeding effect from VCP on pathogenic ataxin-3 aggregation and subsequent toxicity (Ristic, 2018).

Li, Y. X., Sibon, O. C. M. and Dijkers, P. F. (2018). Inhibition of NF-kappaB in astrocytes is sufficient to delay neurodegeneration induced by proteotoxicity in neurons. J Neuroinflammation 15(1): 261. PubMed ID: 30205834

Abstract
This study examined responses in astrocytes induced by expression of disease-associated, aggregation-prone proteins in other cells. A role was examined for intracellular astrocytic responses in a Drosophila model for Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN3 gene. In this Drosophila SCA3 model, eye-specific expression of a biologically relevant portion of the ATXN3 gene, containing expanded polyQ repeats (SCA3(polyQ78)) was expressed. Eye-specific expression of SCA3(polyQ78) resulted in the presence of astrocytes in the eye, suggesting putative involvement of astrocytes in SCA3. In a candidate RNAi screen, genes in astrocytes were identified that can enhance or suppress SCA3(polyQ78)-induced eye degeneration. Relish, a conserved NF-kappaB transcription factor, was identified as an enhancer of degeneration. Activity of Relish was upregulated in the SCA3 model. Relish can exert its effect via Relish-specific AMPs, since downregulation of these AMPs attenuated degeneration. Relish signaling was examined in astrocytes on neurodegeneration. Selective inhibition of Relish expression specifically in astrocytes extended lifespan of flies that expressed SCA3(polyQ78) exclusively in neurons. Inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer's disease. These data demonstrate that astrocytes respond to proteotoxic stress in neurons, and that these astrocytic responses are important contributors to neurodegeneration. The data provide direct evidence for cell-non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases (Li, 2018). Edenharter, O., Schneuwly, S. and Navarro, J. A. (2018). Mitofusin-dependent ER stress triggers glial dysfunction and nervous system degeneration in a Drosophila model of Friedreich's ataxia. Front Mol Neurosci 11: 38. PubMed ID: 29563863

Abstract
Friedreich's ataxia (FRDA) is the most important recessive ataxia in the Caucasian population. It is caused by a deficit of the mitochondrial<.a> protein frataxin. A forward genetic screen was carried out to analyze genetic interactions between genes controlling mitochondrial homeostasis and Drosophila frataxin. The screen has identified silencing of Drosophila mitofusin (Marf) as a suppressor of FRDA phenotypes in glia. Drosophila Marf is known to play crucial roles in mitochondrial fusion, mitochondrial degradation and in the interface between mitochondria and endoplasmic reticulum (ER). The results indicated that frataxin-deficiency had a small impact on mitochondrial morphology but enhanced mitochondrial clearance and altered the ER stress response in Drosophila. Remarkably, it was demonstrated that downregulation of Marf suppresses ER stress in frataxin-deficient cells and this is sufficient to improve locomotor dysfunction, brain degeneration and lipid dyshomeostasis in the FRDA model. In agreement, chemical reduction of ER stress by means of two different compounds was sufficient to ameliorate the effects of frataxin deficiency in three different fly FRDA models. Altogether, these results strongly suggest that the protection mediated by Marf knockdown in glia is mainly linked to its role in the mitochondrial-ER tethering and not to mitochondrial dynamics or mitochondrial degradation and that ER stress is a novel and pivotal player in the progression and etiology of FRDA. This work might define a new pathological mechanism in FRDA, linking mitochondrial dysfunction due to frataxin deficiency and mitofusin-mediated ER stress, which might be responsible for characteristic cellular features of the disease and also suggests ER stress as a therapeutic target (Edenharter, 2018).

Avery, A. W., Thomas, D. D. and Hays, T. S. (2018). Caffeic acid and resveratrol ameliorate cellular damage in cell and Drosophila models of spinocerebellar ataxia type 3 through upregulation of Nrf2 pathway. Free Radic Biol Med 115: 309-317. PubMed ID: 29247688

Abstract
Polyglutamine (polyQ)-expanded mutant ataxin-3 protein, which is prone to misfolding and aggregation, leads to cerebellar neurotoxicity in spinocerebellar ataxia type 3 (SCA3), an inherited PolyQ neurodegenerative disease. Although the exact mechanism is unknown, the pathogenic effects of mutant ataxin-3 are associated with dysregulation of transcription, protein degradation, mitochondrial function, apoptosis, and antioxidant potency. This study explored the protective role and possible mechanism of caffeic acid (CA) and resveratrol (Res) in cells and Drosophila expressing mutant ataxin-3. Treatment with CA and Res increased the levels of antioxidant and autophagy protein expression with consequently corrected levels of reactive oxygen species, mitochondrial membrane potential, mutant ataxin-3, and the aggregation of mutant ataxin-3 in SK-N-SH-MJD78 cells. Moreover, in SK-N-SH-MJD78 cells, CA and Res enhanced the transcriptional activity of nuclear factor erythroid-derived-2-like 2 (Nrf2), a master transcription factor that upregulates the expression of antioxidant defense genes and the autophagy gene p62. CA and Res improved survival and motor performance in SCA3 Drosophila. Additionally, the above-mentioned protective effects of CA were also observed in CA-supplemented SCA3 Drosophila. Notably, blockade of the Nrf2 pathway by use of small interfering RNA annulled the health effects of CA and Res on SCA3, which affirmed the importance of the increase in Nrf2 activation by CA and Res. Additional studies are need to dissect the protective role of CA and Res in modulating neurodegenerative progression in SCA3 and other polyQ diseases (Wu, 2018). Rimkus, S. A. and Wassarman, D. A. (2018). A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant. PLoS One 13(1): e0190821. PubMed ID: 29338042

Abstract
Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, was performed for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. Further studies of Ronnel were carried out because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T (Rimkus, 2018).
Sowa, A. S., Martin, E., Martins, I. M., Schmidt, J., Depping, R., Weber, J. J., Rother, F., Hartmann, E., Bader, M., Riess, O., Tricoire, H. and Schmidt, T. (2018). Karyopherin alpha-3 is a key protein in the pathogenesis of spinocerebellar ataxia type 3 controlling the nuclear localization of ataxin-3. Proc Natl Acad Sci U S A 115(11): E2624-e2633. PubMed ID: 29476013

Abstract
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, this study investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, focus was placed on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-alpha3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-alpha3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3 (Sowa, 2018).

Avery, A. W., Thomas, D. D. and Hays, T. S.(2017). beta-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization. Proc Natl Acad Sci U S A 114(44): E9376-E9385. PubMed ID: 29078305

Abstract
A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of β-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. This study shows in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, this study provides evidence that the mutation alters the mobility and recruitment of β-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, a Drosophila SCA5 model was developed in which an equivalent mutant Drosophila β-spectrin is expressed in neurons that extend complex dendritic arbors, such as Purkinje cells, targeted in SCA5 pathogenesis. The mutation causes a proximal shift in arborization coincident with decreased β-spectrin localization in distal dendrites. SCA5 β-spectrin dominantly mislocalizes α-spectrin and ankyrin-2, components of the endogenous spectrin cytoskeleton. The data suggest that high-affinity actin binding by SCA5 β-spectrin interferes with spectrin-actin cytoskeleton dynamics, leading to a loss of a cytoskeletal mechanism in distal dendrites required for dendrite stabilization and arbor outgrowth (Avery, 2017).

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that causes gait and limb ataxia, slurred speech, and abnormal eye movements. SCA5 stems from autosomal dominant mutations in the SPTBN2 gene that encodes β-III-spectrin, a cytoskeletal protein predominantly expressed in the brain and enriched in cerebellar Purkinje cells. A necessary function of β-III-spectrin in Purkinje cells was demonstrated by β-III-spectrin-null mice, which show ataxic phenotypes and decreased Purkinje cell dendritic arborization. β-III-spectrin consists of an N-terminal actin-binding domain (ABD) followed by 17 spectrin-repeat domains and a C-terminal pleckstrin homology domain. SCA5 mutations that result in single amino acid substitutions or small in-frame deletions have been identified in the ABD and neighboring spectrin-repeat domains. In a SCA5 mouse model, expression in Purkinje cells of a β-III-spectrin transgene containing a spectrin-repeat domain mutation, E532_M544del, causes ataxic phenotypes and thinning of the cerebellar molecular layer that contains Purkinje cell dendrites. This suggests that the cellular mechanism underlying SCA5 pathogenesis is a Purkinje cell deficit linked to the loss of dendritic arborization (Avery, 2017).

The functional unit of β-III-spectrin is considered to be a heterotetrameric complex containing two β-spectrin subunits and two α-spectrin subunits. Through the β-spectrin subunits the spectrin heterotetramer binds and cross-links actin filaments. Multiple β-spectrin protein isoforms have been shown to form a spectrin-actin cytoskeletal structure that lines the plasma membrane of axons and dendrites. The spectrin-actin lattice is a highly conserved neuronal structure identified in the axons of a broad range of neuron types in mammals and in invertebrates, including Drosophila. A spectrin-actin lattice containing β-III-spectrin, or the homolog β-II-spectrin, was identified in the dendrites of hippocampal neurons. Recent studies suggest that the dendritic spectrin-actin cytoskeleton is a ubiquitous feature of neurons, prominent in both dendritic shafts and spines. The widespread localization of β-III-spectrin within the Purkinje cell dendritic arbor suggests that similar spectrin-actin interactions are important for Purkinje cell dendritic function (Avery, 2017).

The spectrin-actin cytoskeleton functions to organize integral membrane proteins through the spectrin adaptor ankyrin and provides mechanical stability to neuronal processes. A form of erythrocyte ankyrin, ankyrin-R, is expressed in Purkinje cells and appears to be required for Purkinje cell health and normal motor function. A hypomorphic ankyrin-R mutation, termed 'normoblastosis', causes Purkinje cell degeneration and ataxia in mice. The subcellular localization of ankyrin-R in the Purkinje cell soma and dendrites mirrors the distribution of β-III-spectrin, and recently β-III-spectrin was shown to physically interact with ankyrin-R. In β-III-spectrin-null mice, ankyrin-R is present in the soma but absent in Purkinje cell dendrites, suggesting that Purkinje cell degeneration and ataxic phenotypes observed in the absence of β-III-spectrin may be linked to a loss of ankyrin-R function in dendrites. A SCA5 mutation that results in a leucine 253-to-proline (L253P) substitution in the ABD of β-III-spectrin causes ectopically expressed β-III-spectrin and ankyrin-R to colocalize internally in HEK293T cells, in contrast to control cells where wild-type β-III-spectrin colocalizes with ankyrin-R at the plasma membrane. This previous study suggests that neurotoxicity caused by the L253P mutation may be connected to spectrin mislocalization and the concomitant mislocalization of ankyrin-R. However, it has not been established whether the L253P mutation affects the dendritic localization of β-spectrin or ankyrin proteins in any neuronal system (Avery, 2017).

This report extends an analysis of the β-III-spectrin L253P mutation, which was recently demonstrated to cause an ∼1,000-fold increase in the binding affinity of the β-III-spectrin ABD for actin filaments in vitro. The mutation is also destabilizing in vitro, causing the ABD to begin to unfold near physiological temperature. Given these results, a key question with important implications for the SCA5 disease mechanism is whether the previously described mislocalization of L253P β-III-spectrin in mammalian cells is driven by a loss of ABD-binding activity, as originally proposed, or instead is the consequence of increased ABD-binding activity. To address the mechanistic basis of β-III-spectrin dysfunction, this study has characterized the L235P mutant protein behavior in mammalian cells. In addition, a Drosophila SCA5 model was generated in which a Drosophila β-spectrin transgene containing the equivalent mutation is conditionally expressed in dendritic arborization sensory neurons. This study used the Drosophila model to analyze the impact of the mutation on dendritic morphology, an aspect of Purkinje cell dysfunction that potentially underlies SCA5 pathology. In living, fully intact larvae, the consequence were examined of the ABD mutation on dendritic arborization, β-spectrin subcellular localization, and the functional interaction of β-spectrin and ankyrin in dendrites (Avery, 2017).

The morphology of dendritic arbors dictates the connectivity of neuronal networks, integrating inputs and propagating signals. The question of how neurons modulate dendritic morphology is of keen interest in the study of neuronal function and neurodegeneration. For example, the molecular and cell biological mechanisms that control branch stability and remodeling within a dendritic field remain largely elusive. This report describes the consequence of a SCA5 mutation on the binding of β-III-spectrin to actin in mammalian cells and leveraged the Drosophila model system to reveal the impact of the SCA5 disease mutation on the neuronal spectrin-actin cytoskeleton and dendritic arborization. This work identifies an important cytoskeletal mechanism in distal dendrites required for formation of large, complex arbors, critical to the function of Purkinje cells targeted in hereditary ataxias (Avery, 2017).

The data suggest that high-affinity actin binding acts dominantly as a driver of L253P β-III-spectrin neurotoxicity by impacting the dynamics of the spectrin-actin network. Drosophila SCA5 β-spectrin containing the equivalent L253P mutation accumulates in the da neuron soma and is absent in distal dendritic regions, in contrast to wild-type β-spectrin that localizes throughout the arbor. In the axons of mammalian neurons the spectrin-actin lattice initially forms near the soma and propagates distally, suggesting that the loss of Drosophila SCA5 β-spectrin in distal dendrites reflects a defect in expansion of the spectrin-actin cytoskeleton from the soma into dendrites. Such an expansion defect may be a consequence of a slow dissociation rate that is typical of high-affinity molecular interactions. Specifically, high-affinity actin binding caused by the mutation may limit the pool of free β-spectrin molecules available to be recruited to an expanding cytoskeleton. Like the loss of Drosophila SCA5 β-spectrin in da neuron dendritic extensions, a reduction was observed of human L253P β-III-spectrin in HEK293T cell plasma membrane protrusions. The absence of L253P β-III-spectrin in filopodium-like and lamellipodium-like extensions, despite abundant localization elsewhere at the plasma membrane, suggests a partitioning between structurally dynamic and stable membrane regions. This partitioning supports the idea that high-affinity actin binding reduces the availability of β-III-spectrin to be recruited to newly formed membrane structures. The data predict that high-affinity binding of L253P β-III-spectrin to actin filaments within the neuronal spectrin-actin lattice negatively impacts Purkinje cell arborization and function by impeding the expansion of the spectrin-actin cytoskeleton in dynamically growing or remodeling dendritic branches and spines (Avery, 2017).

In addition to increasing actin-binding affinity, the L253P mutation destabilizes β-III-spectrin, causing the ABD to begin to unfold near physiological temperature in vitro. This denaturation raised the possibility that cellular phenotypes in mammalian cells are the consequence of ABD protein unfolding and loss of ABD-binding activity rather than elevated ABD-binding activity. Experiments do not support this interpretation, showing instead through co-IP assays that the mutant ABD retains high-affinity actin binding in cells. Indeed, in cultured mammalian cells, protein unfolding reflected in minor degradation products was detected only when the mutant ABD was highly overexpressed. Significantly, the high-affinity actin binding observed for the L253P mutation is mimicked by the alternative substitution, L253A, which, like the L253P mutation, is predicted to disrupt the normal hydrophobic contacts of leucine 253 in the β-III-spectrin ABD. In this case, no degradation of the L253A mutant is detected, and the increased protein stability of the L253A mutation was confirmed in vitro. In light of these results, the observation that the L253A and L253P mutations cause the same β-III-spectrin subcellular localization phenotypes in HEK293T cells supports the conclusion that the behavior of L253P β-III-spectrin is driven by increased ABD-binding activity. These results support the hypothesis that high-affinity actin binding contributes to the L253P β-III-spectrin neurotoxicity that underlies SCA5 pathology (Avery, 2017).

How do the L253P and L253A substitutions account for elevated actin-binding affinity? The location of the mutations in the CH2 domain is consistent with a suspected regulatory role for the domain in mediating actin binding. Biochemical studies of the isolated CH domains of β-spectrin or of the related α-actinin ABD previously documented actin-binding activity for the CH1 domain but not for the CH2 domain. Confinement of binding activity to the CH1 domain is further supported by a structural model for α-actinin ABD-actin complexes in which only a single CH domain is bound to actin filaments. Consistent with the idea that the L253P mutation disrupts a CH2 domain regulatory function, leucine 253 in the CH2 domain is predicted to interface with the CH1 domain and physically bridge the two domains through hydrophobic contacts. The decrease in hydrophobicity introduced by the L253P or L253A substitution is thus predicted to disrupt inter-CH domain contacts and relieve CH2 inhibition. Significantly, disease-causing mutations located in the CH2 domain of α-actinin or filamin also increase actin-binding affinity (Avery, 2017).

In addition to binding filaments of conventional actin, the β-III-spectrin ABD also interacts with ARP1, a component of the dynactin complex that facilitates transport mediated by microtubule motors. Consistent with an ARP1 interaction, a previous study reported that expression of SCA5 β-spectrin in Drosophila motoneurons impairs axonal transport. Given the ~75% similarity in actin and ARP1 primary structures, it is predicted that the L253P mutation will similarly enhance the binding of β-III-spectrin to ARP1. Current studies have not directly addressed this prediction. However, in a previous study, a bimolecular fluorescence complementation assay conducted in HEK293T cells overexpressing ARP1 concluded that the L253P mutation reduces the interaction of β-III-spectrin ABD with ARP1. Nonetheless, a direct test of how L253P β-III-spectrin impacts ARP1 binding is lacking. Indeed, ARP1-binding studies are not straightforward; the native ARP1 filament is difficult to purify, ARP1-specific antibodies are not available, and there is a strong propensity for ARP1 to form nonnative structures when overexpressed in cells. Further experiments are needed to fully understand the impact of the L253P mutation on ARP1 binding and intracellular vesicular transport (Avery, 2017).

In Drosophila, the lone homolog of β-III-spectrin, β-spectrin, localizes to both dendrites and axons, where the function of the spectrin cytoskeleton has been extensively studied. The impact of SCA5/L246P β-spectrin expression on synaptic organization at the neuromuscular junction (NMJ) has been reported. Spectrin RNAi also disrupts synaptic bouton organization but further leads to NMJ retraction, a phenotype not observed in SCA5 motoneurons. The current work shows that SCA5 β-spectrin is absent not only in distal dendrites but also at the axon terminus of da neurons. Thus, SCA5 β-spectrin may dominantly mislocalize the spectrin cytoskeleton not only in dendrites but also in distal axons. In light of these findings, the reduced size of the Drosophila NMJ reported in motor neurons expressing SCA5 β-spectrin may in part reflect a disruption of the spectrin cytoskeleton and associated ankyrin-2 (Avery, 2017).

This work points to a molecular mechanism in the somatodendritic compartment of neurons that enables the formation of large, complex dendritic arbors. As the dendritic arbor grows, dynamic actin assembly in the distal dendrites drives the formation of new terminal branches. It is suggested that during arbor growth expansion of the spectrin-actin cytoskeleton is required to stabilize terminal branches and allow for continued expansion of the arbor. To explain the mutant SCA5 arbor phenotypes, it is proposed that high-affinity binding of the mutant β-spectrin decreases spectrin-actin dynamics and consequently constrains expansion of the spectrin-actin cytoskeleton and stabilization of growing dendrites. The spectrin cytoskeleton has similarly been implicated in axonal growth and stabilization of synaptic junctions. In support of this cytoskeletal mechanism regulating dendritic arbor stability and potentially underlying SCA5 pathology, this study shows that the loss of β-spectrin, as well as ankyrin-2, in the distal dendrites of Drosophila da neurons correlates with a proximal shift in dendritic branching. Importantly, expression of mutant SCA5 β-spectrin and ankyrin-2 RNAi resulted in similar dendritic arborization defects, and SCA5 β-spectrin causes a loss of ankyrin-2 XL in distal dendrites. This study characterizes a progressive elimination of distal dendrites at segmental boundaries in SCA5 arbors. Moreover, lacking expansion of the spectrin-actin cytoskeleton in terminal dendrites, dynamic actin-based assembly drives complex terminal branching at the periphery of SCA5 arbors. However, the stability of the SCA5 terminal branching is compromised, and the outward growth of the arbor field is defective. One possibility is that impaired expansion of the spectrin-actin cytoskeleton and loss of ankyrin-2 in dendrites impacts localization of neuroglian, a cell-adhesion molecule required for arborization and which may mediate stability of dendritic branching in Drosophila da neurons. In Purkinje cells, it is predicted that L253P β-III-spectrin will similarly impair expansion of the spectrin-actin lattice, disrupting dendritic localization of critical membrane proteins ankyrin-R, EAAT4, and mGluR1α, and in consequence promoting defects in arborization and postsynaptic signaling that characterize SCA5 pathology (Avery, 2017).

Significantly, this model for the impact of a SCA5 mutation on cytoskeletal dynamics and distal arborization is similar to a disease model proposed for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) in which a decrease in mitochondrial dynamics is suggested to disrupt distal Purkinje cell arborization. Like the mislocalization of SCA5 β-spectrin in da neurons, loss of function of the ARSACS disease protein sacsin, a mitochondrial protein, causes mitochondria to accumulate in the soma and proximal dendrites but fail to reach distal dendrites in mammalian neurons. Neurons such as Purkinje cells and da neurons that extend complex arbors appear to be especially vulnerable to disruptions to pathways in distal dendrites that support arborization, and this sensitivity possibly explains the cerebellar specificity of SCA5 pathology (Avery, 2017).

Wu, Y. L., Chang, J. C., Lin, W. Y., Li, C. C., Hsieh, M., Chen, H. W., Wang, T. S., Liu, C. S. and Liu, K. L. (2017). Treatment with caffeic acid and resveratrol alleviates oxidative stress induced neurotoxicity in cell and Drosophila models of Spinocerebellar ataxia type3. Sci Rep 7(1): 11641. PubMed ID: 28912527

Abstract
Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a polyglutamine (polyQ) repeat in the protein ataxin-3 which is involved in susceptibility to mild oxidative stress induced neuronal death. This study shows that caffeic acid (CA) and resveratrol (Res) decreased reactive oxygen species (ROS), mutant ataxin-3 and apoptosis and increased autophagy in the pro-oxidant tert-butyl hydroperoxide (tBH)-treated SK-N-SH-MJD78 cells containing mutant ataxin-3. Furthermore, CA and Res improved survival and locomotor activity and decreased mutant ataxin-3 and ROS levels in tBH-treated SCA3 Drosophila. CA and Res also altered p53 and nuclear factor-kappaB (NF-kappaB) activation and expression in tBH-treated cell and fly models of SCA3, respectively. Blockade of NF-kappaB activation annulled the protective effects of CA and Res on apoptosis, ROS, and p53 activation in tBH-treated SK-N-SH-MJD78 cells, which suggests the importance of restoring NF-kappaB activity by CA and Res. These findings suggest that CA and Res may be useful in the management of oxidative stress induced neuronal apoptosis in SCA3 (Wu, 2017).

Avery, A. W., Thomas, D. D. and Hays, T. S. (2017). beta-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization. Proc Natl Acad Sci U S A 114(44): E9376-e9385. PubMed ID: 29078305

Abstract
A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of beta-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. This study shows in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, evidence is provided that the mutation alters the mobility and recruitment of beta-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, a Drosophila SCA5 model was developed in which an equivalent mutant Drosophila beta-spectrin is expressed in neurons that extend complex dendritic arbors, such as Purkinje cells, targeted in SCA5 pathogenesis. The mutation causes a proximal shift in arborization coincident with decreased beta-spectrin localization in distal dendrites. SCA5 beta-spectrin dominantly mislocalizes alpha-spectrin and ankyrin-2, components of the endogenous spectrin cytoskeleton. These data suggest that high-affinity actin binding by SCA5 beta-spectrin interferes with spectrin-actin cytoskeleton dynamics, leading to a loss of a cytoskeletal mechanism in distal dendrites required for dendrite stabilization and arbor outgrowth (Avery, 2017).
Khare, S., et al. (2017). A KCNC3 mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking. PLoS One 12(5): e0173565. PubMed ID: 28467418

Abstract
The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. This study study focused on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). The clinical phenotype of four SCA13 kindreds are detailed that confirm causation of the KCNC3R423H allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3R423H in Drosophila triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in Drosophila, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. Ocular co-expression of KCNC3R423H with Drosophila epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. These results therefore confirm the KCNC3R423H allele as causative for SCA13, through a dominant negative effect on KCNC3WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology (Khare, 2017).

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Chen, K., Lin, G., Haelterman, N.A., Ho, T.S., Li, T., Li, Z., Duraine, L., Graham, B.H., Jaiswal, M., Yamamoto, S., Rasband, M.N. and Bellen, H.J. (2016). Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration. Elife [Epub ahead of print]. PubMed ID: 27343351

Abstract
Mutations in Frataxin (FXN) cause Friedreich's ataxia (FRDA), a recessive neurodegenerative disorder. Previous studies have proposed that loss of FXN causes mitochondrial dysfunction, which triggers elevated reactive oxygen species (ROS) and leads to the demise of neurons. This study describes a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants. Loss of frataxin homolog (fh) in Drosophila was shown to leads to iron toxicity, which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2). Dampening iron toxicity, inhibiting sphingolipid synthesis by Myriocin, or reducing Pdk1 or Mef2 levels, all effectively suppress neurodegeneration in fh mutants. Moreover, increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved. These results indicate that an iron/sphingolipid/PDk1/Mef2 pathway may play a role in FRDA.

Tsou, W. L., Qiblawi, S. H., Hosking, R. R., Gomez, C. M. and Todi, S. V. (2016). Polyglutamine length-dependent toxicity from alpha1ACT in Drosophila models of spinocerebellar ataxia type 6. Biol Open 5(12): 1770-1775. PubMed ID: 27979829

Abstract
Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that results from abnormal expansion of a polyglutamine (polyQ) repeat. SCA6 is caused by CAG triplet repeat expansion in the gene CACNA1A, resulting in a polyQ tract of 19-33 in patients. CACNA1A, a bicistronic gene, encodes the α1A calcium channel subunit and the transcription factor, alpha1ACT. PolyQ expansion in α1ACT causes degeneration in mice. The first Drosophila models of SCA6 have been described that express α1ACT with a normal (11Q) or hyper-expanded (70Q) polyQ. This study reports additional alpha1ACT transgenic flies, which express full-length α1ACT with a 33Q repeat. α1ACT33Q is toxic in Drosophila, but less so than the 70Q version. When expressed everywhere, α1ACT33Q-expressing adults die earlier than flies expressing the normal allele. α1ACT33Q causes retinal degeneration and leads to aggregated species in an age-dependent manner, but at a slower pace than the 70Q counterpart. According to western blots, α1ACT33Q localizes less readily in the nucleus than α1ACT70Q, providing clues into the importance of polyQ tract length on α1ACT localization and its site of toxicity. These new lines are expected to be highly valuable for future work on SCA6 (Tsou, 2016). Parinejad, N., Peco, E., Ferreira, T., Stacey, S.M. and van Meyel, D.J. (2016). Disruption of an EAAT-mediated chloride channel in a Drosophila model of Ataxia. J Neurosci 36: 7640-7647. PubMed ID: 27445142

Abstract
Patients with Type 6 episodic ataxia (EA6) have mutations of the excitatory amino acid transporter EAAT1 (also known as GLAST), but the underlying pathophysiological mechanism for EA6 is not known. EAAT1 is a glutamate transporter expressed by astrocytes and other glia, and it serves dual function as an anion channel. One EA6-associated mutation is a P>R substitution (EAAT1(P>R)) that in transfected cells has a reduced rate of glutamate transport and an abnormal anion conductance. This study expressed this EAAT1(P>R) mutation in glial cells of Drosophila larvae and found that these larvae exhibit episodic paralysis, and their astrocytes poorly infiltrate the CNS neuropil. These defects are not seen in Eaat1-null mutants, and so they cannot be explained by loss of glutamate transport. To explore the role of the abnormal anion conductance of the EAAT1(P>R) mutation, chloride cotransporters were expressed in astrocytes. Like the EAAT1(P>R) mutation, the chloride-extruding K(+)-Cl(-) cotransporter KccB also causes astroglial malformation and paralysis, supporting the idea that the EAAT1(P>R) mutation causes abnormal chloride flow from CNS glia. In contrast, the Na(+)-K(+)-Cl(-) cotransporter Ncc69, which normally allows chloride into cells, rescues the effects of the EAAT1(P>R) mutation. Together, these results indicate that the cytopathology and episodic paralysis in the Drosophila EA6 model stem from a gain-of-function chloride channelopathy of glial cells.

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Azoospermia/Cryptozoospermia

Riera-Escamilla, A., Vockel, M., Nagirnaja, L., Xavier, M. J., Carbonell, A., Moreno-Mendoza, D., Pybus, M., Farnetani, G., Rosta, V., Cioppi, F., Friedrich, C., Oud, M. S., van der Heijden, G. W., Soave, A., Diemer, T., Ars, E., Sanchez-Curbelo, J., Kliesch, S., O'Bryan, M. K., Ruiz-Castañe, E., Azorín, F., Veltman, J. A., Aston, K. I., Conrad, D. F., Tuttelmann, F. and Krausz, C. (2022). Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure. Am J Hum Genet 109(8): 1458-1471. PubMed ID:
35809576
Summary:
Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. This study aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, it is proposed that 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, this study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.

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Berardinelli-Seip congenital lipodystrophy type 2

Ding, L., Yang, X., Tian, H., Liang, J., Zhang, F., Wang, G., Wang, Y., Ding, M., Shui, G. and Huang, X. (2018). Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism. Embo J 37(17). PubMed ID:
30049710

Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca(2+)-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. This study shows that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, these results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism (Ding, 2018).

Impaired lipid metabolism is associated with an imbalance in energy homeostasis and many other disorders. Excessive lipid storage results in obesity, while a lack of adipose tissue leads to lipodystrophy. Clinical investigations reveal that obesity and lipodystrophy share some common secondary effects, especially non-alcoholic fatty liver disease and severe insulin resistance. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/CGL2) is one of the most severe lipodystrophy diseases. Patients with BSCL2 manifest almost total loss of adipose tissue as well as fatty liver, insulin resistance, and myohypertrophy. BSCL2 results from mutation of the Seipin gene, which is highly conserved from yeast to human (Ding, 2018).

To study the function of Seipin, genetic models were established in different organisms, including yeast, fly, and mouse, and in human cells. As a transmembrane protein residing in the endoplasmic reticulum (ER) and in the vicinity of lipid droplet (LD) budding sites, Seipin has been shown to be involved in LD formation, phospholipid metabolism, lipolysis, and ER calcium homeostasis. As a result of the functional studies in these models, several factors that interact with Seipin protein were identified, such as the phosphatidic acid phosphatase lipin, 14-3-3β, and glycerol-3-phosphate acyltransferase (GPAT). Drosophila Seipin (dSeipin) functions tissue autonomously in preventing ectopic lipid accumulation in salivary gland (a non-adipose tissue) and in promoting lipid storage in fat tissue (Tian, 2011). The non-adipose tissue phenotype is likely attributed to the increased level of phosphatidic acid (PA) generated by elevated GPAT activity. In adipose tissue Seipin interacts with the ER Ca2+-ATPase SERCA, whose activity is reduced in dSeipin mutants, leading to reduced ER calcium levels. Further genetic analysis suggested that the perturbed level of intracellular calcium contributes to the lipodystrophy. However, it is not known how the depleted ER calcium pool causes decreased lipid storage (Ding, 2018).

Besides the ER, mitochondria are another important intracellular calcium reservoir. Mitochondrial calcium is mainly derived from the ER through the IP3R channel. IP3R not only releases calcium from the ER into the cytosol, but also provides sufficient Ca2+ at mitochondrion-associated ER membranes (MAMs) for activation of the mitochondrial calcium uniporter. The mitochondrial Ca2+ level varies greatly in different cell types and can be modulated by influx and efflux channel proteins, such as MCU and NCLX, a mitochondrial Na+/Ca2+ exchanger. A proper mitochondrial Ca2+ level is implicated in mitochondrial integrity and function. Mitochondrial calcium is needed to support the activity of the mitochondrial matrix dehydrogenases in the TCA cycle. TCA cycle intermediates are used for the synthesis of important compounds, including glucose, amino acids, and fatty acids. Acetyl-CoA, as the basic building block of fatty acids, is generally derived from glycolysis, the TCA cycle, and fatty acid β-oxidation. In mammalian adipocytes, acetyl-CoA derived from the TCA cycle intermediate citrate is crucial for de novo lipid biosynthesis, which contributes significantly to lipid storage (Ding, 2018 and references therein).

This study used multiple comparative omics to analyze the proteomic, transcriptomic, and metabolic differences between larval fat cells of dSeipin mutants and wild type. The results reveal an impairment in channeling glycolytic metabolites to mitochondrial metabolism in dSeipin mutant fat cells, and scarcity of mitochondrial Ca2+, are the causative factors of this metabolic dysregulation. Evidence is provided showing that dSeipin lipodystrophy is rescued by restoring mitochondrial calcium or replenishing citrate. It is proposed that the low ER Ca2+ level in dSeipin mutants cannot maintain a sufficiently high mitochondrial Ca2+ concentration to support the TCA reactions. This in turn leads to reduced lipogenesis in dSeipin mutants (Ding, 2018).

Seipin promotes fat tissue lipid storage via calcium-dependent mitochondrial metabolism. Defective ER calcium homeostasis in dSeipin mutants is associated with reduced mitochondrial calcium and impaired mitochondrial function, such as low production of TCA cycle metabolites. Restoring mitochondrial calcium levels or replenishing citrate, a key TCA cycle product and also an important precursor of lipogenesis, rescues the lipid storage defects in dSeipin mutant fat cells (Ding, 2018).

This study investigated the underlying causes of Seipin-dependent lipodystrophy by integrating multiple omic analyses, including RNA-seq, quantitative proteomics, and metabolomic analysis. Compared to previous studies based on genetics and traditional cellular phenotypic analysis, these combinatory omic approaches provide an unprecedented spectrum of molecular phenotypes, which not only add new information but also pinpoint logical directions for further investigations (Ding, 2018).

Omics analyses, in particular lipidomic analysis, have been utilized to investigate the underlying mechanisms in several previous Seipin studies and led to the finding that PA is elevated in several Seipin mutant models. In this study, based on genetic rescuing assays and quantitative proteomics analysis, it was initially proposed that downregulated glycolysis is the cause of lipodystrophy. However, both the RNA-seq results and metabolomic data argue against this possibility and suggest a new mechanism. Despite reduced levels of glycolytic enzymes, transcription of the corresponding genes is not affected, and glycolytic metabolites, in particular pyruvate, are increased in dSeipin mutants compared to wild type. Metabolomic data further show that citrate and isocitrate, which are the products of the first two steps of the mitochondrial TCA cycle, are dramatically decreased in dSeipin mutants, suggesting a defective metabolic flow downstream of pyruvate. These results lead to a new possibility that the lipid storage defects in dSeipin mutants are caused by a defective TCA cycle and this is indeed supported by the metabolic flux analysis. These findings further suggest the involvement of mitochondria. In line with this, the previous discovery that fatty acid β-oxidation is elevated in dSeipin mutant fat cells may reflect compensation for the reduced TCA cycle and lipogenesis. This possibility is supported by the results of genetic and citrate-supplement rescue experiments and by citrate measurements (Ding, 2018).

It is known that glycolytic enzymes and metabolites are regulated by a metabolic feedback loop, which may complicate the explanation of genetic interactions. The current findings highlight that although genetic analysis and rescue results provide important clues, multiple lines of evidence are critical for unraveling complex intracellular pathways. In this case, the combination of omic results and genetic analysis led to the finding that mitochondrial metabolism is important in Seipin-associated lipodystrophy (Ding, 2018).

Mitochondria are hubs in key cellular metabolic processes, including the TCA cycle, ATP production, and amino acid catabolism. Mitochondria also play a central role in lipid homeostasis by controlling two seemingly opposite metabolic pathways, lipid biosynthesis, and fatty acid breakdown. Therefore, impairment of mitochondrial function in different tissues may lead to different, even opposite, phenotypes in lipid storage. In tissues where lipid biosynthesis is the major pathway, defective mitochondria might result in reduced lipid storage, whereas in tissues where fatty acid oxidation prevails, the same defect might lead to increased lipid storage. Reduced lipid storage in dSeipin mutants suggests the former case. The reduced level of citrate and other TCA cycle products in dSeipin mutants suggests an impairment of mitochondrial function. The reduction of OCR and ATP production, the decreased Rhod-2 staining, and the aberrant enrichment of mitochondria within autophagosomes all further support this notion. Interestingly, in mouse brown adipose tissue, Seipin mutation increases mitochondrial respiration along with normal MitoTracker labeling (Zhou, 2016). The discrepancies suggest that Seipin may have cell type-specific functions. Unlike white adipose tissue, which favors lipid storage/biosynthesis, brown adipose tissue is prone to fatty acid breakdown (Ding, 2018).

The link between mitochondria and Seipin was concealed in several previous studies. GPATs, which are recently reported Seipin-interacting proteins, participate in many mitochondrial processes. For example, mitochondria from brown adipocytes that are deficient in GPAT4 exhibit high oxidative levels, and mitochondrial GPAT is required for mitochondrial dynamics. PA, which is elevated in Seipin mutants, is required for mitochondrial morphology and function. Similarly, mitochondrial impairments were also observed in various lipodystrophic conditions. Downregulation of mitochondrial transcription and altered mitochondrial function were indicated in type III congenital generalized lipodystrophy. Multiple mitochondrial metabolic processes are altered in mice with lipodystrophy caused by Zmpste24 mutation. HIV patients treated with anti-retroviral therapy manifest partial lipodystrophy and impaired mitochondria in adipocytes. Moreover, mitochondrial dysfunction in adipose tissue triggers lipodystrophy and systemic disorders in mice. Therefore, the contribution of mitochondrial dysfunction to the cause or development of lipodystrophic conditions warrants further examination (Ding, 2018).

It has been previously reported that dSeipin/SERCA-mediated ER calcium homeostasis is critical for lipid storage (Bi, 2014). Consistent with this, transcripts encoding calcium signaling factors are enriched in the genes that are differentially expressed between dSeipin mutants and wild type. Mitochondrial calcium is transported from the ER through the ER-resident channel IP3R. The reduction of mitochondrial calcium in dSeipin mutant fat cells suggests that the decreased ER calcium leads to an insufficient level of mitochondrial calcium. Importantly, RNAi of a putative Drosophila mitochondrial calcium efflux channel (NCLX/CG18660) not only restores the mitochondrial calcium level but also rescues the lipid storage defects in dSeipin mutants, indicating that mitochondrial calcium is key for dSeipin-mediated lipid storage. This explains the previous finding that the lipid storage defects in dSeipin mutants are rescued by RNAi of RyR, which is not required for ER-mitochondrion calcium transport, but not by RNAi of IP3R (Ding, 2018).

Cellular calcium has been linked to lipid storage and related diseases in recent studies. Comprehensive genetic screening in Drosophila showed that ER calcium-related proteins are key regulators of lipid storage. In particular, SERCA, as the sole ER calcium influx channel and an interacting partner of Seipin, has been repeatedly implicated in lipid metabolism. Dysfunctional lipid metabolism can disrupt ER calcium homeostasis by inhibiting SERCA and further disturbing systemic glucose homeostasis. Increased SERCA expression was shown to have dramatic anti-diabetic benefits in mouse models. In a genomewide association study, SERCA was been found to be associated with obesity. In addition, cellular calcium influx is important for transcriptional programming of lipid metabolism, including lipolysis in mice. The current study further elucidates that ER calcium and mitochondrial calcium are important for cellular lipid homeostasis. It also provides a new insight into the pathogenic mechanism of congenital lipodystrophy (Ding, 2018).

Since Seipin mutations lead to opposite effects on lipid storage in adipose tissue (lipodystrophy) and non-adipose tissues (ectopic lipid storage), numerous studies have been carried out to understand the underlying mechanisms. In Seipin mutants, elevated GPAT activity leads to an increased level of PA. This may cause the formation of supersized lipid droplets in non-adipose cells because of the fusogenic property of PA in lipid leaflets, and may also lead to adipogenesis defects due to the potential role of PA as an inhibitor of preadipocyte differentiation. The Seipin-mediated lipid storage phenotype is further complicated by the role of Seipin in lipid droplet formation, which is mainly studied in unicellular eukaryotic yeast or in cultured cells from multicellular eukaryotic organisms. Seipin has been found in the ER-LD contact sites, which are considered as essential subcellular foci for LD formation/maturation. Moreover, in mammalian adipose tissue, the role of Seipin in lipogenesis or lipolysis may also be masked by the defect in early adipogenesis (Ding, 2018).

How can previous findings in different model organisms and different cell types be reconciled? Seipin has been characterized as a tissue-autonomous lipid modulator. It is likely that Seipin participates in lipid metabolism via distinct mechanisms in different tissues. Alternatively, the metabolic processes that involve Seipin may have different outcomes in different tissues. For example, mitochondria have a different impact on lipid metabolism in different tissues: In non-fat cells, mitochondria mainly direct energy mobilization, whereas in fat cells, mitochondria mainly lead anabolism. The molecular role of Seipin and the phenotypic outcomes in Seipin mutants may rely on specific cellular and developmental contexts (Ding, 2018).

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Birt-Hogg-Dubé (BHD) syndrome

Wang, X., Wu, H., Zhao, L., Liu, Z., Qi, M., Jin, Y. and Liu, W. (2021). FLCN regulates transferrin receptor 1 transport and iron homeostasis. J Biol Chem: 100426. PubMed ID:
33609526 Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previous work identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. This study used human kidney-derived HEK293 cells as a model, and it is reported that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, it was further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome.

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Cardiac Lipotoxicity Disease

Guida, M. C., Birse, R. T., Dall'Agnese, A., Toto, P. C., Diop, S. B., Mai, A., Adams, P. D., Puri, P. L. and Bodmer, R. (2019). Intergenerational inheritance of high fat diet-induced cardiac lipotoxicity in Drosophila. Nat Commun 10(1): 193. PubMed ID:
30643137

Obesity is strongly correlated with lipotoxic cardiomyopathy, heart failure and thus mortality. The incidence of obesity has reached alarming proportions worldwide, and increasing evidence suggests that the parents' nutritional status may predispose their offspring to lipotoxic cardiomyopathy. However, to date, mechanisms underlying intergenerational heart disease risks have yet to be elucidated. This study reports that cardiac dysfunction induced by high-fat-diet (HFD) persists for two subsequent generations in Drosophila and is associated with reduced expression of two key metabolic regulators, adipose triglyceride lipase (ATGL/bmm) and transcriptional cofactor PGC-1. Evidence is provided that targeted expression of ATGL/bmm in the offspring of HFD-fed parents protects them, and the subsequent generation, from cardio-lipotoxicity. Furthermore, it was found that intergenerational inheritance of lipotoxic cardiomyopathy correlates with elevated systemic H3K27 trimethylation. Lowering H3K27 trimethylation genetically or pharmacologically in the offspring of HFD-fed parents prevents cardiac pathology. This suggests that metabolic homeostasis is epigenetically regulated across generations (Guida, 2019).

Several studies have established a critical role for triacylglyceride (TAG) hydrolysis in cardiac metabolism and function, in both healthy and diseased hearts. This study shows that acute HFD (5 days of food supplemented with 30% coconut oil) induces lipotoxic cardiomyopathy that can be inherited by the next two generations, via the parental germlines, even when the offspring are raised on normal food diet (NFD). Similar to what has been described by epidemiological studies on the offspring of obese pregnant women, this study found in the fly model that parental HFD led to first generation progeny with increased adult body weight and increased fat content in late-stage embryos. This was no longer the case in second generation offspring, and adult progeny did not exhibit an increase in fat content relative to body weight in either generations. In contrast, metabolic reprogramming across generations was particularly evident in the systemic reduction in the transcript levels of ATGL/bmm lipase and its downstream target PGC-1/spargel, a key regulator of energy metabolism. The intergenerational lipotoxic cardiomyopathy model was further validated by genetically reducing PGC-1 expression in the parents (PGC-1xp heterozygotes), which causes similar cardiac lipotoxicity as HFD exposure. This was sufficient to alter the +/+ offspring's metabolic state, leading to lipotoxic cardiomyopathy later in life, even though these flies carry two wild-type copies of PGC-1. Of note, the partial reduction of PGC-1 induced by acute and parental HFD in control flies and PGC-1XP heterozygous mutant flies is likely having a profound effect on mitochondrial biogenesis that could be underlying the observed heart dysfunction (Guida, 2019).

The experiments presented in this study indicate that HFD and reduced PGC-1 expression levels have the ability to modify the offspring's metabolism leading to heart dysfunction. Thus, it is speculated that parental HFD-dependent metabolic reprogramming and associated lipotoxic cardiomyopathy in the progeny could be prevented by increasing ATGL/bmm levels. Indeed, targeted transgenic expression of bmm is able to reset the altered metabolic state induced by parental HFD, and thus protects the progeny from cardiac lipotoxicity. Remarkably, induction of bmm expression in the early embryo is sufficient to render the adult progeny (G1), as well as the following generation (G2), resistant to acute HFD (Guida, 2019).

The presented data support the hypothesis that the quality of nutrition during gestation leads to fetal programming that functions as a key determinant in establishing predisposition and/or susceptibility to metabolic and cardiovascular disorders later in life. While this hypothesis is sustained by several epidemiological studies, including the Dutch Hunger Winter studies, and many animal models with different environmental stressors, the underling mechanisms on the intergenerational inheritance of lipotoxic cardiomyopathy induced by parental HFD remain mostly unknown. The Drosophila model in this study indicates that the inheritance of altered histone modifications is a key mechanism in the propagation of lipotoxic cardiomyopathy across generations (Guida, 2019).

Histone modifications are essential in regulating chromatin packaging and gene expression for proper development and cell function. In turn, metabolites serve as co-factors for chromatin modifying enzymes, allowing protein activity and gene expression to match the specific energy requirements. It was found that HFD, and potentially its maladaptive fluctuation in metabolites, can lead to changes in chromatin structure and gene expression that are transmitted to the next generation. Indeed, a major finding presented in this study is the involvement of the PRC2 complex in regulating metabolism and heart function in response to a HFD, via H3K27me3 gene repression across generations. The increase in H3K27me3 global levels in the adult flies upon acute or parental HFD supports the hypothesis that HFD causes changes in the epigenome that have lifelong consequences. The results are in line with previous findings in Caenorhabditis elegans and mice, which provide evidence that PRC2-mediated epigenetic modifications in the germ cells can be transmitted to embryos by sperm and/or oocytes. In addition, this study found that reduction of H3K27me3, either by overexpression of UTX or inhibition of EzH2, can prevent the deleterious effects of a parental HFD on heart function and metabolism. Of note, at this point it cannot be ruled out that UTX overexpression and EzH2 inhibition might also have H3K27me3-independent effects (Guida, 2019).

Overall, using the genetic model of Drosophila, this study provides first evidence that early embryonic and tissue-specific modulation of lipolysis in myocardial progenitors, adipose tissue, and the germline leads to tissue-specific and/or systemic metabolic reprogramming or pre-programming that persists into adulthood. Importantly, the imposed metabolic state appears to be inherited by the next two generations as either a predisposition to metabolic imbalance and cardiac dysfunction, or a protection from HFD insult when bmm is overexpressed. Moreover, this study provides evidence that metabolic re-programming that leads to fat accumulation and cardiac lipotoxicity correlates with overall levels of H3K27me3 epigenetic marks, which can be reversed by genetic or pharmacological reduction of H3K27me3 levels. The findings shed light on possible causes of obesogenic heritability and early adult onset of cardiovascular disease and diabetes, which appear to have their roots in the diet and overall metabolic state of the parents. Importantly, this study demonstrates that targeted genetic or pharmacological interventions in the progeny are able to counteract the detrimental effects on cardiac function of parental dietary insults, a protection that persists even in the subsequent generation against acute HFD. These findings provide new perspectives for tackling metabolic syndrome effects across generations and preventing lipotoxic cardiomyopathies (Guida, 2019).

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Wen, D. T., Zheng, L., Lu, K. and Hou, W. Q. (2021). Activation of cardiac Nmnat/NAD+/SIR2 pathways mediates endurance exercise resistance to lipotoxic cardiomyopathy in aging Drosophila. J Exp Biol 224(18). PubMed ID: 34495320

Endurance exercise is an important way to resist and treat high-fat diet (HFD)-induced lipotoxic cardiomyopathy, but the underlying molecular mechanisms are poorly understood. This study used Drosophila to identify whether cardiac Nmnat/NAD+/SIR2 pathway activation mediates endurance exercise-induced resistance to lipotoxic cardiomyopathy. The results showed that endurance exercise activated the cardiac Nmnat/NAD+/SIR2/FOXO pathway and the Nmnat/NAD+/SIR2/PGC-1α pathway, including up-regulating cardiac Nmnat, SIR2, FOXO and PGC-1α expression, superoxide dismutase (SOD) activity and NAD+ levels, and it prevented HFD-induced or cardiac Nmnat knockdown-induced cardiac lipid accumulation, malondialdehyde (MDA) content and fibrillation increase, and fractional shortening decrease. Cardiac Nmnat overexpression also activated heart Nmnat/NAD+/SIR2 pathways and resisted HFD-induced cardiac malfunction, but it could not protect against HFD-induced lifespan reduction and locomotor impairment. Exercise improved lifespan and mobility in cardiac Nmnat knockdown flies. Therefore, the current results confirm that cardiac Nmnat/NAD+/SIR2 pathways are important antagonists of HFD-induced lipotoxic cardiomyopathy. Cardiac Nmnat/NAD+/SIR2 pathway activation is an important underlying molecular mechanism by which endurance exercise and cardiac Nmnat overexpression give protection against lipotoxic cardiomyopathy in Drosophila (Wen, 2021).

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Karekar, P., Jensen, H. N., Russart, K. L. G., Ponnalagu, D., Seeley, S., Sanghvi, S., Smith, S. A., Pyter, L. M., Singh, H. and Gururaja Rao, S. (2021). Tumor-Induced Cardiac Dysfunction: A Potential Role of ROS. Antioxidants (Basel) 10(8). PubMed ID: 34439547
Summary:

Cancer and heart diseases are the two leading causes of mortality and morbidity worldwide. Many cancer patients undergo heart-related complications resulting in high incidences of mortality. It is generally hypothesized that cardiac dysfunction in cancer patients occurs due to cardiotoxicity induced by therapeutic agents, used to treat cancers and/or cancer-induced cachexia. However, it is not known if localized tumors or unregulated cell growth systemically affect heart function before treatment, and/or prior to the onset of cachexia, hence, making the heart vulnerable to structural or functional abnormalities in later stages of the disease. This study incorporated complementary mouse and Drosophila models to establish if tumor induction indeed causes cardiac defects even before intervention with chemotherapy or onset of cachexia. Focus was placed on one of the key pathways involved in irregular cell growth, the Hippo-Yorkie (Yki) pathway. The transcriptional co-activator of the Yki signaling pathway was overexpressed to induce cellular overgrowth; Yki overexpression in the eye tissue of Drosophila results in compromised cardiac function. These cardiac phenotypes were rescued using antioxidant treatment, with which it is concluded that the Yki induced tumorigenesis causes a systemic increase in ROS affecting cardiac function. These results show that systemic cardiac dysfunction occurs due to abnormal cellular overgrowth or cancer elsewhere in the body; identification of specific cardiac defects associated with oncogenic pathways can facilitate the possible early diagnosis of cardiac dysfunction (Karekar, 2021).

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Cerebellar hypoplasia and dysplasia

Accogli, A., Lu, S., Musante, I., Scudieri, P., Rosenfeld, J. A., ..., Bellen, H. J., Lalani, S. R., Zara, F., Striano, P. and Salpietro, V. (2022). Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development. Cerebellum. PubMed ID:
35218524.
Summary:

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. This study identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, evidence is provided linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, this study shows that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, these results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

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Chediak-Higashi syndrome

Lattao, R., Rangone, H., Llamazares, S. and Glover, D. M. (2021). Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins. Dev Cell 56(7): 1000-1013. PubMed ID:
33725482
Summary:

Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. This study showed that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients (Lattao, 2021).

Autosomal recessive Chediak-Higashi syndrome (CHS) results from a mutation in the lysosomal trafficking regulator (LYST) or CHS1 gene and leads to partial albinism, neurological abnormalities, and recurrent bacterial infections. CHS cells have giant lysosome-related organelles (LROs), compartments that, in addition to lysosomal proteins, contain cell-type-specific proteins. LROs include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and pigment granules of Drosophila. Whether the giant LROs of CHS form through the excessive fusion of LROs or by inhibition of their fission is unclear (Lattao, 2021).

The compromised immune system in CHS is associated with enlarged LROs in natural-killer (NK) cells. NK cells normally become polarized with centrosomes close to their contact site with antigen-presenting cells, the immunological synapse (IS). Despite the formation of a mature IS in CHS NK cells, centrosomes do not correctly polarize and the enlarged LROs neither converge at the centrosome nor translocate to the synapse. Such findings could reflect defective microtubule (MT) organization by the centrosomes in CHS cells, and while some groups describe CHS centrosomes to nucleate fewer MTs, others report normal MT numbers, lengths, and distributions. Thus, the consequence of mutation in LYST for centrosome and MT function is unclear (Lattao, 2021).

Drosophila's LYST counterpart is encoded by mauve (mv) (CG42863). mv mutants show a characteristic eye color due to larger pigment granules, defective cellular immunity through large phagosomes, and enlarged starvation-induced autophagosomes, indicating several types of LRO are affected. The embryo's LROs are the yolk granules (YGs), which provide nutrition and energy during early development. YGs are produced and stored in the egg chamber when the yolk proteins (YPs) of follicle cells are internalized by clathrin-mediated endocytosis and trafficked through the endocytic pathway of the growing oocyte. YGs are present at the periphery of the egg until the early nuclear division cycles of the syncytial embryo, when they translocate to the interior as nuclei migrate to the embryo's cortex in nuclear division cycles 8 and 9. Nurse cells of the egg chamber also supply eggs with endoplasmic-reticulum-derived lipid droplets (LDs), which store maternally provided proteins and neutral lipids for energy and membrane biosynthesis (Lattao, 2021).

This study reveals Mauve's role in regulating LRO/YGs and MT nucleation from centrosomes through the maternal effect lethal (MEL) phenotypes of two new mutant alleles of mauve, mvrosario (mvros) and mv3. Embryos derived from mutant mv females have enlarged YGs that fuse with LDs, and this can be reverted by reducing Rab5 activity. mv-derived embryos also show compromised MT nucleation leading to defects in the embryo's mitotic cycles and cytoskeletal organization. Moreover, a requirement for Mauve in regulating MTs through the TACC/Msps pathway suggests a role for endosomal trafficking in the recruitment or maintenance of pericentriolar material (PCM) components at centrosomes (Lattao, 2021).

Previous studies of Drosophila mv mutants suggested a role for Mauve in suppressing the homotypic fusion of LROs (Rahman, 2012). This study has extended those observations by showing that Mauve also regulates heterotypic fusion between LROs and LDs and by showing that Mauve interacts with molecules that regulate the behavior of interphase and mitotic MTs. This study also shows that dominant-negative Rab5 not only rescues the LRO enlargement defect in mv-derived embryos but also ameliorates recruitment of Msps and PCM at centrosomes. The participation of LDs in LRO fusion that this study now describes could have been previously overlooked because of the lower numbers of LDs in other tissues compared with those in embryos or through specific differences in the mutant alleles under study (Lattao, 2021).

The finding that high levels of Mauve did not induce the formation of smaller sized vesicles together with live imaging of excessive fusion events of autofluorescent vesicles during oogenesis in mv mutant females are consistent with a role for Mauve as a negative regulator of vesicle fusion. The behavior of LDs and the incorporation of their content into the dramatically enlarged YGs of mv-derived embryos is also consistent with this model (Lattao, 2021).

Several lines of evidence support a role for Drosophila Mauve protein in regulating MT nucleation. First, this study found an enrichment of Mv-mCherry around the spindle and centrosomes during mitosis. Second, Mauve co-purifies with γ-tubulin and Msps. Third, the rosario phenotype of mauve-derived embryos is enhanced by mutations in d-tacc or msps, suggesting co-involvement of Mauve and the D-TACC:Msps complex in establishing and/or maintaining the MT-mediated organization of the syncytium that ensures dividing nuclei are at the cortex and endoreduplicating yolk nuclei in the interior. Fourth, embryos derived from mv mutant mothers have reduced amounts of both Msps and γ-tubulin at centrosomes, in accord with the diminished MT nucleating capacity of these centrosomes. Fifth, in line with the reduced amounts of MT nucleating molecules at centrosomes, the regrowth of de-polymerized MTs from centrosomes is compromised in mv-derived embryos (Lattao, 2021).

Mauve's co-purification with Msps, but not its D-TACC partner protein, is another indicator that Msps can exist independently of D-TACC. Indeed, Msps is present in several separate pools: independent of D-TACC at the centrosome; in complex with the D-TACC: Clathrin complex on the spindle; with the MT minus-end protein Patronin to assemble perinuclear non-centrosomal MTOCs (ncMTOCs); with the Augmin complex at kinetochores; and in complex with endosomal proteins such as Mauve. It is speculated that mutations affecting the constitution of Msps complexes at any one of these sites can affect another (Lattao, 2021).

The finding of defects in mitotic MT nucleation by centrosomes in mv-derived embryos suggests that there might be similar requirements at later developmental stages that may have been overlooked because flies can progress through most of the development without functional centrosomes (Lattao, 2021).

The increased NUF seen in mv-derived embryos is likely to be a secondary consequence of disruption to either or both membrane trafficking and mitosis. NUF was first described for the mutant of the nuf gene encoding an ADP ribosylation factor effector that associates with Rab11. Nuf protein is required to organize recycling endosomes in the coordinated processes of membrane trafficking and actin remodeling and embryos deficient for Rab11 also show a strong NUF phenotype. Together this suggests the possibility that NUF in mv mutants could result from the accumulation of endosomal components in the enlarged YGs, which would diminish numbers of recycling endosomes and their associated Rab11-Nuf complex. NUF can also occur as a Chk2 protein kinase-mediated response to DNA damage (DSBs), activated by DNA lesions at mitotic onset. However, this study found no evidence for DNA damage marked by the accumulation of phosphorylated γ-H2Av at DSBs. Finally, NUF also occurs in response to a wide range of primary or secondary mitotic defects. Indeed, failure of the sequestration of histone H2Av to LDs results in embryos that display mitotic defects, nuclear fallout, and reduced viability (Lattao, 2021).

Dominant-negative Rab5 suppresses enlarged YG formation and the mitotic defects of mv-derived embryos in accord with known roles of Rab5 at the early endosome and growing indications of a requirement for Rab5 in mitosis. Rab5 also mediates transient interactions between LDs and early endosomes that enable the transport of lipids between the two without resulting in their fusion. The possibility that Msps transiently localizes to LROs in wild-type embryos cannot be reuled out because LD-YG associations were observed in wild-type embryos and Msps is a component of LDs. The incorporation of Msps and LD markers into the enlarged YGs in mv-derived embryos is also rescued by a dominant-negative form of Rab5 and reciprocally, levels of Msps at centrosomes are restored. This suggests that mutation in mauve leads to mislocalization of Msps around YGs at the expense of its localization at the centrosome and so its availability for mitosis. Suppression of these mv phenotypes by dominant-negative Rab5 could therefore either reflect a passive restoration of the balance of Msps between YGs and spindle poles once YG fusion is prevented or a more active role of Rab5 in organizing the spindle poles (Lattao, 2021).

These findings add to a small but growing body of evidence for the roles of endocytic membrane trafficking in regulating centrosomal function. There are no reports of a membrane-independent role of Rab5, although other groups have reported examples of trafficking proteins involved in MT nucleation in a membrane-independent manner, such as ALIX, a PCM component in human and fly cells, whose recruitment depends on Cnn/Cep215 and D-Spd2/Cep192. The late endosome marker Rab11 also appears to be a part of a dynein-dependent retrograde transport pathway bringing MT nucleating factors and spindle pole proteins to mitotic spindle poles. It is not clear whether Rab5-associated structures mature to Rab11-associated structures in mitosis as they do in interphase but it seems that the two vesicle types might have overlapping functions at centrosomes in mitosis. It will be of future interest to put these current findings into context with these earlier demonstrations of roles of Rab5- and Rab11-containing endosomes in spindle function (Lattao, 2021).

The dynamic relationship between endosomal trafficking and recruitment of MT nucleating molecules onto centrosomes may all have relevance for the role of LYST at the IS and how this is affected in CHS. Thus, it is conceivable that there may be a convergence of the two functions of the LYST protein in lymphocytes, both in regulating the size of LROs and in facilitating the correct positing of centrosomes and membraneous structures. Further studies will be required to clarify the precise roles of LYST in regulating vesicle trafficking and MT nucleation in this particular cell type (Lattao, 2021).

Although the results strongly indicate Mauve to act as a negative regulator of vesicle fusion, this study did not directly assess the fusion ability of LROs. In part, this was limited by the autofluorescent nature of YGs and LDs that restricted the extent to which fluorescently tagged proteins could be used to visualize membrane components of these bodies in dynamic studies. Future work should aim to complement these findings in cell culture and in cell-free systems to determine whether the involvement of both LROs and LDs is widespread. In a similar vein, it will be important to assess whether the roles of LYST proteins in regulating MT dynamics are conserved as implied by these findings. This would require carrying out studies of MT dynamics in other cell types, particularly in mammalian cells (Lattao, 2021).

Rahman, M., Haberman, A., Tracy, C., Ray, S. and Kramer, H. (2012). Drosophila mauve mutants reveal a role of LYST homologs late in the maturation of phagosomes and autophagosomes. Traffic 13(12): 1680-1692. PubMed ID: 22934826

Chediak-Higashi syndrome (CHS) is a lethal disease caused by mutations that inactivate the lysosomal trafficking regulator protein (LYST). Patients suffer from diverse symptoms including oculocutaneous albinism, recurrent infections, neutropenia and progressive neurodegeneration. These defects have been traced back to over-sized lysosomes and lysosome-related organelles (LROs) in different cell types. This study explored mutants in the Drosophila mauve gene as a new model system for CHS. The mauve gene (CG42863) encodes a large BEACH domain protein of 3535 amino acids similar to LYST. This reflects a functional homology between these proteins as mauve mutants also display enlarged LROs, such as pigment granules. This Drosophila model also replicates the enhanced susceptibility to infections, and a defect is shown in the cellular immune response. Early stages of phagocytosis proceed normally in mauve mutant hemocytes but, unlike in wild type, late phagosomes fuse and generate large vacuoles containing many bacteria. Autophagy is similarly affected in mauve fat bodies as starvation-induced autophagosomes grow beyond their normal size. Together these data suggest a model in which Mauve functions to restrict homotypic fusion of different pre-lysosomal organelles and LROs (Rahman, 2012).

Mutations that interfere with the function of human LYST are the molecular cause underlying CHS. This study shows that phenotypes similar to those in CHS patients result from loss-of-functions mutations in mv, which encodes the closest homolog to LYST in the Drosophila genome. The original identification of the mv gene was based on its effect on eye color, which is changed in mv mutants due to their oversized pigment granules. This mirrors the oculocutaneous albinism of CHS patients caused by oversized and clumped melanosomes. Similarly, an important clinical symptom of CHS is the susceptibility to bacterial infections, which is also shared by mv mutants. Furthermore, in mv hemocytes an increased tubular morphology of lysosomes was observed, similar to the changes observed in beige macrophages. Together these morphological and phenotypic similarities support the notion that aspects of CHS can be modeled in mv flies. The unique set of molecular and genetic tools available in Drosophila suggests that this fly model will be useful for the analysis of the molecular mechanisms by which LYST homologs regulate membrane trafficking (Rahman, 2012).

Recurring bacterial infections are among the most frequent clinical complications observed in CHS patients, but is not well understood how the cell biological defects cause the enhanced susceptibility of patients for infections. Defects in phagocytosis have been considered as a possible cause. For example, changes in the phagocytosis of Staphylococcus aureus by leukocytes have been detected in some CHS patients, but several other studies found no loss of phagocytic activity in leukocytes from CHS patients. To gain further insight into the role of LYST homologs in phagocytosis this study used primary hemocytes cultured from Drosophila larvae. These cells have proven to be a useful, genetically tractable model system with markers available for different stages of phagocytosis (Rahman, 2012).

The current data indicate that Mauve is not required for the initial phagocytic uptake of bacteria into hemocytes, in agreement with previous work in human or mouse cells lacking LYST function. However, this study observed that phagocytosed bacteria were amassing in oversized late phagosomes of mv hemocytes. Accumulation of bacteria in phagosomes of CHS leukocytes has previously been observed and primarily attributed to intravacuolar bacterial proliferation. This study shows that even when heat-killed bacteria were used in phagocytosis assays, many more bacteria populated late phagosomes of mv compared to wild-type hemocytes. Furthermore, this difference was not reflective of an altered mode of initial uptake of the bacteria, as phagosomes positive for the early Avl and the intermediate Rab7 markers exhibit the normal distribution of bacterial content. Interestingly, enhanced homotypic fusion and the resulting formation of ‘megasomes’ is a hallmark of monocyte phagosomes containing Helicobacter pylori. This strategy is thought to contribute to the ability of these bacteria to evade the immune system and persist lifelong in human hosts. It is not known by which molecular mechanism H. pylori induces phagosome fusion and thus it is intriguing to speculate that these bacteria may inactivate LYST or an associated factor to promote the formation of megasomes (Rahman, 2012).

Similar to observations with mv, the LYST homolog LvsB has been proposed to function by preventing homotypic fusions of contractile vacuoles in Dictyostelium. Although cells mutant for lvsb displayed significantly enlarged contractile vacuoles, delivery of phagocytosed cargo to these vacuoles was not altered. Observations on phagosome maturation also parallel those on the maturation of secretory lysosomes in CHS cytotoxic T lymphocytes; early steps in the biogenesis of these organelles proceeded indistinguishably from wild-type. Only late in their maturation did secretory lysosomes fuse to form the giant LROs characteristic of CHS (Rahman, 2012).

Therefore, a straightforward explanation for the current data is a function of Mauve, and other LYST homologs, late during the maturation of different LROs to either directly or indirectly suppress their homotypic fusion (Rahman, 2012).

An equivalent function of Mauve may also explain the phenotypes observed during starvation-induced autophagy in mv larval fat bodies. Two key observations are the reduced intensity of LTR staining and the increase in the area of mCherry-Atg8-positive structures. The latter is unlikely to reflect simply an increase in expression of mCherry-Atg8, which is driven by Gal4 under control of a heterologous promoter. Furthermore, an increase in size was visible for mCherry-Atg8 and Rab7-positive structures by immunofluorescence and for autophagosomes morphologically identified by electron microcopy. Instead, these observations are straightforward to reconcile with the notion of Mauve functioning to restrain homotypic fusions of autophagosomes and the resulting increase in autophagosome size and ease of their detection in mv fat body cells (Rahman, 2012).

Other observations do not easily fit the notion of increased fusion events in starved mv fat bodies, however. First, reduced LTR staining that typically labels acidic amphisomes and autolysosomes was observed. One explanation for such an observation would be a reduced rate of the fusion of autophagosomes with late endosomes and lysosomes that yield the acidified amphisomes and autolysosomes. Defects in these fusion events have been observed for subunits of the HOPS complex, which is necessary for the fusion of lysosomes with different organelles. However, none of the experimental systems in which its function has been probed has indicated a requirement of LYST for fusion with lysosomes. Alternatively, after their fusion with lysosomes, the significantly increased size of autophagosomes may result in a dampened or delayed acidification of the resulting autolysosomes thus resulting in a reduced trapping of LTR dye in those hybrid organelles (Rahman, 2012).

Such a change in acidification, whether due to the increased size of autophagosomes or due to another effect of Mauve function, could also explain the observation of a green-shift of mCherry-GFP-Atg8-labeled autophagosomes. This chimeric protein has been developed to measure cargo flux by following quenching of the pH-dependent GFP fluorescence as this indicator moves from autophagosomes, which have a pH similar to the cytosol, to acidified lysosomes. If mv autolysosomes fail to acidify as efficiently as in wild-type, the mCherry-GFP-Atg8 indicator is predicted to exhibit the observed green-shift. Thus, both the reduced LTR staining and green-shift of mCherry-GFP-Atg8-labeled autolysosomes are consistent with an acidification defect that may be a secondary effect of the exceptional size of mv autolysosomes (Rahman, 2012).

A direct inhibitory effect of LYST homologs on limiting membrane fusion is a compelling model to explain the recurring theme of oversized organelles that are observed in the diverse CHS models ranging from CHS patient cells and beige mice to lvsB mutant Dictyostelium and now the Drosophila mv mutant. However, the biochemical mechanism by which LYST homologs execute this function is not clear. One set of possible mechanisms has been suggested based on results of two-hybrid screens that detected interactions between LYST and several proteins involved in regulating membrane fusion events, including subunits of SNARE complexes. Whether LYST homologs engage these or other elements of fusion machineries in vivo to suppress inappropriate fusion of LROs remains to be discovered (Rahman, 2012).

The size of organelles is not only determined by the rate of membrane addition by fusions events, but also by the rate at which membranes are removed by fission events. The notion that LYST may contribute to membrane fission from lysosomes was first supported by the observation that LYST overexpression causes a reduction in the size of lysosomes. Furthermore, in lvsB mutant Dictyostelium cells, a defect in fission may also contribute to the failure of lysosomes to mature to post-lysosomes that fuse with the plasma membrane and recycle internalized cell surface proteins. Defects in fission also emerged as the major difference between beige and wild-type mouse cells when the kinetics were observed with which lysosomes restored their steady-state size after acute disturbances (Rahman, 2012).

The data do not help to distinguish between these two models for the molecular function of LYST homologs. The dynamics of the appearance of oversized late phagosomes strongly points to a role of Mauve in suppressing homotypic fusion or promoting fission late during phagosome maturation. Similarly, oversized autophagosomes and pigment granules may reflect a direct role of Mauve in suppressing inappropriate homotypic fusion during maturation of these organelles in wild-type cells. Alternatively, such phenotypes may be an indirect consequence of altered lysosome and LRO physiology. Distinguishing between these possibilities will require a better understanding of the molecular mechanism by which Mauve affects LRO size. The availability of the fly model may open new genetic and molecular approaches toward this goal (Rahman, 2012).

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Chronic Kidney Disease

BLubojemska, A., Stefana, M. I., Sorge, S., Bailey, A. P., Lampe, L., Yoshimura, A., Burrell, A., Collinson, L. and Gould, A. P. (2021). Adipose triglyceride lipase protects renal cell endocytosis in a Drosophila dietary model of chronic kidney disease. PLoS Biol 19(5): e3001230. PubMed ID:
33945525
Summary:

Abstract

Obesity-related renal lipotoxicity and chronic kidney disease (CKD) are prevalent pathologies with complex aetiologies. One hallmark of renal lipotoxicity is the ectopic accumulation of lipid droplets in kidney podocytes and in proximal tubule cells. Renal lipid droplets are observed in human CKD patients and in high-fat diet (HFD) rodent models, but their precise role remains unclear. This study establish a HFD model in Drosophila that recapitulates renal lipid droplets and several other aspects of mammalian CKD. Cell type-specific genetic manipulations show that lipid can overflow from adipose tissue and is taken up by renal cells called nephrocytes. A HFD drives nephrocyte lipid uptake via the multiligand receptor Cubilin (Cubn), leading to the ectopic accumulation of lipid droplets. These nephrocyte lipid droplets correlate with endoplasmic reticulum (ER) and mitochondrial deficits, as well as with impaired macromolecular endocytosis, a key conserved function of renal cells. Nephrocyte knockdown of diglyceride acyltransferase 1 (DGAT1), overexpression of adipose triglyceride lipase (ATGL; brummer), and epistasis tests together reveal that fatty acid flux through the lipid droplet triglyceride compartment protects the ER, mitochondria, and endocytosis of renal cells. Strikingly, boosting nephrocyte expression of the lipid droplet resident enzyme ATGL is sufficient to rescue HFD-induced defects in renal endocytosis. Moreover, endocytic rescue requires a conserved mitochondrial regulator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α). This study demonstrates that lipid droplet lipolysis counteracts the harmful effects of a HFD via a mitochondrial pathway that protects renal endocytosis. It also provides a genetic strategy for determining whether lipid droplets in different biological contexts function primarily to release beneficial or to sequester toxic lipids.

Odenthal, J., Dittrich, S., Ludwig, V., Merz, T., Reitmeier, K., Reusch, B., H0hne, M., Cosgun, Z. C., Hohenadel, M., Putnik, J., Gobel, H., Rinschen, M. M., Altmuller, J., Koehler, S., Schermer, B., Benzing, T., Beck, B. B., Brinkkotter, P. T., Habbig, S. and Bartram, M. P. (2023). Modeling of ACTN4-Based Podocytopathy Using Drosophila Nephrocytes. Kidney Int Rep 8(2): 317-329. PubMed ID: 36815115
Summary:
Genetic disorders are among the most prevalent causes leading to progressive glomerular disease and, ultimately, end-stags renal disease (ESRD) in children and adolescents. Identification of underlying genetic causes is indispensable for targeted treatment strategies and counseling of affected patients and their families. This study reports on a boy who presented at 4 years of age with proteinuria and biopsy-proven focal segmental glomerulosclerosis (FSGS) that was temporarily responsive to treatment with ciclosporin A. Molecular genetic testing identified a novel mutation in alpha-actinin-4 (p.M240T). A feasible and efficient experimental approach is descibed to test its pathogenicity by combining in silico, in vitro, and in vivo analyses. The de novo p.M240T mutation led to decreased alpha-actinin-4 stability as well as protein mislocalization and actin cytoskeleton rearrangements. Transgenic expression of wild-type human alpha-actinin-4 in Drosophila melanogaster nephrocytes was able to ameliorate phenotypes associated with the knockdown of endogenous actinin. In contrast, p.M240T, as well as other established disease variants p.W59R and p.K255E, failed to rescue these phenotypes, underlining the pathogenicity of the novel alpha-actinin-4 variant. These data highlight that the newly identified alpha-actinin-4 mutation indeed encodes for a disease-causing variant of the protein and promote the Drosophila model as a simple and convenient tool to study monogenic kidney disease in vivo.

Odenthal, J., Dittrich, S., Ludwig, V., Merz, T., Reitmeier, K., Reusch, B., H0hne, M., Cosgun, Z. C., Hohenadel, M., Putnik, J., Gobel, H., Rinschen, M. M., Altmuller, J., Koehler, S., Schermer, B., Benzing, T., Beck, B. B., Brinkkotter, P. T., Habbig, S. and Bartram, M. P. (2023). Modeling of ACTN4-Based Podocytopathy Using Drosophila Nephrocytes. Kidney Int Rep 8(2): 317-329. PubMed ID: 36815115

Title

Genetic disorders are among the most prevalent causes leading to progressive glomerular disease and, ultimately, end-stage renal disease (ESRD) in children and adolescents. Identification of underlying genetic causes is indispensable for targeted treatment strategies and counseling of affected patients and their families. This study reports on a boy who presented at 4 years of age with proteinuria and biopsy-proven focal segmental glomerulosclerosis (FSGS) that was temporarily responsive to treatment with ciclosporin A. Molecular genetic testing identified a novel mutation in alpha-actinin-4 (p.M240T). A feasible and efficient experimental approach is descibed to test its pathogenicity by combining in silico, in vitro, and in vivo analyses. The de novo p.M240T mutation led to decreased alpha-actinin-4 stability as well as protein mislocalization and actin cytoskeleton rearrangements. Transgenic expression of wild-type human alpha-actinin-4 in Drosophila melanogaster nephrocytes was able to ameliorate phenotypes associated with the knockdown of endogenous actinin. In contrast, p.M240T, as well as other established disease variants p.W59R and p.K255E, failed to rescue these phenotypes, underlining the pathogenicity of the novel alpha-actinin-4 variant. These data highlight that the newly identified alpha-actinin-4 mutation indeed encodes for a disease-causing variant of the protein and promote the Drosophila model as a simple and convenient tool to study monogenic kidney disease in vivo.

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CerTra Syndrome

Gehin, C., Lone, M. A., Lee, W., Capolupo, L., Ho, S., Adeyemi, A. M., Gerkes, E. H., Stegmann, A. P., ..., Hornemann, T., D'Angelo, G. and Gennarino, V. A. (2023). CERT1 mutations perturb human development by disrupting sphingolipid homeostasis. J Clin Invest. PubMed ID:
36976648

Abstract

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. This study characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which was called CerTra syndrome. These findings uncover a central role for CERT autoregulation in the control of the sphingolipid biosynthetic flux, provide unexpected insight into the structural organisation of CERT, and suggest a possible therapeutic approach for CerTra syndrome patients.

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Charcot-Marie-Tooth Disease

Bharadwaj, R., Cunningham, K. M., Zhang, K. and Lloyd, T. E. (2015). FIG4 regulates lysosome membrane homeostasis independent of phosphatase function. Hum Mol Genet 25(4): 681-92. PubMed ID:
26662798

Abstract

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, Drosophila models were generated of FIG4-related diseases. Fig4 null mutant flies are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS.

Ermanoska, B., Asselbergh, B., Morant, L., Petrovic-Erfurth, M. L., Hosseinibarkooie, S., Leitao-Goncalves, R., Almeida-Souza, L., Bervoets, S., Sun, L., Lee, L., Atkinson, D., Khanghahi, A., Tournev, I., Callaerts, P., Verstreken, P., Yang, X. L., Wirth, B., Rodal, A. A., Timmerman, V., Goode, B. L., Godenschwege, T. A. and Jordanova, A.(2023). Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling. Nat Commun 14(1): 999. PubMed ID: 36890170
Summary:

Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, YARS1 dysfunction was linked to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, this work shows that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration (Ermanoska, 2023).

Kang, K. H., Han, J. E., Kim, H., Kim, S., Hong, Y. B., Yun, J., Nam, S. H., Choi, B. O. and Koh, H.(2023). PINK1 and Parkin Ameliorate the Loss of Motor Activity and Mitochondrial Dysfunction Induced by Peripheral Neuropathy-Associated HSPB8 Mutants in Drosophila Models. Biomedicines 11(3). PubMed ID: 36979812
Summary:

Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral nerve disorders characterized by progressive muscle weakness and atrophy, sensory loss, foot deformities and steppage gait. Missense mutations in the gene encoding the small heat shock protein HSPB8 (HSP22) have been associated with hereditary neuropathies, including CMT. HSPB8 is a member of the small heat shock protein family sharing a highly conserved α-crystallin domain that is critical to its chaperone activity. This study modeled HSPB8 mutant-induced neuropathies in Drosophila. The overexpression of human HSPB8 mutants in Drosophila neurons produced no significant defect in fly development but led to a partial reduction in fly lifespan. Although these HSPB8 mutant genes failed to induce sensory abnormalities, they reduced the motor activity of flies and the mitochondrial functions in fly neuronal tissue. The motor defects and mitochondrial dysfunction were successfully restored by PINK1 and parkin, which are Parkinson's disease-associated genes that have critical roles in maintaining mitochondrial function and integrity. Consistently, kinetin riboside, a small molecule amplifying PINK1 activity, also rescued the loss of motor activity in the HSPB8 mutant model (Kang, 2023).

Han, J. E., Kang, K. H., Kim, H., Hong, Y. B., Choi, B. O., Koh, H. (2023). PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models. Biochem Biophys Res Commun, 682:71-76 PubMed ID: 37804589
Summary:

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). This study introduced the HSPB3 Y118H (HSPB3(Y118H)) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3(Y118H). Surprisingly, PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology (Han, 2023).

Zuko, A., Mallik, M., Thompson, R., Spaulding, E. L., Wienand, A. R., Been, M., Tadenev, A. L. D., van Bakel, N., Sijlmans, C., Santos, L. A., Bussmann, J., Catinozzi, M., Das, S., Kulshrestha, D., Burgess, R. W., Ignatova, Z. and Storkebaum, E.(2021). tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase. Science 373(6559): 1161-1166. PubMed ID: 34516840
Summary:

Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. This study found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, this study found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. These findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential (Zuko, 2021).

Muraoka, Y., Nikaido, A., Kowada, R., Kimura, H., Yamaguchi, M. and Yoshida, H..(2021). Identification of Rpd3 as a novel epigenetic regulator of Drosophila FIG 4, a Charcot-Marie-Tooth disease-causing gene. Neuroreport 32(7): 562-568. PubMed ID: 33850086
Summary:

Mutations in the factor-induced-gene 4 (FIG 4) gene are associated with multiple disorders, including Charcot-Marie-Tooth disease (CMT), epilepsy with polymicrogyria, Yunis-Varon syndrome and amyotrophic lateral sclerosis. The wide spectrum of disorders associated with FIG 4 may be related to the dysregulated epigenetics. Using Gene Expression Omnibus, this study found that HDAC1 binds to the FIG 4 gene locus in the genome of human CD4+ T cells. Rpd3 is a well-known Drosophila homolog of human HDAC1. Previous work established Drosophila models targeting Drosophila FIG 4 (FIG 4) that exhibited defective locomotive ability, abnormal synapse morphology at neuromuscular junctions, enlarged vacuoles in the fat body and aberrant compound eye morphology. Genetic crossing experiments followed by physiological and immunocytochemical analyses revealed that Rpd3 mutations suppressed these defects induced by dFIG 4 knockdown. This demonstrated that Rpd3 is an important epigenetic regulator of dFIG 4, suggesting that the inhibition of HDAC1 represses the pathogenesis of FIG 4-associated disorders, including CMT. Defects in epigenetic regulators, such as HDAC1, may also explain the diverse symptoms of FIG 4-associated disorders.

Kushimura, Y., Azuma, Y., Mizuta, I., Muraoka, Y., Kyotani, A., Yoshida, H., Tokuda, T., Mizuno, T. and Yamaguchi, M. (2018). Loss-of-function mutation in Hippo suppressed enlargement of lysosomes and neurodegeneration caused by dFIG4 knockdown. Neuroreport 29(10): 856-862. PubMed ID: 29742619

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome-lysosome homeostasis. It was hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. The interactions were examined between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, this study identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. The regulation of endosome-lysosome homeostasis was also identified as a novel probable function of Hippo/MST (Kushimura, 2018).

Ali, M. S., Suda, K., Kowada, R., Ueoka, I., Yoshida, H. and Yamaguchi, M. (2020). Neuron-specific knockdown of solute carrier protein SLC25A46a induces locomotive defects, an abnormal neuron terminal morphology, learning disability, and shortened lifespan. IBRO Rep 8: 65-75. PubMed ID: 32140609

Abstract

Various mutations in the SLC25A46 gene have been reported in mitochondrial diseases that are sometimes classified as type 2 Charcot-Marie-Tooth disease, optic atrophy, and Leigh syndrome. Two Drosophila genes, dSLC25A46a and dSLC25A46b have been identified as candidate homologs of human SLC25A46. In the present study, pan-neuron-specific dSLC25A46a knockdown flies were developed and their phenotypes examined. Neuron-specific dSLC25A46a knockdown resulted in reduced mobility in larvae as well as adults. An aberrant morphology for neuromuscular junctions (NMJs), such as a reduced synaptic branch length and decreased number and size of boutons, was observed in dSLC25A46a knockdown flies. Learning ability was also reduced in the larvae of knockdown flies. In dSLC25A46a knockdown flies, mitochondrial hyperfusion was detected in NMJ synapses together with the accumulation of reactive oxygen species and reductions in ATP. These phenotypes were very similar to those of dSLC25A46b knockdown flies, suggesting that dSLC25A46a and dSLC25A46b do not have redundant roles in neurons. Collectively, these results show that the depletion of SLC25A46a leads to mitochondrial defects followed by an aberrant synaptic morphology, resulting in locomotive defects and learning disability. Thus, the dSLC25A46a knockdown fly summarizes most of the phenotypes in patients with mitochondrial diseases, offering a useful tool for studying these diseases (Ali, 2020).

Bervoets, S., Wei, N., Erfurth, M. L., Yusein-Myashkova, S., Ermanoska, B., Mateiu, L., Asselbergh, B., Blocquel, D., Kakad, P., Penserga, T., Thomas, F. P., Guergueltcheva, V., Tournev, I., Godenschwege, T., Jordanova, A. and Yang, X. L. (2019).Transcriptional dysregulation by a nucleus-localized aminoacyl-tRNA synthetase associated with Charcot-Marie-Tooth neuropathy. Nat Commun 10(1): 5045. PubMed ID: 31695036

Abstract

Charcot-Marie-Tooth disease (CMT) is a length-dependent peripheral neuropathy. The aminoacyl-tRNA synthetases constitute the largest protein family implicated in CMT. Aminoacyl-tRNA synthetases are predominantly cytoplasmic, but are also present in the nucleus. This study shows that a nuclear function of tyrosyl-tRNA synthetase (TyrRS) is implicated in a Drosophila model of CMT. CMT-causing mutations in TyrRS induce unique conformational changes, which confer capacity for aberrant interactions with transcriptional regulators in the nucleus, leading to transcription factor E2F1 hyperactivation. Using neuronal tissues, this study revealed a broad transcriptional regulation network associated with wild-type TyrRS expression, which is disturbed when a CMT-mutant is expressed. Pharmacological inhibition of TyrRS nuclear entry with embelin reduces, whereas genetic nuclear exclusion of mutant TyrRS prevents hallmark phenotypes of CMT in the Drosophila model. These data highlight that this translation factor may contribute to transcriptional regulation in neurons, and suggest a therapeutic strategy for CMT (Bervoets, 2019).

Atkinson, D., Nikodinovic Glumac, J., Asselbergh, B., Ermanoska, B., Blocquel, D., Steiner, R., Estrada-Cuzcano, A., Peeters, K., Ooms, T., De Vriendt, E., Yang, X. L., Hornemann, T., Milic Rasic, V. and Jordanova, A. (2017). Sphingosine 1-phosphate lyase deficiency causes Charcot-Marie-Tooth neuropathy. Neurology [Epub ahead of print]. PubMed ID: 28077491

Abstract

This study sought to identify the unknown genetic cause in a family with an axonal form of peripheral neuropathy and atypical disease course. Both patients presented an atypical form of axonal peripheral neuropathy, characterized by acute or subacute onset and episodes of recurrent mononeuropathy. Compound heterozygous mutations were identified cosegregating with disease that were absent in controls in the SGPL1 gene, encoding sphingosine 1-phosphate lyase (SPL). The p.Ser361* mutation triggers nonsense-mediated mRNA decay. The missense p.Ile184Thr mutation causes partial protein degradation. The plasma levels of sphingosine 1-phosphate and sphingosine/sphinganine ratio were increased in the patients. Neuron-specific downregulation of the Drosophila orthologue, Sphingosine-1-phosphate lyase impaired the morphology of the neuromuscular junction and caused progressive degeneration of the chemosensory neurons innervating the wing margin bristles. It is suggested that SPL deficiency is a cause of a distinct form of Charcot-Marie-Tooth disease in humans, thus extending the currently recognized clinical and genetic spectrum of inherited peripheral neuropathies. These data emphasize the importance of sphingolipid metabolism for neuronal function.

Kyotani, A., Azuma, Y., Yamamoto, I., Yoshida, H., Mizuta, I., Mizuno, T., Nakagawa, M., Tokuda, T. and Yamaguchi, M. (2015). Knockdown of the Drosophila FIG4 induces deficient locomotive behavior, shortening of motor neuron, axonal targeting aberration, reduction of life span and defects in eye development. Exp Neurol [Epub ahead of print]. PubMed ID: 26708557

Abstract
Mutations in Factor-Induced-Gene 4 (FIG4) gene have been identified in Charcot-Marie-Tooth disease type 4J (CMT4J), Yunis-Varon syndrome and epilepsy with polymicrogyria. FIG4 protein regulates a cellular abundance of phosphatidylinositol 3,5-bisphosphate (PI3,5P2), a signaling lipid on the cytosolic surface of membranes of the late endosomal compartment. PI3,5P2 is required for retrograde membrane trafficking from lysosomal and late endosomal compartments to the Golgi. However, it is still unknown how the neurodegeneration that occurs in these diseases is related to the loss of FIG4 function. Drosophila has CG17840 (dFIG4) as a human FIG4 homologue. This study specifically knocked down dFIG4 in various tissues, and investigated their phenotypes. Neuron-specific knockdown of dFIG4 results in axonal targeting aberrations of photoreceptor neurons, shortened presynaptic terminals of motor neurons in 3rd instar larvae and reduced climbing ability in adulthood and life span. Fat body-specific knockdown of dFIG4 results in enlarged lysosomes in cells that were detected by staining with LysoTracker. In addition, eye imaginal disc-specific knockdown of dFIG4 disrupts differentiation of pupal ommatidial cell types, such as cone cells and pigment cells, suggesting an additional role of dFIG4 during eye development (Kyotani, 2015).

El Fissi, N., Rojo, M., Aouane, A., Karatas, E., Poliacikova, G., David, C., Royet, J. and Rival, T. (2018). Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy. EMBO Rep. PubMed ID: 29898954

Abstract

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by dominant alleles of the mitochondrial pro-fusion factor Mitofusin 2 (MFN2; see Drosophila Marf). To address the consequences of these mutations on mitofusin activity and neuronal function, this study generate Drosophila models expressing in neurons the two most frequent substitutions (R94Q and R364W, the latter never studied before) and two others localizing to similar domains (T105M and L76P). All alleles trigger locomotor deficits associated with mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism and increased mtDNA mutations, but they differently alter mitochondrial morphology and organization. Substitutions near or within the GTPase domain (R94Q, T105M) result in loss of function and provoke aggregation of unfused mitochondria. In contrast, mutations within helix bundle 1 (R364W, L76P) enhance mitochondrial fusion, as demonstrated by the rescue of mitochondrial alterations and locomotor deficits by over-expression of the fission factor DRP1. In conclusion, this study shows that both dominant negative and dominant active forms of mitofusin can cause CMT2A-associated defects and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients (El Fissi, 2018).

Lopez Del Amo, V., Palomino-Schatzlein, M., Seco-Cervera, M., Garcia-Gimenez, J. L., Pallardo, F. V., Pineda-Lucena, A. and Galindo, M. I. (2017). A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration. Biochim Biophys Acta 1863(3): 801-809. PubMed ID: 28065847

Abstract
Charcot-Marie-Tooth disease is a rare peripheral neuropathy for which there is no specific treatment. Some forms of Charcot-Marie-Tooth are due to mutations in the GDAP1 gene. A striking feature of mutations in GDAP1 is that they have a variable clinical manifestation, according to disease onset and progression, histology and mode of inheritance. Studies in cellular and animal models have revealed a role of GDAP1 in mitochondrial morphology and distribution, calcium homeostasis and oxidative stress. To get a better understanding of the disease mechanism, this study generated models of over-expression and RNA interference of the Drosophila Gdap1 gene. In order to get an overview about the changes that Gdap1 mutations cause in this disease model, a comprehensive determination of the metabolic profile in the flies by nuclear magnetic resonance spectroscopy was combined with gene expression analyses and biophysical tests. The results revealed that both up- and down-regulation of Gdap1 results in an early systemic inactivation of the insulin pathway before the onset of neuromuscular degeneration, followed by an accumulation of carbohydrates and an increase in the beta-oxidation of lipids. These findings are in line with emerging reports of energy metabolism impairments linked to different types of neural pathologies caused by defective mitochondrial function, which is not surprising given the central role of mitochondria in the control of energy metabolism. The relationship of mitochondrial dynamics with metabolism during neurodegeneration opens new avenues to understand the cause of the disease, and for the discovery of new biomarkers and treatments.

Grice, S. J., Sleigh, J. N. and Zameel Cader, M. (2018). Plexin-semaphorin signaling modifies neuromuscular defects in a Drosophila model of peripheral neuropathy. Front Mol Neurosci 11: 55. PubMed ID: 29520219

Abstract

Dominant mutations in GARS, encoding the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and Charcot-Marie-Tooth disease type 2D (CMT2D). This genetic disorder exemplifies a recurring paradigm in neurodegeneration, in which mutations in essential genes cause selective degeneration of the nervous system. Recent evidence suggests that the mechanism underlying CMT2D involves extracellular neomorphic binding of mutant GlyRS to neuronally-expressed proteins. Consistent with this, previous studies indicate a non-cell autonomous mechanism, whereby mutant GlyRS is secreted and interacts with the neuromuscular junction (NMJ). In this Drosophila model for CMT2D, it was previously shown that mutant gars expression decreases viability and larval motor function, and causes a concurrent build-up of mutant GlyRS at the larval neuromuscular presynapse. This study reports additional phenotypes that closely mimic the axonal branching defects of Drosophila plexin transmembrane receptor mutants, implying interference of plexin signaling in gars mutants. Individual dosage reduction of two Drosophila Plexins, plexin A (plexA) and B (plexB) enhances and represses the viability and larval motor defects caused by mutant GlyRS, respectively. However, plexB levels, but not plexA levels, modify mutant GlyRS association with the presynaptic membrane. Furthermore, increasing availability of the plexB ligand, Semaphorin-2a (Sema2a), alleviates the pathology and the build-up of mutant GlyRS, suggesting competition for PlexB binding may be occurring between these two ligands. This toxic gain-of-function and subversion of neurodevelopmental processes indicate that signaling pathways governing axonal guidance could be integral to neuropathology and may underlie the non-cell autonomous CMT2D mechanism (Grice, 2018).

Kushimura, Y., Azuma, Y., Mizuta, I., Muraoka, Y., Kyotani, A., Yoshida, H., Tokuda, T., Mizuno, T. and Yamaguchi, M. (2018). Loss-of-function mutation in Hippo suppressed enlargement of lysosomes and neurodegeneration caused by dFIG4 knockdown. Neuroreport 29(10): 856-862. PubMed ID: 29742619

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome-lysosome homeostasis. It was hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. The interactions were examined between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, this study identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. The regulation of endosome-lysosome homeostasis was also identified as a novel probable function of Hippo/MST (Kushimura, 2018).

Muraoka, Y., Nakamura, A., Tanaka, R., Suda, K., Azuma, Y., Kushimura, Y., Lo Piccolo, L., Yoshida, H., Mizuta, I., Tokuda, T., Mizuno, T., Nakagawa, M. and Yamaguchi, M. (2018). Genetic screening of the genes interacting with Drosophila FIG4 identified a novel link between CMT-causing gene and long noncoding RNAs. Exp Neurol 310: 1-13. PubMed ID: 30165075

Abstract

Neuron-specific knockdown of the dFIG4 gene, a Drosophila homologue of human FIG4 and one of the causative genes for Charcot-Marie-Tooth disease (CMT), reduces the locomotive abilities of adult flies, as well as causing defects at neuromuscular junctions, such as reduced synaptic branch length in presynaptic terminals of the motor neurons in third instar larvae. Eye imaginal disc-specific knockdown of dFIG4 induces abnormal morphology of the adult compound eye, the rough eye phenotype. A modifier screening of the dFIG4 knockdown-induced rough eye phenotype was carried out using a set of chromosomal deficiency lines on the second chromosome. By genetic screening, 9 and 15 chromosomal regions were detected whose deletions either suppressed or enhanced the rough eye phenotype induced by the dFIG4 knockdown. By further genetic screening with mutants of individual genes in one of these chromosomal regions, the gene CR18854 was identified that suppressed the rough eye phenotype and the loss-of-cone cell phenotype. The CR18854 gene encodes a long non-coding RNA (lncRNA) consisting of 2566 bases. Mutation and knockdown of CR18854 patially suppressed the enlarged lysosome phenotype induced by Fat body-specific knockdown of dFIG4. Further characterization of CR18854, and a few other lncRNAs in relation to dFIG4 in neuron, using neuron-specific dFIG4 knockdown flies indicated a genetic link between the dFIG4 gene and lncRNAs including CR18854 and hsromega. Data was obtained indicating genetic interaction between CR18854 and Cabeza, a Drosophila homologue of human FUS, which is one of the causing genes for amyotrophic lateral sclerosis (ALS). These results suggest that lncRNAs such as CR18854 and hsromega are involved in a common pathway in CMT and ALS pathogenesis (Muraoka, 2018).

Shimada, S., Muraoka, Y., Ibaraki, K., Takano-Shimizu-Kouno, T., Yoshida, H. and Yamaguchi, M. (2019). Identification of CR43467 encoding a long non-coding RNA as a novel genetic interactant with dFIG4, a CMT-causing gene. Exp Cell Res: 111711. PubMed ID: 31704059
Abstract
The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. A previous modifier screening on the dFIG4 knockdown-induced rough eye phenotype identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA), as genetic interactants with dFIG4. The present study, in more extensive genetic screening, found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, a CR43467-specific knockdown line was established using the CRISPR-dCas9 system. By using this system, it was demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. This study has identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.

Kodani, A., Yamaguchi, M., Itoh, R., Huynh, M. A. and Yoshida, H. (2022). A Drosophila model of the neurological symptoms in Mpv17-related diseases. Sci Rep 12(1): 22632. PubMed ID: 36587049
Abstract
Mutations in the Mpv17 gene are responsible for MPV17-related hepatocerebral mitochondrial DNA depletion syndrome and Charcot-Marie-Tooth (CMT) disease. CG11077 (Drosophila Mpv17; dMpv17), an ortholog of human MPV17, was knocked down in the nervous system in Drosophila melanogaster and the behavioral and cellular phenotypes were investigated. The resulting dMpv17 knockdown larvae showed impaired locomotor activity and learning ability consistent with mitochondrial defects suggested by the reductions in mitochondrial DNA and ATP production and the increases in the levels of lactate and reactive oxygen species. Furthermore, an abnormal morphology of the neuromuscular junction, at the presynaptic terminal, was observed in dMpv17 knockdown larvae. These results reproduce well the symptoms of human diseases and partially reproduce the phenotypes of Mpv17-deficient model organisms. Therefore, it is suggested that neuron-specific dMpv17 knockdown in Drosophila is a useful model for investigation of MPV17-related hepatocerebral mitochondrial DNA depletion syndrome and CMT caused by Mpv17 dysfunction.

Ermanoska, B., Asselbergh, B., Morant, L., Petrovic-Erfurth, M. L., Hosseinibarkooie, S., Leitao-Gonçalves, R., Almeida-Souza, L., Bervoets, S., Sun, L., Lee, L., Atkinson, D., Khanghahi, A., Tournev, I., Callaerts, P., Verstreken, P., Yang, X. L., Wirth, B., Rodal, A. A., Timmerman, V., Goode, B. L., Godenschwege, T. A. and Jordanova, A. (2023). Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling. Nat Commun 14(1): 999. PubMed ID: 36890170
Summary:

Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, YARS1 dysfunction was linked to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, this work shows that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.

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Chronic Obstructive Pulmonary Disease

Rouka, E., Gourgoulianni, N., Lupold, S., Hatzoglou, C., Gourgoulianis, K. I. and Zarogiannis, S. G. (2022). Prediction and enrichment analyses of the Homo sapiens-Drosophila melanogaster COPD-related orthologs: potential for modeling of human COPD genomic responses with the fruit fly. Am J Physiol Regul Integr Comp Physiol 322(1): R77-r82. PubMed ID:
34877887

Abstract
The significant similarities in airway epithelial cells between mammals and the fruit fly Drosophila melanogaster have rendered the latter an important model organism for studies of chronic inflammatory lung diseases. Focusing on the chronic obstructive pulmonary disease (COPD), this study mapped human gene orthologs associated with this disease in D. melanogaster to identify functionally equivalent genes for immediate, further screening with the fruit fly model. The DIOPT-DIST tool was accessed for the prediction of the COPD-associated orthologs between humans and Drosophila. Enrichment analyses with respect to pathways of the retrieved functional homologs were performed using the ToppFun and FlyMine tools, identifying 73 unique human genes as well as 438 fruit fly genes. The ToppFun analysis verified that the human gene list is associated with COPD phenotypes. Furthermore, the FlyMine investigation highlighted that the Drosophila genes are functionally connected mainly with the "ABC-family proteins mediated transport" and the "β-catenin-independent WNT signaling pathway." These results suggest an evolutionarily conserved role toward responses to inhaled toxicants and CO(2) in both species. It is reasoned that the predicted orthologous genes should be further studied in the Drosophila models of cigarette smoke-induced COPD.

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Ciliopathy

Hou, Y., Wu, Z., Zhang, Y., Chen, H., Hu, J., Guo, Y., Peng, Y. and Wei, Q. (2020). Functional Analysis of Hydrolethalus Syndrome Protein HYLS1 in Ciliogenesis and Spermatogenesis in Drosophila. Front Cell Dev Biol 8: 301. PubMed ID:
32509774

Abstract
Cilia and flagella are conserved subcellular organelles, which arise from centrioles and play critical roles in development and reproduction of eukaryotes. Dysfunction of cilia leads to life-threatening ciliopathies. HYLS1 is an evolutionarily conserved centriole protein, which is critical for ciliogenesis, and its mutation causes ciliopathy-hydrolethalus syndrome. However, the molecular function of HYLS1 remains elusive. This study investigated the function of HYLS1/CG42231 in cilia formation using the Drosophila model. Drosophila HYLS1 is a conserved centriole and basal body protein. Deletion of HYLS1 led to sensory cilia dysfunction and spermatogenesis abnormality. Importantly, this study found that Drosophila HYLS1 is essential for giant centriole/basal body elongation in spermatocytes and is required for spermatocyte centriole to efficiently recruit pericentriolar material and for spermatids to assemble the proximal centriole-like structure (the precursor of the second centriole for zygote division). Hence, by taking advantage of the giant centriole/basal body of Drosophila spermatocyte, this study uncovered previously uncharacterized roles of HYLS1 in centriole elongation and assembly (Hou, 2020).

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Cocaine Use Disorder

Huggett, S. B., Hatfield, J. S., Walters, J. D., McGeary, J. E., Welsh, J. W., Mackay, T. F. C., Anholt, R. R. H. and Palmer, R. H. C. (2021). Ibrutinib as a potential therapeutic for cocaine use disorder. Transl Psychiatry 11(1): 623. PubMed ID:
34880215
Summary:
Cocaine use presents a worldwide public health problem with high socioeconomic cost. No current pharmacologic treatments are available for cocaine use disorder (CUD) or cocaine toxicity. To explore pharmaceutical treatments for this disorder and its sequelae gene expression data was analyzed from post-mortem brain tissue of individuals with CUD who died from cocaine-related causes with matched cocaine-free controls (n = 71, M(age) = 39.9, 100% male, 49% with CUD, 3 samples/brain regions). To match molecular signatures from brain pathology with potential therapeutics, this study leveraged the L1000 database honing in on neuronal mRNA profiles of 825 repurposable compounds (e.g., FDA approved). 16 compounds were identified that were negatively associated with CUD gene expression patterns across all brain regions, all of which outperformed current targets undergoing clinical trials for CUD. An additional 43 compounds were positively associated with CUD expression. An in silico follow-up potential therapeutics was performed using independent transcriptome-wide in vitro and in vivo (mouse cocaine self-administration) datasets to prioritize candidates for experimental validation. Among these medications, ibrutinib was consistently linked with the molecular profiles of both neuronal cocaine exposure and mouse cocaine self-administration. The therapeutic efficacy of ibrutinib was assessed using the Drosophila melanogaster model. Ibrutinib reduced cocaine-induced startle response and cocaine-induced seizures, despite increasing cocaine consumption. These results suggest that ibrutinib could be used for the treatment of cocaine use disorder.

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Colorectal cancer

Bangi, E., Murgia, C., Teague, A.G., Sansom, O.J. and Cagan, R.L. (2016). Functional exploration of colorectal cancer genomes using Drosophila. Nat Commun 7: 13615. PubMed ID:
27897178

Abstract

The multigenic nature of human tumours presents a fundamental challenge for cancer drug discovery. This study used Drosophila to generate 32 multigenic models of colon cancer using patient data from The Cancer Genome Atlas. These models recapitulate key features of human cancer, often as emergent properties of multigenic combinations. Multigenic models such as ras p53 pten apc exhibit emergent resistance to a panel of cancer-relevant drugs. Exploring one drug in detail, a mechanism of resistance for the PI3K pathway inhibitor BEZ235 was identified. Based on this, a combinatorial therapy that circumvents this resistance through a two-step process of emergent pathway dependence and sensitivity termed 'induced dependence' was developed. This approach is effective in cultured human tumour cells, xenografts and mouse models of colorectal cancer. These data demonstrate how multigenic animal models that reference cancer genomes can provide an effective approach for developing novel targeted therapies (Bangi, 2016).

A Personalized Therapeutics Approach Using an In Silico Drosophila Patient Model Reveals Optimal Chemo- and Targeted Therapy Combinations for Colorectal Cancer. Front Oncol 11: 692592. PubMed ID: 34336681

Abstract

In silico models of biomolecular regulation in cancer, annotated with patient-specific gene expression data, can aid in the development of novel personalized cancer therapeutic strategies. Drosophila melanogaster is a well-established animal model that is increasingly being employed to evaluate such preclinical personalized cancer therapies. This study reports five Boolean network models of biomolecular regulation in cells lining the Drosophila midgut epithelium and annotate them with colorectal cancer patient-specific mutation data to develop an in silico Drosophila Patient Model (DPM). Cell-type-specific RNA-seq gene expression data from the FlyGut-seq database were employed to annotate and then validate these networks. Next, three literature-based colorectal cancer case studies were used to evaluate cell fate outcomes from the model. Results obtained from analyses of the proposed DPM help: (i) elucidate cell fate evolution in colorectal tumorigenesis, (ii) validate cytotoxicity of nine FDA-approved CRC drugs, and (iii) devise optimal personalized treatment combinations. The personalized network models helped identify synergistic combinations of paclitaxel-regorafenib, paclitaxel-bortezomib, docetaxel-bortezomib, and paclitaxel-imatinib for treating different colorectal cancer patients. Follow-on therapeutic screening of six colorectal cancer patients from cBioPortal using this drug combination demonstrated a 100% increase in apoptosis and a 100% decrease in proliferation. In conclusion, this work outlines a novel roadmap for decoding colorectal tumorigenesis along with the development of personalized combinatorial therapeutics for preclinical translational studies (Gondal, 2021).

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Congenital disorders of glycosylation

Parkinson, W. M., Dookwah, M., Dear, M. L., Gatto, C. L., Aoki, K., Tiemeyer, M. and Broadie, K. (2016). Neurological roles for phosphomannomutase type 2 in a new Drosophila congenital disorder of glycosylation disease model. Dis Model Mech [Epub ahead of print]. PubMed ID:
26940433

Abstract

The most common Congenital disorders of glycosylation (CDGs), CDG-Ia or PMM2-CDG, arises from phosphomannomutase type 2 (PMM2) mutations. This study reports the generation and characterization of the first Drosophila PMM2-CDG model. CRISPR-generated Drosophila pmm2 null mutants display severely disrupted glycosylation and early lethality, while RNAi-targeted neuronal PMM2 knockdown results in a strong shift in pauci-mannose glycan abundance, progressive incoordination and later lethality, closely paralleling human CDG-Ia symptoms of shortened lifespan, movement impairments and defective neural development. Analyses of the well-characterized Drosophila neuromuscular junction (NMJ) reveal synaptic glycosylation loss accompanied by structural architecture and functional neurotransmission defects. NMJ synaptogenesis is driven by intercellular signals traversing an extracellular synaptomatrix co-regulated by glycosylation and matrix metalloproteinases (MMPs). Specifically, Wnt Wingless (Wg) trans-synaptic signaling depends on the heparan sulfate proteoglycan (HSPG) co-receptor Dally-like protein (Dlp), which is regulated by synaptic MMP activity. Loss of synaptic MMP2, Wg ligand, Dlp co-receptor and downstream trans-synaptic signaling occurs with PMM2 knockdown. Taken together, this Drosophila CDG disease model provides a new avenue for the dissection of cellular and molecular mechanisms underlying neurological impairments and a means to discover and test novel therapeutic treatment strategies.

Frappaolo, A., Sechi, S., Kumagai, T., Robinson, S., Fraschini, R., Karimpour-Ghahnavieh, A., Belloni, G., Piergentili, R., Tiemeyer, K.H., Tiemeyer, M., Giansanti, M.G. (2016). (2017). COG7 deficiency in Drosophila generates multifaceted developmental, behavioral and protein glycosylation phenotypes. J. Cell Sci. 130(21): 3637--3649. PubMed ID: 28883096

Abstract

Congenital disorders of glycosylation (CDG) comprise a family of human multisystemic diseases caused by recessive mutations in genes required for protein N-glycosylation. More than 100 distinct forms of CDGs have been identified and most of them cause severe neurological impairment. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2 and COG4-COG8 cause monogenic forms of inherited, autosomal recessive CDGs. This study generated a Drosophila COG7-CDG model that closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects associated with altered N-glycome profiles. Consistent with these alterations, larval neuromuscular junctions of Cog7 mutants exhibit a significant reduction in bouton numbers. The COG complex was shown to cooperate with Rab1 and Golgi phosphoprotein 3 to regulate Golgi trafficking; overexpression of Rab1 can rescue the cytokinesis and locomotor defects associated with loss of Cog7. These results suggest that the Drosophila COG7-CDG model can be used to test novel potential therapeutic strategies by modulating trafficking pathways (Frappaolo, 2017).

Han, S. Y., Pandey, A., Moore, T., Galeone, A., Duraine, L., Cowan, T. M. and Jafar-Nejad, H. (2020). A conserved role for AMP-activated protein kinase in NGLY1 deficiency. PLoS Genet 16(12): e1009258. PubMed ID: 33315951

Abstract

Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, this study reports that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/- mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKα levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease (Han, 2020).

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Covid

Herrera, P. and Cauchi, R. J.
(2023). Functional characterisation of the ACE2 orthologues in Drosophila provides insights into the neuromuscular complications of COVID-19.Biochim Biophys Acta Mol Basis Dis 1869(8): 166818. PubMed ID: 37495086

SARS-CoV-2, the virus responsible for the coronavirus disease of 2019 (COVID-19), gains cellular entry via interaction with the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. Although SARS-CoV-2 mainly targets the respiratory system, the neuromuscular system also appears to be affected in a large percentage of patients with acute or chronic COVID-19. The cause of the well-described neuromuscular manifestations resulting from SARS-CoV-2 infection remains unresolved. These may result from the neuromuscular-invasive capacity of the virus leading to direct injury. Alternatively, they may be the consequence of ACE2 inactivation either due to viral infection, ACE2 autoantibodies or both. This study made use of the Drosophila model to investigate whether ACE2 downregulation is sufficient to induce neuromuscular phenotypes. Moderate gene silencing of ACE2 orthologues Ance or Ance3 was shown o diminish survival on exposure to thermal stress only upon induction of neuromuscular fatigue driven by increased physical activity. A strong knockdown of Ance or Ance3 directed to muscle reduced or abolished adult viability and caused obvious motoric deficits including reduced locomotion and impaired flight capacity. Selective knockdown of Ance and Ance3 in neurons caused wing defects and an age-dependent decline in motor behaviour, respectively, in adult flies. Interestingly, RNA sequencing led to the discovery of several differentially spliced genes that are required for synaptic function downstream of Ance or Ance3 depletion. These findings are therefore supportive of the notion that loss of a RAS-independent function for ACE2 contributes to the neuromuscular manifestations associated with SARS-CoV-2 infection (Herrera, 2023).

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Creutzfeldt-Jakob Disease and Prion Disease

Thackray, A. M., Cardova, A., Wolf, H., Pradl, L., Vorberg, I., Jackson, W. S. and Bujdoso, R.
(2017). Genetic human prion disease modelled in PrP transgenic Drosophila. Biochem J 474(19): 3253-3267. PubMed ID: 28814578

Abstract

Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. Site-directed mutagenesis was used to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, these novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host (Thackray, 2017).


Thackray, A. M., Lam, B., Shahira Binti Ab Razak, A., Yeo, G. and Bujdoso, R. (2020). Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease. Biochem J 477(4): 833-852. PubMed ID: 32108870

Abstract

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. This study used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, these data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology (Thackray, 2020).


Thackray, A. M., Lam, B., McNulty, E. E., Nalls, A. V., Mathiason, C. K., Magadi, S. S., Jackson, W. S., Andreoletti, O., Marrero-Winkens, C., Schatzl, H. and Bujdoso, R. (2022). Clearance of variant Creutzfeldt-Jakob disease prions in vivo by the Hsp70 disaggregase system. Brain 145(9): 3236-3249. PubMed ID: 35446941

Abstract
The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. This study tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so prion protein (PrP) transgenic Drosophila were used that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was downregulated in prion-exposed PrP Drosophila. RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas overexpression mitigated toxicity. Strikingly, prion seeding activity in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 overexpression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease.

Myers, R. R., John, A., Zhang, W., Zou, W. Q., Cembran, A. and Fernandez-Funez, P. (2023). Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila. J Biol Chem 299(7): 104881. PubMed ID: 37269948

Abstract

Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. This study describes the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. First, human PrP-Y225A was examined by molecular dynamics simulations. Next, human PrP was introduced in Drosophila, and the toxicity of human PrP-WT and Y225A was compared in the eye and in brain neurons. Y225A stabilizes the β2-α2 loop into a 3(10)-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, this study determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain (Myers, 2023).

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Cystic fibrosis

Kim, K., Lane, E. A., Saftien, A., Wang, H., Xu, Y., Wirtz-Peitz, F. and Perrimon, N.
(2020). Drosophila as a model for studying cystic fibrosis pathophysiology of the gastrointestinal system. Proc Natl Acad Sci U S A. PubMed ID: 32345720

Abstract

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common symptoms include progressive lung disease and chronic digestive conditions. CF is the first human genetic disease to benefit from having five different species of animal models. Despite the phenotypic differences among the animal models and human CF, these models have provided invaluable insight into understanding disease mechanisms at the organ-system level. This study identified a member of the ABCC4 family, CG5789, that has the structural and functional properties expected for encoding the Drosophila equivalent of human CFTR, and thus it is refered to as Drosophila CFTR (Dmel\CFTR). Knockdown of Dmel\CFTR in the adult intestine disrupts osmotic homeostasis and displays CF-like phenotypes that lead to intestinal stem cell hyperplasia. Expression of wild-type human CFTR, but not mutant variants of CFTR that prevent plasma membrane expression, rescues the mutant phenotypes of Dmel\CFTR. Furthermore, RNA sequencing (RNA-Seq)-based transcriptomic analysis was performed using Dmel\CFTR fly intestine and a mucin gene, Muc68D, was identified that is required for proper intestinal barrier protection. Altogether, these findings suggest that Drosophila can be a powerful model organism for studying CF pathophysiology (Kim, 2020).

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Epilepsy and seizure disorders

Praschberger, R., Lowe, S. A., Malintan, N. T., Giachello, C. N. G., Patel, N., Houlden, H., Kullmann, D. M., Baines, R. A., Usowicz, M. M., Krishnakumar, S. S., Hodge, J. J. L., Rothman, J. E. and Jepson, J. E. C.
(2017). Mutations in Membrin/GOSR2 reveal stringent secretory pathway demands of dendritic growth and synaptic integrity. Cell Rep 21(1): 97-109. PubMed ID: 28978487

Abstract

Mutations in the Golgi SNARE (SNAP [soluble NSF attachment protein] receptor) protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitous and essential protein mediating ER-to-Golgi membrane fusion. Thus, it is unclear how mutations in Membrin result in a disorder restricted to the nervous system. This study used a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. The pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in Drosophila models of GOSR2-PME. Furthermore, Membrin mutations were shown to cause fragmentation of the presynaptic cytoskeleton coupled with transsynaptic instability and hyperactive neurotransmission. This study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME (Praschberger, 2017).

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Du, S., Zeng, S., Song, L., Ma, H., Chen, R., Luo, J., Wang, X., Ma, T., Xu, X., Sun, H., Yi, P., Guo, J., Huang, Y., Liu, M., Wang, T., Liao, W. P., Zhang, L., Liu, J. Y. and Tang, B. (2023). b>Functional characterization of novel NPRL3 mutations identified in three families with focal epilepsy. Sci China Life Sci. PubMed ID: 37071290
Summary:
Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study, three novel mutations in NPRL3 (nitrogen permease regulator-like 3), c.937_945del, c.1514dupC and 6,706-bp genomic DNA (gDNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing (WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of mTOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced mTOR signaling in cultured cells, possibly due to impaired inhibition of mTORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.

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Majlish, A. N. K., Lye, S. H., Cytron, E., Bolus, H., Marotto, K. and Chtarbanova, S. (2021). The Drosophila K(+) -dependent Na(+) /Ca(2+) exchanger Nckx30C is implicated in temperature sensitive paralysis and age-dependent neurodegeneration. Alzheimers Dement 17 Suppl 12: e058564. PubMed ID: 34971107
Summary:
Increasing evidence links epileptic seizures to neurodegenerative disorders such as Alzheimer's disease (AD). Perturbed cellular Ca(2+) homeostasis is known to be implicated in AD pathogenesis. Previous studies in the model organism Drosophila have established a connection between mutations linked to temperature-sensitive (TS) paralysis, which is reminiscent of vertebrate epileptiform behavior, and neurodegeneration. An unbiased genetic screen was performed to identify mutants that exhibit paralytic behavior at 38°C. Gene mapping and DNA sequencing were used to locate the mutation in the Nckx30C gene. Mutant line 426 exhibited TS paralysis and progressive neurodegeneration in comparison to controls. The mutation was mapped to the Nckx30C locus, in the region encoding the predicted ion-binding domain. In comparison to wild type, 426 flies showed reduced lifespan, impaired climbing and changes in NMJ morphology, indicative of synaptic dysfunction. Lifespan reduction and early-onset neurodegeneration were found in flies carrying two other Nckx30C alleles. Neuron- but not glia-specific knockdown of Nckx30C recapitulates the TS-paralytic phenotype of 426 flies. Knockdown of Nckx30C in glia and neurons led to early onset climbing defects. It is concluded that the Drosophila Nckx30C gene encodes for a K(+) -dependent Na(+) /Ca(2+) exchanger with enriched expression in brain tissue. Nckx30C is homologous to mammalian Solute Carrier Family 24 (SLC24) proteins of which pathophysiological involvement and function in the brain is poorly understood. These studies of Nckx30C in flies could help to decipher the function of SLC24 proteins in both seizures and neurodegenerative conditions such as AD.

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Yap, Z. Y., Efthymiou, S., Seiffert, S., ..., Houlden, H. and Yoon, W. H. (2021). Bi-allelic variants in OGDHL cause a neurodevelopmental spectrum disease featuring epilepsy, hearing loss, visual impairment, and ataxia. Am J Hum Genet. PubMed ID: 34800363
Summary:
The 2-oxoglutarate dehydrogenase-like (OGDHL) protein is a rate-limiting enzyme in the Krebs cycle that plays a pivotal role in mitochondrial metabolism. OGDHL expression is restricted mainly to the brain in humans. This study reports nine individuals from eight unrelated families carrying bi-allelic variants in OGDHL with a range of neurological and neurodevelopmental phenotypes including epilepsy, hearing loss, visual impairment, gait ataxia, microcephaly, and hypoplastic corpus callosum. The variants include three homozygous missense variants (p.Pro852Ala, p.Arg244Trp, and p.Arg299Gly), three compound heterozygous single-nucleotide variants (p.Arg673Gln/p.Val488Val, p.Phe734Ser/p.Ala327Val, and p.Trp220Cys/p.Asp491Val), one homozygous frameshift variant (p.Cys553Leufs*16), and one homozygous stop-gain variant (p.Arg440Ter). To support the pathogenicity of the variants, a novel CRISPR-Cas9-mediated tissue-specific knockout was developed with cDNA rescue system for dOgdh, the Drosophila ortholog of human OGDHL. Pan-neuronal knockout of dOgdh led to developmental lethality as well as defects in Krebs cycle metabolism, which was fully rescued by expression of wild-type dOgdh. Studies using the Drosophila system indicate that p.Arg673Gln, p.Phe734Ser, and p.Arg299Gly are severe loss-of-function alleles, leading to developmental lethality, whereas p.Pro852Ala, p.Ala327Val, p.Trp220Cys, p.Asp491Val, and p.Arg244Trp are hypomorphic alleles, causing behavioral defects. Transcript analysis from fibroblasts obtained from the individual carrying the synonymous variant (c.1464T>C [p.Val488Val]) in family 2 showed that the synonymous variant affects splicing of exon 11 in OGDHL. Human neuronal cells with OGDHL knockout exhibited defects in mitochondrial respiration, indicating the essential role of OGDHL in mitochondrial metabolism in humans. Together, these data establish that the bi-allelic variants in OGDHL are pathogenic, leading to a Mendelian neurodevelopmental disease in humans.

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Tapia, A., Giachello, C. N., Palomino-Schatzlein, M., Baines, R. A. and Galindo, M. I. (2021). Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome. Life (Basel) 11(11). PubMed ID: 34833136

Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. A knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, was made by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, an NMR metabolomic profiling of heads was performed that revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.

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Manivannan, S. N., Roovers, J., Smal, N., Myers, C. T., Turkdogan, D., Roelens, F., Kanca, O., Chung, H. L., Scholz, T., Hermann, K., Bierhals, T., Caglayan, H. S., Stamberger, H., Mefford, H., de Jonghe, P., Yamamoto, S., Weckhuysen, S. and Bellen, H. J. (2021). De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies. Brain. PubMed ID: 34788397

FZR1, which encodes the Cdh1 subunit of the Anaphase Promoting Complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homolog of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype Myoclonic Atonic Epilepsy. Functional evidence is provided that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homolog fzr and overexpression studies, it was shown that patient variants can affect proper neurodevelopment. This study consolidates the relationship between FZR1 and developmental epileptic encephalopathy, and expands the associated phenotype. It is concluded that heterozygous loss-of-function of FZR1 leads to developmental epileptic encephalopathies associated with a spectrum of neonatal to childhood onset seizure types, developmental delay and mild ataxia. In summary, this approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental epileptic encephalopathy cases.

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Byers, N., Hahm, E. T. and Tsunoda, S. (2021). Slo2/K(Na) Channels in Drosophila Protect Against Spontaneous and Induced Seizure-like Behavior Associated with an Increased Persistent Na(+) Current. J Neurosci. PubMed ID: 34544836

Na(+)-sensitivity is a unique feature of Na(+)-activated K(+) (K(Na)) channels, making them naturally suited to counter a sudden influx in Na(+) ions. As such, it has long been suggested that K(Na) channels may serve a protective function against excessive excitation associated with neuronal injury and disease. This study examined K(Na) channels encoded by the Drosophila Slo2 (dSlo2) gene in males and females. dSlo2/K(Na) channels are selectively expressed in cholinergic neurons in the adult brain, as well as in glutamatergic motor neurons, where dampening excitation may function to inhibit global hyperactivity and seizure-like behavior. Indeed, this study shows that effects of feeding Drosophila a cholinergic agonist are exacerbated by the loss of dSlo2/K(Na) channels. dSlo2/K(Na) channels encode a TTX-sensitive K(+) conductance, indicating that dSlo2/K(Na) channels can be activated by Na(+) carried by voltage-dependent Na(+) channels. The role of dSlo2/K(Na) channels was tested in established genetic seizure models in which the voltage-dependent persistent Na(+) current (I(Nap)) was elevated. The absence of dSlo2/K(Na) channels increased susceptibility to mechanically-induced seizure-like behavior. Finally, this study showed that loss of dSlo2/K(Na) channels in both genetic and pharmacologically-primed seizure models resulted in the appearance of spontaneous seizures. Together, these results support a model in which dSlo2/K(Na) channels, activated upon neuronal over-excitation, contribute to a protective threshold to suppress the induction of seizure-like activity (Byers, 2021).

Roemmich, A. J., Vu, T., Lukacsovich, T., Hawkins, C., Schutte, S. S. and O'Dowd, D. K. (2021).Seizure Phenotype and Underlying Cellular Defects in Drosophila Knock-In Models of DS (R1648C) and GEFS+ (R1648H) SCN1A Epilepsy. eNeuro 8(5). PubMed ID: 34475263

Abstract

Mutations in the voltage-gated sodium channel gene SCN1A are associated with human epilepsy disorders, but how most of these mutations alter channel properties and result in seizures is unknown. This study focuses on two different mutations occurring at one position within SCN1A R1648C (R-C) is associated with the severe disorder Dravet syndrome, and R1648H (R-H), with the milder disorder GEFS+. To explore how these different mutations contribute to distinct seizure disorders, Drosophila lines with the R-C or R-H mutation, or R1648R (R-R) control substitution in the fly sodium channel gene para were generated by CRISPR-Cas9 gene editing. The R-C and R-H mutations are homozygous lethal. Animals heterozygous for R-C or R-H mutations displayed reduced life spans and spontaneous and temperature-induced seizures not observed in R-R controls. Electrophysiological recordings from adult GABAergic neurons in R-C and R-H mutants revealed the appearance of sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. The only significant change observed in underlying sodium currents in both R-C and R-H mutants was a hyperpolarized deactivation threshold at room and elevated temperature compared with R-R controls. Since this change is constitutive, it is likely to interact with heat-induced changes in other cellular properties to result in the heat-induced increase in sustained depolarizations and seizure activity. Further, the similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines, suggests that disease symptoms of different severity associated with these mutations in humans could be due in large part to differences in genetic background (Roemmich, 2021).

Mituzaite, J., Petersen, R., Claridge-Chang, A. and Baines, R. A. (2021).Characterization of Seizure Induction Methods in Drosophila. eNeuro 8(4). PubMed ID: 34330816

Abstract

Epilepsy is one of the most common neurologic disorders. Around one third of patients do not respond to current medications. This lack of treatment indicates a need for better understanding of the underlying mechanisms and, importantly, the identification of novel targets for drug manipulation. The fruit fly Drosophila melanogaster has a fast reproduction time, powerful genetics, and facilitates large sample sizes, making it a strong model of seizure mechanisms. To better understand behavioral and physiological phenotypes across major fly seizure genotypes this study systematically measured seizure severity and secondary behavioral phenotypes at both the larval and adult stage. Comparison of several seizure-induction methods; specifically electrical, mechanical and heat induction, show that larval electroshock is the most effective at inducing seizures across a wide range of seizure-prone mutants tested. Locomotion in adults and larvae was found to be non-predictive of seizure susceptibility. Recording activity in identified larval motor neurons revealed variations in action potential (AP) patterns, across different genotypes, but these patterns did not correlate with seizure susceptibility. To conclude, while there is wide variation in mechanical induction, heat induction, and secondary phenotypes, electroshock is the most consistent method of seizure induction across known major seizure genotypes in Drosophila (Mituzaite, 2021).

Iyengar, A. and Wu, C. F. (2021). Fly seizure EEG: field potential activity in the Drosophila brain. J Neurogenet: 1-11. PubMed ID: 34278939

Abstract

Hypersynchronous neural activity is a characteristic feature of seizures. Although many Drosophila mutants of epilepsy-related genes display clear behavioral spasms and motor unit hyperexcitability, field potential measurements of aberrant hypersynchronous activity across brain regions during seizures have yet to be described. This study reports a straightforward method to observe local field potentials (LFPs) from the Drosophila brain to monitor ensemble neural activity during seizures in behaving tethered flies. High frequency stimulation across the brain reliably triggers a stereotypic sequence of electroconvulsive seizure (ECS) spike discharges readily detectable in the dorsal longitudinal muscle (DLM) and coupled with behavioral spasms. During seizure episodes, the LFP signal displayed characteristic large-amplitude oscillations with a stereotypic temporal correlation to DLM flight muscle spiking. ECS-related LFP events were clearly distinct from rest- and flight-associated LFP patterns. The LFP activity was further characterized during different types of seizures originating from genetic and pharmacological manipulations. In the 'bang-sensitive' sodium channel mutant bangsenseless (bss), the LFP pattern was prolonged, and the temporal correlation between LFP oscillations and DLM discharges was altered. Following administration of the pro-convulsant GABA(A) blocker picrotoxin, a qualitatively different LFP activity pattern was uncovered that consisted of a slow (1-Hz), repetitive, waveform, closely coupled with DLM bursting and behavioral spasms. This approach to record brain LFPs presents an initial framework for electrophysiological analysis of the complex brain-wide activity patterns in the large collection of Drosophila excitability mutants (Iyengar, 2021).

Kasture, A. S., Fischer, F. P., Kunert, L., Burger, M. L., Burgstaller, A. C., El-Kasaby, A., Hummel, T. and Sucic, S. (2022) . >Drosophila melanogaster as a model for unraveling unique molecular features of epilepsy elicited by human GABA transporter 1 variants (2022). Front Neurosci 16: 1074427. PubMed ID: 36741049
Summary:
Mutations in the human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1: see Drosophila VGAT) can instigate myoclonic-atonic and other generalized epilepsies in the afflicted individuals. This study systematically examined fifteen hGAT-1 disease variants, all of which dramatically reduced or completely abolished GABA uptake activity. Many of these loss-of-function variants were absent from their regular site of action at the cell surface, due to protein misfolding and/or impaired trafficking machinery (as verified by confocal microscopy and de-glycosylation experiments). A modest fraction of the mutants displayed correct targeting to the plasma membrane, but nonetheless rendered the mutated proteins devoid of GABA transport, possibly due to structural alterations in the GABA binding site/translocation pathway. This study focused on a folding-deficient A288V variant. In flies, A288V reiterated its impeded expression pattern, closely mimicking the ER-retention demonstrated in transfected HEK293 cells. Functionally, A288V presented a temperature-sensitive seizure phenotype in fruit flies. Diverse small molecules were employed to restore the expression and activity of folding-deficient hGAT-1 epilepsy variants, in vitro (in HEK293 cells) and in vivo (in flies). This study identified three compounds (chemical and pharmacological chaperones) conferring moderate rescue capacity for several variants. These data grant crucial new insights into: (1) the molecular basis of epilepsy in patients harboring hGAT-1 mutations, and (2) a proof-of-principle that protein folding deficits in disease-associated hGAT-1 variants can be corrected using the pharmacochaperoning approach. Such innovative pharmaco-therapeutic prospects inspire the rational design of novel drugs for alleviating the clinical symptoms triggered by the numerous emerging pathogenic mutations in hGAT-1.

Kunduri, G., Turner-Evans, D., Konya, Y., Izumi, Y., Nagashima, K., Lockett, S., Holthuis, J., Bamba, T., Acharya, U. and Acharya, J. K. (2018). Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants. Proc Natl Acad Sci U S A. PubMed ID: 30185559

Abstract

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. This study shows an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, this study shows the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype (Kunduri, 2018).

Hope, K. A., Johnson, D., Miller, P. W., Lopez-Ferrer, D., Kakhniashvili, D. and Reiter, L. T. (2020). Transcriptomic and proteomic profiling of glial versus neuronal Dube3a overexpression reveals common molecular changes in gliopathic epilepsies. Neurobiol Dis: 104879. PubMed ID: 32344153

Abstract

Epilepsy affects millions of individuals worldwide and many cases are pharmacoresistant. Duplication 15q syndrome (Dup15q) is a genetic disorder caused by duplications of the 15q11.2-q13.1 region. Phenotypes include in a high rate of pharmacoresistant epilepsy. This study developed a Dup15q model in Drosophila melanogaster that recapitulates seizures in Dup15q by over-expressing fly Dube3a or human UBE3A in glial cells, but not neurons, implicating glia in the Dup15q epilepsy phenotype. Dube3a overexpression in glia (repo>Dube3a) versus neurons (elav>Dube3a) using transcriptomics and proteomics of whole fly head extracts. 851 transcripts differentially regulated in repo>Dube3a were identified, including an upregulation glutathione S-transferase (GST) genes that occurred cell autonomously within glial cells. Approximately 2,500 proteins were reliably measured by proteomics, most of which were also quantified at the transcript level. Combined transcriptomic and proteomic analysis revealed an enrichment of 21 synaptic transmission genes downregulated at the transcript and protein in repo>Dube3a indicating synaptic proteins change in a cell non-autonomous manner in repo>Dube3a flies. Six additional glia originating bang-sensitive seizure lines were identified, and upregulation of GSTs in 4 out of these 6 lines was identified. These data suggest GST upregulation is common among gliopathic seizures and may ultimately provide insight for treating epilepsy (Hope, 2020).

Luthy, K., et al. (2019). TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model. Brain 142(8): 2319-2335. PubMed ID: 31257402

Abstract
mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. Through whole genome/exome sequencing compound heterozygous mutations, R360H and G501R, within the TLDc domain, were identified in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (OMIM). Six patients were discovered to harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. The crystal structure of the conserved Drosophila TLDc domain was solved in this study. The functional consequences were characterized of a strong and a weak TLDc mutation using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model, neuronally expressing human TBC1D24, it was demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects. The neuronal phenotypes of the mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating mutant animals with antioxidants N-acetylcysteine amide or alpha-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. These data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. It is proposed that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations (Luthy, 2019).

Savitsky, M., Solis, G. P., Kryuchkov, M. and Katanaev, V. L. (2019). Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies. Biomedicines 8(10). PubMed ID: 33036271

Abstract

Several hundred genes have been identified to contribute to epilepsy-the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, this study describes the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the Drosophila functions. This genetic model will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics (Savitsky, 2020).

Wang, J., Qiao, J. D., Liu, X. R., Liu, D. T., Chen, Y. H., Wu, Y., Sun, Y., Yu, J., Ren, R. N., Mei, Z., Liu, Y. X., Shi, Y. W., Jiang, M., Lin, S. M., He, N., Li, B., Bian, W. J., Li, B. M., Yi, Y. H., Su, T., Liu, H. K., Gu, W. Y. and Liao, W. P. (2019). UNC13B variants associated with partial epilepsy with favourable outcome. Brain. PubMed ID: 33876820

Abstract

The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein, mammalian uncoordinated 13-2 (Munc13-2), that is highly expressed in the brain-predominantly in the cerebral cortex-and plays an essential role in synaptic vesicle priming and fusion, potentially affecting neuronal excitability. However, the functional significance of UNC13B mutation in human disease is not known. This study screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy without acquired causes by trio-based whole-exome sequencing. UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. The eight probands all had focal seizures and focal discharges in EEG recordings, including two patients who experienced frequent daily seizures and one who showed abnormalities in the hippocampus by brain MRI; however, all of the patients showed favorable outcome without intellectual or developmental abnormalities. The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant, and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database. Computational modeling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a; Paralytic) knockdown Drosophila. Electrophysiologic recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results suggest that UNC13B is potentially associated with epilepsy. The frequent daily seizures and hippocampal abnormalities but ultimately favorable outcome under antiepileptic therapy in patients indicate that partial epilepsy caused by UNC13B variant is a clinically manageable condition (Wang, 2021).

Chi, W., Iyengar, A. S. R., Fu, W., Liu, W., Berg, A. E., Wu, C. F. and Zhuang, X. (2022). Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes. Proc Natl Acad Sci U S A 119(9). PubMed ID: 35217610
Summary:

Abstract
Pyridox(am)ine 5 (') -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 (') -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, four knock-in Drosophila alleles, h(WT) , h(R) (116) (Q) , h(D) (33) (V) , and h(R) (95) (H) were generated, in which the endogenous Drosophila PNPO (sugarlethal) was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. These knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and characterized molecular defects. Diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, it was found that h(R95H) had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.

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5q35-microduplication

Quintero-Rivera, F., Eno, C. C., Sutanto, C., Jones, K. L., Nowaczyk, M. J. M., Wong, D., Earl, D., Mirzaa, G., Beck, A. and Martinez-Agosto, J. A. (2021). 5q35 duplication presents with psychiatric and undergrowth phenotypes mediated by NSD1 overexpression and mTOR signaling downregulation. Hum Genet. PubMed ID:
33389145

Abstract

Nuclear receptor binding SET domain protein 1, NSD1, encodes a histone methyltransferase H3K36. NSD1 is responsible for the phenotype of the reciprocal 5q35.2q35.3 microdeletion-microduplication syndromes. This study expanded the phenotype and demonstrated the functional role of NSD1 (Drosophila homolog: NSD) in microduplication 5q35 syndrome. Through an international collaboration, this study reports nine new patients, contributing to the emerging phenotype, highlighting psychiatric phenotypes in older affected individuals. Focusing specifically on the undergrowth phenotype, the effects of Mes-4/NSD overexpression were modeled in Drosophila melanogaster. The individuals (including a family) from diverse backgrounds with duplications ranging in size from 0.6 to 4.5 Mb, have a consistent undergrowth phenotype. Mes-4 overexpression in the developing wing causes undergrowth, increased H3K36 methylation, and increased apoptosis. Altering the levels of insulin receptor (IR) rescues the apoptosis and the wing undergrowth phenotype, suggesting changes in mTOR pathway signaling. Leucine supplementation rescued Mes-4/NSD induced cell death, demonstrating decreased mTOR signaling caused by NSD1. Given that this study shows mTOR inhibition as a likely mechanism and amelioration of the phenotype by leucine supplementation in a fly model, it is suggested further studies should evaluate the therapeutic potential of leucine or branched chain amino acids as an adjunct possible treatment to ameliorate human growth and psychiatric phenotypes, and inclusion of 5q35-microduplication as part of the differential diagnosis for children and adults with delayed bone age, short stature, microcephaly, developmental delay, and psychiatric phenotypes is proposed (Quintero-Rivera, 2021).

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Fanconi Syndrome

Marchesin, V., Perez-Marti, A., Le Meur, G., Pichler, R., Grand, K., Klootwijk, E. D., Kesselheim, A., Kleta, R., Lienkamp, S. and Simons, M. (2019). Molecular basis for autosomal-dominant renal Fanconi Syndrome caused by HNF4A. Cell Rep 29(13): 4407-4421. PubMed ID:
31875549

Abstract

HNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). This study finds that expression of the conserved fly ortholog HNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type HNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. These data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors.

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Fatty acid hydroxylase-associated neurodegeneration (FAHN)

Mandik, F., Kanana, Y., Rody, J., Misera, S., Wilken, B., Laabs von Holt, B. H., Klein, C. and Vos, M. (2022). A new model for fatty acid hydroxylase-associated neurodegeneration reveals mitochondrial and autophagy abnormalities. Front Cell Dev Biol 10: 1000553. PubMed ID:
36589738

Abstract
Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare disease that exhibits brain modifications and motor dysfunctions in early childhood. The condition is caused by a homozygous or compound heterozygous mutation in fatty acid 2 hydroxylase (FA2H), whose encoded protein synthesizes 2-hydroxysphingolipids and 2-hydroxyglycosphingolipids and is therefore involved in sphingolipid metabolism. Drosophila is an excellent model for many neurodegenerative disorders; hence, this study has characterized and validated the first FAHN Drosophila model. The investigation of loss of dfa2h lines revealed behavioral abnormalities, including motor impairment and flying disability, in addition to a shortened lifespan. Furthermore, alterations in mitochondrial dynamics, and autophagy were identified. Analyses of patient-derived fibroblasts, and rescue experiments with human FA2H, indicated that these defects are evolutionarily conserved. This study thus presents a FAHN Drosophila model organism that provides new insights into the cellular mechanism of FAHN.

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Floating-Harbor syndrome

Messina, G., Prozzillo, Y., Delle Monache, F., Santopietro, M. V., Atterrato, M. T. and Dimitri, P. (2021). The ATPase SRCAP is associated with the mitotic apparatus, uncovering novel molecular aspects of Floating-Harbor syndrome. BMC Biol 19(1): 184. PubMed ID:
34474679

Abstract
Floating-Harbor syndrome is a rare genetic disease affecting human development caused by dominant truncating mutations in the SRCAP gene, which encodes the ATPase SRCAP, the core catalytic subunit of the homonymous chromatin-remodeling complex. The main function of the SRCAP complex is to promote the exchange of histone H2A with the H2A.Z variant. According to the canonical role played by the SRCAP protein in epigenetic regulation, the Floating-Harbor syndrome is thought to be a consequence of chromatin perturbations. However, additional potential physiological functions of SRCAP have not been sufficiently explored. This study combined cell biology, reverse genetics, and biochemical approaches to study the subcellular localization of the SRCAP protein and assess its involvement in cell cycle progression in HeLa cells. Surprisingly, SRCAP was found to associate with components of the mitotic apparatus (centrosomes, spindle, midbody), interacts with a plethora of cytokinesis regulators, and positively regulates their recruitment to the midbody. Remarkably, SRCAP depletion perturbs both mitosis and cytokinesis. Similarly, DOM-A, the functional SRCAP orthologue in Drosophila melanogaster, is found at centrosomes and the midbody in Drosophila cells, and its depletion similarly affects both mitosis and cytokinesis. These findings provide first evidence suggesting that SRCAP plays previously undetected and evolutionarily conserved roles in cell division, independent of its functions in chromatin regulation. SRCAP may participate in two different steps of cell division: by ensuring proper chromosome segregation during mitosis and midbody function during cytokinesis. Moreover, these findings emphasize a surprising scenario whereby alterations in cell division produced by SRCAP mutations may contribute to the onset of Floating-Harbor syndrome.

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Galactosemia

Jumbo-Lucioni, P. P., Parkinson, W. M., Kopke, D. L. and Broadie, K. (2016). Coordinated movement, neuromuscular synaptogenesis and trans-synaptic signaling defects in Drosophila galactosemia models. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27466186

Abstract

The multiple galactosemia disease states manifest long-term neurological symptoms. Galactosemia I results from loss of galactose-1-phosphate uridyltransferase (GALT), which converts galactose-1-phosphate + UDP-glucose to glucose-1-phosphate + UDP-galactose. Galactosemia II results from loss of galactokinase (GALK), phosphorylating galactose to galactose-1-phosphate. Galactosemia III results from the loss of UDP-galactose 4'-epimerase (GALE), which interconverts UDP-galactose and UDP-glucose, as well as UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. UDP-glucose pyrophosphorylase (UGP) alternatively makes UDP-galactose from uridine triphosphate and galactose-1-phosphate. All four UDP-sugars are essential donors for glycoprotein biosynthesis with critical roles at the developing neuromuscular synapse. Drosophila galactosemia I (dGALT) and II (dGALK) disease models genetically interact; manifesting deficits in coordinated movement, neuromuscular junction (NMJ) development, synaptic glycosylation, and Wnt trans-synaptic signaling. Similarly, dGALE and dUGP mutants display striking locomotor and NMJ formation defects, including expanded synaptic arbors, glycosylation losses, and differential changes in Wnt trans-synaptic signaling. In combination with dGALT loss, both dGALE and dUGP mutants compromise the synaptomatrix glycan environment that regulates Wnt trans-synaptic signaling that drives 1) presynaptic Futsch/MAP1b microtubule dynamics and 2) postsynaptic Frizzled nuclear import (FNI). Taken together, these findings indicate UDP-sugar balance is a key modifier of neurological outcomes in all three interacting galactosemia disease models, suggest that Futsch homolog MAP1B and the Wnt Frizzled receptor may be disease-relevant targets in epimerase and transferase galactosemias, and identify UGP as promising new potential therapeutic target for galactosemia neuropathology (Jumbo-Lucioni, 2016).

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Galactosemia

Marchesin, V., Perez-Marti, A., Le Meur, G., Pichler, R., Grand, K., Klootwijk, E. D., Kesselheim, A., Kleta, R., Lienkamp, S. and Simons, M.
(2019). Molecular basis for autosomal-dominant renal Fanconi Syndrome caused by HNF4A. Cell Rep 29(13): 4407-4421. PubMed ID: 31875549

Abstract

HNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). This study finds that expression of the conserved fly ortholog HNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type HNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. These data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors (Marchesin, 2019).

HNF4A is specifically expressed in the kidney in proximal tubules and is required for the terminal differentiation of proximal tubular cells. However, out of all of the known mutations, only the R85W mutation causes FRTS. Using fly nephrocytes as a model for proximal tubules, this study shows that both dHNF4 KD and overexpression of the FRTS mutation (R167W in flies) causes mitochondrial alterations and LD accumulation. By using a dHNF4-GFP as a reporter protein, this study shows that the FRTS mutation provokes the nuclear export of the wild-type protomer in a dominant-negative manner, possibly explaining the similarity to the KD. The nuclear export is additionally associated with cytoplasmic aggregate formation, increased autophagy, severe ER morphology changes, and eventually cell death. All of these phenotypes are not seen in the KD, suggesting that they are the result of the aggregate formation. However, they could be observed when expressing wild-type dHNF4 at high levels, suggesting dominant-negative effects in this condition as well. For both dHNF4R167W/FRTS and dHNF4highOE, these effects could be rescued by the increase in nuclear levels through dephosphorylation at S169 (Marchesin, 2019).

Crucial for the understanding of the investigated mutations are structural studies of the HNF4A homodimer/DNA complex, in which the homodimer forms multiple domain-domain junctions and a convergence zone or 'nerve center,' in which the two LBDs, the upstream positioned DBD, and the hinge region meet. S87 maps precisely to this center, leading to unfavorable charge repulsion upon phosphorylation and disengagement of the quaternary structure needed for DNA binding. In another nuclear receptor, constitutive androstane receptor (CAR), the corresponding phosphorylation leads to the formation of inactive homodimers, providing support for this model. As R85 is in close contact with the DNA bases and backbone of the dipartite DR1 element in both upstream and downstream protomers of the homodimer, the lack of DNA binding in the R85W mutant may promote phosphorylation-dependent conversion of active to inactive homodimers and, finally, nuclear export of both mutant and wild-type protomers (Marchesin, 2019).

By contrast, the effects of the MODY mutation R171W resembled those of the phosphorylation-deficient S169A mutant, which is supported by two phosphorylation prediction algorithms, showing that R171W/MODY (or R89W/MODY) but not R167W/FRTS (or R85W/FRTS) reduces the probability for phosphorylation at S169 (or S87). In addition, the available HNF4A structure shows that R89 is closer to the S87 convergence zone compared to R85, which may explain that at the R171 (or R89) position, the substitution with the bulky tryptophan could interfere with the S169 (or the mammalian S87) phosphorylation site. Unlike for R167W (or R85W), the reduced DNA binding of R171W (or R89W) may therefore not result in phosphorylation-dependent dominant-negative effects, which is consistent with the haploinsufficiency that has been proposed for most MODY1 mutations. The reverse conclusion would be that haploinsufficiency alone is not enough to cause FRTS and that dominant-negative effects are required in addition (Marchesin, 2019).

As a consequence of the nuclear export due to the dominant-negative mechanism, this study uncovered cytotoxic effects that were not seen in the dHNF4 KD and hence are most likely linked with the aggregation of potentially misfolded proteins in the cytoplasm. Co-labeling with the nuclear envelope marker lamin D shows that the aggregate formation already commences during nuclear export. It can therefore be assumed that the small nuclear pore size causes an accumulation of dHNF4 beneath the nuclear envelope, which in turn favors aggregate formation. Although the order of cytosolic events is not entirely clear, it is speculated that the aggregates first pose a challenge for the clearance capacity of the proteasome, which is the normal site of HNF4A degradation. As a result, the ER may help by translocating chaperones into the cytoplasm to stimulate autophagy, as has been described for BiP and PDI. Consequently, the ability of the ER to address its own misfolded membrane-bound or luminal proteins could be weakened, thereby causing ER stress. Previously, cytoplasmic aggregates due to dominant-negative mutations or polyQ expansions have been observed in two other nuclear receptors, the thyroid receptor and the androgen receptor, respectively. In the latter case, the cytoplasmic aggregates were also associated with ER stress (Marchesin, 2019).

While dHNF4 has been shown to act as a regulator of mitochondria gene expression in the fly, the role of mammalian HNF4A in regulating mitochondrial function has so far been poorly investigated. Using the iREC system, this study showed that the R85W mutation affects mitochondria by decreasing the transcription of genes for mitochondrial structure and function. This was accompanied by a reduction in β-oxidation, leading to LD accumulation. As the HNF4A R85W also showed lower mRNA levels, it was not possible to assess the contribution of any dominant-negative effects in this model. Also, it was not possible to directly measure any effects of HNF4A R85W on DNA binding as was done in the COS-7 cell system. Nevertheless, both the nephrocyte and iREC results suggest that the effect on mitochondria could be one of the reasons why HNF4A R85W affects the fatty acid-consuming proximal tubules. In this manner, HNF4A-associated FRTS would be related to mitochondriopathies with isolated Fanconi syndrome or Fanconi syndrome as renal manifestation in more widespread disease. Given that HNF4A may be regulated by free fatty acids, it would be interesting to explore how extensively HNF4A is controlled by the reabsorption of exogenous fatty acids in proximal tubules (or nephrocytes) and whether fatty acid re-esterification and storage in LDs counteracts HNF4A activation (Marchesin, 2019).

In conclusion, these findings establish HNF4A as a master regulator of lipid metabolism in nephrocytes with high relevance for proximal tubular cells. This study further provides insight into the molecular basis of HNF4A-associated FRTS that has remained enigmatic since the identification of the first patient with the R85W mutation. The results suggest a rationale for treatments aimed at preventing the nuclear export of HNF4A in such patients. As serine phosphorylation in the DBD has been shown to be conserved in many nuclear hormone receptors, blocking this phosphorylation as a means to increase nuclear activity should also have more general implications (Marchesin, 2019).


Galloway-Mowat syndrome - a severe, inherited kidney-neurological disease

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Galloway-Mowat syndrome - a severe, inherited kidney-neurological disease


Krausel, V., Pund, L., Nusse, H., Bachir, H., Ricker, A., Klingauf, J., Weide, T., Pavenstadt, H., Krahn, M. P. and Braun, D. A. (2022). The transcription factor ATF4 mediates endoplasmic reticulum stress-related podocyte injury and slit diaphragm defects. Kidney Int. PubMed ID:
36587794
Summary:
Mutations in OSGEP and four other genes that encode subunits of the KEOPS complex cause Galloway-Mowat syndrome, a severe, inherited kidney-neurological disease. The complex catalyzes an essential posttranscriptional modification of tRNA and its loss of function induces endoplasmic reticulum (ER) stress. Using Drosophila garland nephrocytes and cultured human podocytes, this study aimed to elucidate the molecular pathogenic mechanisms of KEOPS-related glomerular disease and to test pharmacological inhibition of ER stress-related signaling as a therapeutic principle. ATF4, an ER stress-mediating transcription factor, or its fly orthologue Crc, were upregulated in both fly nephrocytes and human podocytes. Knockdown of Tcs3, a fly orthologue of OSGEP, caused slit diaphragm defects, recapitulating the human kidney phenotype. OSGEP cDNA with mutations found in patients lacked the capacity for rescue. Genetic interaction studies in Tcs3-deficient nephrocytes revealed that Crc mediates not only cell injury, but surprisingly also slit diaphragm defects, and that genetic or pharmacological inhibition of Crc activation attenuates both phenotypes. These findings are conserved in human podocytes where ATF4 inhibition improved the viability of podocytes with OSGEP knockdown, with chemically induced ER stress, and where ATF4 target genes and pro-apoptotic gene clusters are upregulated upon OSGEP knockdown. Thus, these data identify ATF4-mediated signaling as a molecular link among ER stress, slit diaphragm defects, and podocyte injury, and the data suggest that modulation of ATF4 signaling may be a potential therapeutic target for certain podocyte diseases.

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Glioma and Glioblastoma

Portela, M., Segura-Collar, B., Argudo, I., Saiz, A., Gargini, R., Sanchez-Gomez, P. and Casas-Tinto, S. (2018). Oncogenic dependence of glioma cells on kish/TMEM167A regulation of vesicular trafficking. Glia. PubMed ID:
30506943

Abstract

Genetic lesions in glioblastoma (GB) include constitutive activation of PI3K and EGFR pathways to drive cellular proliferation and tumor malignancy. An RNAi genetic screen, performed in Drosophila melanogaster to discover new modulators of GB development, identified a member of the secretory pathway: kish/TMEM167A. Downregulation of kish/TMEM167A impaired fly and human glioma formation and growth, with no effect on normal glia. Glioma cells increased the number of recycling endosomes, and reduced the number of lysosomes. In addition, EGFR vesicular localization was primed toward recycling in glioma cells. kish/TMEM167A downregulation in gliomas restored endosomal system to a physiological state and altered lysosomal function, fueling EGFR toward degradation by the proteasome. These endosomal effects mirrored the endo/lysosomal response of glioma cells to Brefeldin A (BFA), but not the Golgi disruption and the ER collapse, which are associated with the undesirable toxicity of BFA in other cancers. These results suggest that glioma growth depends on modifications of the vesicle transport system, reliant on kish/TMEM167A. Noncanonical genes in GB could be a key for future therapeutic strategies targeting EGFR-dependent gliomas (Portela, 2018).

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Kotian, N., Troike, K. M., Curran, K. N., Lathia, J. D. and McDonald, J. A. (2021). A Drosophila RNAi screen reveals conserved glioblastoma-related adhesion genes that regulate collective cell migration. G3 (Bethesda) 12(1). PubMed ID: 34849760
Summary:
Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. This study first identified cell adhesion, cell matrix, cell junction, and associated regulatory genes that are expressed in human brain tumors. RNAi knockdown of the Drosophila orthologs was performed in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, eight adhesion-related genes were identified that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

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Liu, J., Tao, X., Zhu, Y., Li, C., Ruan, K., Diaz-Perez, Z., Rai, P., Wang, H. and Zhai, R. G. (2021). NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis. Elife 10. PubMed ID: 34919052
Summary:

Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD(+) has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD(+) synthesis have had limited success due to insufficient mechanistic understanding. This study adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD(+) synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD(+)-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. These findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD(+) synthetic enzyme NMNAT and NAD(+)-dependent PTM enzymes that regulates glioma growth.

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Maravat, M., Bertrand, M., Landon, C., Fayon, F., Morisset-Lopez, S., Sarou-Kanian, V. and Decoville, M. (2021). Complementary Nuclear Magnetic Resonance-Based Metabolomics Approaches for Glioma Biomarker Identification in a Drosophila melanogaster Model. J Proteome Res. PubMed ID: 34286978

Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort. Glioma has been induced in Drosophila by co-activating the epidermal growth factor receptor and the phosphatidyl-inositol-3 kinase signaling pathways. Complementary nuclear magnetic resonance (NMR) techniques were used to obtain metabolic profiles in the third instar larvae brains. Fresh organs were directly studied by (1)H high resolution-magic angle spinning (HR-MAS) NMR, and brain extracts were analyzed by solution-state (1)H-NMR. Statistical analyses revealed differential metabolic signatures, impacted metabolic pathways, and glioma biomarkers. Each method was efficient to determine biomarkers. The highlighted metabolites including glucose, myo-inositol, sarcosine, glycine, alanine, and pyruvate for solution-state NMR and proline, myo-inositol, acetate, and glucose for HR-MAS show very good performances in discriminating samples according to their nature with data mining based on receiver operating characteristic curves. Combining results allows for a more complete view of induced disturbances and opens the possibility of deciphering the biochemical mechanisms of these tumors. The identified biomarkers provide a means to rebalance specific pathways through targeted metabolic therapy and to study the effects of pharmacological treatments using Drosophila as a model organism (Maravat, 2021).

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Formica, M., Storaci, A. M., Bertolini, I., Carminati, F., Knævelsrud, H., Vaira, V. and Vaccari, T. (2021). V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis. Autophagy: 1-11. PubMed ID: 33978540 ]

Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, an established Drosophila melanogaster model of glioma was used based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H(+)-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, these data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM.

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Goh, G. H., Blache, D., Mark, P. J., Kennington, W. J. and Maloney, S. K. (2021). Daily temperature cycles prolong lifespan and have sex-specific effects on peripheral clock gene expression in Drosophila melanogaster. J Exp Biol. PubMed ID: 33758022

Circadian rhythms optimize health by coordinating the timing of physiological processes to match predictable daily environmental challenges. The circadian rhythm of body temperature is thought to be an important modulator of molecular clocks in peripheral tissues, but how daily temperature cycles impact physiological function is unclear. This study examined the effect of constant (25°C, T(CON)) and cycling (28°C/22°C during light/dark, T(CYC)) temperature paradigms on lifespan of Drosophila melanogaster, and the expression of clock genes, Heat shock protein 83 (Hsp83), Frost (Fst), and Senescence-associated protein 30 (smp-30). Male and female Drosophila housed at T(CYC) had longer median lifespans than those housed at T(CON) T(CYC) induced robust Hsp83 rhythms and rescued the age-related decrease in smp-30 expression that was observed in flies at T(CON), potentially indicating an increased capacity to cope with age-related cellular stress. Ageing under T(CON) led to a decrease in the amplitude of expression of all clock genes in the bodies of male flies, except for cyc, which was non-rhythmic, and for per and cry in female flies. Strikingly, housing under T(CYC) conditions rescued the age-related decrease in amplitude of all clock genes, and generated rhythmicity in cyc expression, in the male flies, but not the female flies. The results suggest that ambient temperature rhythms modulate Drosophila lifespan, and that the amplitude of clock gene expression in peripheral body clocks may be a potential link between temperature rhythms and longevity in male Drosophila Longevity due to T(CYC) appeared predominantly independent of clock gene amplitude in female Drosophila.

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Conte, M., Casas-Tinto, S. and Soler, J.. (2021). Modeling invasion patterns in the glioblastoma battlefield. PLoS Comput Biol 17(1): e1008632. PubMed ID: 33513131

Abstract

Glioblastoma is the most aggressive tumor of the central nervous system, due to its great infiltration capacity. Understanding the mechanisms that regulate the Glioblastoma invasion front is a major challenge with preeminent potential clinical relevances. In the infiltration front, the key features of tumor dynamics relate to biochemical and biomechanical aspects, which result in the extension of cellular protrusions known as tumor microtubes. The coordination of metalloproteases expression, extracellular matrix degradation, and integrin activity emerges as a leading mechanism that facilitates Glioblastoma expansion and infiltration in uncontaminated brain regions. This paper proposes a novel multidisciplinary approach, based on in vivo experiments in Drosophila and mathematical models, that describes the dynamics of active and inactive integrins in relation to matrix metalloprotease concentration and tumor density at the Glioblastoma invasion front. The mathematical model is based on a non-linear system of evolution equations in which the mechanisms leading chemotaxis, haptotaxis, and front dynamics compete with the movement induced by the saturated flux in porous media. This approach is able to capture the relative influences of the involved agents and reproduce the formation of patterns, which drive tumor front evolution. These patterns have the value of providing biomarker information that is related to the direction of the dynamical evolution of the front and based on static measures of proteins in several tumor samples. Furthermore, biomechanical elements, like the tissue porosity, are considered as indicators of the healthy tissue resistance to tumor progression (Conte, 2021).

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Portela, M., Venkataramani, V., Fahey-Lozano, N., Seco, E., Losada-Perez, M., Winkler, F. and Casas-Tinto, S. (2019). Glioblastoma cells vampirize WNT from neurons and trigger a JNK/MMP signaling loop that enhances glioblastoma progression and neurodegeneration. PLoS Biol 17(12): e3000545. PubMed ID: 31846454

Abstract
Glioblastoma (GB) is the most lethal brain tumor, and Wingless (Wg)-related integration site (WNT) pathway activation in these tumors is associated with a poor prognosis. Clinically, the disease is characterized by progressive neurological deficits. However, whether these symptoms result from direct or indirect damage to neurons is still unresolved. Using Drosophila and primary xenografts as models of human GB, this study describes a mechanism that leads to activation of WNT signaling (Wg in Drosophila) in tumor cells. GB cells display a network of tumor microtubes (TMs) that enwrap neurons, accumulate Wg receptor Frizzled1 (Fz1), and, thereby, deplete Wg from neurons, causing neurodegeneration. This process has been defined process as "vampirization." Furthermore, GB cells establish a positive feedback loop to promote their expansion, in which the Wg pathway activates cJun N-terminal kinase (JNK) in GB cells, and, in turn, JNK signaling leads to the post-transcriptional up-regulation and accumulation of matrix metalloproteinases (MMPs), which facilitate TMs' infiltration throughout the brain, TMs' network expansion, and further Wg depletion from neurons. Consequently, GB cells proliferate because of the activation of the Wg signaling target, beta-catenin, and neurons degenerate because of Wg signaling extinction. These findings reveal a molecular mechanism for TM production, infiltration, and maintenance that can explain both neuron-dependent tumor progression and also the neural decay associated with GB.

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Sarthy, J. F., Meers, M. P., Janssens, D. H., Henikoff, J. G., Feldman, H., Paddison, P. J., Lockwood, C. M., Vitanza, N. A., Olson, J. M., Ahmad, K. and Henikoff, S. (2020). Histone deposition pathways determine the chromatin landscapes of H3.1 and H3.3 K27M oncohistones. Elife 9. PubMed ID: 32902381

Abstract

Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. This study shows that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNA replication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, this study demonstrated that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. It is proposed that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis (Sarthy, 2020).

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Gangwani, K., Snigdha, K. and Kango-Singh, M. (2020). Tep1 Regulates Yki Activity in Neural Stem Cells in Drosophila Glioma Model. Front Cell Dev Biol 8: 306. PubMed ID: 32457905

Abstract

Glioblastoma Multiforme (GBM) is the most common form of malignant brain tumor with poor prognosis. Amplification of Epidermal Growth Factor Receptor (EGFR), and mutations leading to activation of Phosphatidyl-Inositol-3 Kinase (PI3K) pathway are commonly associated with GBM. Using a previously published Drosophila glioma model generated by coactivation of PI3K and EGFR pathways [by downregulation of Pten and overexpression of oncogenic Ras] in glial cells, this study showed that the Drosophila Tep1 gene (ortholog of human CD109) regulates Yki (the Drosophila ortholog of human YAP/TAZ) via an evolutionarily conserved mechanism. Oncogenic signaling by the YAP/TAZ pathway occurs in cells that acquire CD109 expression in response to the inflammatory environment induced by radiation in clinically relevant models. Further, downregulation of Tep1 caused a reduction in Yki activity and reduced glioma growth. A key function of Yki in larval CNS is stem cell renewal and formation of neuroblasts. Other reports suggest different upstream regulators of Yki activity in the optic lobe versus the central brain regions of the larval CNS. It was hypothesized that Tep1 interacts with the Hippo pathway effector Yki to regulate neuroblast numbers. Tests were performed to see whether Tep1 acts through Yki to affect glioma growth and if in normal cells Tep1 affects neuroblast number and proliferation. These data suggests that Tep1 affects Yki mediated stem cell renewal in glioma, as reduction of Tep significantly decreases the number of neuroblasts in glioma. Thus, this study identifies Tep1-Yki interaction in the larval CNS that plays a key role in glioma growth and progression (Gangwani, 2020).

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Xie, S., Xu, C., Wu, C., Lou, Y., Duan, J., Sang, R., Lou, Z., Hou, J., Ge, W., Xi, Y. and Yang, X. (2023). Co-dependent regulation of p-BRAF and potassium channel KCNMA1 levels drives glioma progression. Cell Mol Life Sci 80(3): 61. PubMed ID: 36763212

Abstract

BRAF mutations have been found in gliomas which exhibit abnormal electrophysiological activities, implying their potential links with the ion channel functions. This study identified the Drosophila potassium channel, Slowpoke (Slo), the ortholog of human KCNMA1, as a critical factor involved in dRafGOF glioma progression. Slo was upregulated in dRafGOF glioma. Knockdown of slo led to decreases in dRafGOF levels, glioma cell proliferation, and tumor-related phenotypes. Overexpression of slo in glial cells elevated dRaf expression and promoted cell proliferation. Similar mutual regulations of p-BRAF and KCNMA1 levels were then recapitulated in human glioma cells with the BRAF mutation. Elevated p-BRAF and KCNMA1 were also observed in HEK293T cells upon the treatment of 20 mM KCl, which causes membrane depolarization. Knockdown KCNMA1 in these cells led to a further decrease in cell viability. Based on these results, it is concluded that the levels of p-BRAF and KCNMA1 are co-dependent and mutually regulated. It is proposed that, in depolarized glioma cells with BRAF mutations, high KCNMA1 levels act to repolarize membrane potential and facilitate cell growth. This study provides a new strategy to antagonize the progression of gliomas as induced by BRAF mutations.

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Heart Disease

Lovato, T. L., Blotz, B., Bileckyj, C., Johnston, C. A., Cripps, R. M. (2023). Modeling a variant of unknown significance in the Drosophila ortholog of the human cardiogenic gene NKX2.5. Disease models & mechanisms, 16(9) PubMed ID:
37691628

Abstract

Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. This study used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene NKX2.5 (also known as NKX2-5). An R321N allele of the NKX2.5 ortholog tinman (tin) was generated to model a human K158N variant, and its function was tested in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila T-box cardiac factor named Dorsocross1. A tinR321N allele was generated using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. It is proposed that the human K158N variant is pathogenic through causing a deficiency in DNA binding and a reduced ability to interact with a cardiac co-factor, and that cardiac defects might arise later in development or adult life (Lovato, 2023).

Birker, K., Ge, S., Kirkland, N. J., Theis, J. L., Marchant, J., Fogarty, Z. C., Missinato, M. A., Kalvakuri, S., Grossfeld, P., Engler, A. J., Ocorr, K., Nelson, T. J., Colas, A. R., Olson, T. M., Vogler, G. and Bodmer, R. (2023). Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity. Elife 12. PubMed ID: 37404133

Abstract

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. This study performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, 60 additional prioritized candidate genes from these patients were tested for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs (Birker, 2023).

Hypoplastic left heart syndrome (HLHS) is a birth defect that accounts for 2-4% of congenital heart defects (CHDs), equal to 1000-2000 HLHS births in the United States per year. HLHS has been proposed to be caused by genetic, epigenetic, or environmental factors. The severe cardiac characteristics of HLHS include aortic and mitral stenosis or atresia, and reduced size of the left ventricle and aorta; however, there is a spectrum of cardiac phenotypes that can underly HLHS pathophysiology. If not treated with reconstructive heart surgeries or cardiac transplantation, infants born with HLHS will not survive. To date, the standard treatment for this disease is a three-stage surgical procedure, which begins neonatally and aims overall to achieve right ventricle-dependent systemic circulation and deliver oxygen-poor blood more directly to the lungs. Although the surgical procedures correctly divert left ventricular function to the right ventricle, there is a subgroup of HLHS patients who are at risk of latent heart failure, which is often preceded by reduced ejection fraction (Birker, 2023).

Although several studies have examined the molecular underpinnings of HLHS, the number of genes associated with this disease is small (e.g. NKX2-5, NOTCH1, ETS1, MYH6, LRP2, and CELSR1), and they are not yet conclusively determined as causal for HLHS. Defining pathogenic mechanisms has proved elusive given the oligogenic complexity of HLHS. Overall, there is a great need to functionally evaluate newly emerging HLHS candidate genes to understand how they may contribute to the molecular, cellular, and morphological processes underlying HLHS (Birker, 2023).

Drosophila is well-suited for modeling genetic underpinnings of CHDs: many of the genes and gene programs found in the Drosophila heart are evolutionarily conserved, including a core set of cardiogenic transcription factors and inductive factors (e.g. Nkx2-5/tinman), approximately 75% of known human disease-causing genes having fly orthologs, and the developing mammalian and Drosophila hearts share developmental similarities, such as their origin within the mesoderm (Birker, 2023).

Mitochondria have been postulated to play a critical role in HLHS pathogenesis. For example, a recent study reported that cardiomyocytes derived from iPSCs of HLHS patients (iPSC-CM), who later developed right ventricular failure, had reduced mitochondrial concentration, ATP production, and contractile force. This study revealed downregulated expression of genes involved in mitochondrial processes, such as ATP synthesis coupled electron transport. Another study of HLHS patient-derived iPSC-CMs revealed reduced mitochondrial size, number, and malformed mitochondrial inner membranes using transmission electron microscopy. Similarly, an HLHS mouse model with Sap130 and Pcdha9 mutations showed mitochondrial defects manifested as reduced cristae density and smaller mitochondrial size. Despite a lack of understanding of the exact mitochondrial mechanisms underlying HLHS pathogenesis, recent experimental and bioinformatic data suggest an underlying role of mitochondria in HLHS (Birker, 2023).

In this study, a cohort of 183 HLHS proband-parent trios underwent whole genome sequencing (WGS) to identify candidate genes, including a prioritized consanguineous family where genes harboring rare, predicted damaging homozygous variants were investigated. Among the resulting candidate HLHS genes tested in Drosophila, cardiac-specific knockdown (KD) of Chchd3/6 (coiled-coil-helix-coiled-coil-helix-domain-containing protein 6) of the MICOS (mitochondrial contact site and cristae organization system) complex exhibited severe heart structure and function defects. The MICOS complex is an eight-subunit complex in mammals (five in Drosophila) located in the inner mitochondrial membrane that is necessary to maintain cristae morphology and ATP production. It is closely associated and interacts with SAMM50 (sorting and assembly machinery, CG7639), which is located in the outer mitochondrial membrane. The MICOS complex's role in cardiac development and functional homeostasis is not known but is likely important for efficient ATP production. Reduced contractility was observed upon cardiac-specific Chchd3/6 KD, diminished sarcomeric Actin and Myosin levels, as well as severe mitochondrial morphology defects, which manifested as fragmented and aggregated structures. Similar phenotypes were observed upon cardiac KD of other MICOS complex genes, as well as other mitochondrial genes such as ATP synthase (complex V), specifically ATP synthase B and β. Significantly diminished proliferation of human induced pluripotent stem cell (iPSC)-derived ventricular-like cardiomyocytes (VCMs) was found upon KD of MICOS genes. Finally, a family-based candidate gene interaction screen in Drosophila revealed three genes that genetically interact with Chchd3/6: Cdk12 (activator RNA polymerase II activator), RNF149 (goliath, gol, E3 ubiquitin ligase), SPTBN1 (β Spectrin, scaffolding protein). In summary, Chchd3/6 and other components important for mitochondrial homeostasis were identified as critical for establishing and maintaining cardiac structure and function, and likely contribute to HLHS and/or latent heart failure following surgical palliation (Birker, 2023).

HLHS is characterized by a small left heart, including reduced left ventricle size and mitral and/or atrial atresia or stenosis, and aortic hypoplasia, collectively obstructing systemic blood flow. As a consequence, newborns cannot sustain systemic blood flow for more than a few days and therefore require treatment soon after birth. There is a need for improved therapies to treat HLHS patients, and this requires a better understanding of the biology behind HLHS pathogenesis. This study probed the genetic basis of HLHS using WGS and powerful bioinformatic gene variant prioritization in a large cohort of HLHS proband-parent trios combined with model system validation (Birker, 2023).

The 11 H family was prioritized because of consanguinity, implicating a homozygous recessive mode of inheritance that resulted in a short list of nine candidate genes. These candidate genes were probed in Drosophila and iPSC-CMs for a potential role in cardiomyocyte development and function, to gain new insights into HLHS and CHDs in general. Among these HLHS gene candidates, this study focused on CHCHD3/6, which has not been previously studied in the heart, and which had striking cardiac functional and structural defects in Drosophila. Specifically, the preliminary gene screen demonstrated that Chchd3/6 cardiac-specific KD caused reduced contractility and decreased sarcomeric F-Actin and Myosin staining (Birker, 2023).

The data suggest that Chchd3/6 is necessary during larval and early adult stages to maintain contractility in the adult heart. This is relevant since patients with HLHS have both structural heart disease and risk for later myocardial failure. The prevailing 'no flow, no grow' hypothesis for HLHS pathogenesis surmises that reduced blood flow in the fetal heart causes underdevelopment of the left ventricle. A reduced ability for the heart to contract in utero, due to reduced CHCHD6 activity, could contribute to decreased ventricular blood flow in the embryo, resulting in an abnormally small left ventricle. Moreover, reduced CHCHD6 activity could compromise right ventricular function later in life. In fact, the 11 H proband exhibited mildly reduced right ventricular ejection fraction several years after successful surgical palliation. Consistent with the model system, CHCHD6 deficiency could result in cumulative impairment of mitochondrial function, leading to contractile dysfunction. Why a mitochondrial defect would have a preferential effect on the left ventricle is still an enigma. It is speculated that some of the patient-specific variants that potentially contribute to this likely polygenic disease are in genes that may have a higher expression level or functional importance in the left ventricle, thus in combination with MICOS variants preferentially affecting left-ventricular growth and differentiation, leading to decreased contractility, then again compounded by impaired blood flow feeding back to diminishing growth. Future studies investigating the polygenic basis of HLHS are needed to address this question (Birker, 2023).

Chchd3/6 KD in the fly heart led to mitochondrial fission-fusion defects, with reduced ATP synthase (complex V) levels, and consequently impaired ATP production. It has previously been reported that CHCHD3 KD in HeLa cells resulted in fragmented mitochondria that was due to improper mitochondrial fusion. It has also been demonstrated in yeast that individual or combinatorial loss of MICOS complex proteins disrupt cristae morphology, thus suggesting a mechanism by which CHCHD3/6 loss could mediate HLHS pathogenesis. Furthermore, a genetic interaction between was identified SAMM50 and CHCHD3/6 that leads to a contractile deficit and diminished sarcomeric F-Actin. Recent findings demonstrate that SAMM50 directly interacts mammalian CHCHD3, to mediate inner and outer membrane bridging and cristae morphology (Birker, 2023).

The data further suggest that ETC Complex V/ ATP synthase is a potential downstream effector of CHCHD3/6 and MICOS complex function. Individual KD of ATP synthase subunits resulted in reduced fractional shortening and reduced sarcomeric actin. As a result, reduced CHCHD3/6 expression is hypothesized to affects ETC function, specifically ATP synthase, leading to reduced ATP production. Since the MICOS complex is in involved ETC assembly in cristae, ATPase subunits may not be assembled correctly causing mitochondrial dysfunction, accompanied by reduced/abnormal mito-GFP staining. OXPHOS complex assembly has been shown to be disrupted upon MICOS depletion, and it is speculated that ATP synthase function may be disrupted when CHCHD3/6 is reduced. Consistent with this, depletion of ATP synthase levels was observed upon Chchd3/6 KD (Birker, 2023).

Finally, a potential oligogenic basis of HLHS was tested in the family-based CHCHD3 and CHCHD6 interaction screen and identified three hits that reduced fractional shortening only in conjunction with CHCHD3/6, but not on their own. Co-KD of Cdk12 and Chchd3/6 also reduced fractional shortening, and caused greater lethality relative to Cdk12 KD alone. Cdk12 activates RNA polymerase II to regulate transcription elongation. It was postulated that since Chchd3/6 is a nuclear-encoded gene, reducing transcription with Cdk12 KD could decrease CHCHD3/6 levels in a background where CHCHD3/6 activity is already compromised. Alternatively, reduced transcription of other nuclear genes associated with ATP production in combination with Chchd3/6 KD could further reduce ATP levels enough to cause contractility defects. In support of this, a study examining the effects of RMP (RNA polymerase II subunit 5-mediating protein) found that mice with cardiac-specific Rpm KO exhibited reduced fractional shortening and ATP levels, which were attributed to a reduction in mRNA and protein levels of the mitochondrial biogenesis factor PGC1α. The second hit, goliath, is an endosomal ubiquitin E3 ligase. Although goliath has been implicated in endosomal recycling, its role in Drosophila mitophagy in vivo has not been examined. Reduced cardiac contractility with co-KD of gol and Chchd3/6 could result from impaired mitophagy and reduced mitochondrial biogenesis. Together, the accumulation of damaged mitochondria can reduce ATP content required for contraction. The third hit, β-Spectrin, acts as a scaffolding protein. Recent data suggests that the human ortholog, SPTBN1 (Nonerythroid spectrin β) influences SPTAN1 (Nonerythroid spectrin α) levels, which has a calmodulin binding domain. Therefore, decreased β-Spec expression could reduce Calmodulin levels, thereby reducing contractility due to the combined reduction in Ca2+ handling and Chchd3/6 KD-induced reduced ATP levels (Birker, 2023).

In summary, this study has identified a novel mechanism potentially involved HLHS pathogenesis, starting by analyzing WGS data from a prioritized family and large cohort of HLHS patients, followed by functional testing in vivo using the Drosophila heart model and in vitro using human iPSC-derived CMs. Compromised contractile capacity, diminished sarcomeric F-Actin and Myosin accumulation, and mitochondrial dysfunction in Chchd3/6 KD Drosophila hearts are promising phenotypes that could contribute to early HLHS manifestations or heart failure complications later in life. Further examination of the interactions between the MICOS complex and other emerging candidate genes will identify novel gene functions and pathways that contribute to HLHS pathogenesis. Furthermore, a detailed elucidation of novel candidate genes and genetic interactions based on patient-specific rare potentially damaging variants is expected to lead to gene networks that are relevant for HLHS and other CHDs (Birker, 2023). Schroeder, A. M., Allahyari, M., Vogler, G., Missinato, M. A., Nielsen, T., Yu, M. S., Theis, J. L., Larsen, L. A., Goyal, P., Rosenfeld, J., Nelson, T. J., Olson, T. M., Colas, A. R., Grossfeld, P. and Bodmer, R. (2019). Model system identification of novel congenital heart disease gene candidates: focus on RPL13. Hum Mol Genet. PubMed ID: 31625562

Abstract
Genetics is a significant factor contributing to congenital heart disease (CHD), but understanding of the genetic players and networks involved in CHD pathogenesis is limited. This study searched for de novo Copy Number Variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. This search focused on new candidate disease genes within 19 deleted de novo CNVs that did not cover known CHD genes. For this study, an integrated high-throughput phenotypical platform was developed to probe for defects in cardiogenesis and cardiac output in human iPSC-derived multipotent cardiac progenitor cells (MCPs) and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 knockdown, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. Knockdown of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, this study identified new genes as candidates for causing human congenital heart disease, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease (Schroeder, 2019).

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Auxerre-Plantie, E., Nielsen, T., Grunert, M., Olejniczak, O., Perrot, A., Ozcelik, C., Harries, D., Matinmehr, F., Dos Remedios, C., Muhlfeld, C., Kraft, T., Bodmer, R., Vogler, G. and Sperling, S. R. (2020). Identification of MYOM2 as a candidate gene in hypertrophic cardiomyopathy and tetralogy of fallot and its functional evaluation in the Drosophila heart. Dis Model Mech. PubMed ID: 33033063

Abstract

The causal genetic underpinnings of congenital heart diseases, which are often complex and with multigenic background, are still far from understood. Moreover, there are also predominantly monogenic heart defects, such as cardiomyopathies, with known disease genes for the majority of cases. This study identified mutations in myomesin 2 (MYOM2) in patients with Tetralogy of Fallot (TOF), the most common cyanotic heart malformation, as well as in patients with hypertrophic cardiomyopathy (HCM), who do not exhibit any mutations in the known disease genes. MYOM2 is a major component of the myofibrillar M-band of the sarcomere and a hub gene within interactions of sarcomere genes. This study shows that patient-derived cardiomyocytes exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths. Moreover, a comprehensive functional analyses in the Drosophila animal model reveal that the so far uncharacterized fly gene CG14964 may be an ortholog of MYOM2, as well as other myosin binding proteins (henceforth named as Drosophila Myomesin and Myosin Binding protein (dMnM)). Its partial loss-of-function or moderate cardiac knockdown results in cardiac dilation, whereas more severely reduced function causes a constricted phenotype and an increase in sarcomere myosin protein. Moreover, compound heterozygous combinations of CG14964 and the sarcomere gene Mhc (MYH6/7) exhibited synergistic genetic interactions. In summary, these results suggest that MYOM2 not only plays a critical role in maintaining robust heart function but may also be a candidate gene for heart diseases such as HCM and TOF, as it is clearly involved in the development of the heart (Auxerre-Plantie, 2020).

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Manivannan, S. N., Darouich, S., Masmoudi, A., Gordon, D., Zender, G., Han, Z., Fitzgerald-Butt, S., White, P., McBride, K. L., Kharrat, M. and Garg, V. (2020). Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy. PLoS Genet 16(5): e1008639. PubMed ID: 32453731

Abstract
Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. This study reports a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, one of the affected children was discovered to have a homozygous frameshift variant in Myosin light chain 2, which alters the last 20 amino acids of the protein and is predicted to impact the most C-terminal of the three EF-hand domains in MYL2. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and the infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed a marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant and three other MYL2 stop-gain variants that result in loss of the EF domains are stably expressed but show impaired localization. The degradation of the MYL2-fs can be rescued by inhibiting the cell's proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither the MYL2-fs nor the MYL2:p.Gly162Arg variant supports normal cardiac function. The tools that have been generated for this study provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. This study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants in infantile HCM and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathy.

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Huang, K., Miao, T., Chang, K., Kim, J., Kang, P., Jiang, Q., Simmonds, A. J., Di Cara, F. and Bai, H. (2020). Impaired peroxisomal import in Drosophila oenocytes causes cardiac dysfunction by inducing upd3 as a peroxikine. Nat Commun 11(1): 2943. PubMed ID: 32523050

Abstract
Aging is characterized by a chronic, low-grade inflammation, which is a major risk factor for cardiovascular diseases. It remains poorly understood whether pro-inflammatory factors released from non-cardiac tissues contribute to the non-autonomous regulation of age-related cardiac dysfunction. This study reports that age-dependent induction of cytokine unpaired 3 (upd3) in Drosophila oenocytes (hepatocyte-like cells) is the primary non-autonomous mechanism for cardiac aging. upd3 is significantly up-regulated in aged oenocytes. Oenocyte-specific knockdown of upd3 is sufficient to block aging-induced cardiac arrhythmia. It was further shown that the age-dependent induction of upd3 is triggered by impaired peroxisomal import and elevated JNK signaling in aged oenocytes. Hormonal factors induced by peroxisome dysfunction are referred to as peroxikines. Intriguingly, oenocyte-specific overexpression of Pex5, the key peroxisomal import receptor, blocks age-related upd3 induction and alleviates cardiac arrhythmicity. Thus, these studies identify an important role of hepatocyte-specific peroxisomal import in mediating non-autonomous regulation of cardiac aging (Huang, 2020).

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Akasaka, T., Ocorr, K., Lin, L., Vogler, G., Bodmer, R. and Grossfeld, P. . (2020). Overexpression of Kif1A in the Developing Drosophila Heart Causes Valvar and Contractility Defects: Implications for Human Congenital Heart Disease. J Cardiovasc Dev Dis 7(2). PubMed ID: 32498427

Abstract

Left-sided congenital heart defects (CHDs) are among the most common forms of congenital heart disease, but a disease-causing gene has only been identified in a minority of cases. This study identified a candidate gene for CHDs, KIF1A, that was associated with a chromosomal balanced translocation t(2;8)(q37;p11) in a patient with left-sided heart and aortic valve defects. The breakpoint was in the 5' untranslated region of the KIF1A gene at 2q37, which suggested that the break affected the levels of Kif1A gene expression. Transgenic fly lines overexpressing Kif1A specifically in the heart muscle (or all muscles) caused diminished cardiac contractility, myofibrillar disorganization, and heart valve defects, whereas cardiac knockdown had no effect on heart structure or function. Overexpression of Kif1A also caused increased collagen IV deposition in the fibrous network that normally surrounds the fly heart. Kif1A overexpression in C2C12 myoblasts resulted in specific displacement of the F-actin fibers, probably through a direct interaction with G-actin. These results point to a Kif1A-mediated disruption of F-actin organization as a potential mechanism for the pathogenesis in at least some human CHDs (Akasaka, 2020).

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lmen, L., Volpato, C. B., Kervadec, A., Pineda, S., Kalvakuri, S., Alayari, N. N., Foco, L., Pramstaller, P. P., Ocorr, K., Rossini, A., Cammarato, A., Colas, A. R., Hicks, A. A. and Bodmer, R. (2020). (2020). Silencing of CCR4-NOT complex subunits affect heart structure and function. Dis Model Mech. PubMed ID: 32471864

Abstract

Genome wide association studies (GWAS) have identified variants that associate with QT-interval length. Three of the strongest associating variants (SNPs) are located in the putative promotor region of CNOT1, a gene encoding the central subunit of CCR4-NOT, a multi-functional, conserved complex regulating gene expression and mRNA stability and turnover. The minimum fragment of the CNOT1 promoter containing all three variants was isolated from individuals homozygous for the QT-risk alleles and it was then demonstrated that the haplotype associating with longer QT-interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression may contribute to abnormal QT-intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-CNOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes, CNOT1/not1 and CNOT7/8/pop2, in vivo, was either lethal or resulted in dilated cardiomyopathy, reduced contractility, or a propensity for arrhythmia. Silencing CNOT2/not2, CNOT4/not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/not1 and CNOT7/8/pop2 are required during cardiac remodeling from larval to adult stages. In sum, this study has demonstrated how disease associated genes identified by GWAS can be investigated, by combining human cardiomyocyte cell-based and whole organism in vivo heart models. These results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a critical role of the CCR4-NOT complex in heart development and function (Elmen, 2020).

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Hereditary neuropathy

Cortese, A., Zhu, Y., Rebelo, A. P.....Zhai, R. G. and Zuchner, S. (2020). Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes. Nat Genet 52(5): 473-481. PubMed ID: 32367058

Abstract

This study report biallelic mutations in the sorbitol dehydrogenase gene (SORD) as the most frequent recessive form of hereditary neuropathy. 45 individuals were identified from 38 families across multiple ancestries carrying the nonsense c.757delG (p.Ala253GlnfsTer27) variant in SORD, in either a homozygous or compound heterozygous state. SORD is an enzyme that converts sorbitol into fructose in the two-step polyol pathway previously implicated in diabetic neuropathy. In patient-derived fibroblasts, a complete loss of SORD protein and increased intracellular sorbitol were found. Furthermore, the serum fasting sorbitol levels in patients were dramatically increased. In Drosophila, loss of SORD orthologs caused synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient-derived fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a novel and potentially treatable cause of neuropathy and may contribute to a better understanding of the pathophysiology of diabetes (Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes (Cortese, 2020).

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Human papillomavirus E6-induced malignancy

Padash Barmchi, M., Gilbert, M., Thomas, M., Banks, L., Zhang, B. and Auld, V. J. (2016). A Drosophila model of HPV E6-induced malignancy reveals essential roles for Magi and the Insulin Receptor. PLoS Pathog 12: e1005789. PubMed ID: 27537218

Abstract

The causative agents of cervical cancers, high-risk human papillomaviruses (HPVs), cause cancer through the action of two oncoproteins, E6 and E7. The E6 oncoprotein cooperates with an E3 ubiquitin ligase (UBE3A; see Drosophila Ube3a) to target the p53 tumour suppressor and important polarity and junctional PDZ proteins for proteasomal degradation. Hozwever, the causative link between degradation of PDZ proteins and E6-mediated malignancy is largely unknown. An in vivo model of HPV E6-mediated cellular transformation was developed using Drosophila as model. Co-expression of E6 and human UBE3A in wing and eye epithelia results in severe morphological abnormalities. Furthermore, E6, via its PDZ-binding motif and in cooperation with UBE3A, targets a suite of PDZ proteins, including Magi, Dlg and Scribble. Similar to human epithelia, Drosophila Magi is a major degradation target. Magi overexpression rescues the cellular abnormalities caused by E6+UBE3A coexpression and this activity of Magi is PDZ domain-dependent. Tumorigenesis occurred when E6+UBE3A are expressed in conjunction with activated/oncogenic forms of Ras or Notch. This study identified the insulin receptor signaling pathway as being required for E6+UBE3A induced hyperplasia. These results suggest a highly conserved mechanism of HPV E6 mediated cellular transformation (Padash Barmchi, 2016).

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Hypertrophic Cardiomyopathy

Bell, K. M., Kronert, W. A., Huang, A., Bernstein, S. I. and Swank, D. M. (2019). The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production. J Physiol. PubMed ID: 30950055

Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, an HCM myosin mutation, R249Q, was expressed in Drosophila indirect flight muscle (IFM), and myofibril structure, skinned fibre mechanical properties, and flight ability were assessed. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. R249Q myosin slows attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, these results do not support the increased contractility hypothesis. Instead, these results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output (Bell, 2019).

Tallo, C. A., Duncan, L. H., Yamamoto, A. H., Slaydon, J. D., Arya, G. H., Turlapati, L., Mackay, T. F. C. and Carbone, M. A. (2021). Heat shock proteins and small nucleolar RNAs are dysregulated in a Drosophila model for feline hypertrophic cardiomyopathy. G3 (Bethesda) 11(1). PubMed ID: 33561224

Abstract

In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. This study establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. RNA-seq was used to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans (Tallo, 2021).

Suggs, J. A., Melkani, G. C., Glasheen, B. M., Detor, M. M., Melkani, A., Marsan, N. P., Swank, D. M. and Bernstein, S. I. (2017). A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects. Dis Model Mech 10(6): 761-771. PubMed ID: 28258125

Abstract

Inclusion body myopathy type 3 (IBM-3) patients display congenital joint contractures with early-onset muscle weakness that becomes more severe in adults. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. The corresponding myosin mutation (E701K) in Drosophila Myosin was expressed in homozygous Drosophila indirect flight muscles and the myofibrillar degeneration and inclusion bodies observed in the human disease was recapitulated. Purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM-3 is a dominant condition, the disease state was examined in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild-type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed an 85% decrease in maximum oscillatory power generation and an approximately 6-fold reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, it is hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro motility, reduced muscle power output and focal myofibrillar disorganization similar to that seen in human IBM-3 patients (Suggs, 2017).

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Inflamatory Gut Disease

Madi, J. R., Outa, A. A., Ghannam, M., Hussein, H. M., Shehab, M., Hasan, Z., Fayad, A. A., Shirinian, M. and Rahal, E. A. (2021)
(2021). Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases. Front Immunol 12: 586930. PubMed ID: 33828545
Summary:

Abstract

The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since immune-stimulating capabilities of EBV DNA has been reported in various models, this study investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. Inflamed gastrointestinal tracts were generated in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. These findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators.

Keshav, N., Ammankallu, R., Shashidhar, Paithankar, J. G., Baliga, M. S., Patil, R. K., Kudva, A. K. and Raghu, S. V. (2022). Dextran sodium sulfate alters antioxidant status in the gut affecting the survival of Drosophila melanogaster. 3 Biotech 12(10): 280. PubMed ID: 36275361

Abstract

Inflammatory bowel disease (IBD) is a group of disorders characterized by chronic inflammation in the intestine. Several studies confirmed that oxidative stress induced by an enormous amount of reactive free radicals triggers the onset of IBD. Currently, there is an increasing trend in the global incidence of IBD and it is coupled with a lack of adequate long-term therapeutic options. At the same time, progress in research to understand the pathogenesis of IBD has been hampered due to the absence of adequate animal models. Currently, the toxic chemical Dextran Sulfate Sodium (DSS) induced gut inflammation in rodents is widely perceived as a good model of experimental colitis or IBD. Drosophila melanogaster, a genetic animal model, shares ~ 75% sequence similarity to genes causing different diseases in humans and also has conserved digestion and absorption features. Therefore, the current study used Drosophila as a model system to induce and investigate DSS-induced colitis. Anatomical, biochemical, and molecular analyses were performed to measure the levels of inflammation and cellular disturbances in the gastrointestinal (GI) tract of Drosophila. This study shows that DSS-induced inflammation lowers the levels of antioxidant molecules, affects the life span, reduces physiological activity and induces cellular damage in the GI tract mimicking pathophysiological features of IBD in Drosophila. Such a DSS-induced Drosophila colitis model can be further used for understanding the molecular pathology of IBD and screening novel drugs (Keshav, 2022).

Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Dextran sodium sulfate alters antioxidant status in the gut affecting the survival of Drosophila melanogaster. 3 Biotech 12(10): 280. PubMed ID: 36275361

Abstract

. Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701

Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila

Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs (Yan, 2023).

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Hyperuricemia

Lang, S., Hilsabeck, T. A., Wilson, K. A., Sharma, A., Bose, N., Brackman, D. J., Beck, J. N., Chen, L., Watson, M. A., Killilea, D. W., Ho, S., Kahn, A., Giacomini, K., Stoller, M. L., Chi, T. and Kapahi, P.
(2019). A conserved role of the insulin-like signaling pathway in diet-dependent uric acid pathologies in Drosophila melanogaster. PLoS Genet 15(8): e1008318. PubMed ID: 31415568

Abstract

Elevated uric acid (UA) is a key risk factor for many disorders, including metabolic syndrome, gout and kidney stones. Despite frequent occurrence of these disorders, the genetic pathways influencing UA metabolism and the association with disease remain poorly understood. In humans, elevated UA levels resulted from the loss of the of the urate oxidase (Uro) gene around 15 million years ago. Therefore, this study used a Drosophila melanogaster model with reduced expression of the orthologous Uro gene to study the pathogenesis arising from elevated UA. Reduced Uro expression in Drosophila resulted in elevated UA levels, accumulation of concretions in the excretory system, and shortening of lifespan when reared on diets containing high levels of yeast extract. Furthermore, high levels of dietary purines, but not protein or sugar, were sufficient to produce the same effects of shortened lifespan and concretion formation in the Drosophila model. The insulin-like signaling (ILS) pathway has been shown to respond to changes in nutrient status in several species. This study observed that genetic suppression of ILS genes reduced both UA levels and concretion load in flies fed high levels of yeast extract. Further support for the role of the ILS pathway in modulating UA metabolism stems from a human candidate gene study identifying SNPs in the ILS genes AKT2 and FOXO3 being associated with serum UA levels or gout. Additionally, inhibition of the NADPH oxidase (NOX) gene rescued the reduced lifespan and concretion phenotypes in Uro knockdown flies. Thus, components of the ILS pathway and the downstream protein NOX represent potential therapeutic targets for treating UA associated pathologies, including gout and kidney stones, as well as extending human healthspan (Lang, 2019).

Hilsabeck, T. A. U., Liu-Bryan, R., Guo, T., Wilson, K. A., Bose, N., Raftery, D., Beck, J. N., Lang, S., Jin, K., Nelson, C. S., Oron, T., Stoller, M., Promislow, D., Brem, R. B., Terkeltaub, R. and Kapahi, P. (2022). A fly GWAS for purine metabolites identifies human FAM214 homolog medusa, which acts in a conserved manner to enhance hyperuricemia-driven pathologies by modulating purine metabolism and the inflammatory response. Geroscience. PubMed ID: 35381951

Abstract
Elevated serum urate (hyperuricemia) promotes crystalline monosodium urate tissue deposits and gout, with associated inflammation and increased mortality. To identify modifiers of uric acid pathologies, a fly Genome-Wide Association Study (GWAS) was performed on purine metabolites using the Drosophila Genetic Reference Panel strains. The candidate genes were tested using the Drosophila melanogaster model of hyperuricemia and uric acid crystallization ("concretion formation") in the kidney-like Malpighian tubule. Medusa (mda) activity increased urate levels and inflammatory response programming. Conversely, whole-body mda knockdown decreased purine synthesis precursor phosphoribosyl pyrophosphate, uric acid, and guanosine levels; limited formation of aggregated uric acid concretions; and was sufficient to rescue lifespan reduction in the fly hyperuricemia and gout model. Levels of mda homolog FAM214A were elevated in inflammatory M1- and reduced in anti-inflammatory M2-differentiated mouse bone marrow macrophages, and influenced intracellular uric acid levels in human HepG2 transformed hepatocytes. In conclusion, mda/FAM214A (see Drosophila CG9005) acts in a conserved manner to regulate purine metabolism, promotes disease driven by hyperuricemia and associated tissue inflammation, and provides a potential novel target for uric acid-driven pathologies.

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Hypotonia

Kim, J. H., Singh, M., Pan, G., Lopez, A., Zito, N., Bosse, B. and Ye, B.
(2020). Frameshift mutations of YPEL3 alter the sensory circuit function in Drosophila. Dis Model Mech 13(6). PubMed ID: 32461240

Abstract
A frameshift mutation in Yippee-like (YPEL) 3 was recently found from a rare human disorder with peripheral neurological conditions including hypotonia and areflexia. The YPEL gene family is highly conserved from yeast to human, but its members' functions are poorly defined. Moreover, the pathogenicity of the human YPEL3 variant is completely unknown. This study generated a Drosophila model of human YPEL3 variant and a genetic null allele of Drosophila homolog of YPEL3 (referred to as dYPEL3/CG15309). Gene-trap analysis suggests that dYPEL3 is predominantly expressed in subsets of neurons, including larval nociceptors. Analysis of chemical nociception induced by allyl-isothiocyanate (AITC), a natural chemical stimulant, revealed reduced nociceptive responses in both dYPEL3 frameshift and null mutants. Subsequent circuit analysis showed reduced activation of second-order neurons (SONs) in the pathway without affecting nociceptor activation upon AITC treatment. Although the gross axonal and dendritic development of nociceptors was unaffected, the synaptic contact between nociceptors and SONs was decreased by the dYPEL3 mutations. Furthermore, expressing dYPEL3 in larval nociceptors rescued the behavioral deficit in dYPEL3 frameshift mutants, suggesting a presynaptic origin of the deficit. Together, these findings suggest that the frameshift mutation results in YPEL3 loss of function and may cause neurological conditions by weakening synaptic connections through presynaptic mechanisms (Kim,2020).

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Insomnia

Belfer, S. J., Bashaw, A. G., Perlis, M. L. and Kayser, M. S.
(2019). A Drosophila model of sleep restriction therapy for insomnia. Mol Psychiatry. PubMed ID: 30824866

Abstract

Insomnia is the most common sleep disorder among adults, especially affecting individuals of advanced age or with neurodegenerative disease. Insomnia is also a common comorbidity across psychiatric disorders. Cognitive behavioral therapy for insomnia (CBT-I) is the first-line treatment for insomnia; a key component of this intervention is restriction of sleep opportunity, which optimizes matching of sleep ability and opportunity, leading to enhanced sleep drive. Despite the well-documented efficacy of CBT-I, little is known regarding how CBT-I works at a cellular and molecular level to improve sleep, due in large part to an absence of experimentally-tractable animals models of this intervention. Guided by human behavioral sleep therapies, this study developed a Drosophila model for sleep restriction therapy (SRT) of insomnia. It was demonstrated that restriction of sleep opportunity through manipulation of environmental cues improves sleep efficiency in multiple short-sleeping Drosophila mutants. The response to sleep opportunity restriction requires ongoing environmental inputs, but is independent of the molecular circadian clock. This sleep opportunity restriction paradigm was applied to aging and Alzheimer's disease fly models; sleep impairments in these models are reversible with sleep restriction, with associated improvement in reproductive fitness and extended lifespan. This work establishes a model to investigate the neurobiological basis of CBT-I, and provides a platform that can be exploited toward novel treatment targets for insomnia (Belfer, 2019).

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Intellectual Disability associated with WAC

Lugtenberg, D., et al. (2016). De novo loss-of-function mutations in WAC cause a recognizable intellectual disability syndrome and learning deficits in Drosophila. Eur J Hum Genet. PubMed ID: 26757981

Abstract

Recently WAC was reported as a candidate gene for intellectual disability (ID) based on the identification of a de novo mutation in an individual with severe ID. WAC regulates transcription-coupled histone H2B ubiquitination and has previously been implicated in the 10p12p11 contiguous gene deletion syndrome. This study reports on 10 individuals with de novo WAC mutations which were identified through routine (diagnostic) exome sequencing and targeted resequencing of WAC in 2326 individuals with unexplained ID. All but one mutation was expected to lead to a loss-of-function of WAC. Clinical evaluation of all individuals revealed phenotypic overlap for mild ID, hypotonia, behavioral problems and distinctive facial dysmorphisms, including a square-shaped face, deep set eyes, long palpebral fissures, and a broad mouth and chin. These clinical features were also previously reported in individuals with 10p12p11 microdeletion syndrome. To investigate the role of WAC in ID, the importance of the Drosophila WAC orthologue (CG8949) was studied in habituation, a non-associative learning paradigm. Neuronal knockdown of Drosophila CG8949 resulted in impaired learning, suggesting that WAC is required in neurons for normal cognitive performance. In conclusion, this study has defined a clinically recognizable ID syndrome, caused by de novo loss-of-function mutations in WAC. Independent functional evidence in Drosophila further supported the role of WAC in ID. On the basis of these data WAC can be added to the list of ID genes with a role in transcription regulation through histone modification (Lugtenberg, 2016).

David-Morrison, G., Xu, Z., Rui, Y.N., Charng, W.L., Jaiswal, M., Yamamoto, S., Xiong, B., Zhang, K., Sandoval, H., Duraine, L., Zuo, Z., Zhang, S. and Bellen, H.J. (2016). WAC regulates mTOR activity by acting as an adaptor for the TTT and Pontin/Reptin complexes. Dev Cell 36: 139-151. PubMed ID: 26812014

Abstract

The ability to sense energy status is crucial in the regulation of metabolism via the mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTT-Pontin/Reptin complex is responsive to changes in energy status. Under energy-sufficient conditions, the TTT-Pontin/Reptin complex promotes mTORC1 dimerization and mTORC1-Rag interaction, which are critical for mTORC1 activation. This study shows that WAC is a regulator of energy-mediated mTORC1 activity. In a Drosophila screen designed to isolate mutations that cause neuronal dysfunction, wacky, the homolog of WAC, was identified. Loss of Wacky leads to neurodegeneration, defective mTOR activity, and increased autophagy. Wacky and WAC have conserved physical interactions with mTOR and its regulators, including Pontin and Reptin, which bind to the TTT complex to regulate energy-dependent activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating mTORC1 dimerization and mTORC1-Rag interaction (David-Morrison, 2016).

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Intellectual Disability associated with WAC

Gunaseelan, S., Wang, Z., Tong, V. K. J., Ming, S. W. S., Razar, R., Srimasorn, S., Ong, W. Y., Lim, K. L. and Chua, J. J. E.
(2021). Loss of FEZ1, a gene deleted in Jacobsen syndrome, causes locomotion defects and early mortality by impairing motor neuron development. Hum Mol Genet. PubMed ID: 33395696

Abstract
e

FEZ1-mediated axonal transport plays important roles in central nervous system development but its involvement in the peripheral nervous system is not well-characterised. FEZ1 is deleted in Jacobsen syndrome (JS), an 11q terminal deletion developmental disorder. JS patients display impaired psychomotor skills, including gross and fine motor delay, suggesting that FEZ1 deletion may be responsible for these phenotypes, given its association with the development of motor-related circuits. Supporting this hypothesis, the data shows that FEZ1 is selectively expressed in the rat brain and spinal cord. Its levels progressively increase over the developmental course of human motor neurons derived from embryonic stem cells. Deletion of FEZ1 strongly impaired axon and dendrite development, and significantly delayed the transport of synaptic proteins into developing neurites. Concurring with these observations, Drosophila unc-76 mutants showed severe locomotion impairments, accompanied by a strong reduction of synaptic boutons at neuromuscular junctions. These abnormalities were ameliorated by pharmacological activation of UNC-51/ATG1, a FEZ1-activating kinase, with rapamycin and metformin. Collectively, the results highlight a role for FEZ1 in motor neuron development and implicate its deletion as an underlying cause of motor impairments in JS patients (Gunaseelan, 2021).

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Kohlschutter-Tonz syndrome

Kim, M., Jang, D., Yoo, E., Oh, Y., Sonn, J. Y., Lee, J., Ki, Y., Son, H. J., Hwang, O., Lee, C., Lim, C. and Choe, J. (2017). Rogdi defines GABAergic control of a wake-promoting dopaminergic pathway to sustain sleep in Drosophila. Sci Rep 7(1): 11368. PubMed ID:
28900300

Abstract

Kohlschutter-Tonz syndrome (KTS) is a rare genetic disorder with neurological dysfunctions including seizure and intellectual impairment. Mutations at the Rogdi locus have been linked to development of KTS, yet the underlying mechanisms remain elusive. This study demonstrates that a Drosophila homolog of Rogdi acts as a novel sleep-promoting factor by supporting a specific subset of gamma-aminobutyric acid (GABA) transmission. Rogdi mutant flies displayed insomnia-like behaviors accompanied by sleep fragmentation and delay in sleep initiation. The sleep suppression phenotypes were rescued by sustaining GABAergic transmission primarily via metabotropic GABA receptors or by blocking wake-promoting dopaminergic pathways. Transgenic rescue further mapped GABAergic neurons as a cell-autonomous locus important for Rogdi-dependent sleep, implying metabotropic GABA transmission upstream of the dopaminergic inhibition of sleep. Consistently, an agonist specific to metabotropic but not ionotropic GABA receptors titrated the wake-promoting effects of dopaminergic neuron excitation. Taken together, these data provide the first genetic evidence that implicates Rogdi in sleep regulation via GABAergic control of dopaminergic signaling. Given the strong relevance of GABA to epilepsy, it is proposed that similar mechanisms might underlie the neural pathogenesis of Rogdi-associated KTS (Kim, 2017).

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Laminopathy

Bhide, S., Trujillo, A. S., O'Connor, M. T., Young, G. H., Cryderman, D. E., Chandran, S., Nikravesh, M., Wallrath, L. L. and Melkani, G. C. (2018). Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan. Aging Cell: e12747. PubMed ID:
29575479

Abstract

Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. This study modeled human disease-causing mutations in the Drosophila Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Simultaneous over-expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies (Bhide, 2018).

Mutations in the human LMNA gene are associated with a collection of diseases called laminopathies in which the most common manifestation is progressive cardiac disease. This study has generated Drosophila melanogaster models of age-dependent cardiac dysfunction. In these models, mutations synonymous with those causing disease in humans were introduced into Drosophila LamC. Cardiac-specific expression of mutant LamC resulted in (1) cardiac contractility, conduction, and physiological defects, (2) abnormal nuclear envelope morphology, (3) cytoplasmic LamC aggregation, (4) nuclear enrichment of the redox transcriptional regulator CncC (mammalian Nrf2), (5) and upregulation of autophagy cargo receptor Ref(2)P (mammalian p62). These cardiac defects were enhanced with age and accompanied by increased adipose tissue in the adult fat bodies and a shortened lifespan (Bhide, 2018).

To understand the mechanistic basis of cardiolaminopathy and identify genetic suppressors, advantage was taken of powerful genetic tools available in Drosophila. The presence of cytoplasmic LamC aggregates prompted a determination of whether increasing autophagy would suppress the cardiac defects. Cardiac-specific upregulation of autophagy (Atg1 OE) suppressed G489V-induced cardiac defects. Consistent with this, decreased autophagy due to expression of Atg1 DN resulted in enhanced deterioration of G489V-induced cardiac dysfunction. Interestingly, cardiac-specific Atg5 OE and Atg8a OE, two factors that also promote autophagy, showed little to no suppression of G489V-induced heart dysfunction, suggesting that Atg1 might be rate limiting in this context. These findings are consistent with studies in mouse laminopathy models in which rapamycin and temsirolimus had beneficial effects on heart and skeletal muscle through inhibition of AKT/mTOR signaling. These findings are depicted in a model (see Model for the interactions between the autophagy and CncC/Keap1 signaling pathway in mutant lamin-induced cardiac disease) in which cytoplasmic aggregation of mutant LamC results in upregulation of p62, which in turn inhibits autophagy via activation of TOR and inactivation of AMPK. AMPK inactivation leads to the activation of PI3K/Akt/mTOR pathway and inhibition of autophagy Atg1 OE promoted clearance of the LamC aggregates and restored proteostasis in these Drosophila models. Thus, the data suggest that mutant LamC reduces autophagy, resulting in impairment of cellular proteostasis that leads to cardiac dysfunction (Bhide, 2018).

Cardiac-specific expression of mutant LamC altered CncC subcellular localization. Previously, Drosophila larval body wall muscles expressing G489V were shown to experience reductive stress, an atypical redox state characterized by high levels of reduced glutathione and NADPH, and upregulation CncC target genes (Dialynas, 2015). Cardiac-specific CncC RNAi in the wild-type LamC background did not produce major cardiac defects. Consistent with this, Nrf2 deficiency in mice does not compromise cardiac and skeletal muscle performance. Cardiac-specific CncC RNAi suppressed G489V-induced cardiac dysfunction and reduced cytoplasmic LamC aggregation, but not R205W-induced defects. However, cardiac-specific RNAi against CncC did not affect G489V-induced adipose tissue accumulation and lifespan shortening. Similar to the nuclear enrichment of CncC in hearts expressing G489V, human muscle biopsy tissue from an individual with a point mutation in the LMNA gene that results in G449V (analogous to Drosophila G489V) showed nuclear enrichment of Nrf2 (Dialynas, 2015). Disruption of Nrf2/Keap1 signaling has also been reported for Hutchinson-Gilford progeria, an early-onset aging disease caused by mutations in LMNA. In this case, however, the thickened nuclear lamina traps Nrf2 at the nuclear envelope that results in a failure to activate Nrf2 target genes, leading to oxidative stress. In these studies, CncC nuclear enrichment was observed; however, a redox imbalance was not readily observed at the three-time points investigated. This might indicate that there is a window of time in disease progression in which redox imbalance occurs and that mechanisms are in place to re-establish homeostasis (Bhide, 2018).

It has been postulated that there is cross-talk between autophagy and Nrf2/Keap1 signaling. This was tested by manipulating autophagy and CncC (Nrf2) alone and in combination. CncC RNAi suppressed the cardiac defects caused by G489V, but not the lipid accumulation and lifespan shortening, suggesting the latter two phenotypes are not specifically due to loss of cardiac function. In contrast, Atg1 OE suppressed the cardiac and adipose tissue defects and lengthened the lifespan. The double treatment (simultaneous Atg1 OE and RNAi knockdown of CncC) gave the most robust suppression of the mutant phenotypes and completely restored the lifespan. Interestingly, Atg1 DN and RNAi knockdown of CncC simultaneously did not further deteriorate or improve the mutant phenotypes. Taken together, these data suggest that autophagy plays a key role in suppression of the G498V-induced phenotypes and that knockdown on CncC enhances this suppression (Bhide, 2018).

These findings support a model whereby autophagy and Nrf2 signaling are central to cardiac health. It is proposed that cytoplasmic aggregation of LamC increases levels of Ref(2)P (p62), which competitively binds to Keap1, resulting in CncC (Nrf2) translocation to the nucleus. Inside the nucleus, Nrf2 regulates genes involved in detoxification. Continued expression of antioxidant genes results in the disruption of redox homeostasis, defective mitochondria, and dysregulation of energy homeostasis/energy sensor such as AMPK and its downstream targets. Simultaneously, upregulation of Ref(2)P (p62) causes inhibition of autophagy via activation of TOR, which leads to the inactivation of AMPK. AMPK inactivation in combination with activation of the TOR pathway causes cellular and metabolic stress that leads to cardiomyopathy. In support of this model, transcriptomics data from muscle tissue of an individual with muscular dystrophy expressing Lamin A/C G449V (analogous to Drosophila G489V) showed (1) upregulation of transcripts from Nrf2 target genes, (2) upregulation of genes encoding subunits of the mTOR complex, and (3) downregulation of AMPK, further demonstrating relevance of the Drosophila model for providing insights on human pathology (Bhide, 2018).

Coombs, G. S., Rios-Monterrosa, J. L., Lai, S., Dai, Q., Goll, A. C., Ketterer, M. R., Valdes, M. F., Uche, N., Benjamin, I. J. and Wallrath, L. L. (2021). Modulation of muscle redox and protein aggregation rescues lethality caused by mutant lamins. Redox Biol 48: 102196. PubMed ID: 34872044

Abstract

Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, new Drosophila models and human iPS cell-derived cardiomyocytes were generated. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.

Chandran, S., Suggs, J. A., Wang, B. J., Han, A., Bhide, S., Cryderman, D. E., Moore, S. A., Bernstein, S. I., Wallrath, L. L. and Melkani, G. C. (2018). Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling. Hum Mol Genet. PubMed ID: 30239736

Abstract

Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus, and regulate gene expression. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization, and up-regulation of the autophagy cargo receptor p62. It was hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMPK/4E-BP1/autophagy/proteostatic pathways. S6K mRNA levels were increased and AMPKalpha and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKalpha and down-stream targets 4E-BP, Foxo and PGC1alpha, as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects, and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy (Chandran, 2018).

Laminopathies are a collection of diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane where they provide structural support for the nucleus and regulate gene expression. Laminopathies include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD2, OMIM #181350), Limb-Girdle muscular dystrophy type 1B (LGMD1B, 159001), congenital muscular dystrophy (MDC, OMIM #613205), dilated cardiomyopathy type 1A (CMD1A, OMIM #115200), familial partial lipodystrophy type 2 (FPLD2, OMIM #151660) and early on-set aging syndromes such as Hutchinson-Gilford progeria syndrome (HGPS; OMIM #176670). It is unclear how LMNA mutations result in tissue-specific defects when mutant lamins are expressed in nearly all tissue. The pathogenic mechanisms of laminopathies are not well defined; hence, a greater understanding is needed to support the development of therapeutic interventions (Chandran, 2018).

Over 400 distinct mutations have been identified in the LMNA gene, among the highest number of mutations discovered in a single human gene. The majority of these are point mutations throughout the gene that give rise to single amino acid substitutions in lamins A and C, two isoforms derived from alternatively spliced LMNA messenger RNA (mRNA). Amino acid substitutions that give rise to skeletal muscular dystrophy are often accompanied by congenital muscular dystrophy (CMD). EDMD2 in particular is characterized by progressive muscle weakness, joint contractures and CMD with conduction defects. While much is known about the functions of lamins in the nucleus where they play a role in maintaining nuclear envelope (NE) integrity and organizing the genome, their functions in signaling pathways are becoming equally important with respect to disease mechanisms. For example, mutant lamins cause perturbations of the mammalian target of rapamycin (mTOR) pathway, which can be partially reversed with mTOR inhibitors such as rapamycin and temsiromilus. Genetic ablation of S6K1 (encoding ribosomal protein S6 protein kinase 1), a downstream substrate of mTOR, improved muscle function and extended lifespan of Lmna-/- mice. mTOR activity inversely correlates with the rate of autophagy, which plays a role in regulating nuclear-to-cytoplasmic transport and degradation of Lamin B1. Consistent with these findings, activation of autophagy suppressed cardiac laminopathy in a Drosophila model. Thus, regulation of the mTOR pathway is critical for muscle health in the context of laminopathies, however, which factors upstream and downstream of mTOR play key roles needed further investigation (Chandran, 2018).

To evaluate the role of TOR signaling and autophagy in lamin-associated muscle disease, Drosophila melanogaster (fruit fly) models of laminopathies were established. Drosophila models have proved to be powerful in defining the mechanistic basis of human disease, including muscle disorders associated with cytoskeletal defects. In addition, Drosophila models have been used to identify potential therapeutic targets for human aging disorders. Relevant for this study, Drosophila indirect flight muscle (IFM) models have been successfully used to define the molecular basis for muscle organization and disorganization. Importantly, expression of dominant negative (DN) mutants and knock-down (KD) of IFM-specific genes does not cause lethality in flies, allowing evaluation of pathophysiological aspects of progressive muscle degeneration without effects on the remainder of the organism. A dominant flightless phenotype with abnormal wing position provides powerful visual markers of defective IFM function. D. melanogaster, with its high degree of genome conservation to humans and manipulability through versatile genetic techniques, is an excellent model for understanding the molecular mechanisms of mutant lamin-induced skeletal muscle defects (Chandran, 2018).

The expression of D. melanogaster Lamin C (LamC) gene is developmentally regulated and nearly ubiquitously expressed, similar to the human LMNA gene. LamC shares amino acid sequence identity with human lamins A and C. Lamins have a conserved protein domain structure with a globular head, coiled-coil rod and a tail domain possessing an immunoglobulin-fold (Ig-fold). In addition, LamC localizes to the NE in all Drosophila tissues investigated including cardiac and larval body wall muscle tissue, supporting Drosophila as a useful model. Furthermore, the pathogenic genes and pathways described in this study are highly conserved between Drosophila and humans, offering the possibilities for the identification of conserved drug targets. The genetic and pharmacological manipulation of these pathways will provide mechanistic tests for potential skeletal muscle laminopathy therapies (Chandran, 2018).

To address the molecular basis of skeletal muscle laminopathies, mutations were made in Drosophila LamC analogous to those that cause muscle disease in humans. Muscle-specific expression of mutant LamC resulted in muscle functional defects that were accompanied by a plethora of cellular abnormalities including cytoplasmic aggregation of NE proteins. It was hypothesized that these cytoplasmic aggregates trigger signaling pathways and alter cellular and metabolic homeostasis, which results in muscle dysfunction. In support of this hypothesis and to reveal relevance to human pathology, transcriptomics data obtained from human muscle biopsy tissue showed misregulation of genes in the AMP-activated protein kinase (AMPK)/TOR/autophagy signaling pathways. Genetic manipulation of these pathways in Drosophila IFM suppressed the muscle defects, suggesting that misregulation of these pathways was causal to the muscle pathology. Overall, this analysis identified potential new therapeutic targets for lamin-associated skeletal myopathies and possibly other laminopathies (Chandran, 2018).

Although several hundred mutations in the LMNA gene have been identified and many studies have been performed on lamins, the pathogenic mechanisms of laminopathies remain not well understood. Greater insights are needed for therapeutic interventions. To address the molecular pathology of laminopathies and to understand the functions of lamins, Drosophila models of skeletal myopathies were developed. Mutations in Drosophila LamC were generated that are analogous to human LMNA mutations and expressed exclusively in the IFM, a muscle that produces a readily visible held-up wing phenotype upon muscle dysfunction (Chandran, 2018).

Three of the four LamC mutants examined in this study (R205W, G489V and V528P) caused severe muscle defects upon expression with Act88F and Fln Gal4 drivers (expressed before sarcomere assembly/maturation). In contrast, A177P caused only moderate functional defects when expressed with the same drivers, despite similar levels of LamC protein. These data demonstrate that the severity of the abnormal phenotypes is mutation-specific. This is similar to the human disease condition in which individuals with different LMNA mutations exhibit a wide range of disease severity depending on the location of the amino acid substitution. Expression of the mutant lamins after sarcomere assembly/maturation via the DJ-694 Gal4 driver resulted in only moderate functional defects. Taken together, these data suggested that mutant LamC interfered with sarcomere assembly/maturation. This idea was supported by TEM images showing disruption of sarcomere organization when using the Act88F and Fln Gal4 drivers. During sarcomere assembly, several proteins are produced de novo and presence of cytoplasmic LamC aggregates might interfere with the formation of multi-protein complexes that are associated with the contractile apparatus. It is also possible that cytoplasmic LamC aggregates interfere with proteostasis by sequestering chaperone proteins that facilitate protein folding following de novo synthesis. Loss of sarcomere structure and mitochondrial defects are known to cause the held-up wing phenotype and loss of flight. Both of these phenotypes are useful phenotypes for drug screens. The fact that this study identified mutation-specific variation in muscle disease severity further suggest that the Drosophila models will be useful for identifying modifier genes, which provide another level of complexity with regard to the range of disease severity observed in individuals, including family members with the same LMNA mutation (Chandran, 2018).

To better understand the molecular and cellular basis of the muscle pathology, an in-depth analysis of the Drosophila models was performed. Cytological analysis revealed cytoplasmic aggregates of LamC and nuclear pore proteins, nuclear blebbing, disruption of the cytoskeletal organization and mitochondrial morphology. During the natural aging process, accumulation of aggregates often results from defective proteostasis. Interestingly, protein aggregation in Huntington disease leads to amyloids that cause sarcomeric assembly defects due to loss of proteostasis. It is proposed that the abnormal accumulation of cytoplasmic NE protein aggregates leads to an impairment of proteostasis, causing loss of muscle function. These findings are consistent with cytoplasmic aggregation of NE proteins in muscle biopsies from individuals with skeletal muscle laminopathy. Thus, the results show that the IFM defects in the Drosophila models share characteristics with the human diseased muscle (Chandran, 2018).

To further define the pathological mechanisms of mutant LamC in skeletal muscle, the effects of mutant LamC on autophagy and metabolic signaling was examined. Ref(2)P, the Drosophila homologue of mammalian polyubiquitin binding protein p62, is up-regulated in IFM expressing mutant LamC relative to controls. Misregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Keap1 redox signaling mediated by p62 has also been associated with muscle atrophy and cardiomyopathy, and this pathway is predicted to influence autophagy. p62 is an adaptor protein that binds protein aggregates and targets them for autophagy and proteasome-based destruction. However, it is unknown how p62 influences laminopathy-mediated autophagic defects. Studies in mice show that loss of A-type lamins leads to cardiac and muscle defects due to alterations in mTOR signaling, which influences the rate of authophagy. Autophagy is responsible for the regulation of lamin B1 levels. Thus, it is possible that autophagy flux is impaired by NE protein aggregates. This might lead to the persistence of defective mitochondria, resulting in the up-regulation of the TOR pathway, which in turn contributes to the down-regulation of autophagy (Chandran, 2018).

Transcriptomic data from human laminopathy muscle allowed (1) obtaining unbiased insights into the gene expression profile of human diseased muscle, (2) comparing the data obtained with Drosophila to human diseased muscle to validate the use of the current models and (3) establishing the translational potential of the Drosophila models. Based upon the knowledge gained from the RNA sequencing of human muscle biopsy tissue, this study identified pathogenic pathways and then modulated those pathways using the rapid genetics offered by Drosophila. Through these genetic manipulations, it was possible to reduced (and eliminated in some cases) NE protein aggregation and alter intracellular signaling to ameliorate muscle dysfunction. Through modulation of AMPK, PGC1α, Foxo, S6K and 4E-BP, key players were identified that regulate autophagy in suppressing laminopathy-induced skeletal myopathy and mitochondrial dysmorphology. The pathway components identified might serve as valuable disease markers and provide new targets for the development of rational therapeutic strategies (Chandran, 2018).

Based on human muscle transcriptomics and genetic manipulations in Drosophila, this study has shown that activation of AMPK suppressed muscle laminopathy. AMPK is a sensor of cellular energy and metabolism that is linked to regulating autophagy, proteostasis and mitochondrial function. AMPK has conserved functions in many species, including Drosophila, and occurs universally as heterotrimeric complexes containing catalytic α-subunits and regulatory β-and γ-subunits. Increased expression of AMPK prevented age-related phenotypes in old mice, such as weight gain and decline of mitochondrial function. Activation of the AMPK pathway improved lamin-induced myopathy by removing abnormal aggregates, achieving autophagic and mitochondrial homeostasis. Consistent with these findings, the AMPK activator metformin lowered progerin (a specific mutant form of lamin A/C) levels and suppressed defects in the HGPS-induced pluripotent stem cell model (Chandran, 2018).

The data extend these findings by showing that the positive effects of AMPK activation are mainly through PGC1α, with contributions from Foxo, both of which maintain metabolic and cellular homeostasis. Previously, it was shown that Foxo/4E-BP signaling regulates age-induced proteostasis, including suppression of age-associated aggregation in skeletal muscle. As observed with rapamycin treatment in mouse models, activation of 4E-BP, a key downstream effector of the mTOR complex, is thought to reduce TOR activity. Muscle-specific expression of 4E-BP suppressed age-related protein aggregates and metabolic defects in Drosophila and mouse models. However, whole-body OE of 4E-BP1 shortened the lifespan of Lmna-/- mice possibly by enhancing lipolysis. In the Drosophila IFM models, activation of S6K enhanced muscle deterioration and a DN version of S6K suppressed muscle dysfunction, presumably by activating autophagy as evidenced by the reduction of cytoplasmic aggregation of NE proteins. Overall, this study identified specific downstream targets of AMPK that suppress muscle laminopathy (Chandran, 2018).

Based upon these findings and those in the literature, a model is proposed that describes how cytoplasmic aggregates of NE proteins impact autophagy and signaling pathways and contribute to muscle pathology. According to this model, cytoplasmic aggregation of NE proteins lead to increased levels of Ref(2)P/p62, which bind to the protein aggregates. Accumulation of p62 causes up-regulation of the TOR pathway that leads to inhibition of autophagy in the skeletal muscle. Accumulation of Ref(2)P/p62 also causes up-regulation of the regulatory associated protein of MTOR complex 1 (RPTOR), which binds mTOR and inhibits autophagy. Thus, autophagy is down-regulated by two mechanisms, causing a disruption in proteostasis. Moreover, up-regulation of the mTOR pathway causes increased S6K activity, which leads to imbalance in energy homeostasis. Consistent with this model, the transcriptomic data from the laminopathy muscle biopsy tissue showed up-regulation of RPTOR and S6K, implying that autophagy is down-regulated. Also, in support of this model, KD of S6K in Drosophila IFM suppressed the muscle defects. Inhibition of autophagy is predicted to cause a reduction in AMPK activity. Consistent with this idea, all three AMPKα transcripts were down-regulated in the laminopathy muscle biopsy tissue. OE of AMPKα in Drosophila IFM suppressed the muscle defects. AMPK inactivation leads to the activation of PI3K/AKT/mTOR pathway, which was also up-regulated in the human muscle biopsy. Another important function of AMPK is to control the expression of genes involved in energy metabolism and aging by enhancing the activity of sirtuin 1 (SIRT1). SIRT1 controls the activity of downstream targets such as PGC-1α, the master regulator of mitochondrial biogenesis, and Foxo, which is involved in delaying the aging process, by reducing protein aggregation through controlling its target 4E-BP. The transcriptomic data showed that SIRT1 and downstream targets, PGC-1α, Foxo and 4E-BP, were down-regulated, which would cause an imbalance in cellular energy metabolism leading to cellular stress and compromising skeletal muscle function. In agreement, OE of dPGC-1, Foxo and 4E-BP in the Drosophila models suppressed the abnormal muscle phenotypes. Several kinases were up-regulated in the human muscle biopsy tissue. Up-regulation of these kinases has been observed in lamin-associated cardiomyopathy. Genetic modulation of these kinases is needed to test their effectiveness in suppressing muscle laminopathy and other lamin-based disorders (Chandran, 2018).

Overall, the data provide new insights on potential targets for small molecular screens. As proof-of-principle, dietary supplementation of rapamycin (TOR inhibitor) or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), activator of AMPK, in Drosophila media suppressed the mutant LamC-induced muscle defects. Drosophila allows evaluation of the effects of these compounds on functional and cellular defects caused by mutant LamC in the context of a whole organism. Promising compounds can then be tested in pre-clinical mouse laminopathy models. Given that lamin-associated muscular dystrophies have pathophysiological features shared with other laminopathies and diseases such as diabetes, these findings have the potential for broad impact (Chandran, 2018).

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Leigh Syndrome

Loewen, C. A. and Ganetzky, B. (2018). Mito-nuclear interactions affecting lifespan and neurodegeneration in a Drosophila model of Leigh Syndrome. Genetics 208(4):1535-1552. PubMed ID:
29496745

Abstract

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. This study describes the characterization of a mutation in Drosophila ND23, a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh Syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23. A key feature of Leigh Syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. It was discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23, including a variant affecting a mitochondrially-encoded component of complex I. Thus, this work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease (Loewen, 2018).

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Limb-girdle muscular dystrophy D2

Blazquez-Bernal, A., Fernandez-Costa, J. M., Bargiela, A. and Artero, R. (2021). Inhibition of autophagy rescues muscle atrophy in a LGMDD2 Drosophila model. Faseb j 35(10): e21914. PubMed ID:
34547132

Abstract

Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. This study reports the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog, Tnpo-SR. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model. Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, this study provides a proof of concept of autophagy as a target to treat disease phenotypes, and a neurogenic component is proposed to explain mutant TNPO3 pathogenicity in diseased muscles (Blazquez-Bernal, 2021).

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Lysosomal storage disease

Hindle, S. J., Hebbar, S., Schwudke, D., Elliott, C. J. and Sweeney, S. T. (2017). A saposin deficiency model in Drosophila: Lysosomal storage, progressive neurodegeneration and sensory physiological decline. Neurobiol Dis. PubMed ID:
27913291

Abstract

Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. Mutations causing an absence or impaired function of saposins in humans lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. A Drosophila model of saposin deficiency has been generated and characterised that shows striking similarities to the human diseases. Drosophila Saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. These data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing understanding of the cellular pathology implicated in saposin deficiency and related LSDs (Hindle, 2016).

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De Filippis, C., Napoli, B., Rigon, L., Guarato, G., Bauer, R., Tomanin, R. and Orso, G. (2017). Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes. Cells 11(1). PubMed ID: 35011691
Summary:
Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. This paper reports a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). This study showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. These results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations.

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Sellin, J., et al. (2017). Characterization of Drosophila saposin-related mutants as a model for lysosomal sphingolipid storage diseases. Dis Model Mech [Epub ahead of print]. PubMed ID: 28389479

Abstract

Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs), which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. The digestion of intra-lumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins), which are cleaved from a Prosaposin precursor. prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression.This study identified the Drosophila Prosaposin orthologue Saposin-related (Sap-r) as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with enlarged endo-lysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. sap-r mutants show reduced viability with approximately 50% adult survivors, allowing study of progressive neurodegeneration and analysis thelipid profile in young and aged flies. Additionally, a defect was observed in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, autophagy was found to be increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, this study establishes Drosophila sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological signaling events (Sellin, 2017).

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>De Filippis, C., Napoli, B., Rigon, L., Guarato, G., Bauer, R., Tomanin, R. and Orso, G. (2021). Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes. Cells 11(1). PubMed ID: 35011691
Summary:
Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. This paper reports a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). This study showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. These results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations.

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Maple syrup urine disease

Tsai, H. Y., Wu, S. C., Li, J. C., Chen, Y. M., Chan, C. C. and Chen, C. H. (2020). Loss of the Drosophila branched-chain α-keto acid dehydrogenase complex (BCKDH) results in neuronal dysfunction. Dis Model Mech. PubMed ID:
32680850

Abstract

Maple syrup urine disease (MSUD) is an inherited error in the metabolism of branched-chain amino acids (BCAAs) caused by a severe deficiency of the branched chain keto-acid dehydrogenase (BCKDH) enzyme, which ultimately leads to neurological disorders. The limited therapies, including protein-restricted diets and liver transplants, are not as effective as they could be for the treatment of MSUD due to the current lack of molecular insights into the disease pathogenesis. To address this issue, a Drosophila model of MSUD was developed by knocking out the dDBT (CG5599) gene, an ortholog of the human dihydrolipoamide branched chain transacylase (DBT) subunit of BCKDH. The homozygous dDBT mutant larvae recapitulate an array of MSUD phenotypes, including aberrant BCAA accumulation, developmental defects, poor mobile behavior, and disrupted L-glutamate homeostasis. Moreover, the dDBT mutation causes neuronal apoptosis during the developmental progression of larval brains. The genetic and functional evidence generated by in vivo depletion of dDBT expression in the eye shows severe impairment of retinal rhadomeres. Further, the dDBT mutant shows elevated oxidative stress and higher lipid peroxidation accumulation in the larval brain. Therefore it is concluded from in vivo evidence that the loss of dDBT results in oxidative brain damage that may led to neuronal cell death and contribute to aspects of MSUD pathology. Importantly, when the dDBT mutants were administrated with Metformin, the aberrances in BCAA levels and motor behavior were ameliorated. This intriguing outcome strongly merits the use of the dDBT mutant as a platform for developing MSUD therapies (Tsai, 2020).

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Meier-Gorlin syndrome

McDaniel, S. L., Hollatz, A. J., Branstad, A. M., Gaskill, M. M., Fox, C. A. and Harrison, M. M. (2019).Tissue-specific DNA replication defects in Drosophila melanogaster caused by a Meier-Gorlin syndrome mutation in Orc4. Genetics. PubMed ID:
31818869

Abstract

Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct tissue-specific developmental defects. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, MCM5, and DONSON) are mutated in individuals diagnosed with MGS. The essential role of ORC is to license origins during the G1 phase of the cell cycle, but ORC has also been implicated in several non-replicative functions. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in genes encoding ORC lead to the tissue-specific defects associated with the disease. To begin to address these issues, Cas9-mediated genome engineering was used to generate a Drosophila melanogaster model of individuals carrying a specific Meier-Gorlin syndrome mutation in ORC4 along with control strains. Together these strains provide the first metazoan model for an MGS mutation in which the mutation was engineered at the endogenous locus along with precisely defined control strains. Flies homozygous for the engineered MGS allele reach adulthood, but with several tissue-specific defects. Genetic analysis revealed that this Orc4 allele was a hypomorph. Mutant females were sterile, and phenotypic analyses suggested that defects in DNA replication was an underlying cause. By leveraging the well-studied Drosophila system, this study provides evidence that a disease-causing mutation in Orc4 disrupts DNA replication, and it is proposed that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.

Balasov, M., Akhmetova, K. and Chesnokov, I. (2020). Humanized Drosophila Model of the Meier-Gorlin Syndrome Reveals Conserved and Divergent Features of the Orc6 Protein. Genetics. PubMed ID: 33037049.

Abstract

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears and skeletal abnormalities. Patients with MGS often carry mutations in genes encoding the subunits of the Origin Recognition Complex (ORC), components of the pre-replicative complex (pre-RC) and replication machinery. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. A mutation in the conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. Recently, a new mutation in Orc6 was also identified however, it is localized in the N-terminal domain of the protein. In order to study the functions of Orc6, the human gene was used to rescue the orc6 deletion in Drosophila. Using this "humanized" Orc6-based Drosophila model of the Meier-Gorlin syndrome it was discovered that unlike the previous Y225S MGS mutation in Orc6, the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. These studies revealed the importance of evolutionarily conserved and variable domains of Orc6 protein and allowed the studies of human protein functions and the analysis of the critical amino acids in live animal heterologous system as well as provided novel insights into the mechanisms underlying MGS pathology (Balasov, 2020).

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Microcephaly

Yu, L., Li, G., Deng, J., Jiang, X., Xue, J., Zhu, Y., Huang, W., Tang, B. and Duan, R. (2020). The UFM1 cascade times mitosis entry associated with microcephaly. Faseb j 34(1): 1319-1330. PubMed ID:
31914610

Abstract

Posttranslational modifications enhance the functional diversity of the proteome by modifying the substrates. The UFM1 cascade is a novel ubiquitin-like modification system. The mutations in UFM1, its E1 (UBA5) and E2 (UFC1), have been identified in patients with microcephaly. However, its pathological mechanisms remain unclear. This study observed the disruption of the UFM1 cascade in Drosophila neuroblasts (NBs) decreased the number of NBs, leading to a smaller brain size. The lack of ufmylation in NBs resulted in an increased mitotic index and an extended G2/M phase, indicating a defect in mitotic progression. In addition, live imaging of the embryos revealed an impaired E3 ligase (Ufl1) function resulted in premature entry into mitosis and failed cellularization. Even worse, the embryonic lethality occurred as early as within the first few mitotic cycles following the depletion of Ufm1. Knockdown of ufmylation in the fixed embryos exhibited severe phenotypes, including detached centrosomes, defective microtubules, and DNA bridge. Furthermore, the UFM1 cascade could alter the level of phosphorylation on tyrosine-15 of CDK1 (pY15-CDK1), which is a negative regulator of the G2 to M transition. These findings yield unambiguous evidence suggesting that the UFM1 cascade is a microcephaly-causing factor that regulates the progression of the cell cycle at mitosis phase entry (Yu, 2020).

Lim, N. R., Shohayeb, B., Zaytseva, O., Mitchell, N., Millard, S. S., Ng, D. C. H. and Quinn, L. M. (2017). Glial-specific functions of microcephaly protein WDR62 and interaction with the mitotic kinase AURKA are essential for Drosophila brain growth. Stem Cell Reports. PubMed ID: 28625535

Abstract

The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. This study used Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. It was further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA (Aurora A). Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly (Lim, 2017).

Genome-wide exome sequencing of microcephaly (MCPH) patients identified wd40-repeat protein 62 (wdr62) as the second most commonly mutated gene. WDR62 is a ubiquitously expressed cytoplasmic protein in interphase and localizes to the spindle pole in mitosis. A feature of many WDR62 MCPH-associated alleles is an inability to localize to the mitotic spindle pole, and wdr62 depletion is also associated with defects in spindle and centrosomal integrity, mitotic delay, and reduced brain growth in rodents. The neural stem cell (NSC) population gives rise to all neuronal cells in the adult brain. NSC behavior is governed by both cell intrinsic factors and extrinsic factors from the supporting stem cell niche, including the glial lineage, which acts non-autonomously to control stem cell renewal and differentiation of daughters (Lim, 2017).

The connection between NSCs and their niche, and the importance of spindle integrity to asymmetric division, has been best defined for Drosophila NSCs/neuroblasts (NB). WDR62 scaffolds kinases that are important mitotic regulators including c-Jun N-terminal kinase (JNK) members of the mitogen-activated protein kinase superfamily and Aurora kinase A (AURKA). In flies, AURKA regulates NB proliferation and is required for the localization of mitotic NB polarity complex protein Bazooka (mammalian Par3) to the apical Par complex (comprising the Par anchor, Inscuteable [Insc] adaptor protein, and Gαi/Pins/Mud complex). This establishes the apical-basal NB axis essential for self-renewal and differentiation. As a consequence, mutant aurka causes NB overproliferation and tissue overgrowth. The WDR62 ortholog in Drosophila (dWDR62) is required for brain growth, but whether signaling between WDR62 and AURKA modulates brain development has not been reported (Lim, 2017).

In addition to the NB lineage, Drosophila studies suggest that the glial lineage governs overall brain volume through regulation of cell-cycle re-entry and neuroepithelial expansion of NBs. However, potential contribution(s) of individual brain lineage(s) (NB or glia) to the defective brain growth associated with global depletion of wdr62 or aurka is currently unclear. This study confirmed that WDR62 is required for spindle orientation in NBs, however, wdr62 depletion specifically in NBs does not significantly retard brain growth. Rather, control of brain growth predominantly depends upon glial lineage function, as depletion of either aurka or wdr62 specifically in the glial lineage significantly reduces brain volume. Moreover, although wdr62 depletion suppressed brain overgrowth associated with aurka depletion in NBs, wdr62 knockdown specifically in the glial lineage enhanced the small brain phenotype associated with aurka depletion. Collectively, these data suggest that WDR62 function is negatively regulated by AURKA in NBs but positively regulated by AURKA in glia, and thus demonstrates that lineage-specific signaling functions of AURKA-WDR62 in Drosophila orchestrate larval brain growth and development (Lim, 2017).

In the mammalian brain, radial glia behave as NSCs that are supported by outer radial glia through cell-cell contact and secretion of growth factors required for maintenance of a stem cell niche. Another class of glial cells, the microglia population, regulates neural precursor cell numbers to govern final neuronal numbers residing in the cortex. However, whether MCPH genes such as wdr62 are important for glial cell fate is unclear. This study dissected the lineage-specific contribution of WDR62 to brain development and revealed that loss of WDR62 function specifically in the glial, but not the NB lineage, profoundly altered brain growth. Moreover, wdr62 depletion in glia likely impairs brain growth autonomously (i.e., through depletion of glia), and also results in NSC loss, suggesting that a focus on WDR62 function in glia will be integral to elucidating how wdr62 loss of function contributes to MCPH. That depletion of wdr62 in the NB lineage was not associated with reduced brain volume, despite reducing NB number, also provides a likely explanation for the recent observation that NB defects associated with global wdr62 depletion fail to account for reduced brain size (Lim, 2017).

Hypomorphic wdr62 mutant mice have reduced brain size, with associated mitotic defects and an overall decrease in neural progenitor cells. In Drosophila, spindle orientation defects following wdr62 loss of function likely underlie the G2 delay and increased mitotic figures in NBs. This phenotype is also reminiscent of the cleavage plane misorientation observed in NSCs in wdr62-depleted rat brains. In Drosophila NBs, WDR62 regulates the interphase localization of Centrosomin (CNN, mammalian CDK5RAP2) to the apical centrosome, and thus centrosomal maturation and positioning. Interestingly, CNN is also an AURKA target that governs spindle orientation independently from cortical polarity establishment during mitosis. Similar to the phenotype observed for wdr62 depletion in NBs, cnn loss of function is associated with spindle orientation defects and reduced NB number. Thus, it is tempting to speculate that WDR62 and CNN function in the same AURKA-dependent signaling complex during mitosis (Lim, 2017).

Ex vivo studies have demonstrated that AURKA phosphorylation of WDR62 promotes spindle pole localization during mitosis. Mouse models suggest AURKA and WDR62 interact in vivo to control brain growth. Compound heterozygous wdr62+/-;aurka+/- mice have a much smaller body size than single heterozygotes but, although the mitotic index of the cerebral cortex was significantly increased and NSCs were reduced, consistent with a mitotic delay radial glia, potential changes to brain volume were not measured. This study, demonstrates that the brain overgrowth associated with aurka depletion specifically in NBs was suppressed by co-depletion of wdr62, bringing brain volume to within the control range. In contrast, the small brain phenotypes, due to glial-specific depletion of either aurka or wdr62, were further reduced by co-knockdown. Thus, in the context of normal brain development, AURKA likely acts to promote WDR62-dependent glial proliferation, but antagonizes WDR62 function in the NB lineage (Lim, 2017).

These findings indicate that WDR62 likely functions in AURKA-mediated regulation of spindle orientation but not in the establishment of cortical polarity. One reason for the differential output of AURKA regulation of WDR62 (between NB and glia) could stem from the symmetrical nature of glial division, where there is no evidence for cortical polarization. In contrast to the in vivo mammalian studies and previous Drosophila studies, which employed global depletion strategies for wdr62, these studies have enabled dissection of the relative contribution of wdr62 loss-of-function from each of the major brain lineages. In particular, the observation that depletion in either the NB or glial lineage is associated with reduced cell number, but an overall reduction in brain size was only observed when wdr62 was reduced in glia, places great interest in examining the relative contribution of glial-specific depletion of wdr62 in mice to brain size. Moreover, future studies of the pathogenic wdr62 mutations, and identified AURKA phosphorylation sites on WDR62, in the glial lineage are likely to inform on the contribution of this lineage to impaired brain growth in microcephaly (Lim, 2017).

Lim, N. R., Shohayeb, B., Ho, U., Yeap, Y. Y., Parton, R. G., Millard, S. S., Xu, Z., Piper, M. and Ng, D. C. H. (2020). The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development. Hum Mol Genet 29(2): 248-263. PubMed ID: 31816041

Abstract
WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial losion of WDR62 function to the growth of major brain lineages is unknowns-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. This study utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, deficient recruitment was observed of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

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Mitochondrial disease

Fogle, K.J., Hertzler, J.I., Shon, J.H. and Palladino, M.J. (2016). The ATP-sensitive K channel is seizure protective and required for effective dietary therapy in a model of mitochondrial encephalomyopathy. J Neurogenet [Epub ahead of print]. PubMed ID:
27868454

Abstract

Effective therapies are lacking for mitochondrial encephalomyopathies (MEs). MEs are devastating diseases that predominantly affect the energy-demanding tissues of the nervous system and muscle, causing symptoms such as seizures, cardiomyopathy, and neuro- and muscular degeneration. Even common anti-epileptic drugs which are frequently successful in ameliorating seizures in other diseases tend to have a lower success rate in ME, highlighting the need for novel drug targets, especially those that may couple metabolic sensitivity to neuronal excitability. Furthermore, alternative epilepsy therapies such as dietary modification are gaining in clinical popularity but have not been thoroughly studied in ME. Using the Drosophila ATP61 model of ME, this study analyzed dietary therapy throughout disease progression and found that it is highly effective against the seizures of ME, especially a high fat/ketogenic diet, and that the benefits are dependent upon a functional KATP channel complex. Further experiments with KATP show that it is seizure-protective in this model, and that pharmacological promotion of its open state also ameliorates seizures. These studies represent important steps forward in the development of novel therapies for a class of diseases that is notoriously difficult to treat, and lay the foundation for mechanistic studies of currently existing therapies in the context of metabolic disease (Fogle, 2016).


Foriel, S., Beyrath, J., Eidhof, I., Rodenburg, R. J., Schenck, A. and Smeitink, J. A. M. (2017). Feeding difficulties, a key feature of the Drosophila NDUFS4 mitochondrial disease model. Dis Model Mech 11(3). PubMed ID: 29590638

Abstract

Mitochondrial diseases are associated with a wide variety of clinical symptoms and variable degrees of severity. Patients with such diseases generally have a poor prognosis and often an early fatal disease outcome. With an incidence of 1 in 5000 live births and no curative treatments available, relevant animal models to evaluate new therapeutic regimes for mitochondrial diseases are urgently needed. By knocking down ND-18, the unique Drosophila ortholog of NDUFS4, an accessory subunit of the NADH:ubiquinone oxidoreductase (Complex I), this study developed and characterized several dNDUFS4 models that recapitulate key features of mitochondrial disease. Like in humans, the dNDUFS4 KD flies display severe feeding difficulties, an aspect of mitochondrial disorders that has so far been largely ignored in animal models. The impact of this finding, and an approach to overcome it, are discussed in the context of interpreting disease model characterization and intervention studies (Foriel, 2018).

Brischigliaro, M., Corra, S., Tregnago, C., Fernandez-Vizarra, E., Zeviani, M., Costa, R. and De Pitta, C. (2019). Knockdown of APOPT1/COA8 causes Cytochrome c oxidase deficiency, neuromuscular impairment, and reduced resistance to oxidative stress in Drosophila melanogaster. Front Physiol 10: 1143. PubMed ID: 31555154

Abstract

Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. This study reportd that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. These data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects (Brischigliaro, 2019).

Yap, Z. Y., Park, Y. H., Wortmann, S. B., Gunning, A. C., Ezer, S., Lee, S., Duraine, L., Wilichowski, E., Wilson, K., Mayr, J. A., Wagner, M., Li, H., Kini, U., Black, E. D., Monaghan, K. G., Lupski, J. R., Ellard, S., Westphal, D. S., Harel, T. and Yoon, W. H. (2019). Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes. Genome Med 13(1): 55. PubMed ID: 33845882

Abstract

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, exome sequencing was performed for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants. This study reports 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, Phe50Leu, Gly236Val, and Arg327Pro were shown to cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles. These findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation (Yap, 2021).

Kowada, R., Kodani, A., Ida, H., Yamaguchi, M., Lee, I. S., Okada, Y. and Yoshida, H. (2021). The function of Scox in glial cells is essential for locomotive ability in Drosophila. Sci Rep 11(1): 21207. PubMed ID: 34707123

Abstract
Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. This study demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans.

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Mucopolysaccharidosis

Rigon, L., Kucharowski, N., Eckardt, F. and Bauer, R. (2020). Modeling Mucopolysaccharidosis Type II in the Fruit Fly by Using the RNA Interference Approach. Life (Basel) 10(11). PubMed ID:
33142967

Abstract

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that occurs due to the deficit of the lysosomal enzyme iduronate 2-sulfatase (IDS) that leads to the storage of the glycosaminoglycan heparan- and dermatan-sulfate in all organs and tissues. It is characterized by important clinical features and the severe form presents with a heavy neurological involvement. However, almost nothing is known about the neuropathogenesis of MPS II. To address this issue, a ubiquitous, neuronal, and glial-specific knockdown model was developed in Drosophila melanogaster by using the RNA interference (RNAi) approach. Knockdown of the Ids/CG12014 gene resulted in a significant reduction of the Ids gene expression and enzymatic activity. However, glycosaminoglycan storage, survival, molecular markers (Atg8a, Lamp1, Rab11), and locomotion behavior were not affected. Even strongly reduced, IDS-activity was enough to prevent a pathological phenotype in a MPS II RNAi fruit fly. Thus, a Drosophila MPS II model requires complete abolishment of the enzymatic activity (Rigon, 2020).

Basu, I., Bar, S., Prasad, M. and Datta, R. (2022). Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR. Biochim Biophys Acta Mol Basis Dis 1868(7): 166399. PubMed ID: 35318126

Abstract
Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. This issue was addressed using a recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of body fat. This study reports that the lean phenotype of the CG2135-/- larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. The data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135-/- larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135-/- larvae. Interestingly, treatment of the CG2135-/- larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. These finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.

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Myeloproliferative neoplasms

Terriente-Félix, A., Pérez, L., Bray, S.J., Nebreda, A.R. and Milán, M. (2017). Drosophila model of myeloproliferative neoplasm reveals a feed-forward loop in the JAK pathway mediated by p38 MAPK signalling. Dis Model Mech [Epub ahead of print]. PubMed ID:
28237966

Abstract

Myeloproliferative neoplasms (MPNs) of the Philadelphia-negative class comprise polycythemia vera, essential thrombocythemia and primary myelofibrosis (PMF). They are associated with aberrant amounts of myeloid lineage cells in the blood, and in the case of overt PMF, with the development of myelofibrosis in the bone marrow and the failure to produce normal blood cells. These diseases are usually caused by gain-of-function mutations in the kinase JAK2. This study used Drosophila to investigate the consequences of activation of the JAK2 ortholog in hematopoiesis. The maturing hemocytes in the lymph gland, the major hematopoietic organ in the fly, was identified as the cell population susceptible to induce hypertrophy upon targeted overexpression of JAK. JAK was shown to activate a feed-forward loop including the cytokine-like ligand Upd3 and its receptor Domeless, which are required to induce lymph gland hypertrophy. Moreover, p38 MAPK signalling plays a key role in this process by inducing the expression of the ligand Upd3. Interestingly, forced activation of the p38 MAPK pathway in maturing hemocytes suffices to generate hypertrophic organs and the appearance of melanotic tumours. These results illustrate a novel pro-tumorigenic cross-talk between the p38 MAPK pathway and JAK signalling in a Drosophila model of MPNs. Based on the shared molecular mechanisms underlying MPNs in flies and humans, the interplay between Drosophila JAK and p38 signalling pathways unravelled in this work might have translational relevance for human MPNs (Terriente-Félix, 2017).

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Bernardoni, R., Giordani, G., Signorino, E., Monticelli, S., Messa, F., Pradotto, M., Rosso, V., Bracco, E., Giangrande, A., Perini, G., Saglio, G. and Cilloni, D. (2018). A new BCR-ABL1 Drosophila model as a powerful tool to elucidate pathogenesis and progression of chronic myeloid leukemia. Haematologica. PubMed ID: 30409797

Abstract

The oncoprotein BCR-ABL1 triggers Chronic Myeloid Leukemia. It is clear that the disease relies on the constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. This study describes and validates a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue specific promoters and thought as a versatile tool to perform genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1 dependent phenotypes. Importantly, in patients with leukemia decrease of human Dab1 and Dab2 expression correlates with a more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate the Drosophila model that promises to be an excellent system to perform unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators to better elucidate the mechanism of leukemia onset and progression (Bernardoni, 2018).

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Muscular Dystrophies

Cerro-Herreros, E., Chakraborty, M., Perez-Alonso, M., Artero, R. and Llamusi, B.
(2017). Expanded CCUG repeat RNA expression in Drosophila heart and muscle trigger Myotonic Dystrophy type 1-like phenotypes and activate autophagocytosis genes. Sci Rep 7(1): 2843. PubMed ID: 28588248

Abstract

Myotonic dystrophies (DM1-2) are neuromuscular genetic disorders caused by the pathological expansion of untranslated microsatellites. DM1 and DM2, are caused by expanded CTG repeats in the 3'UTR of the DMPK gene and CCTG repeats in the first intron of the CNBP gene, respectively. Mutant RNAs containing expanded repeats are retained in the cell nucleus, where they sequester nuclear factors and cause alterations in RNA metabolism. However, for unknown reasons, DM1 is more severe than DM2. To study the differences and similarities in the pathogenesis of DM1 and DM2, model flies were generated by expressing pure expanded CUG ([250]x) or CCUG ([1100]x) repeats, respectively, and compared them with control flies expressing either 20 repeat units or GFP. Surprisingly, severe muscle reduction and cardiac dysfunction were observed in CCUG-expressing model flies. The muscle and cardiac tissue of both DM1 and DM2 model flies showed DM1-like phenotypes including overexpression of autophagy-related genes, RNA mis-splicing and repeat RNA aggregation in ribonuclear foci along with the Muscleblind protein. These data reveal, for the first time, that expanded non-coding CCUG repeat-RNA has similar in vivo toxicity potential as expanded CUG RNA in muscle and heart tissues and suggests that specific, as yet unknown factors, quench CCUG-repeat toxicity in DM2 patients (Cerro-Herreros, 2017).


Coni, S., Falconio, F. A., Marzullo, M., Munafo, M., Zuliani, B., Mosti, F., Fatica, A., Ianniello, Z., Bordone, R., Macone, A., Agostinelli, E., Perna, A., Matkovic, T., Sigrist, S., Silvestri, G., Canettieri, G. and Ciapponi, L. (2021). Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function. Elife 10. PubMed ID: 34517941.

Abstract

Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. This study shows that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. This study demonstrated that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, this study showed a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, evidence is provided that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. It is suggested that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions (, 2021).


Chakraborty, M., Sellier, C., Ney, M., Pascal, V., Charlet-Berguerand, N., Artero, R. and Llamusi, B. (2018). Daunorubicin reduces MBNL1 sequestration caused by CUG-repeat expansion and rescues cardiac dysfunctions in a Drosophila model of myotonic dystrophy. Dis Model Mech 11(4). PubMed ID: 29592894

Abstract

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder caused by expression of mutant myotonin-protein kinase (DMPK) transcripts containing expanded CUG repeats. Pathogenic DMPK RNA sequesters the muscleblind-like (MBNL) proteins, causing alterations in metabolism of various RNAs. Cardiac dysfunction represents the second most common cause of death in DM type 1 (DM1) patients. However, the contribution of MBNL sequestration in DM1 cardiac dysfunction is unclear. This study overexpressed Muscleblind (Mbl), the Drosophila MBNL orthologue, in cardiomyocytes of DM1 model flies and observed a rescue of heart dysfunctions, which are characteristic of these model flies and resemble cardiac defects observed in patients. A drug - daunorubicin hydrochloride - was identified that directly binds to CUG repeats and alleviates Mbl sequestration in Drosophila DM1 cardiomyocytes, resulting in mis-splicing rescue and cardiac function recovery. These results demonstrate the relevance of Mbl sequestration caused by expanded-CUG-repeat RNA in cardiac dysfunctions in DM1, and highlight the potential of strategies aimed at inhibiting this protein-RNA interaction to recover normal cardiac function (Chakraborty, 2018).


Lee, J., Bai, Y., Chembazhi, U. V., Peng, S., Yum, K., Luu, L. M., Hagler, L. D., Serrano, J. F., Chan, H. Y. E., Kalsotra, A. and Zimmerman, S. C. (2019). Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1. Proc Natl Acad Sci U S A. PubMed ID: 30975744

Abstract

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. This study reports intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of this multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics (Lee, 2019).


Kiss, A. A., Somlyai-Popovics, N., Kiss, M., Boldogkoi, Z., Csiszar, K. and Mink, M. (2019). Type IV collagen is essential for proper function of integrin-mediated adhesion in Drosophila muscle fibers. Int J Mol Sci 20(20). PubMed ID: 31623094

Abstract

Congenital muscular dystrophy (CMD), a subgroup of myopathies is a genetically and clinically heterogeneous group of inherited muscle disorders and is characterized by progressive muscle weakness, fiber size variability, fibrosis, clustered necrotic fibers, and central myonuclei present in regenerating muscle. Type IV collagen (COL4A1) mutations have recently been identified in patients with intracerebral, vascular, renal, ophthalmologic pathologies and congenital muscular dystrophy, consistent with diagnoses of Walker-Warburg Syndrome or Muscle-Eye-Brain disease. Morphological characteristics of muscular dystrophy have also been demonstrated Col4a1 mutant mice. Yet, several aspects of the pathomechanism of COL4A1-associated muscle defects remained largely uncharacterized. Based on the results of genetic, histological, molecular, and biochemical analyses in an allelic series of Drosophila col4a1 mutants, evidence is provided that col4a1 mutations arise by transitions in glycine triplets, associate with severely compromised muscle fibers within the single-layer striated muscle of the common oviduct, characterized by loss of sarcomere structure, disintegration and streaming of Z-discs, indicating an essential role for the COL4A1 protein. Features of altered cytoskeletal phenotype include actin bundles traversing over sarcomere units, amorphous actin aggregates, atrophy, and aberrant fiber size. The mutant COL4A1-associated defects appear to recapitulate integrin-mediated adhesion phenotypes observed in RNA-inhibitory Drosophila. These results provide insight into the mechanistic details of COL4A1-associated muscle disorders and suggest a role for integrin-collagen interaction in the maintenance of sarcomeres (Kiss, 2019).


Auxerre-Plantie, E., Nakamori, M., Renaud, Y., Huguet, A., Choquet, C., Dondi, C., Miquerol, L., Takahashi, M. P., Gourdon, G., Junion, G., Jagla, T., Zmojdzian, M. and Jagla, K. (2019). Straightjacket/alpha2delta3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1. Elife 8. PubMed ID: 31829940

Abstract

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. This study simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. Deregulations of several genes controlling cellular calcium levels were detected, including increased expression of straightjacket/ alpha2delta3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. It was also shown that ventricular alpha2delta3 expression is significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/alpha2delta3 levels could offer a strategy to prevent conduction defects in DM1.


>Souidi, A., Nakamori, M., Zmojdzian, M., Jagla, T., Renaud, Y. and Jagla, K. . Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1. EMBO Rep 24(4): e56616. PubMed ID: 36852954

Abstract
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. This study used Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. miR-1 down-regulation in the heart was found to lead to dilated cardiomyopathy (DCM), a DM1-associated phenotype. In silico screening for miR-1 targets was combined with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM.

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Myopathy

Dahl-Halvarsson, M., Olive, M., Pokrzywa, M., Ejeskar, K., Palmer, R. H., Uv, A. E. and Tajsharghi, H.
(2018). Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member. Proc Natl Acad Sci U S A. PubMed ID: 29946036

Abstract

Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/beta-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the alpha-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. A Drosophila model of Laing distal myopathy was developed by genomic engineering of the Drosophila Mhc locus. Flies expressing only Mhc(K1728del) in indirect flight and jump muscles, and heterozygous Mhc(K1728del) animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of Mhc(K1728del) mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous Mhc(K1728del) larvae also demonstrated structural and functional impairments in heart muscle. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with Mhc(K1728del) were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. These observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy (Dahl-Halvarsson, 2018).

Dahl-Halvarsson, M., Olive, M., Pokrzywa, M., Norum, M., Ejeskar, K. and Tajsharghi, H. (2020). Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase. Dis Model Mech 13(12). PubMed ID: 33234710

Abstract

Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/β-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. This study developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. A higher expression level of the mutated allele was shown to be concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients (Dahl-Halvarsson, 2020).

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Nephrotic Syndrome

Hermle, T., Braun, D.A., Helmstädter, M., Huber, T.B. and Hildebrandt, F.
(2016). Modeling monogenic human nephrotic syndrome in the Drosophila garland cell nephrocyte J Am Soc Nephrol [Epub ahead of print]. PubMed ID: 27932481

Abstract

Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. Drosophila garland cell nephrocytes are podocyte-like cells and thus provide a potential in vivo model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. This study discovered that two Drosophila slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, it was found that this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes requires Cubilin and reflects size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, Drosophila orthologs of human monogenic causes of nephrotic syndrome were screened and conservation of the central pathogenetic alterations was observed. It was found that the silencing of the coenzyme Q10 (CoQ10) biosynthesis gene Coq2 disrupts slit diaphragm morphology. Restoration of CoQ10 synthesis by vanillic acid partially rescues the phenotypic and functional alterations induced by Coq2-RNAi. Notably, Coq2 colocalizes with mitochondria, and Coq2 silencing increases the formation of reactive oxygen species (ROS). Silencing of ND75, a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ10, phenocopies the effect of Coq2-RNAi. Moreover, the ROS scavenger glutathione partially rescues the effects of Coq2-RNAi. In conclusion, Drosophila garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of COQ2-nephropathy (Hermle, 2016).



Kampf, L. L. et al. (2019). TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome. J Am Soc Nephrol 30(12): 2338-2353. PubMed ID: 31732614

Abstract

Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. This study report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. This study expanded the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology.

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Neurological disorder with regression, abnormal movements, loss of speech, and seizures (NEDAMSS)

Sinha Ray, S., Dutta, D., Dennys, C., Powers, S., Roussel, F., Lisowski, P., Glazar, P., Zhang, X., Biswas, P., Caporale, J. R., Rajewsky, N., Bickle, M., Wein, N., Bellen, H. J., Likhite, S., Marcogliese, P. C. and Meyer, K. C. (2022). Mechanisms of IRF2BPL-related disorders and identification of a potential therapeutic strategy. Cell Rep 41(10): 111751. PubMed ID:
36476864

Abstract
The recently discovered neurological disorder with regression, abnormal movements, loss of speech, and seizures (NEDAMSS) is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. This study reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. These findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.

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Neurofibromatosis

King, L. B., Boto, T., Botero, V., Aviles, A. M., Jomsky, B. M., Joseph, C., Walker, J. A. and Tomchik, S. M.
(2020). Developmental loss of neurofibromin across distributed neuronal circuits drives excessive grooming in Drosophila. PLoS Genet 16(7): e1008920. PubMed ID: 32697780
Summary:

Abstract
Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, grooming was examined in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1.

Botero, V., Stanhope, B. A., Brown, E. B., Grenci, E. C., Boto, T., Park, S. J., King, L. B., Murphy, K. R., Colodner, K. J., Walker, J. A., Keene, A. C., Ja, W. W. and Tomchik, S. M. (2021). Neurofibromin regulates metabolic rate via neuronal mechanisms in Drosophila. Nat Commun 12(1): 4285. PubMed ID: 34257279

Abstract

Neurofibromatosis type 1 is a chronic multisystemic genetic disorder that results from loss of function in the neurofibromin protein. Neurofibromin may regulate metabolism, though the underlying mechanisms remain largely unknown. This study shows that neurofibromin regulates metabolic homeostasis in Drosophila via a discrete neuronal circuit. Loss of neurofibromin increases metabolic rate via a Ras GAP-related domain-dependent mechanism, increases feeding homeostatically, and alters lipid stores and turnover kinetics. The increase in metabolic rate is independent of locomotor activity, and maps to a sparse subset of neurons. Stimulating these neurons increases metabolic rate, linking their dynamic activity state to metabolism over short time scales. These results indicate that neurofibromin regulates metabolic rate via neuronal mechanisms, suggest that cellular and systemic metabolic alterations may represent a pathophysiological mechanism in neurofibromatosis type 1, and provide a platform for investigating the cellular role of neurofibromin in metabolic homeostasis (Botero, 2021).

Georganta, E. M., Moressis, A. and Skoulakis, E. M. (2021). Associative learning requires Neurofibromin to modulate GABAergic inputs to Drosophila Mushroom Bodies. J Neurosci. PubMed ID: 33972401

Abstract

Cognitive dysfunction, is among the hallmark symptoms of Neurofibromatosis 1, and accordingly, loss of the Drosophila melanogaster ortholog of Neurofibromin 1 (dNf1), precipitates associative learning deficits. However, the affected circuitry in the adult CNS remained unclear and the compromised mechanisms debatable. Although the main evolutionarily conserved function attributed to Nf1 is to inactivate Ras, decreased cAMP signalling upon its loss has been thought to underlie impaired learning. Using mixed sex populations, it was determined that dNf1 loss results in excess GABAergic signaling to the center for associative learning Mushroom Body (MB) neurons, apparently suppressing learning. dNf1 is necessary and sufficient for learning within these non-MB neurons, as a dAlk and Ras1-dependent, but PKA-independent modulator of GABAergic neurotransmission. Surprisingly, this study also uncovered and discuss a postsynaptic Ras1-dependent, but dNf1-independnet signaling within the MBs that apparently responds to presynaptic GABA levels and contributes to the learning deficit on the mutants.

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Neuronal ceroid lipofuscinosis

Imler, E., Pyon, J. S., Kindelay, S., Torvund, M., Zhang, Y. Q., Chandra, S. S. and Zinsmaier, K. E.
(2019). A Drosophila model of neuronal ceroid lipofuscinosis CLN4 reveals a hypermorphic gain of function mechanism. Elife 8. PubMed ID: 31663851

Abstract

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPalpha. Animal models of CLN4 were developed by expressing CLN4 mutant human CSPalpha (hCSPalpha) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPalpha and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPalpha accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, it is suggested that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking (Imler, 2019).

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Diabetic Neuropathy

Kim, K., Cha, S. J., Choi, H. J., Kang, J. S. and Lee, E. Y.
(2021). Dysfunction of Mitochondrial Dynamics in Drosophila Model of Diabetic Nephropathy. Life (Basel) 11(1). PubMed ID: 33477666

Abstract

Although mitochondrial dysfunction is associated with the development and progression of diabetic nephropathy (DN), its mechanisms are poorly understood, and it remains debatable whether mitochondrial morphological change is a cause of DN. In this study, a Drosophila DN model was established by treating a chronic high-sucrose diet that exhibits similar phenotypes in animals. Results showed that flies fed a chronic high-sucrose diet exhibited a reduction in lifespan, as well as increased lipid droplets in fat body tissue. Furthermore, the chronic high-sucrose diet effectively induced the morphological abnormalities of nephrocytes in Drosophila. High-sucrose diet induced mitochondria fusion in nephrocytes by increasing Opa1 and Marf expression. These findings establish Drosophila as a useful model for studying novel regulators and molecular mechanisms for imbalanced mitochondrial dynamics in the pathogenesis of DN. Furthermore, understanding the pathology of mitochondrial dysfunction regarding morphological changes in DN would facilitate the development of novel therapeutics.

Dabbara, H., Schultz, A. and Im, S. H. (2021). Drosophila insulin receptor regulates diabetes-induced mechanical nociceptive hypersensitivity. MicroPubl Biol 2021. PubMed ID: 34549177

Abstract

Painful diabetic neuropathy (PDN) is one of the predominant complications of diabetes that causes numbness, tingling, and extreme pain sensitivity. Understanding the mechanisms of PDN pathogenesis is important for patient treatments. This study reports Drosophila models of diabetes-induced mechanical nociceptive hypersensitivity. Type 2 diabetes-like conditions and loss of insulin receptor function in multidendritic sensory neurons lead to mechanical nociceptive hypersensitivity. Furthermore, it was found that restoring insulin signaling in multidendritic sensory neurons can block diabetes-induced mechanical nociceptive hypersensitivity. This work highlights the critical role of insulin signaling in nociceptive sensory neurons in the regulation of diabetes-induced nociceptive hypersensitivities (Dabbara, 2021).

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N-Glycanase 1 deficiency

Owings, K. G., Lowry, J. B., Bi, Y., Might, M. and Chow, C. Y.
(2018). Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches. Hum Mol Genet. PubMed ID: 29346549

Abstract

Autosomal recessive loss-of-function mutations in N-Glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction, and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, a Drosophila model of NGLY1 deficiency was developed. Loss of NGLY1 function resulted in developmental delay and lethality. RNAseq was used to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. This study shows that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches (Owings, 2018).

Han, S. Y., Pandey, A., Moore, T., Galeone, A., Duraine, L., Cowan, T. M. and Jafar-Nejad, H. (2020). A conserved role for AMP-activated protein kinase in NGLY1 deficiency. PLoS Genet 16(12): e1009258. PubMed ID: 33315951

Abstract
Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1).. Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, this study reports that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/- mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKα levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease (Han, 2020).

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Nicotine addiction

El-Merhie, N., Kruger, A., Uliczka, K., Papenmeier, S., Roeder, T., Rabe, K. F., Wagner, C., Angstmann, H. and Krauss-Etschmann, S.
(2021). Sex dependent effect of maternal e-nicotine on F1 Drosophila development and airways. Sci Rep 11(1): 4441. PubMed ID: 33627715

Abstract

E-cigarettes are heavily advertised as healthier alternative to common tobacco cigarettes, leading more and more women to switch from regular cigarettes to ENDS (electronic nicotine delivery system) during pregnancy. While the noxious consequences of tobacco smoking during pregnancy on the offspring health are well-described, information on the long-term consequences due to maternal use of e-cigarettes do not exist so far. Therefore, this study aimed to investigate how maternal e-nicotine influences offspring development from earliest life until adulthood. To this end, virgin female Drosophila melanogaster flies were exposed to nicotine vapor (8 μg nicotine) once per hour for a total of eight times. Following the last exposure, e-nicotine or sham exposed females were mated with non-exposed males. The F1-generation was then analyzed for viability, growth and airway structure. Maternal exposure to e-nicotine not only leads to reduced maternal fertility, but also negatively affects size and weight, as well as tracheal development of the F1-generation, lasting from embryonic stage until adulthood. These results not only underline the need for studies investigating the effects of maternal vaping on offspring health, but also propose this established model for analyzing molecular mechanisms and signaling pathways mediating these intergenerational changes (El-Merhie, 2021)

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Nonalcoholic fatty liver disease

Liu, Z., Huang, Y., Hu, W., Huang, S., Wang, Q., Han, J. and Zhang, Y. Q.
(2014). dAcsl, the Drosophila ortholog of acyl-CoA synthetase long-chain family member 3 and 4, inhibits synapse growth by attenuating bone morphogenetic protein signaling via endocytic recycling. J Neurosci 34(8): 2785-2796. PubMed ID: 24553921

Abstract

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. This study reports that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated Mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, these results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation (Liu, 2014).

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Noonan Syndrome

Leahy, S. N., Song, C., Vita, D. J. and Broadie, K. (2023). FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission. PLoS Biol 21(1): e3001969. PubMed ID:
36701299

Abstract
Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, this study found both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. Presynaptic pERK activation was found in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.

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Obesity

Vaziri, A., Khabiri, M., Genaw, B. T., May, C. E., Freddolino, P. L. and Dus, M. (2020). Persistent epigenetic reprogramming of sweet taste by diet. Sci Adv 6(46). PubMed ID:
33177090

Abstract

Diets rich in sugar, salt, and fat alter taste perception and food preference, contributing to obesity and metabolic disorders, but the molecular mechanisms through which this occurs are unknown. This study shows that in response to a high sugar diet, the epigenetic regulator Polycomb Repressive Complex 2.1 (PRC2.1) persistently reprograms the sensory neurons of Drosophila melanogaster flies to reduce sweet sensation and promote obesity. In animals fed high sugar, the binding of PRC2.1 to the chromatin of the sweet gustatory neurons is redistributed to repress a developmental transcriptional network that modulates the responsiveness of these cells to sweet stimuli, reducing sweet sensation. Half of these transcriptional changes persist despite returning the animals to a control diet, causing a permanent decrease in sweet taste. These results uncover a new epigenetic mechanism that, in response to the dietary environment, regulates neural plasticity and feeding behavior to promote obesity (Vaziri, 2020).

In this study set out to understand how dietary experience alters the gustatory system to promote food intake and weight gain. Specifically, advantage was taken of the simple sensory system of D. melanogaster and its exquisite genetic and neural tools to identify the molecular mechanisms through which diet composition changes neural physiology and behavior. Previous work has shown high dietary sugar decreased the responsiveness of the sensory neurons to sugar stimuli, leading to a dulled sense of sweet taste, independently of fat accumulation. This study show sthat the decrease in sweet taste sensation that flies experience after chronic exposure to a high sugar diet is caused by the cell-autonomous action of the PRC2.1 in the sweet gustatory neurons. Mutations and pharmacological inhibition of PRC2.1 blocked the effects of the food environment on neural activity, behavior, and obesity. While the possibility that PRC1 and Pho RC may also be involved, this study found that mutations or knockdown in these complexes had no effect on taste. To this point, it was observed that in Pcl mutants, even if the neural responses to sucrose were identical in control and sugar diet fed flies, they were of lower in magnitude than those of control flies, suggesting that PRC2.1 may modulate plasticity bidirectionally in response to the nutrient environment. This also suggests that, within limits, it is the relative rather than the absolute output of the sensory neurons that is important for taste sensation and diet-induced obesity (Vaziri, 2020).

In the high sugar food environment, PRC2.1 chromatin occupancy was redistributed, leading to the repression of transcription factors, neural, signaling, and metabolic genes that decreased the responsiveness of the Gr5a+ neurons and the fly's sensory experience of sweetness. However, it was found that PRC2.1 did not directly bind to neuronal genes in these cells and that, instead, it targeted transcription factors involved in processes such as sensory neuron development, synaptic function, and axon targeting. Specifically, on a high sugar diet Pcl binding was increased at the loci of transcription factors cad, GATAe, nub/pdm, Ptx1 and decreased at the scro locus and lead to corresponding changes in the mRNA levels of these genes (Vaziri, 2020).

Computational analysis revealed that in the Gr5a+ cells, these five transcriptional factors regulate a network of ~658 candidate target genes implicated in synaptic function, signal transduction, and metabolism. Changes in the levels of the five transcription factors on a high sugar diet resulted in the overall repression of their target genes, providing a possible explanation for the alterations in the responsiveness of the Gr5a+ cells that were observed. Several positive and negative regulatory loops were predicted among the five transcription factors, suggesting that they could form a regulatory hub that is responsive to changes in the dietary environment. Knockdown of the four activators and a few of their targets and overexpression of scro resulted in a decrease in sweet taste on a control diet. However, overexpression of cad, Ptx1, and nub alone and knockdown of scro did not rescue taste deficiencies in animals fed a high sugar diet. Since there is (i) overlap in the predicted targets between the repressor scro and each of the four activators and (ii) overlap among the targets of the four activators, one possibility is that, as long as scro levels are higher because of the sugar diet, the repressive drive is so strong that overcoming it requires collaborative binding among the activators. Together, these findings suggest that this transcriptional hub and the gene battery it controls are necessary for sweet taste and reshaped by high dietary sugar (Vaziri, 2020).

How do these transcription factors and their targets modulate the physiology of these gustatory neurons? Several of these transcription factors (Ptx1, scro, and nub/pdm) control the proper branching, synaptic connectivity, and activity of sensory neurons, while others (cad and nub/pdm) play a role in neuroblast development; PRC2 also functions as a competence factor in neural proliferation, differentiation, and sensory neurons. It is proposed that the gene battery of ~658 genes controlled by this transcriptional hub may define the intrinsic properties of the sweet sensing neurons. It was observed that the four activators that are repressed by Pcl under the high sugar condition are enriched in the Gr5a+ cells while scro is depleted. Further, many of the target genes are involved in signaling, synaptic function, and cell adhesion, including the kinase haspin, the adenylate cyclase ACXD, syt-alpha, Arc1, and the tetraspanin, jonan, and innexin proteins among others. These genes were part of a highly interconnected network, which could affect the responsiveness and wiring of the sweet gustatory neurons. Since no change in the expression of the sweet taste receptors, or the misexpression of other taste receptors was detected, the data are not consistent with a complete 'loss' of identity of the Gr5a+ neurons with a high sugar diet. Instead, it is hypothesized that PRC2.1 tunes these sensory neurons to the dietary environment by altering a developmental transcriptional network that controls the intrinsic properties of the Gr5a+ cells, particularly those involved in signal transduction, connectivity, synaptic function, and metabolism. Studies that test the effects of this network on the wiring, morphology, and signal transduction of the sweet sensory neurons will shed light on how exactly the transcriptional remodeling caused by PRC2.1 found in this study affects the physiology of Gr5a+ cells (Vaziri, 2020).

While the experiments show that PRC2.1 chromatin occupancy shifts with the dietary environment, this study did not define the signaling mechanisms through which this change in binding occurs. Thus, the question of how exactly PRC2.1 binding is altered in response to the food environment remains open. Recent studies suggest that the activity of Polycomb Group Proteins is directly and indirectly linked to cellular metabolism, including kinase signaling cascades, the post-translational modification O-linked-N-acetylglucosaminylation (GlcNAcylation), and the availability of cofactors for histone modifications. Previous work showed that the hexosamine biosynthesis pathway enzyme O-GlcNAc transferase (OGT) acts in the Gr5a+ neurons to mediate the effects of high dietary sugar on sweet taste; whether the interaction between OGT and PRC2 is what promotes the repressive activity of the latter in these sensory neurons is a question worth investigating. Notably, the dysregulation of Polycomb-associated chromatin has been found in mice and humans with diet-induced obesity, suggesting that the mechanisms found in this study could also underlie the chemosensory alterations reported in mammals (Vaziri, 2020).

More broadly, this work opens up the exciting possibility that PRC2 may modulate neural plasticity in response to environmental conditions by reengaging developmental programs. Despite its central role in development and maintenance of neural identity, studies have not directly linked PRC2.1 to neural plasticity. However, in other postmitotic cells such as muscle, Polycomb Group Proteins are known to reshape transcriptional programs according to environmental stressors, such as oxidative stress, injury, temperature, and light. These findings advance the conceptual understanding of the role of Polycomb Group Proteins in the nervous system and suggest that they could also modulate 'neural states' and metaplasticity in response to environment stimuli. Using neuroepigenetic mechanisms such as those used by Polycomb Group Proteins to tune neural states to external conditions could provide several advantages compared to the medley of other cellular-, receptor-, or synaptic plasticity-based mechanisms. Specifically, it would allow cells to (i) orchestrate a coordinated response, (ii) create a memory of the environment, and (iii) buffer small fluctuations until a substantial challenge is perceived. It is particularly fascinating to think about the molecular mechanisms through which these neural states may be established. The need of neurons to constantly maintain their identity may mean that environmental signals such as the extent of sensory stimulation could alter the expression of developmental gene batteries and affect neural physiology. It has been speculated that some forms of plasticity may reengage developmental programs that specify the intrinsic properties of neurons. In this study it was observed that the regulators of the transcriptional network that was uncovered functions in sensory neuron development and are enriched in the Gr5a+ cells. Thus, it could be a hallmark of neuroepigenetic plasticity to exploit developmental programs, linking the known role of PRC2 in establishing cell fates with this newly found function in modulating cell states (Vaziri, 2020).

Incidentally, reengaging developmental programs could be the reason why some environments and experiences leave a memory that leads to the persistent expression of the phenotype beyond the presence of the triggering stimulus, as these could target neural connectivity and set synaptic weight thresholds. This study found that the changes in taste sensation and half of the sugar diet neural state set by PRC2.1 remained even after animals were moved back to the control diet for up to 20 days. A limitation of this study is that because of the small number of Gr5a+ neurons and their anatomically inaccessible location, it was not possible to measure the identity of the molecular memory in these cells alone. However, it was seen that the phenotypic memory of the high sugar food environment was dependent on the constitutive action of PRC2.1. Thus, on the basis of other studies showing that the H3K27 methyl mark acts as a molecular memory during development, it is speculated that this is likely to be the memory signal in the Gr5a+ cells too. Stable maintenance of the memory requires active recruitment of PRC2; while this study did not measure Pcl occupancy and chromatin accessibility at PREs in the recovery diet with and without the inhibitor, the findings that PRC1.2 is actively required for the maintenance of the taste phenotype and that 47% of its indirect targets are still repressed indicate that PRC2.1 may be stably recruited to the transcription factors loci. Perhaps, conditions that lead to metabolic remodeling such as prolonged fasting could reset its binding. Last, it is not known whether the diet-induced chemosensory plasticity observed in humans and rodents is persistent or reversible. Unlike in D. melanogaster, mammalian taste cells are not postmitotic neurons, and so, they regenerate every few weeks. Thus, the persistence of chemosensory plasticity in mammals, if it exists, may involve different mechanisms in the taste cells, such as a decrease in their renewal or changes in their wiring to sensory neurons. However, a decrease in the responses of the chorda tympani to sweetness has been observed in rats fed a 30% sucrose diet, and thus, the current findings may be applicable to the sensory nerves (Vaziri, 2020).

In conclusion, this study shows that PRC2.1 mediates the effects of high dietary sugar on sweet taste by establishing persistent alterations in the taste neurons that remain as a phenotypic and transcriptional memory of the previous food environment. It is speculated that this memory may lock animals into patterns of feeding behavior that become maladaptive and promote obesity. Thus, dietary experience, in ways like trauma, can induce lasting molecular alterations that restrict the behavioral plasticity of animals and affect disease risk. Since the content of sugar in processed foods is similar to that fed flies in this study and the function of Polycomb Group Proteins is conserved from plants to humans, this work is broadly relevant to understanding the effects of processed food on the mammalian taste system and its impact on food intake and a whole range of diet-related conditions and diseases that affect billions of people around the globe (Vaziri, 2020). Livelo, C., Guo, Y., Abou Daya, F., Rajasekaran, V., Varshney, S., Le, H. D., Barnes, S., Panda, S. and Melkani, G. C. (2023). Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models. Nat Commun 14(1): 949. PubMed ID: 36810287

Abstract

Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. This study demonstrates that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, these data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments (Livelo, 2023).

Obesity is a global and public health problem linked to various comorbidities. Major contributors to obesity include living a lifestyle comprised of high-caloric diets and having a genetic predisposition to the disease. The skeletal muscle plays a crucial role in metabolism as it is the major tissue responsible for glucose uptake from the blood. Muscle dysfunction due to obesity can lead to insulin resistance and reduced energy levels. Indeed, intramyocellular lipids or intramyocellular triglycerides (IMCL/IMTG) catalyzed by diacylglyceride acyltransferase 2 (DGAT2) deposited within skeletal muscle cells can be harmful if not routinely depleted as observed in athletes. In addition, truncal adiposity has been associated with increased levels of S-adenosylmethionine (SAM) in overfed humans. SAM is a universal methyl donor involved in various physiological processes and increased levels have been observed to be a pathogenic catalyst that requires regulation from entities such as glycine N-methyltransferase (GNMT). GNMT converts SAM to sarcosine with the help of glycine, which can be produced via sarcosine dehydrogenase (SARDH) (Livelo, 2023).

The primary driving force behind muscle metabolism relates to supplying energy needed for muscular contractions. Adenosine triphosphate (ATP) helps maintain muscle fiber contraction and ATP is regulated by metabolic pathways such as AMPK-signaling and the purine cycle. AMPK generally acts as a central sensor of energy status (AMP/ATP and ADP/ATP ratios) and maintains energy balance by regulating downstream anabolic and catabolic pathways. In skeletal muscle, activation of AMPK has been shown to improve glucose uptake and insulin sensitivity11 under obesogenic pressure, while chronic activation of AMPK increases muscle fiber oxidative capability by enhancing mitochondrial biogenesis. Meanwhile, the purine cycle helps maintain appropriate energy levels during exercise through ATP formation in the adenylate kinase reaction, enhancement of glycolysis, and anaplerosis (metabolic pathways that replenish citric acid cycle intermediate) through the production of fumurate. Insight into muscle function, metabolism, and energy production continues to be a growing topic of interest as new studies continue to emerge (Livelo, 2023).

Drosophila melanogaster is an amenable model for studying human metabolic diseases as mechanisms associated with nutrient sensing, energy utilization, and energy storage are mostly conserved. Obese Drosophila were previously studied by using a high-fat diet-induced obesity model (HFD) and a genetic-induced obesity model (flies lack sphingosine kinase 2; Sk2 mutant). Both obesity models displayed skeletal muscle dysfunction, accumulation of aberrant lipids, insulin resistance as well as mitochondrial defects. An intervention known as time-restricted feeding (TRF) has been shown to regulate gene expression and gene rhythmicity leading to the amelioration of obesity and metabolic dysfunction. Imposing TRF on Drosophila subjected to obesogenic challenges attenuated the adverse effects of obesity shown by improved muscle performance, reduced intramuscular fat, lowered phospho-AKT levels, in addition to the reduction in a marker of insulin resistance. A recent human study of 11 men with obesity in a randomized cross-over design demonstrated that short-term TRF was sufficient to modulate rhythmic metabolism of lipids, amino acids and improve nocturnal glucose levels and insulin profiles in skeletal muscle during daytime20,21. This study indicates that TRF is potentially impactful in managing pathologies related to metabolism and obesity while providing a natural and affordable form of alternate therapy. TRF has proved to be beneficial in various animal models of obesity shown in mouse liver, Drosophila heart, and muscle18,19,22. However, there is little information regarding the mechanistic impacts of TRF on skeletal muscle in different obesity models (Livelo, 2023).

This study investigates the mechanistic basis for TRF improvement in skeletal muscle by assessing transcriptomic data of WT, HFD, and Sk2 models under TRF. It was demonstrate dthat the expression levels of genes related to glycine production (Sardh and CG5955) and utilization (Gnmt) are upregulated under TRF in WT, HFD, and Sk2 models. Furthermore, the expression level of a key enzyme involved in triglyceride synthesis (Dgat2) was downregulated in all TRF conditions. Interestingly, TRF induces upregulation in genes and increases in metabolites related to the purine cycle in HFD model. On the other hand, upregulation of genes and increases in metabolites relating to glycolysis, glycogen metabolism, tricarboxylic acid cycle (TCA), and electron transport chain (ETC) connected by AMP kinase (AMPK) signaling are observed under TRF in Sk2 model. Muscle functional assessments, cytological and biochemical assays, and metabolomic analyses were further performed to validate the pathways and their biological significance in muscle function. Taken together, this study elucidates potential mechanisms behind TRF's protective properties against skeletal muscle dysfunction and metabolic impairment induced by obesity, which may pave the way for future TRF studies in muscle (Livelo, 2023).

The prevalence of obesity continues to be a worldwide growing issue associated with crippling healthcare and economic burdens. Skeletal muscle plays a primary role in energy and protein metabolism, glucose uptake and storage, and essential daily physiological tasks such as breathing and locomotion. Interestingly, studies have demonstrated that TRF, a natural non-pharmaceutical intervention, protects against obesity, aging, and circadian disruption in peripheral tissues such as the skeletal muscle. This study explores potential mechanisms responsible for TRF-mediated improvement of muscle function under conditions of obesity (HFD and Sk2). From transcriptomic analyses, common up/downregulated genes under TRF having orthologs to humans were found to be related to glycine production (Sardh, and CG5955), SAM regulation (Gnmt), and triglyceride synthesis (Dgat2). Flies under HFD-TRF predominantly showed upregulated genes related to the purine cycle. In contrast, Sk2-TRF flies showed upregulation of the gene encoding the catalytic subunit of AMPK (AMPKα) and downstream pathways involved in glycolysis, glycogen metabolism, TCA cycle, and ETC (Livelo, 2023).

Previous studies have observed that GNMT allows the universal methyl donor, SAM, to be converted to SAH by transferring a methyl group to glycine. Interestingly, higher SAM levels in older adults correlated with increased fat mass and truncal adiposity, suggesting a role in obesity. Furthermore, a related study observed that SAM was increased in overfed humans. TRF-mediated upregulation of Gnmt, together with Sardh and CG5955, glycine producers that can assist SAM catabolism. Measured glycine levels were significantly increased in Sk2-TRF while HFD-TRF and WT-TRF did not mirror the same level of increase. It is hypothesizes that glycine increases in HFD-TRF may not be measurable because of HFD-TRF-induced purine cycle activation, where glycine is consumed by phosphoribosylglycinamide transformylase (encoded by Gart). Both essential purine cycle genes Gart and Nmdmc were found to be significantly upregulated in HFD-TRF but not in Sk2-TRF, suggesting that glycine consumption through the purine pathway occurred only in HFD. Interestingly, inducing Gart KD in HFD-TRF led to glycine levels being significantly increased compared to HFD-ALF. While glycine levels were largely unchanged in the WT condition, it is uncertain if further aging is required to show the effects of TRF on glycine levels in WT flies which are generally healthier compared to HFD and Sk2. IFM-specific KD of Gnmt, Sardh and CG5955 using Act88F displayed a significant reduction in muscle performance. Furthermore, cytology of Gnmt, Sardh, and CG5955 KD flies displayed ectopic infiltration of lipids in the skeletal muscle. Interestingly, upon IFM-specific KD of Gnmt, Sardh, and CG5955, previously observed TRF-mediated muscle improvements were abolished signifying the three genes' importance in TRF-mediated muscle improvement (Livelo, 2023).

Dgat2 downregulation was observed in all TRF conditions. A recent study found that overexpression of Dgat2 in glycolytic type 2 muscle led to increased lipid accumulation and insulin resistance in mice. This aligns with the observation that muscle performance during aging was retained in IFM-specific Dgat2 KD flies, and significantly less lipid accumulation in muscle was seen. Moreover, IFM-specific overexpression of human Dgat2 (hDgat2) resulted in reduced muscle performance (and increased lipid accumulation in muscle, signifying a conserved role of Dgat2 and its translational potential in humans. Interestingly, subjecting Dgat2 KD flies to RD- and HFD-TRF showed additional improvements in muscle performance, accompanied by a further reduction of Dgat2 levels. Moreover, flies with hDgat2 overexpression demonstrated improved muscle performance under TRF compared to their ALF counterparts, and interestingly, with reduced expression of endogenous Dgat2. Taken together, these results suggest the possibility that TRF-mediated reduction of Dgat2 provides benefits to muscle performance in both Dgat2 KD and hDgat2 overexpression flies. This does not however, preclude any other pleiotropic effects of TRF which may have played contributing roles in the observed muscle improvement. It is known that Dgat2 knockdown increases de novo synthesis of fatty acids from glucose towards a TAG pool, which is simultaneously hydrolyzed, yielding fatty acids for mitochondrial oxidatio. This may suggest TRF's ability to mediate glucose metabolism in muscle stems from Dgat2 reduction (Livelo, 2023).

Evaluating upregulated genes predominantly in HFD-TRF, genes were observed with functions relating to the purine cycle and immune-related response. Although involved in immune function, the purine cycle also helps balance energy requirements potentially needed for muscular contraction through several ways, including replenishment of the TCA intermediate of fumarate and increasing flux for adenylate kinase. A key gene for both TCA anaplerosis and adenylate kinase flux, AdSL, was significantly upregulated along with most upstream enzymes. Interestingly, KD of adenylosuccinate lyase (AdSL) using Act88F driver showed impairment of flight index, while using DJ694 driver showed normal flight performance but lost the TRF benefits in muscle. In this study, comparing collected metabolites from HFD-TRF to HFD-ALF flies, an increase was found in ADP (a precursor to ATP), fumarate (an entry point for enriching the TCA), and malate (a subsequent product of fumarate upon entering TCA). Furthermore, HFD-ALF flies were found to have higher levels of inosine, hypoxanthine, and xanthine, which are markers of ATP catabolism and ATP exhaustion. This corroborates the observation of reduced ATP levels in HFD-ALF compared to WT-ALF and HFD-TRF displayed increased levels of ATP. This may suggest that ATP is a key component modulated by TRF and involved in mediating muscle improvement under HFD conditions. Although the results indicate purine involvement in HFD-TRF, the results are limited in its ability to show dynamics and changes in metabolite levels over time. To show dynamics, more sophisticated methodology requiring C13 labeling would be required. Interestingly, a recent human study comparing 11 human individuals with obesity under TRF (8-h eating window) and extending feeding (EXF; 15-h eating window) showed purine cycle genes upregulated as well in TRF. These findings provide support for the significance of the purine cycle in humans, however, the cause of obesity from this study was undisclosed and differences existed in TRF duration and eating window period. Another study has shown that ADSL plays a role in ATP generation and a new role of ADSL has been recently uncovered as an insulin secretagogue leading to insulin release and glucose uptake. Taken into conjunction with the downregulation of Dgat2, AdSL may also aid in TRF's ability to combat insulin resistance under HFD conditions (Livelo, 2023).

Furthermore, an entry point of the purine cycle, 10 formyl tetrahydrofolate (10-formylTHF) has been negatively correlated with insulin resistance and obesity. In this study Pug and Nmdmc are significantly upregulated, which helps produce 10-formylTHF. Folate is a crucial component for the final production of 10-formylTHF, which leads to the activation of purine cycle. Folate deficiency can lead to cardiovascular disease, muscle weakness, and difficulty in walking. In addition, folate supplementation demonstrated flight improvement and increased relative ATP levels. This may suggest that the maintenance of optimal folate levels are crucial for purine cycle activation and may subsequently help to improve muscle performance. However, increased ATP levels in Gnmt KD from folic acid supplementation did not mirror the same magnitude of flight improvement seen in Nmdmc KD. In addition, control flies with folic acid supplementation demonstrated flight improvement with only modest increases in ATP levels. This may suggest that not just overall levels of ATP are important but potentially other factors such as ATP flux may play a role in improving flight performance. Interestingly, literature suggests that GNMT may promote purine-related pathways through its aid in the production of 5, 10-methylene tetrahydrofolate with SARDH, which is the entry point of folate into mitochondria. Furthermore, GNMT also modulates purine expression through its translocation to the nucleus in folate-depleted conditions such as HFD (Livelo, 2023).

Differentially expressed genes (DEGs) found in Sk2-TRF compared to Sk2-ALF to be predominantly involved in TCA, glycogen metabolism, glycolysis, and mitochondrial ETC. These pathways are connected through AMPK signaling, further the catalytic domain of AMPK was also observed to be significantly upregulated in Sk2-TRF. It is well known that AMPK acts as an energy sensor able to sense high ratios of AMP/ATP and help regulate lipid metabolism. AMPK is also linked to catabolism and energy production for muscle contraction through ATP production in glycolysis, TCA, and ETC, mediating glucose uptake in tissues such as muscle and mediating fatty acid oxidation. The current results showed consistent upregulation of ETC-related genes, genes encoding TCA key enzymes (aconitase, isocitrate dehydrogenase, α-keto glutarate dehydrogenase) and genes associated with glycolytic and glycogen metabolism. Metabolites were found to increase under Sk2-TRF were L-carnitine, propionyl-carnitine, and acetylcarnitine, key components for assisting the production of acetyl-CoA needed for TCA were also found. Citric acid and malic acid, products of TCA cycle increased under TRF in Sk2. Further, increases in NADH suggest increased NAD+ consumption in TCA cycle while production of NAD+ may suggest increased activity of ETC. In addition, it was found melezitose and melibiose, a trisaccharide and disaccharide increased under Sk2-ALF, suggesting that these two are metabolized in TRF via glycogen metabolism which is also associated with AMPK signaling. The findings of these metabolites support the involvement of AMPK signaling under Sk2-TRF however, is limited in its ability to elucidate the dynamics of these metabolites and changes over time. A previous study has shown that GNMT assists AMPK activation indirectly as SAM's methylation of protein phosphatase 2A (PP2A) leads to inhibition of AMPK activation, therefore, GNMT reduction of SAM may lead to AMPK activation (Livelo, 2023).

In summary, TRF previously exhibited improvement in metabolic and skeletal muscle function in Drosophila, and this current study provides a potential mechanistic basis for the TRF-mediated benefits. It was identified that Gnmt, Sardh, CG5955, and Dgat2 were modulated across all conditions under TRF. IFM-specific KD of these genes impact ectopic lipid deposition and muscle performance. TRF-mediated benefits in IFM are abrogated upon suppression of Gnmt, Sardh, and CG5955, indicating that TRF-mediated upregulation of Gnmt, Sardh, and CG5955 may account for at least a part of the TRF beneficial effect observed in muscle. Furthermore, transcriptomic and metabolomics analyses demonstrated that distinct pathways were modulated under TRF in both HFD and Sk2 obese models. While HFD-TRF displayed activation of the purine cycle, Sk2-TRF displayed activation in AMPK signaling and downstream pathways. In addition, KD of genes associated with the purine cycle and AMPK signaling led to impaired muscle function. As both the purine cycle and AMPK signaling can modulate ATP levels, the results suggest that TRF modulates the purine cycles in HFD and AMPK signaling in Sk2, leading to changes in energy balance and subsequent improvement of muscle function. Overall, these results assess the functional importance of purine cycles and AMPK downstream signaling within the skeletal muscle in different obesity models under TRF potentially initiated by CRTC and FOXO (Livelo, 2023). Bozkurt, B., Terlemez, G., Sezgin, E.. (2023). Basidiomycota species in Drosophila gut are associated with host fat metabolism. Sci Rep, 13(1):13807 PubMed ID: 37612350

Abstract

The importance of bacterial microbiota on host metabolism and obesity risk is well documented. However, the role of fungal microbiota on host storage metabolite pools is largely unexplored. This study investigated the role of microbiota on D. melanogaster fat metabolism, and examine interrelatedness between fungal and bacterial microbiota, and major metabolic pools. Fungal and bacterial microbiota profiles, fat, glycogen, and trehalose metabolic pools are measured in a context of genetic variation represented by whole genome sequenced inbred Drosophila Genetic Reference Panel (DGRP) samples. Increasing Basidiomycota, Acetobacter persici, Acetobacter pomorum, and Lactobacillus brevis levels correlated with decreasing triglyceride levels. Host genes and biological pathways, identified via genome-wide scans, associated with Basidiomycota and triglyceride levels were different suggesting the effect of Basidiomycota on fat metabolism is independent of host biological pathways that control fungal microbiota or host fat metabolism. Although triglyceride, glycogen and trehalose levels were highly correlated, microorganisms' effect on triglyceride pool were independent of glycogen and trehalose levels. Multivariate analyses suggested positive interactions between Basidiomycota, A. persici, and L. brevis that collectively correlated negatively with fat and glycogen pools. In conclusion, fungal microbiota can be a major player in host fat metabolism. Interactions between fungal and bacterial microbiota may exert substantial control over host storage metabolite pools and influence obesity risk (Bozkurt, 2023). Santos-Cruz, L. F., Sigrist-Flores, S. C., Castaneda-Partida, L., Heres-Pulido, M. E., Duenas-García, I. E., Piedra-Ibarra, E., Ponciano-Gomez, A., Jimenez-Flores, R. and Campos-Aguilar, M. (2023). Effects of Fructose and Palmitic Acid on Gene Expression in Drosophila melanogaster Larvae: Implications for Neurodegenerative Diseases Int J Mol Sci 24(12). PubMed ID: 37373426

Abstract

One of the largest health problems worldwide is the development of chronic noncommunicable diseases due to the consumption of hypercaloric diets. Among the most common alterations are cardiovascular diseases, and a high correlation between overnutrition and neurodegenerative diseases has also been found. The urgency in the study of specific damage to tissues such as the brain and intestine led this study to use Drosophila melanogaster to examine the metabolic effects caused by the consumption of fructose and palmitic acid in specific tissues. Thus, third instar larvae (96 ± 4 h) of the wild Canton-S strain of D. melanogaster were used to perform transcriptomic profiling in brain and midgut tissues to test for the potential metabolic effects of a diet supplemented with fructose and palmitic acid. The data suggest that this diet can alter the biosynthesis of proteins at the mRNA level that participate in the synthesis of amino acids, as well as fundamental enzymes for the dopaminergic and GABAergic systems in the midgut and brain. These also demonstrated alterations in the tissues of flies that may help explain the development of various reported human diseases associated with the consumption of fructose and palmitic acid in humans. These studies will not only help to better understand the mechanisms by which the consumption of these alimentary products is related to the development of neuronal diseases but may also contribute to the prevention of these conditions (Santos-Cruz, 2023). Livelo, C., Guo, Y., Abou Daya, F., Rajasekaran, V., Varshney, S., Le, H. D., Barnes, S., Panda, S. and Melkani, G. C. (2023). Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models. Nat Commun 14(1): 949. PubMed ID: 36810287

Abstract
Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. This study demonstrates that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, these data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments (Livelo, 2023). Oliveras-Canellas, N., Castells-Nobau, A., de la Vega-Correa, L., Latorre-Luque, J., Motger-Alberti, A., Arnoriaga-Rodriguez, M., Garre-Olmo, J., Zapata-Tona, C., Coll-Martínez, C., Ramio-Torrenta, L., Moreno-Navarrete, J. M., Puig, J., Villarroya, F., Ramos, R., Casado-Anguera, V., Martin-Garcia, E., Maldonado, R., Mayneris-Perxachs, J. and Fernandez-Real, J. M. (2023). Adipose tissue coregulates cognitive function. Sci Adv 9(32): eadg4017. PubMed ID: 37566655

Abstract

Obesity is associated with cognitive decline. Recent observations in mice propose an adipose tissue (AT)-brain axis. This study identified 188 genes from RNA sequencing of AT in three cohorts that were associated with performance in different cognitive domains. These genes were mostly involved in synaptic function, phosphatidylinositol metabolism, the complement cascade, anti-inflammatory signaling, and vitamin metabolism. These findings were translated into the plasma metabolome. The circulating blood expression levels of most of these genes were also associated with several cognitive domains in a cohort of 816 participants. Targeted misexpression of candidate gene ortholog in the Drosophila fat body significantly altered flies memory and learning. Among them, down-regulation of the neurotransmitter release cycle-associated gene SLC18A2 improved cognitive abilities in Drosophila and in mice. Up-regulation of RIMS1 in Drosophila fat body enhanced cognitive abilities. Current results show previously unidentified connections between AT transcriptome and brain function in humans, providing unprecedented diagnostic/therapeutic targets in AT (Oliveras-Canellas, 2023). Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859

Abstract

Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, it was found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. These data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites (Mitchell, 2023). Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain PLoS Biol, 21(11):e3002359 PubMed ID: 37934726

Abstract

Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris (Alassaf, 2023). Mirzoyan, Z., Valenza, A., Zola, S., Bonfanti, C., Arnaboldi, L., Ferrari, N., Pollard, J., Lupi, V., Cassinelli, M., Frattaroli, M., Sahin, M., Pasini, M. E., Bellosta, P. (2023). A Drosophila model targets Eiger/TNFα to alleviate obesity-related insulin resistance and macrophage infiltration. Disease models & mechanisms, 16(11) PubMed ID: 37828911

Abstract

Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. Genetic manipulation was employed to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. Attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, both phenotypes were ameliorated. The data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. This model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease (Mirzoyan, 2023). Gendron, C. M., Chakraborty, T. S., Duran, C., Dono, T. and Pletcher, S. D. (2023). Ring neurons in the Drosophila central complex act as a rheostat for sensory modulation of aging. PLoS Biol 21(6): e3002149. PubMed ID: 37310911

Abstract

Sensory perception modulates aging, yet little is known about how. An understanding of the neuronal mechanisms through which animals orchestrate biological responses to relevant sensory inputs would provide insight into the control systems that may be important for modulating lifespan. This study provides new awareness into how the perception of dead conspecifics, or death perception, which elicits behavioral and physiological effects in many different species, affects lifespan in the fruit fly, Drosophila melanogaster. Previous work demonstrated that cohousing Drosophila with dead conspecifics decreases fat stores, reduces starvation resistance, and accelerates aging in a manner that requires both sight and the serotonin receptor 5-HT2A. This study demonstrated that a discrete, 5-HT2A-expressing neural population in the ellipsoid body (EB) of the Drosophila central complex, identified as R2/R4 neurons, acts as a rheostat and plays an important role in transducing sensory information about the presence of dead individuals to modulate lifespan. Expression of the insulin-responsive transcription factor foxo in R2/R4 neurons and insulin-like peptides dilp3 and dilp5, but not dilp2, are required, with the latter likely altered in median neurosecretory cells (MNCs) after R2/R4 neuronal activation. These data generate new insights into the neural underpinnings of how perceptive events may impact aging and physiology across taxa (Gendron, 2023). Bozkurt, B., Terlemez, G. and Sezgin, E. (2023). Basidiomycota species in Drosophila gut are associated with host fat metabolism. Sci Rep 13(1): 13807. PubMed ID: 37612350

Abstract

The importance of bacterial microbiota on host metabolism and obesity risk is well documented. However, the role of fungal microbiota on host storage metabolite pools is largely unexplored. This study aimed to investigate the role of microbiota on D. melanogaster fat metabolism, and examine interrelatedness between fungal and bacterial microbiota, and major metabolic pools. Fungal and bacterial microbiota profiles, fat, glycogen, and trehalose metabolic pools are measured in a context of genetic variation represented by whole genome sequenced inbred Drosophila Genetic Reference Panel (DGRP) samples. Increasing Basidiomycota, Acetobacter persici, Acetobacter pomorum, and Lactobacillus brevis levels correlated with decreasing triglyceride levels. Host genes and biological pathways, identified via genome-wide scans, associated with Basidiomycota and triglyceride levels were different suggesting the effect of Basidiomycota on fat metabolism is independent of host biological pathways that control fungal microbiota or host fat metabolism. Although triglyceride, glycogen and trehalose levels were highly correlated, microorganisms' effect on triglyceride pool were independent of glycogen and trehalose levels. Multivariate analyses suggested positive interactions between Basidiomycota, A. persici, and L. brevis that collectively correlated negatively with fat and glycogen pools. In conclusion, fungal microbiota can be a major player in host fat metabolism. Interactions between fungal and bacterial microbiota may exert substantial control over host storage metabolite pools and influence obesity risk (Bozkurt, 2023). Zhang, R. X., Li, S. S., Li, A. Q., Liu, Z. Y., Neely, G. G. and Wang, Q. P. (2022). dSec16 Acting in Insulin-like Peptide Producing Cells Controls Energy Homeostasis in Drosophila. Life (Basel) 13(1). PubMed ID: 36676030

Abstract

Many studies show that genetics play a major contribution to the onset of obesity. Human genome-wide association studies (GWASs) have identified hundreds of genes that are associated with obesity. However, the majority of them have not been functionally validated. SEC16B has been identified in multiple obesity GWASs but its physiological role in energy homeostasis remains unknown. This study used Drosophila to determine the physiological functions of dSec16 in energy metabolism. These results showed that global RNAi of dSec16 increased food intake and triglyceride (TAG) levels. Furthermore, this TAG increase was observed in flies with a specific RNAi of dSec16 in insulin-like peptide producing cells (IPCs) with an alteration of endocrine peptides. Together, this study demonstrates that dSec16 acting in IPCs controls energy balance and advances the molecular understanding of obesity (Zhang, 2022).

Jin, J. H., Wen, D. T., Chen, Y. L. and Hou, W. Q. (2023). Muscle FOXO-Specific Overexpression and Endurance Exercise Protect Skeletal Muscle and Heart from Defects Caused by a High-Fat Diet in Young Drosophila. Front Biosci (Landmark Ed) 28(1): 16. PubMed ID: 36722272
Summary:
Obesity appears to significantly reduce physical activity, but it remains unclear whether this is related to obesity-induced damage to skeletal muscle (SM) and heart muscle (HM). Endurance exercise (EE) reduces obesity-induced defects in SM and HM, but its molecular mechanism is poorly understood. The UAS/GAL4 system was used to construct the regulation of SM-specific FOXO gene expression in Drosophila, and the transgenic Drosophila was subjected to EE and high-fat diet (HFD) intervention. The structure and function of SM and HM were impaired by a HFD and muscle-FOXO-specific RNAi (MFSR), including reduced climbing speed and climbing endurance, reduced fractional shortening of the heart, damaged myofibrils, and reduced mitochondria in HM. Besides, a HFD and MFSR increased triglyceride level and malondialdehyde level, decreased the Sirt1 and FOXO protein level, and reduced carnitine palmityl transferase I, superoxide dismutase, and catalase activity level. They down-regulated FOXO and bmm expression level in SM and HM. On the contrary, both muscle FOXO-specific overexpression (MFSO) and EE prevented abnormal changes of SM and HM in function, structure, or physiology caused by HFD and MFSR. Besides, EE also prevented defects of SM and HM induced by MFSR. Current findings confirmed MFSO and EE protected SM and heart from defects caused by a HFD via enhancing FOXO-realated antioxidant pathways and lipid catabolism. FOXO played a vital role in regulating HFD-induced defects in SM and HM, but FOXO was not a key regulatory gene of EE against damages in SM and HM. The mechanism was related to activity of Sirt1/FOXO/SOD (superoxide dismutase), CAT (catalase) pathways and lipid catabolism in SM and HM.

Rivera, O., McHan, L., Konadu, B., Patel, S., Sint Jago, S. and Talbert, M. E. (2019). A high-fat diet impacts memory and gene expression of the head in mated female Drosophila melanogaster. J Comp Physiol B 189(2): 179-198. PubMed ID: 30810797

Abstract

Obesity predisposes humans to a range of life-threatening comorbidities, including type 2 diabetes and cardiovascular disease. Obesity also aggravates neural pathologies, such as Alzheimer's disease, but this class of comorbidity is less understood. When Drosophila melanogaster (flies) are exposed to high-fat diet (HFD) by supplementing a standard medium with coconut oil, they adopt an obese phenotype of decreased lifespan, increased triglyceride storage, and hindered climbing ability. The latter development has been previously regarded as a potential indicator of neurological decline in fly models of neurodegenerative disease. The objective of this study was to establish the obesity phenotype in Drosophila and identify a potential correlation, if any, between obesity and neurological decline through behavioral assays and gene expression microarray. Mated female w(1118) flies exposed to HFD were found to maintain an obese phenotype throughout adult life starting at 7 days, evidenced by increased triglyceride stores, diminished life span, and impeded climbing ability. While climbing ability worsened cumulatively between 7 and 14 days of exposure to HFD, there was no corresponding alteration in triglyceride content. Microarray analysis of the mated female w(1118) fly head revealed HFD-induced changes in expression of genes with functions in memory, metabolism, olfaction, mitosis, cell signaling, and motor function. Meanwhile, an Aversive Phototaxis Suppression assay in mated female flies indicated reduced ability to recall an entrained memory 6 h after training. Overall, these results support the suitability of mated female flies for examining connections between diet-induced obesity and nervous or neurobehavioral pathology, and provide many directions for further investigation (Rivera, 2019).

Guo, X., Yu, Z. and Yin, D. (2023) (2022). Sex-dependent obesogenic effect of tetracycline on Drosophila melanogaster deteriorated by dysrhythmia. J Environ Sci (China) 124: 472-480. PubMed ID: 36182155 Abstract
Antibiotics have been identified as obesogens contributing to the prevalence of obesity. Moreover, their environmental toxicity shows sex dependence, which might also explain the sex-dependent obesity observed. Yet, the direct evidence for such a connection and the underlying mechanisms remain to be explored. In this study, the effects of tetracycline, which is a representative antibiotic found in both environmental and food samples, on Drosophila melanogaster were studied with consideration of both sex and circadian rhythms (represented by the eclosion rhythm). Results showed that in morning-eclosed adults, tetracycline significantly stimulated the body weight of females (AM females) at 0.1, 1.0, 10.0 and 100.0 μg/L, while tetracycline only stimulated the body weight of males (AM males) at 1.0 μg/L. In the afternoon-eclosed adults, tetracycline significantly stimulated the body weight of females (PM females) at 0.1, 1.0 and 100.0 μg/L, while it showed more significant stimulation in males (PM males) at all concentrations. Notably, the stimulation levels were the greatest in PM males among all the adults. The results showed the clear sex dependence of the obesogenic effects, which was diminished by dysrhythmia. Further biochemical assays and clustering analysis suggested that the sex- and rhythm-dependent obesogenic effects resulted from the bias toward lipogenesis against lipolysis. Moreover, they were closely related to the preference for the energy storage forms of lactate and glucose and also to the presence of excessive insulin, with the involvement of glucolipid metabolism. Such relationships indicated potential bridges between the obesogenic effects of pollutants and other diseases, e.g., cancer and diabetes (Guo, 2023).

Liu, J., Zhang, Y., Wang, Q. Q., Zhou, Y., Liu, J. L. (2023). Drosophila melanogaster as a Model Organism for Obesity and Type-2 Diabetes Mellitus by Applying High-Sugar and High-Fat Diets. Fat body-specific reduction of CTPS alleviates HFD-induced obesity. Elife, 12 PubMed ID: 37695169 Abstract

Obesity induced by high-fat diet (HFD) is a multi-factorial disease including genetic, physiological, behavioral, and environmental components. Drosophila has emerged as an effective metabolic disease model. Cytidine 5'-triphosphate synthase (CTPS) is an important enzyme for the de novo synthesis of CTP, governing the cellular level of CTP and the rate of phospholipid synthesis. CTPS is known to form filamentous structures called cytoophidia, which are found in bacteria, archaea, and eukaryotes. This study demonstrates that CTPS is crucial in regulating body weight and starvation resistance in Drosophila by functioning in the fat body. HFD-induced obesity leads to increased transcription of CTPS and elongates cytoophidia in larval adipocytes. Depleting CTPS in the fat body prevented HFD-induced obesity, including body weight gain, adipocyte expansion, and lipid accumulation, by inhibiting the PI3K-Akt-SREBP axis. Furthermore, a dominant-negative form of CTPS also prevented adipocyte expansion and downregulated lipogenic genes. These findings not only establish a functional link between CTPS and lipid homeostasis but also highlight the potential role of CTPS manipulation in the treatment of HFD-induced obesity (Liu, 2023).

Baenas, N. and Wagner, A. E. (2022). Drosophila melanogaster as a Model Organism for Obesity and Type-2 Diabetes Mellitus by Applying High-Sugar and High-Fat Diets. Biomolecules 12(2). PubMed ID: 35204807 Abstract
Several studies have been published introducing Drosophila melanogaster as a research model to investigate the effects of high-calorie diets on metabolic dysfunctions. However, differences between the use of high-sugar diets (HSD) and high-fat diets (HFD) to affect fly physiology, as well as the influence on sex and age, have been seldom described. Thus, the aim of the present work was to investigate and compare the effects of HSD (30% sucrose) and HFD (15% coconut oil) on symptoms of metabolic dysfunction related to obesity and type-2 diabetes mellitus, including weight gain, survival, climbing ability, glucose and triglycerides accumulation and expression levels of Drosophila insulin-like peptides (dIlps). Female and male flies were subjected to HSD and HFD for 10, 20 and 30 days. The obtained results showed clear differences in the effects of both diets on survival, glucose and triglyceride accumulation and dIlps expression, being gender and age determinant. The present study also suggested that weight gain does not seem to be an appropriate parameter to define fly obesity, since other characteristics appear to be more meaningful in the development of obesity phenotypes. Taken together, the results demonstrate a key role for both diets, HSD and HFD, to induce an obese fly phenotype with associated diseases. However, further studies are needed to elucidate the underlying molecular mechanisms how both diets differently affect fly metabolism.

De Groef, S., Wilms, T., Balmand, S., Calevro, F. and Callaerts, P. (2021). Sexual Dimorphism in Metabolic Responses to Western Diet in Drosophila melanogaster. Biomolecules 12(1). PubMed ID: 35053181

Abstract
Obesity is a chronic disease affecting millions of people worldwide. The fruit fly (Drosophila melanogaster) is an interesting research model to study metabolic and transcriptomic responses to obesogenic diets. However, the sex-specific differences in these responses are still understudied and perhaps underestimated. This study exposed adult male and female Dahomey fruit flies to a standard diet supplemented with sugar, fat, or a combination of both. The exposure to a diet supplemented with 10% sugar and 10% fat efficiently induced an increase in the lipid content in flies, a hallmark for obesity. This increase in lipid content was more prominent in males, while females displayed significant changes in glycogen content. A strong effect of the diets on the ovarian size and number of mature oocytes was also present in females exposed to diets supplemented with fat and a combination of fat and sugar. In both males and females, fat body morphology changed and was associated with an increase in lipid content of fat cells in response to the diets. The expression of metabolism-related genes also displayed a strong sexually dimorphic response under normal condi-tions and in response to sugar and/or fat-supplemented diets. This study shows that the exposure of adult fruit flies to an obesogenic diet containing both sugar and fat allowed studying sexual dimorphism in metabolism and the expression of genes regulating metabolism.

Nunes, R. D., Drummond-Barbosa, D. (2023). Development, 150(20) PubMed ID: 37795747

Abstract

Obesity is linked to reduced fertility in various species, from Drosophila to humans. Considering that obesity is often induced by changes in diet or eating behavior, it remains unclear whether obesity, diet, or both reduce fertility. This study shows that Drosophila females on a high-sugar diet become rapidly obese and less fertile as a result of increased death of early germline cysts and vitellogenic egg chambers (or follicles). They also have high glycogen, glucose and trehalose levels and develop insulin resistance in their fat bodies (but not ovaries). By contrast, females with adipocyte-specific knockdown of the anti-obesity genes brummer or adipose are obese but have normal fertility. Remarkably, females on a high-sugar diet supplemented with a separate source of water have mostly normal fertility and glucose levels, despite persistent obesity, high glycogen and trehalose levels, and fat body insulin resistance. These findings demonstrate that a high-sugar diet affects specific processes in oogenesis independently of insulin resistance, that high glucose levels correlate with reduced fertility on a high-sugar diet, and that obesity alone does not impair fertility (Nunes, 2023).

Agrawal, N., Lawler, K., Davidson, C. M., Keogh, J. M., Legg, R., Barroso, I., Farooqi, I. S. and Brand, A. H. (2021). Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila. PLoS Biol 19(11): e3001255. PubMed ID: 34748544

Abstract

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. This study uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, this study identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. This study found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength in this approach in predicting novel human obesity genes and signalling pathways and their site of action (Agreawal, 2021).

Nayak, N. and Mishra, M. (2021). High fat diet induced abnormalities in metabolism, growth, behavior, and circadian clock in Drosophila melanogaster. Life Sci 281: 119758. PubMed ID: 34175317

Abstract
The current lifestyle trend has made people vulnerable to diabetes and related diseases. Years of scientific research have not been able to yield a cure to the disease completely. The current study aims to investigate a link between high-fat diet mediated diabesity and circadian rhythm in the Drosophila model and inferences that might help in establishing a cure to the dreaded disease. Several experimental methods including phenotypical, histological, biochemical, molecular, and behavioral assays were used in the study to detect obesity, diabetes, and changes in the circadian clock in the fly model. The larva and adults of Drosophila melanogaster exposed to high-fat diet (HFD) displayed excess deposition of fat as lipid droplets and micronuclei formation in the gut, fat body, and crop. Larva and adults of HFD showed behavioral defects. The higher amount of triglyceride, glucose, trehalose in the whole body of larva and adult fly confirmed obesity-induced hyperglycemia. The overexpression of insulin gene (Dilp2) and tribble (trbl) gene expression confirmed insulin resistance in HFD adults. Elevated ROS level, developmental delay, altered metal level, growth defects, locomotory rhythms, sleep fragmentation, and expression of circadian genes (per, tim, and clock) were observed in HFD larva and adults. Thus, HFD impairs the metabolism to produce obesity, insulin resistance, disruption of clock, and circadian clock related co-mordities in D. melanogaster. The circadian gene expression provides an innovative perspective to understand and find a new treatment for type-II diabetes and circadian anomalies.

Watanabe, L. P. and Riddle, N. C. (2021). GWAS reveal a role for the central nervous system in regulating weight and weight change in response to exercise. Sci Rep 11(1): 5144. PubMed ID: 33664357

Abstract

Body size and weight show considerable variation both within and between species. This variation is controlled in part by genetics, but also strongly influenced by environmental factors including diet and the level of activity experienced by the individual. Due to the increasing obesity epidemic in much of the world, there is considerable interest in the genetic factors that control body weight and how weight changes in response to exercise treatments. This study addressed this question in the Drosophila model system, utilizing 38 strains of the Drosophila Genetics Reference Panel. GWAS was used to identify the molecular pathways that control weight and weight changes in response to exercise. This study found that there is a complex set of molecular pathways controlling weight, with many genes linked to the central nervous system (CNS). The CNS also plays a role in the weight change with exercise, in particular, signaling from the CNS. Additional analyses revealed that weight in Drosophila is driven by two factors, animal size, and body composition, as the amount of fat mass versus lean mass impacts the density. Thus, while the CNS appears to be important for weight and exercise-induced weight change, signaling pathways are particularly important for determining how exercise impacts weight (Watanabe, 2021).

Vaziri, A., Khabiri, M., Genaw, B. T., May, C. E., Freddolino, P. L. and Dus, M. (2020). Persistent epigenetic reprogramming of sweet taste by diet. Sci Adv 6(46). PubMed ID: 33177090

Abstract

Diets rich in sugar, salt, and fat alter taste perception and food preference, contributing to obesity and metabolic disorders, but the molecular mechanisms through which this occurs are unknown. This study shows that in response to a high sugar diet, the epigenetic regulator Polycomb Repressive Complex 2.1 (PRC2.1) persistently reprograms the sensory neurons of Drosophila melanogaster flies to reduce sweet sensation and promote obesity. In animals fed high sugar, the binding of PRC2.1 to the chromatin of the sweet gustatory neurons is redistributed to repress a developmental transcriptional network that modulates the responsiveness of these cells to sweet stimuli, reducing sweet sensation. Half of these transcriptional changes persist despite returning the animals to a control diet, causing a permanent decrease in sweet taste. These results uncover a new epigenetic mechanism that, in response to the dietary environment, regulates neural plasticity and feeding behavior to promote obesity (Vaziri, 2020).

van Dam, E., van Leeuwen, L. A. G., Dos Santos, E., James, J., Best, L., Lennicke, C., Vincent, A. J., Marinos, G., Foley, A., Buricova, M., Mokochinski, J. B., Kramer, H. B., Lieb, W., Laudes, M., Franke, A., Kaleta, C. and Cocheme, H. M. (2020). Sugar-Induced Obesity and Insulin Resistance Are Uncoupled from Shortened Survival in Drosophila. Cell Metab. PubMed ID: 32197072

Abstract

High-sugar diets cause thirst, obesity, and metabolic dysregulation, leading to diseases including type 2 diabetes and shortened lifespan. However, the impact of obesity and water imbalance on health and survival is complex and difficult to disentangle. This study shows that high sugar induces dehydration in adult Drosophila, and water supplementation fully rescues their lifespan. Conversely, the metabolic defects are water-independent, showing uncoupling between sugar-induced obesity and insulin resistance with reduced survival in vivo. High-sugar diets promote accumulation of uric acid, an end-product of purine catabolism, and the formation of renal stones, a process aggravated by dehydration and physiological acidification. Importantly, regulating uric acid production impacts on lifespan in a water-dependent manner. Furthermore, metabolomics analysis in a human cohort reveals that dietary sugar intake strongly predicts circulating purine levels. This model explains the pathophysiology of high-sugar diets independently of obesity and insulin resistance and highlights purine metabolism as a pro-longevity target (van Dam, 2020).

Kezos, J. N., Phillips, M. A., Thomas, M. D., Ewunkem, A. J., Rutledge, G. A., Barter, T. T., Santos, M. A., Wong, B. D., Arnold, K. R., Humphrey, L. A., Yan, A., Nouzille, C., Sanchez, I., Cabral, L. G., Bradley, T. J., Mueller, L. D., Graves, J. L., Jr. and Rose, M. R. (2019). Genomics of early cardiac dysfunction and mortality in obese Drosophila melanogaster. Physiol Biochem Zool 92(6): 591-611. PubMed ID: 31603376

Abstract

In experimental evolution, functional demands are imposed on laboratory populations of model organisms using selection. After enough generations of such selection, the resulting populations constitute excellent material for physiological research. An intense selection regime for increased starvation resistance was imposed on 10 large outbred Drosophila populations. The selection responses were observed of starvation and desiccation resistance, metabolic reserves, and heart robustness via electrical pacing. Furthermore, the pooled genomes of these populations were sequenced. As expected, significant increases in starvation resistance and lipid content were found in 10 intensely selected SCO populations. The selection regime also improved desiccation resistance, water content, and glycogen content among these populations. Additionally, the average rate of cardiac arrests in 10 obese SCO populations was double the rate of the 10 ancestral CO populations. Age-specific mortality rates were increased at early adult ages by selection. Genomic analysis revealed a large number of single nucleotide polymorphisms across the genome that changed in frequency as a result of selection. These genomic results were similar to those obtained in the laboratory from less direct selection procedures. The combination of extensive genomic and phenotypic differentiation between these 10 populations and their ancestors makes them a powerful system for the analysis of the physiological underpinnings of starvation resistance (Kezos, 2019).

Stobdan, T., Sahoo, D., Azad, P., Hartley, I., Heinrichsen, E., Zhou, D. and Haddad, G. G. (2019). High fat diet induces sex-specific differential gene expression in Drosophila melanogaster. PLoS One 14(3): e0213474. PubMed ID: 30861021

Abstract

Currently about 2 billion adults globally are estimated to be overweight and ~13% of them are obese. High fat diet (HFD) is one of the major contributing factor to obesity, heart disease, diabetes and cancer. Recent findings on the role of HFD in inducing abnormalities in neurocognition and susceptibility to Alzheimer's disease are highly intriguing. Since fundamental molecular pathways are often conserved across species, studies involving Drosophila melanogaster as a model organism can provide insight into the molecular mechanisms involving human disease. In order to study some of such mechanisms in the central nervous system as well in the rest of the body, this study investigated the effect of HFD on the transcriptome in the heads and bodies of male and female flies kept on either HFD or regular diet (RD). Using comprehensive genomic analyses which include high-throughput transcriptome sequencing, pathway enrichment and gene network analyses, this study found that HFD induces a number of responses that are sexually dimorphic in nature. There was a robust transcriptional response consisting of a downregulation of stress-related genes in the heads and glycoside hydrolase activity genes in the bodies of males. In the females, the HFD led to an increased transcriptional change in lipid metabolism. A strong correlation also existed between the takeout gene and hyperphagic behavior in both males and females. It is concluded that a) HFD induces a differential transcriptional response between males and females, in heads and bodies and b) the non-dimorphic transcriptional response that was identified was associated with hyperphagia. Therefore, these data on the transcriptional responses in flies to HFD provides potentially relevant information to human conditions including obesity (Stobdan, 2019).

May, C. E., Vaziri, A., Lin, Y. Q., Grushko, O., Khabiri, M., Wang, Q. P., Holme, K. J., Pletcher, S. D., Freddolino, P. L., Neely, G. G. and Dus, M. (2019). High dietary sugar reshapes sweet taste to promote feeding behavior in Drosophila melanogaster. Cell Rep 27(6): 1675-1685.e1677. PubMed ID: 31067455

Abstract

Recent studies find that sugar tastes less intense to humans with obesity, but whether this sensory change is a cause or a consequence of obesity is unclear. To tackle this question, the effects of a high sugar diet on sweet taste sensation and feeding behavior were studied in Drosophila melanogaster. On this diet, fruit flies have lower taste responses to sweet stimuli, overconsume food, and develop obesity. Excess dietary sugar, but not obesity or dietary sweetness alone, caused taste deficits and overeating via the cell-autonomous action of the sugar sensor O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in the sweet-sensing neurons. Correcting taste deficits by manipulating the excitability of the sweet gustatory neurons or the levels of OGT protected animals from diet-induced obesity. This work demonstrates that the reshaping of sweet taste sensation by excess dietary sugar drives obesity and highlights the role of glucose metabolism in neural activity and behavior (May, 2019).

Musselman, L. P., Fink, J. L. and Baranski, T. J. (2019). Similar effects of high-fructose and high-glucose feeding in a Drosophila model of obesity and diabetes. PLoS One 14(5): e0217096. PubMed ID: 31091299

Abstract

As in mammals, high-sucrose diets lead to obesity and insulin resistance in the model organism Drosophila melanogaster. To explore the relative contributions of glucose and fructose, sucrose's component monosaccharides, their effects on larval physiology were compared. Both sugars exhibited similar effects to sucrose, leading to obesity and hyperglycemia. There were no striking differences resulting from larvae fed high glucose versus high fructose. Some small but statistically significant differences in weight and gene expression were observed that suggest Drosophila is a promising model system for understanding monosaccharide-specific effects on metabolic homeostasis (Musselman, 2019).

Kwon, S. Y., Massey, K., Watson, M. A., Hussain, T., Volpe, G., Buckley, C. D., Nicolaou, A. and Badenhorst, P. (2020). Oxidised metabolites of the omega-6 fatty acid linoleic acid activate dFOXO. Life Sci Alliance 3(2). PubMed ID: 31992650

Abstract

Obesity-induced inflammation, or meta-inflammation, plays key roles in metabolic syndrome and is a significant risk factor in diabetes and cardiovascular disease. To investigate causal links between obesity, meta-inflammation, and insulin signaling a Drosophila model was established to determine how elevated dietary fat and changes in the levels and balance of saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs) influence inflammation. Negligible effect of saturated fatty acid on inflammation was observed but marked enhancement or suppression by omega-6 and omega-3 PUFAs, respectively. Using combined lipidomic and genetic analysis, omega-6 polyunsaturated fatty acid was shown to enhance meta-inflammation by producing linoleic acid-derived lipid mediator 9-hydroxy-octadecadienoic acid (9-HODE). Transcriptome analysis reveals 9-HODE functions by regulating FOXO family transcription factors. 9-HODE activates JNK, triggering FOXO nuclear localisation and chromatin binding. FOXO TFs are important transducers of the insulin signaling pathway that are normally down-regulated by insulin. By activating FOXO, 9-HODE could antagonise insulin signaling providing a molecular conduit linking changes in dietary fatty acid balance, meta-inflammation, and insulin resistance (Kwon, 2020).

Cormier, R. P. J., Champigny, C. M., Simard, C. J., St-Coeur, P. D. and Pichaud, N. (2019). Dynamic mitochondrial responses to a high-fat diet in Drosophila melanogaster. Sci Rep 9(1): 4531. PubMed ID: 30872605

Abstract

Mitochondria can utilize different fuels according to physiological and nutritional conditions to promote cellular homeostasis. However, during nutrient overload metabolic inflexibility can occur, resulting in mitochondrial dysfunctions. High-fat diets (HFDs) are usually used to mimic this metabolic inflexibility in different animal models. However, how mitochondria respond to the duration of a HFD exposure is still under debate. This study investigated the dynamic of the mitochondrial and physiological functions in Drosophila melanogaster at several time points following an exposure to a HFD. The results showed that after two days on the HFD, mitochondrial respiration as well as ATP content of thorax muscles are increased, likely due to the utilization of carbohydrates. However, after four days on the HFD, impairment of mitochondrial respiration at the level of complex I, as well as decreased ATP content were observed. This was associated with an increased contribution of complex II and, most notably of the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH) to mitochondrial respiration. It is suggested that this increased mG3PDH capacity reflects the occurrence of metabolic inflexibility, leading to a loss of homeostasis and alteration of the cellular redox status, which results in senescence characterized by decreased climbing ability and premature death (Cormier, 2019).

A change in dietary resources is considered to be amongst the most important environmental stressors for an organism, impacting several aspects of phenotype. Both nutrient scarcity and abundance have most likely participated in shaping the evolution of cellular processes, and adjustments at the molecular and metabolic levels allowing to restore cellular homeostasis are crucial to survive these types of stress1. At the subcellular level, mitochondria integrate multiple metabolic pathways and produce the majority of adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS), sustaining life itself. The mitochondrial machinery can modulate the utilization of different fuels such as glucose and fat, which enables a metabolic flexibility that is controlled by nutrient availability and physiological conditions. However, during nutrient overload, a metabolic inflexibility characterised by competition between fuels such as carbohydrates and fatty acids can occur, resulting in a lessened ability to select proper mitochondrial substrates. As a consequence, the cell fails to adjust fuel choice in response to nutritional demand resulting in impaired homeostasis and mitochondrial dysfunctions. These mitochondrial dysfunctions are identified by a deficiency to oxidize substrates and/or to produce ATP which induce impaired fuel alternation and energy dysregulation (Cormier, 2019).

Nutrients from the diet are absorbed, converted to substrates in the cell's cytosol, and are then transported into the mitochondrial matrix. Inside the mitochondrial matrix, substrate-derived metabolites are oxidized, leading to the formation of reducing equivalents such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), which are used to supply the electron transport system (ETS) located in the inner mitochondrial membrane. Complex I, the main entry point of electrons into the ETS, oxidizes the mitochondrial NADH generated by the tricarboxylic acid cycle (TCA). Other complexes such as complex II and the electron transferring flavoprotein (ETF) oxidize the FADH2 produced by the TCA via succinate, and by fatty acid oxidation via fatty acylCoA, respectively. Additionally, the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH) allows the entry of electrons into the ETS through the reduction of glycerol-3-phosphate derived from either dihydroxyacetone phosphate (a metabolite from glycolysis) or from the glycerol obtained by triglyceride or diglyceride degradation. The electrons from these different complexes are then sequentially transferred from complex III, to complex IV, before reaching the final acceptor, molecular oxygen. This electron transport generates a proton-motive force used by complex V to drive the phosphorylation of ADP to ATP (Cormier, 2019).

These different electron feeders (among others) enable mitochondria to switch between oxidative substrates depending on their availability. A complex molecular network of effectors and transcriptional factors known as the nutrient sensing pathways is tightly linked to mitochondrial functions and allows the integration and the coordination of the organism's metabolism through hormonal signals. While these nutrient sensing pathways are under thorough investigation and are steadily being characterized in different physiological contexts, the modulation of mitochondrial responses according to nutrient availability is less understood. Nutrient overload has been linked to metabolic inflexibility in which mitochondrial dysfunctions play a central part, leading to a loss of homeostasis and the occurrence of several pathologies. For example, chronic exposure to a high fat diet (HFD) has been shown to result in the modification of mitochondrial quantity and oxidative functions associated with obesity and insulin resistance in different model organisms. Several studies have shown that in rodent skeletal muscle, mitochondrial functions are increased, decreased or unaffected depending on the exposure to different types of HFD. These divergent results suggest that HFD composition as well as the duration of the exposure differently affect mitochondrial functions that adjust accordingly to promote metabolic homeostasis. However, animal models such as rats and/or mice can be problematic to study precise mitochondrial responses, as the experimental time-frame to evaluate the short- and long-term effects of a HFD can be difficult to determine (Cormier, 2019).

Recently, the fruit fly, Drosophila melanogaster, has emerged as a suitable model to understand the fundamental mechanisms that control metabolism. Drosophila fed a HFD display increased triglyceride fat, insulin resistance, deregulation of insulin-TOR signalling, oxidative stress, cardiac dysfunctions as well as alterations in fatty acid, amino acid and carbohydrate metabolisms. However, no studies have investigated mitochondrial oxygen consumption changes in Drosophila fed on such diet at different experimental time points. In this study, this model was used to investigate the physiological and mitochondrial responses at different days following an exposure to a HFD. Specifically, Drosophila were fed either a standard diet (SD) or a HFD (SD supplemented with 20% (w/v) coconut oil as an increased source of saturated fat) and evaluated longevity, as well as climbing abilities, mitochondrial respiration, ATP content, glucose and glycogen concentrations, and enzymatic activities of pyruvate kinase (PK) and citrate synthase (CS) before (Day 0) and at several time points following the exposure to the HFD. It was hypothesized that after a short-term exposure to the HFD, adjustments of mitochondrial functions will occur to maintain cellular homeostasis. However, following long-term exposure, it was suspected that the emergence of mitochondrial dysfunctions will result in loss of homeostasis and physiological dysfunctions, ultimately leading to premature death (Cormier, 2019).

This study evaluated the mitochondrial and physiological responses of Drosophila melanogaster following an exposure to a HFD characterised by an increased content in saturated fatty acids compared to a SD. The results showed that at the mitochondrial level, increased respiration rates driven by an increased capacity of complex I were observed after two days on the HFD which is likely due to utilization of carbohydrates despite the abundance of fatty acids, leading to adjustments of mitochondrial metabolism to maintain metabolic homeostasis and higher ATP content in the thorax muscle. However, after four days on the HFD, an impairment of the mitochondrial respiration at the level of complex I was observed which was associated to a decrease P/L ratio and in ATP content, as well as an increased contribution of complex II and mG3PDH to mitochondrial respiration. These increased contributions reflect the occurrence of metabolic inflexibility, especially after 10 days, leading to a loss of homeostasis which results in physiological defects and senescence (Cormier, 2019).

Nutrient availability is known to influence metabolic homeostasis and transitions between different substrate utilization by the cell occurs during normal energy metabolism. However, it has been suggested that substrate competition at the mitochondrial level during nutrient overload can lead to metabolic deregulation and mitochondrial impairment which may be at the origin of several pathological conditions. As a matter of fact, HFDs with high caloric content have been widely used to induce metabolic inflexibility and to study metabolic diseases such as type 2 diabetes and obesity-related disorders in several mammals. Although it is generally accepted that a long-term increased fat intake might lead to mitochondrial defects, how the mitochondria dynamically respond to nutrient overload caused by an exposure to a HFD is still a question under debate. It has been shown that mitochondrial proteins, fat oxidation capacity, mitochondrial respiration, and expression of genes involved in OXPHOS in rodent skeletal muscle are differently modulated in response to different HFDs. These discrepancies could likely be attributed to different factors such as the animal model (rat vs mice), the diet composition in macronutrients or the fat source (animal vs vegetal, saturated vs unsaturated fatty acids), as well as the duration of the exposure (Cormier, 2019).

To evaluate the effects of a HFD in a time relevant manner, this study used Drosophila melanogaster, an animal model with a relatively short lifespan that has been shown to display metabolic inflexibility after exposure to HFD. When Drosophila are fed a HFD enriched with 20% coconut oil, it has been shown that triglyceride levels as well as total fatty acid abundance were increased in Drosophila after seven days which was associated with a drastically reduced lifespan likely due to impaired metabolism. Consistent with these studies, the results showed that the same HFD also induced an important decrease in lifespan. As most catabolic processes (carbohydrates, amino acids, and fatty acids) converge into the mitochondria for the production of ATP, mitochondrial oxygen consumption was investigated, as well as ATP content in the thorax muscle. The results showed that after two days on the HFD, mitochondrial oxygen consumption was increased using pyruvate, the end product of glycolysis, in combination with malate, an intermediate of the TCA cycle (CI-OXPHOS). The utilization of these substrates reflects an increased capacity of complex I to oxidize the NADH produced from pyruvate oxidation and the TCA cycle, which leads to increased ATP content. In Drosophila, carbohydrates are the main macronutrient sustaining mitochondrial metabolism in muscles. Therefore, the results suggest that Drosophila are increasing their capacity to oxidize carbohydrates despite the abundance of fatty acids during the first days of the HFD exposure. This is confirmed with increased PK enzymatic activity at Day 1, as well as with decreased glucose content from Day 1, and decreased glycogen content from Day 2. Consistent with this hypothesis, glucose levels of flies exposed to either 20% or 30% coconut oil HFD for seven and two days, respectively, are significantly decreased suggesting utilization and depletion of carbohydrates during the first days of the exposure1. In parallel, the mitochondrial coupling (P/L ratio) is not affected and remains constant for the first two days of the HFD exposure, suggesting that mitochondrial homeostasis is maintained and that the increase observed represents efficient adjustments to cope for the dietary change. However, the capacity of the ETS to utilize the other substrates (proline, succinate and G3P) is unaffected (Cormier, 2019).

After this initial adjustment period, CI-OXPHOS is significantly decreased at Day 4 and even more reduced at Day 10, which resulted in a concomitant decrease in the P/L ratio. Although this decrease could be attributed to decreased mitochondrial number, no changes were detected of CS enzymatic activity or of Complex IV capacity, which are considered good markers of mitochondrial density. A previous study showed that Drosophila on a 20% HFD displayed decreased activity of dehydrogenases as well as mitochondrial viability after seven days of exposure, which was also associated with decreased climbing abilities and reduced lifespan. A possible explanation for these results is that fatty acids are efficiently stored on the short-term, but are then transported to the muscle cells and transformed into acetylcoenzyme A (Ac-CoA) for ATP generation, leading to inhibition of the pyruvate dehydrogenase complex (PDH), converting pyruvate to Ac-CoA. This is in accordance with the PK decreased enzymatic activity observed from Day 2, whereas it was increased at Day 1. Moreover, increased capacity was detected of complex II to utilize succinate which is formed by the TCA from Ac-CoA at Day 10. This increase was however not observed at Day 4, when CI-OXPHOS was starting to decrease. Interestingly, mG3PDH capacity to oxidize G3P was also importantly increased at both Days 4 and 10. G3P is a substrate that can be formed by two different pathways. First, it can be produced by glycolysis via conversion of dihydroxyacetone phosphate by the cytosolic G3PDH. Inhibition of PDH by fatty acids should result in pyruvate accumulation, which was demonstrated after 7 days on a 20% coconut oil HFD26. In turn, this accumulation should promote increased metabolite concentrations upstream of glycolysis, including dihydroxyacetone phosphate, and thus, increased G3P should be available for mitochondrial respiration. G3P can also be formed via phosphorylation of the glycerol derived from triglyceride or diglyceride catabolism by the glycerol kinase. Therefore, exposure to the HFD should result in higher G3P production, explaining the important increased contribution of G3P to the mitochondrial respiration (Cormier, 2019).

Thus, a metabolic reprogramming seems to occur at the mitochondrial level after four days on the HFD, mainly driven by decreased pyruvate formation (as seen with PK activity) and oxidation leading to a decreased complex I capacity, and an increased capacity to oxidize G3P by the mG3PDH. Interestingly, it has recently been shown that after seven days on a 20% coconut oil HFD, increased reactive oxygen species (ROS) levels and lipid peroxidation were observed in Drosophila, suggesting the occurrence of oxidative stress that was associated with decreased lifespan and climbing abilities. The mG3PDH has been shown to be an important site of superoxide production in both mammals and Drosophila. Moreover, when respiring with G3P as a substrate, mitochondria from Drosophila are producing higher rate of superoxide than with other substrates. The increased mG3Pdh capacity observed in this study suggests that after four days, higher superoxide rates are produced, possibly leading to oxidative damages. In turn, these damages could explain the decreased lifespan observed, as higher reactive oxygen species production and oxidative damages are associated with aging. This observed premature senescence occurs after the decreased oxygen consumption was detected, indicating that mitochondrial dysfunctions could be the cause of the reduced lifespan. It is therefore suggested that the loss of homeostasis observed after four days on the HFD might be in part due to the mG3PDH capacity. Theoretically, an increased capacity of mG3PDH might help the cell to maintain homeostasis by providing an alternate route for both carbohydrates and fatty acids, diminishing metabolic inflexibility. However, this higher capacity might lead to increased reactive oxygen species production, altering the redox status of the cell and participating in the loss of homeostasis which leads to decreased ATP production, impairment of physiological functions, and premature death. This hypothesis remains to be confirmed, but characterization of mG3PDH regulation following a HFD exposure, as well as specific reactive oxygen species production by the mG3PDH using novel inhibitors of mG3PDH represents interesting research avenues for further studies (Cormier, 2019).

In summary, this study reveals important dynamic changes of mitochondrial functions during the course of a HFD exposure in Drosophila melanogaster. On the short-term, mitochondrial functions and ATP content of the thorax muscle are increased, which likely represents an efficient adjustment to a change in dietary resources. However, after a few days, metabolic inflexibility occurs, likely due to accumulation of fatty acids and depletion of carbohydrates, leading to mitochondrial dysfunctions at the level of complex I, decreased ATP content and loss of homeostasis, which in turn cause physiological defects and decreased lifespan. This provides a potential explanation for the contradicting results obtained in other animal models exposed to a HFD, as the duration of the exposure and the timing of the experiments used with these models might not be able to identify these dynamic changes. This study also showed that mG3PDH might be a key player in the mitochondrial response to a HFD. Drosophila are increasingly used as a relevant model to study the underlying mechanisms of several metabolic diseases such as type 2 diabetes and obesity-related disorders. Therefore, the mechanistic link between increased mG3PDH capacity and loss of homeostasis following the HFD exposure could provide significant new insights for the understanding of these diseases (Cormier, 2019).

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Villanueva, J. E., Livelo, C., Trujillo, A. S., Chandran, S., Woodworth, B., Andrade, L., Le, H. D., Manor, U., Panda, S. and Melkani, G. C. (2021). Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption. Nat Commun 10(1): 2700. PubMed ID: 31221967

Abstract

Summary:

Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. This study shows that Drosophila melanogaster subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes (Villanueva, 2019).

Hofbauer, H. F., Heier, C., Sen Saji, A. K. and Kuhnlein, R. P. (2020). Lipidome remodeling in aging normal and genetically obese Drosophila males. Insect Biochem Mol Biol: 103498. PubMed ID: 33221388

Abstract

Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. This study used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. This comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, the first evidence is presented for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life (Hofbauer, 2020).

Hofbauer, H. F., Heier, C., Sen Saji, A. K. and Kuhnlein, R. P. (2020). Lipidome remodeling in aging normal and genetically obese Drosophila males. Insect Biochem Mol Biol: 103498. PubMed ID: 33221388

Abstract

Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. This study used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. This comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, evidence is presented for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life (Hofbauer, 2020).

Liu, J., Wang, X., Ma, R., Li, T., Guo, G., Ning, B., Moran, T. H. and Smith, W. W. (2020). AMPK signaling mediates synphilin-1-induced hyperphagia and obesity in Drosophila. J Cell Sci. PubMed ID: 33443093

Abstract

Expression of synphilin-1 in neurons induces hyperphagia and obesity in a Drosophila model. However, the molecular pathways underlying synphilin-1-linked obesity remain unclear. This study used the Drosophila model, and genetic tools were used to study the synphilin-1-linked pathways in energy balance by combining molecular biology and pharmacological approaches. Expression of human synphilin-1 in flies increased AMPK phosphorylation at Thr172 compared with non-transgenic flies. Knockdown of AMPK reduced AMPK phosphorylation and food intake in non-transgenic flies, and further suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia, fat storage, and body weight gain in transgenic flies. Expression of constitutively activated AMPK significantly increased food intake and body weight gain in non-transgenic flies, but it did not alter food intake in the synphilin-1 transgenic flies. In contrast, expression of dominant-negative AMPK reduced food intake in both non-transgenic and synphilin-1 transgenic flies. Treatment with STO609 also suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia and body weight gain. These results demonstrated that the AMPKsignaling pathway plays a critical role in synphilin-1-induced hyperphagia and obesity. These findings provide new insights into the mechanisms of synphilin-1 controlled energy homeostasis (Liu, 2020).

Song, T., Qin, W., Lai, Z., Li, H., Li, D., Wang, B., Deng, W., Wang, T., Wang, L. and Huang, R. (2023). Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse. Cell Res. PubMed ID: 37055592

Abstract

Obesity imposes a global health threat and calls for safe and effective therapeutic options. This study found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine increased the production of a neuropeptide FMRFamide (FMRFa). Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. This study also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without xal abnormalities. Therefore, this study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases (Song, 2023).

Song, T., Qin, W., Lai, Z., Li, H., Li, D., Wang, B., Deng, W., Wang, T., Wang, L. and Huang, R. (2023). Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse. Cell Res. PubMed ID: 37055592
Summary:
Obesity imposes a global health threat and calls for safe and effective therapeutic options. This study found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine increased the production of a neuropeptide FMRFamide (FMRFa). Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. This study also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without xal abnormalities. Therefore, this study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases.

Livelo, C., Guo, Y., Abou Daya, F., Rajasekaran, V., Varshney, S., Le, H. D., Barnes, S., Panda, S. and Melkani, G. C. (2023). Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models. Nat Commun 14(1): 949. PubMed ID: 36810287
Summary:
Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. This study demonstrates that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, these data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments.

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Oculopharyngeal muscular dystrophy

Ribot, C., Soler, C., Chartier, A., Al Hayek, S., Nait-Saidi, R., Barbezier, N., Coux, O. and Simonelig, M. (2022). Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy. PLoS Genet 18(1): e1010015. PubMed ID:
35025870

Abstract
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. This study used a Drosophila model of OPMD, expression of alanine-expanded mammalian PABPN1 in Drosophila muscles, to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens a large number of UPS components were identified that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

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Nait-Saidi, R., Chartier, A., Abgueguen, E., Guedat, P. and Simonelig, M. (2023). The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response. Open Biol 13(4): 230008. PubMed ID: 37042114

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. In this study, using Drosophila OPMD models, it was shown that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. This study shows that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD (Nait-Saidi, 2023).

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PeripheralNeuropathy

Kim, Y. Y., Yoon, J. H., Um, J. H., Jeong, D. J., Shin, D. J., Hong, Y. B., Kim, J. K., Kim, D. H., Kim, C., Chung, C. G., Lee, S. B., Koh, H. and Yun, J. (2020). PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy. PLoS One 15(9): e0239126. PubMed ID:
32941465

Abstract

Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. This study examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. The class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. Paclitaxel was found to induce mitochondrial dysfunction in C4da neurons and PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN (Kim, 2020).

Bush, K. M., Barber, K. R., Martinez, J. A., Tang, S. J. and Wairkar, Y. P. (2021). Drosophila model of anti-retroviral therapy induced peripheral neuropathy and nociceptive hypersensitivity. Biol Open 10(1). PubMed ID: 33504470

Abstract

The success of antiretroviral therapy (ART) has improved the survival of HIV-infected patients significantly. However, significant numbers of patients on ART whose HIV disease is well controlled show peripheral sensory neuropathy (PSN), suggesting that ART may cause PSN. Although the nucleoside reverse transcriptase inhibitors (NRTIs), one of the vital components of ART, are thought to contribute to PSN, the mechanisms underlying the PSN induced by NRTIs are unclear. This study developed a Drosophila model of NRTI-induced PSN that recapitulates the salient features observed in patients undergoing ART: PSN and nociceptive hypersensitivity. Furthermore, the data demonstrate that pathways known to suppress PSN induced by chemotherapeutic drugs are ineffective in suppressing the PSN or nociception induced by NRTIs. Instead, it was found that increased dynamics of a peripheral sensory neuron may possibly underlie NRTI-induced PSN and nociception. This model provides a solid platform in which to investigate further mechanisms of ART-induced PSN and nociceptive hypersensitivity (Bush, 2021). Ribot, C., Soler, C., Chartier, A., Al Hayek, S., Nait-Saidi, R., Barbezier, N., Coux, O. and Simonelig, M. (2022). Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy. PLoS Genet 18(1): e1010015. PubMed ID: 35025870
Summary:
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. This study used a Drosophila model of OPMD, expression of alanine-expanded mammalian PABPN1 in Drosophila muscles, to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens a large number of UPS components were identified that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

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Peroxisome biogenesis disorders

Wangler, M. F., Chao, Y. H., Bayat, V., Giagtzoglou, N., Shinde, A. B., Putluri, N., Coarfa, C., Donti, T., Graham, B. H., Faust, J. E., McNew, J. A., Moser, A., Sardiello, M., Baes, M. and Bellen, H. J.
(2017). Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse. PLoS Genet 13(6): e1006825. PubMed ID: 28640802

Abstract

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis this study undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics this study uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. Bioinformatic transcriptional profiling was used to examine gene co-regulation between peroxisomal genes and other metabolic pathways. It was observed that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. These data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD (Wangler, 2017).

Peroxisomes are ubiquitous organelles present in all eukaryotic cells. Peroxisomes perform specific biochemical functions in the cell including fatty acid β-oxidation of very-long-chain fatty acids (VLCFA), α-oxidation of branched chain fatty acids, plasmalogen biosynthesis, and also participate in the metabolism of reactive oxygen species and glyoxylate. Peroxisomes are formed by the action of 14 peroxins encoded by PEX genes, the majority of which are involved in translocation of peroxisomal enzymes into the matrix, with others designating peroxisomal membrane. Human diseases due to autosomal recessive loss of function mutations in the PEX genes comprise a group of severe disorders known as peroxisome biogenesis disorders (PBD) with involvement of brain, bone, kidney and liver and death within the first year of life (Wangler, 2017).

The peroxisome's well documented role in β-oxidation of VLCFA and synthesis of ether lipids has led to considerable focus on lipid metabolism as the key pathogenic factor in disease pathogenesis in PBD. The accumulation of VLCFA has been proposed as the primary pathway influencing severity and as a therapeutic target. A more general alteration of peroxisomal lipids have been proposed as a developmental insult to the brain in PBD (Wangler, 2017).

However, while the increases in VLCFA and loss of plasmalogens in peroxisomal metabolism are likely to be a significant part of the pathogenesis of PBD, other metabolic pathways are also likely to play a role. Indeed, patients with pathogenic variants in PEX2, PEX10 and PEX16 that allow survival into childhood or adulthood have been reported with very mild abnormalities in VLCFA metabolism, and plasmalogen biosynthesis. These studies suggest that additional or even distinct peroxisomal functions are involved in PBD pathogenesis (Wangler, 2017).

Peroxisomal biology is highly conserved across eukaryotes which has allowed this same genetic machinery to be studied across several model organisms. In mice, studies of a spectrum of enzymatic and biogenesis defects in global and conditional knockouts has allowed insight into the role of peroxisomes in vertebrate tissues. Severe early phenotypes affecting brain, growth, and viability have been observed in Pex2, Pex5 and Pex13 knock-out mice. In addition a Pex1 knock-in for a common missense allele in human PBD produces mice with growth failure, cholestasis and retinopathy. Pex genes have been shown to have tissue specific effects. For example, an oligodendrocyte-specific loss of peroxisomal biogenesis produces much of the axonal loss and demyelination seen in PBD suggesting a cell autonomous role of peroxisomes in oligodendrocytes. Hepatocyte knockouts produce effects on mitochondrial morphology and ER stress. Several recent studies have also explored peroxisomal biogenesis in Drosophila demonstrating the evolutionary conservation. Studies of Drosophila pex mutants demonstrated a role for VLCFA in interfering with spermatogenesis leading to infertility (Chen, 2010). In addition, fly pex16 mutants have been shown to have locomotor defects, and shortened lifespan (Nakayama, 2011). Collectively, the study of peroxisomes in flies and mice have provided compelling data that the function of peroxisomes in longevity, locomotion and metabolism are conserved from flies to man (Wangler, 2017).

A key question that has not been addressed by the previous fly studies is whether the phenotype due to loss of peroxisomes is determined by any pathways in metabolism beyond peroxisomal lipids. Indeed, a comprehensive metabolic profile of peroxisomal biogenesis mutants is lacking. This study utilize genetics, transcriptional informatics and untargeted metabolomics to show that Drosophila pex mutants exhibit an unanticipated defect in sugar metabolism and are sensitive to reduced dietary sugar. A strong transcriptional co-regulation between peroxisomal genes was found in the fly and enzymes in glucose metabolism, and similar transcriptional signatures are observed in mice (Wangler, 2017).

Through genetic and pharmacologic studies, this study has identified monocarboxylate transporters (MCTs) and lactate as critical components for Lactate Dehydrogenase (LD) accumulation in flies and mammalian cells. LD accumulation in glia depends on the transfer of lactate from glia to neurons. Lactate is metabolized in neurons to produce AcCoA, a key input for energy production in the TCA cycle. A surplus of AcCoA, which may be caused by a defective TCA cycle or mitochondrial dysfunction, provides the impetus to synthesize lipids, whose transport to glia depends on Fatty acid transport protein and apolipoproteins. This leads to the accumulation of LD in glia (pigment glia in flies, astrocytes in mammals). Interestingly, loss of ApoD in fly glia can be compensated for by human APOE, which is expressed at high levels in human astrocytes. This suggests that a major role of APOE is to promote the transfer of lipids between neuron and glia for lipid storage in LD. Hence, this study provides evidence that weaves together mechanisms of cell-cell communication, metabolic coupling, and neuron-glia feedback in cell death and neurodegeneration. Altogether, these observations may have important implications regarding our understanding of pathogenic mechanisms in AD (Wangler, 2017).

Since the Astrocyte-neuron lactate shuttle (ANLS) hypothesis was proposed, compelling evidence indicates that glia play a critical role in lactate production and release, including Drosophila perineural glia (Volkenhoff, 2015), mammalian astrocytes, and oligodendrocytes. The current studies show that lactate transport from glia to neurons is critical for glial LD accumulation under normal and stress conditions. Glial lactate is transported and taken up into neurons through MCTs, providing further support for the evolutionary conservation of ANLS. Furthermore, genetic or pharmacologic inhibition of various enzymes in the metabolic pathways reduces glial LD accumulation, likely by limiting substrate available for lipid synthesis. These findings demonstrate that neuronal lipid synthesis requires lactate as a building block and that the lipids are synthesized in neurons. This study provides biochemical evidence that extracellular lactate can be incorporated into glial lipids at high levels, demonstrating that lactate is an important source for lipogenesis (Wangler, 2017).

The process of glial lactate transport to neurons is not solely due to elevation of ROS or mitochondrial dysfunction, as restricting lactate in neurons that overexpress JNK or SREBP also reduces glial LD accumulation. This indicates that the pathway of lactate and lipid transport operates under non-pathological conditions as well. Moreover, this study shows that FATPs are necessary for lipid transport in neurons and LD accumulation in glial cells and that this mechanism is conserved in vertebrates. FATPs have been extensively characterized for their biochemical properties in lipid processing and uptake in vitro, but until now their role in the mammalian nervous system has not been explored. The current data show that a set of proteins expressed in neuron and glia function in a coordinated manner to provide and store lipids in the form of LD in glia when ROS are elevated (Wangler, 2017).

An unanticipated discovery is the role of ApoD and ApoE in lipid transport and accumulation. ApoD is an atypical apolipoprotein that does not share significant sequence homology to other apolipoprotein family members, and it is thought to transport lipids in a manner similar to that of proteins in the lipocalin family. In flies, the ApoD homologs Glaz and Nlaz are secreted proteins. The loss of either Glaz or Nlaz in flies, or of their homolog ApoD in mice, leads to increased sensitivity to ROS. Both ApoD and ApoE are highly expressed in the mammalian nervous system and appear to have a potentially compensatory relationship. ApoD is upregulated in the circulating lipoproteins of Apoe null mice and expression of both proteins is altered in AD and after brain injuries. LD formations are lost in Rh-ND42 IR flies when there is heterozygous loss of Glaz, but LD formation is restored when APOE2 or APOE3 is expressed under the Glaz promoter in this context. These findings show that the two variants and Glaz likely play similar roles in lipid transport and respond similarly to oxidative stress (Wangler, 2017).

Overexpressing Glaz, APOE2, or APOE3 in glia results in substantial glial LD accumulation, whereas overexpression of the APOE4 variant does not. This is consistent with previous studies showing that Apoe-deficient mice have fewer cortical fatty acids. Given that the APOE4 allele cannot restore lipid transport and glial LD accumulation, the data strongly suggest that APOE4 is a partial loss-of-function allele in these phenotypic assays. Several studies have shown that elevated levels of ApoD in flies or mice are neuroprotective. Interestingly, Apoe-/- mice have been linked to an altered oxidative stress response and shown to exhibit increased lipid peroxidation when aged, but LDs have not been implicated (Wangler, 2017).

In the presence of low or moderate elevations of ROS, LDs store peroxidated lipid, providing a protective mechanism against low levels of lipid peroxidation. Indeed, the data support protective effects of glial LD accumulation in response to low ROS. Flies that express APOE3 or APOE4 under the control of the Glaz regulatory elements and are fed rotenone exhibit obvious differences: APOE4 flies are unable to accumulate glial LD and exhibit signs of neurodegeneration, whereas APOE3 flies accumulate glial LD and are comparable to wild-type. Furthermore, when aged, the APOE4 flies exhibit significantly more neuronal death than APOE3 flies. These data show that the inability to accumulate lipids into LD in the presence of elevated ROS promotes neurodegeneration (Wangler, 2017).

The context of glial LD accumulation is critical in neurodegeneration. Indeed, LD accumulation itself is not detrimental, as overexpression of SREBP in the absence of ROS does not lead to neurodegeneration. Conversely, in the presence of high ROS, as observed in some mitochondrial mutants, the protective mechanisms of glial LD accumulation are overridden, damaging the glia and negatively affecting neuronal survival. In this context, LDs disappear as neurodegeneration progresses but triglyceride levels continue to rise (Liu, 2015). The disappearance of LDs suggests that the phospholipid monolayer of the organelle is likely no longer intact due to peroxidation, and that peroxidated lipids are no longer contained in LDs, affecting cell health (Wangler, 2017).

The observation that loss of Glaz or Apoe decreases LD accumulation in flies and vertebrate cells and that these animals and cells have a compromised ability to cope with elevated levels of ROS suggests that LD formation provides neuroprotection. These findings are also consistent with the observations that the three human APOE alleles (E2, E3, and E4) have very different abilities to induce LD accumulation. Interestingly, the APOE2 allele, which is protective against AD, is the most efficient in lipid transport and in promoting LD accumulation in these studies. In contrast, the APOE4 allele, which is semi-dominantly linked to the development of AD, is highly inefficient in lipid transport and LD accumulation. It is proposed that protection from damage resulting from age-dependent progressive mitochondrial dysfunction and increased oxidative stress in neurons relies in part on the protection conferred by proper lipid transfer to glia, which sequestrates peroxidated lipids into LDs (Wangler, 2017).

How does the astrocyte/neuron lactate shuttle support the capacity of glial cells to protect neurons against ROS? It is proposed that astrocytes provide the reduced three-carbon metabolite lactate to neurons as a fatty acid precursor, rather than providing fatty acids, ensuring that low-level neuronal fatty acid synthesis continually produces new, undamaged fatty acids. De novo fatty acid and lipid synthesis leads to lipid turnover, with neurons maintaining a constant lipid level by exporting the excess through an ApoE-dependent pathway that steadily removes normal and damaged lipids. Astrocytes take up these lipids and oxidize them for fuel, which provides the reducing equivalents to ensure that the astrocytes export lactate, not pyruvate, to neurons. This arrangement becomes functionally critical for neuroprotection upon exposure to ROS, due to the generation of high levels of peroxidated lipids. Neuronal induction of JNK elevates lipid production, increasing lipid turnover and locally alleviating the detrimental effects of ROS by exporting peroxidated lipids to astrocytes, where they accumulate (relatively safely) in LDs. As such, LDs are a lagging indicator of neuroprotection that has already occurred. After a transient ROS challenge, return to normal metabolism will allow glia to steadily deplete their LDs. During ROS challenges, failure of the glia to provide neurons with lactate (via the ANLS pathway), failure of neuronal fatty acid synthesis, or failure of ApoE-dependent neuronal lipid export will block this lactate/lipid cycle and prevent neuroprotection by allowing neuronal accumulation of ROS products (Wangler, 2017).

Although this work focuses on the role of APOE-dependent lipid transfer between neurons and glia, the APOE alleles are associated with a susceptibility to develop other disease conditions unrelated to the nervous system. Indeed, the APOE alleles contribute most significantly to blood cholesterol variability in humans. For example, although APOE2 carriers are protected from AD, approximately 10% of individuals with two copies of APOE2 will develop type III hyperlipoproteinemia, leading to xanthomas in subcutaneous tissues. Interestingly, the majority of APOE2 carriers have normal to low levels of circulating cholesterol, suggesting that the enhanced transport of lipids by APOE2 results in tissue-specific phenotypes. Thus, it is possible that APOE2's increased ability to transport lipids may result in excessive lipid accumulation, as found in type III hyperlipoproteinemia. Meanwhile, enhanced reverse cholesterol transport due to APOE2 likely promotes cholesterol clearance in the circulatory system, contributing to hypocholesterolemia. On the other hand, APOE4 carriers tend to have higher levels of circulating cholesterols, coronary artery disease, and atherosclerosis. The findings that APOE4 is unable to transport lipids in the CNS may be relevant for the pathogenesis of APOE in coronary health. The inability of APOE4 to efficiently transport lipids may lead to elevated blood lipid content, atherosclerosis, and increased lipid peroxidation, contributing to coronary artery disease risk. In sum, these findings provide key insights into the mechanism of neuron-glia metabolic cooperation and point to the broader implications of APOE allelic functional differences in systemic health and disease (Wangler, 2017).

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Perry Syndrome

Hosaka, Y., Inoshita, T., Shiba-Fukushima, K., Cui, C., Arano, T., Imai, Y. and Hattori, N.
(2017). Reduced TDP-43 expression improves neuronal activities in a Drosophila model of Perry syndrome. EBioMedicine [Epub ahead of print]. PubMed ID: 28625517

Abstract

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. This study reports that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43, TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities (Hosaka, 2017)

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Polycystic kidney disease

Millet-Boureima, C., Chingle, R., Lubell, W. D. and Gamberi, C.
(2019). Cyst reduction in a polycystic kidney disease Drosophila model using Smac mimics. Biomedicines 7(4). PubMed ID: 31635379

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited malady affecting 12.5 million people worldwide. Therapeutic options to treat PKD are limited, due in part to lack of precise knowledge of underlying pathological mechanisms. Mimics of the second mitochondria-derived activator of caspases (Smac), a mitochondrial protein that is released together with cytochrome c from the mitochondria during apoptosis, have exhibited activity as antineoplastic agents and reported recently to ameliorate cysts in a murine ADPKD model, possibly by differentially targeting cystic cells and sparing the surrounding tissue. A first-in-kind Drosophila PKD model has now been employed to probe further the activity of novel Smac mimics. Substantial reduction of cystic defects was observed in the Malpighian (renal) tubules of treated flies, underscoring mechanistic conservation of the cystic pathways and potential for efficient testing of drug prototypes in this PKD model. Moreover, the observed differential rescue of the anterior and posterior tubules overall, and within their physiologically diverse intermediate and terminal regions implied a nuanced response in distinct tubular regions contingent upon the structure of the Smac mimic. Knowledge gained from studying Smac mimics reveals the capacity for the Drosophila model to precisely probe PKD pharmacology highlighting the value for such critical evaluation of factors implicated in renal function and pathology (Millet-Boureima, 2019).

Millet-Boureima, C., Rozencwaig, R., Polyak, F. and Gamberi, C. (2020). Cyst Reduction by Melatonin in a Novel Drosophila Model of Polycystic Kidney Disease. Molecules 25(22). PubMed ID: 33238462

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) causes progressive cystic degeneration of the renal tubules, the nephrons, eventually severely compromising kidney function. ADPKD is incurable, with half of the patients eventually needing renal replacement. Treatments for ADPKD patients are limited and new effective therapeutics are needed. Melatonin, a central metabolic regulator conserved across all life kingdoms, exhibits oncostatic and oncoprotective activity and no detected toxicity. This study used the Bicaudal C (BicC) Drosophila model of polycystic kidney disease to test the cyst-reducing potential of melatonin. Significant cyst reduction was found in the renal (Malpighian) tubules upon melatonin administration and suggest mechanistic sophistication. Similar to vertebrate PKD, the BicC fly PKD model responds to the antiproliferative drugs rapamycin and mimics of the second mitochondria-derived activator of caspases (Smac). Melatonin appears to be a new cyst-reducing molecule with attractive properties as a potential candidate for PKD treatment (Millet-Boureima, 2020).

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Polyglutamine (polyQ) disorders

Yadav, S. and Tapadia, M.G.
(2016). Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster. Dev Biol 409(1): 166-80. PubMed ID: 26517966

Abstract

Polyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. This study expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis. Progressive degeneration, which is the hallmark of neurodegeneration was also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of multi-drug resistance genes following polyQ expression was up regulated. The study gives an insight into the effects of polyQ aggregates in non-neuronal tissues (Yadav, 2016).

Minakawa, E. N., Popiel, H. A., Tada, M., Takahashi, T., Yamane, H., Saitoh, Y., Takahashi, Y., Ozawa, D., Takeda, A., Takeuchi, T., Okamoto, Y., Yamamoto, K., Suzuki, M., Fujita, H., Ito, C., Yagihara, H., Saito, Y., Watase, K., Adachi, H., Katsuno, M., Mochizuki, H., Shiraki, K., Sobue, G., Toda, T., Wada, K., Onodera, O. and Nagai, Y. (2020). Arginine is a disease modifier for polyQ disease models that stabilizes polyQ protein conformation. Brain. PubMed ID: 32436573

Abstract

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal β-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. This study identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. Representative chemical chaperones were screened using an in vitro polyQ aggregation assay, and arginine was identified as a potent polyQ aggregation inhibitor. In vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic β-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.

Chen, Z. S., Wong, A. K. Y., Cheng, T. C., Koon, A. C. and Chan, H. Y. E. (2019). FipoQ/FBXO33, a Cullin-1 based ubiquitin ligase complex component modulates ubiquitination and solubility of polyglutamine disease protein. J Neurochem. PubMed ID: 30685895

Abstract

Polyglutamine (polyQ) diseases describe a group of progressive neurodegenerative disorders caused by the CAG triplet repeat expansion in the coding region of the disease genes. To date, nine such diseases, including spinocerebellar ataxia type 3 (SCA3), have been reported. The formation of SDS-insoluble protein aggregates in neurons causes cellular dysfunctions, such as impairment of the ubiquitin-proteasome system (UPS), and contributes to polyQ pathologies. Recently, the E3 ubiquitin ligases, which govern substrate specificity of the UPS, have been implicated in polyQ pathogenesis. The Cullin (Cul) proteins are major components of Cullin-RING ubiquitin ligases (CRLs) complexes that are evolutionarily conserved in the Drosophila genome. This study examined the effect of individual Culs on SCA3 pathogenesis, and found that the knockdown of Cul1 expression enhances SCA3-induced neurodegeneration and reduces the solubility of expanded SCA3-polyQ proteins. The F-box proteins are substrate receptors of Cul1-based CRL. A genetic modifier screen of the 19 Drosophila F-box genes was performed, and F-box involved in polyQ pathogenesis (FipoQ) was identified as a genetic modifier of SCA3 degeneration that modulates the ubiquitination and solubility of expanded SCA3-polyQ proteins. In the human SK-N-MC cell model, F-box only protein 33 (FBXO33) exerts similar functions as FipoQ in modulating the ubiquitination and solubility of expanded SCA3-polyQ proteins. Taken together, this study demonstrates that Cul1-based CRL and its associated F-box protein, FipoQ/FBXO33, modify SCA3 protein toxicity. These findings will lead to a better understanding of the disease mechanism of SCA3, and provide insights on developing treatments against SCA3 (Chen, 2019).

Chen, Z. S., Li, L., Peng, S., Chen, F. M., Zhang, Q., An, Y., Lin, X., Li, W., Koon, A. C., Chan, T. F., Lau, K. F., Ngo, J. C. K., Wong, W. T., Kwan, K. M. and Chan, H. Y. E. (2018). Planar cell polarity gene Fuz triggers apoptosis in neurodegenerative disease models. EMBO Rep. PubMed ID: 30026307

Abstract

Planar cell polarity (PCP) describes a cell-cell communication process through which individual cells coordinate and align within the plane of a tissue. This study shows that overexpression of Fuz, a PCP gene, triggers neuronal apoptosis via the Dishevelled/Rac1 GTPase/MEKK1/JNK/caspase signalling axis. Consistent with this finding, endogenous Fuz expression is upregulated in models of polyglutamine (polyQ) diseases and in fibroblasts from spinocerebellar ataxia type 3 (SCA3) patients. The disruption of this upregulation mitigates polyQ-induced neurodegeneration in Drosophila. The transcriptional regulator Yin Yang 1 (YY1) associates with the Fuz promoter. Overexpression of YY1 promotes the hypermethylation of Fuz promoter, causing transcriptional repression of Fuz. Remarkably, YY1 protein is recruited to ATXN3-Q84 aggregates, which reduces the level of functional, soluble YY1, resulting in Fuz transcriptional derepression and induction of neuronal apoptosis. Furthermore, Fuz transcript level is elevated in amyloid beta-peptide, Tau and alpha-synuclein models, implicating its potential involvement in other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Taken together, this study unveils a generic Fuz-mediated apoptotic cell death pathway in neurodegenerative disorders (Chen, 2018).

Hong, H., Koon, A. C., Chen, Z. S., Wei, Y., An, Y., Li, W., Lau, M. H. Y., Lau, K. F., Ngo, J. C. K., Wong, C. H., Au-Yeung, H. Y., Zimmerman, S. C. and Chan, H. Y. E. (2018). AQAMAN, a bisamidine-based inhibitor of toxic protein inclusions in neurons, ameliorates cytotoxicity in polyglutamine disease models. J Biol Chem. PubMed ID: 30593503

Abstract

Polyglutamine (polyQ) diseases are a group of dominantly inherited neurodegenerative disorders caused by the expansion of an unstable CAG repeat in the coding region of the affected genes. Hallmarks of polyQ diseases include the accumulation of misfolded protein aggregates, leading to neuronal degeneration and cell death. PolyQ diseases are currently incurable, highlighting the urgent need for approaches that inhibit the formation of or disaggregate cytotoxic polyQ protein inclusions. This study screened for bisamidine-based inhibitors that can inhibit neuronal polyQ protein inclusions. One inhibitor, AQAMAN, prevents polyQ protein aggregation and promotes deaggregation of self-assembled polyQ proteins in several models of polyQ diseases. Using immunocytochemistry, AQAMAN was found to significantly reduce polyQ protein aggregation and specifically suppresses polyQ protein-induced cell death. Using a recombinant and purified polyQ protein (Trx-Huntingtin-Q46), it was further demonstrated that AQAMAN interferes with polyQ self-assembly, preventing polyQ aggregation, and dissociates preformed polyQ aggregates in a cell-free system. Remarkably, AQAMAN feeding of Drosophila expressing expanded polyQ disease protein suppresses polyQ-induced neurodegeneration in vivo. In addition, using inhibitors and activators of the autophagy pathway, it was demonstrated that AQAMAN's cytoprotective effect against polyQ toxicity is autophagy-dependent. In summary, this study has identified AQAMAN as a potential therapeutic for combating polyQ protein toxicity in polyQ diseases. These findings further highlight the importance of the autophagy pathway in clearing harmful polyQ proteins (Hong, 2018).

Xie, J., Han, Y. and Wang, T. (2018). RACK1 modulates polyglutamine-induced neurodegeneration by promoting ERK degradation in Drosophila. PLoS Genet 17(5): e1009558. PubMed ID: 33983927

Abstract

Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. A genome-wide genetic screen was conducted to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington's disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases (Xie, 2021).

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Pontocerebellar Hypoplasia

Morton, D. J., Jalloh, B., Kim, L., Kremsky, I., Nair, R. J., Nguyen, K. B., Rounds, J. C., Sterrett, M. C., Brown, B., Le, T., Karkare, M. C., McGaughey, K. D., Sheng, S., Leung, S. W., Fasken, M. B., Moberg, K. H. and Corbett, A. H. (2020). A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons. PLoS Genet 16(7): e1008901. PubMed ID:
32645003

Abstract
The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, tissue-specific RNAi drivers were used. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, CRISPR/Cas9 gene editing technology was used to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.

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Primary Ciliary Dyskinesia

Lage, P. Z., Xi, Z., Lennon, J., Hunter, I., Chan, W. K., Carrancio, A. B., von Kriegsheim, A. and Jarman, A. P. (2021). The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly. Biol Open. PubMed ID:
34553759

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. This study reports the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly (Lage, 2021).

Abstract

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Renal Fibrosis

Zheng, W., Ocorr, K. and Tatar, M. (2020). Extra-cellular matrix induced by steroids and aging through a G-protein coupled receptor in a Drosophila model of renal fibrosis. Dis Model Mech. PubMed ID:
32461236

Abstract
Aldosterone is produced by the mammalian adrenal cortex to modulate blood pressure and fluid balance, however excessive, prolonged aldosterone promotes fibrosis and kidney failure. How aldosterone triggers disease may involve actions independent of its canonical mineralocorticoid receptor. This study presents a Drosophila model of renal pathology caused by excess extra-cellular matrix formation, stimulated by exogenous aldosterone and by insect ecdysone. Chronic administration of aldosterone or ecdysone induces expression and accumulation of collagen-like Pericardin at adult nephrocytes - podocyte-like cells that filter circulating hemolymph. Excess Pericardin deposition disrupts nephrocyte (glomerular) filtration and causes proteinuria in Drosophila, hallmarks of mammalian kidney failure. Steroid-induced Pericardin production arises from cardiomyocytes associated with nephrocytes, potentially reflecting an analogous role of mammalian myofibroblasts in fibrotic disease. Remarkably, the canonical ecdysteroid nuclear hormone receptor, Ecdysone Receptor EcR, is not required for aldosterone or ecdysone to stimulate Pericardin production or associated renal pathology. Instead, these hormones require a cardiomyocyte-associated G-protein coupled receptor, Dopamine-EcR (DopEcR), a membrane-associated receptor previously characterized in the fly brain as affecting behavior. DopEcR in the brain is known to affect behavior through interactions with the Drosophila epidermal growth factor receptor, dEGFR. This study finds the steroids ecdysone and aldosterone require dEGFR in cardiomyocytes to induce fibrosis of the cardiac-renal system. As well, endogenous ecdysone that becomes elevated with age is found to foster age-associated fibrosis, and to require both cardiomyocyte DopEcR and dEGFR. This Drosophila renal disease model reveals a novel signaling pathway through which steroids may modulate mammalian fibrosis through potential orthologs of DopEcR (Zheng, 2020).

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Rabinow Syndrome

Gignac, S. J., MacCharles, K. R., Fu, K., Bonaparte, K., Akarsu, G., Barrett, T. W., Verheyen, E. M. and Richman, J. M. (2023). Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila. Hum Mol Genet 25(4):651-9. PubMed ID:
26662796 Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome. Dis Model Mech 16(4). PubMed ID: 36916233

Abstract

The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. This study focused on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (β-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. Human wild-type DVL1 or DVL1 variants were expressed alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. These data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.

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Retinitis pigmentosa

Chow, C. Y., Kelsey, K. J., Wolfner, M. F. and Clark, A. G. (2015). Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila. Hum Mol Genet 25(4):651-9. PubMed ID:
26662796

Abstract
Individuals carrying the same pathogenic mutation can present with a broad range of disease outcomes. While some of this variation arises from environmental factors, it is increasingly recognized that the background genetic variation of each individual can have a profound effect on the expressivity of a pathogenic mutation. In order to understand this background effect on disease-causing mutations, studies need to be performed across a wide range of backgrounds. Recent advancements in model organism biology allow testing of mutations across genetically diverse backgrounds and identification of the genes that influence the expressivity of a mutation. This study used the Drosophila Genetic Reference Panel, a collection of approximately 200 wild-derived strains, to test the variability of the retinal phenotype of the Rh1G69D Drosophila model of retinitis pigmentosa (RP). The Rh1G69D retinal phenotype is quite a variable quantitative phenotype. To identify the genes driving this extensive phenotypic variation, a genome-wide association study was performed. One hundred and six candidate genes were identified, including 14 high-priority candidates. Functional testing by RNAi indicates that 10/13 top candidates tested influence the expressivity of Rh1G69D. The human orthologs of the candidate genes have not previously been implicated as RP modifiers and their functions are diverse, including roles in endoplasmic reticulum stress, apoptosis and retinal degeneration and development. This study demonstrates the utility of studying a pathogenic mutation across a wide range of genetic backgrounds. These candidate modifiers provide new avenues of inquiry that may reveal new RP disease mechanisms and therapies (Chow, 2015).


Brandwine, T., Ifrah, R., Bialistoky, T., Zaguri, R., Rhodes-Mordov, E., Mizrahi-Meissonnier, L., Sharon, D., Katanaev, V. L., Gerlitz, O. and Minke, B. (2021). Knockdown of Dehydrodolichyl Diphosphate Synthase in the Drosophila Retina Leads to a Unique Pattern of Retinal Degeneration. Front Mol Neurosci 14: 693967. PubMed ID: 34290587
Park, J. E., Lee, J., Ok, S., Byun, S., Chang, E. J., Yoon, S. E., Kim, Y. J. and Kang, M. J. (2023). Wg/Wnt1 and Erasp link ER stress to proapoptotic signaling in an autosomal dominant retinitis pigmentosa model. Exp Mol Med 55(7): 1544-1555. PubMed ID: 37464094

The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, an unbiased in vivo RNAi screen was performed with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1(P37H)) toxicity, alleviating retinal degeneration in the Drosophila ADRP model. In contrast, overexpression of Erasp resulted in enhanced caspase activity in Drosophila S2 cells treated with apoptotic inducers and the stabilization of the initiator caspase Dronc (Death regulator Nedd2-like caspase) by stimulating DIAP1 (Drosophila inhibitor of apoptosis protein 1) degradation. These findings helped identify a novel cell death signaling pathway involved in retinal degeneration in an autosomal dominant retinitis pigmentosa model (Park, 2023).
Brandwine, T., Ifrah, R., Bialistoky, T., Zaguri, R., Rhodes-Mordov, E., Mizrahi-Meissonnier, L., Sharon, D., Katanaev, V. L., Gerlitz, O. and Minke, B. (2021). Knockdown of Dehydrodolichyl Diphosphate Synthase in the Drosophila Retina Leads to a Unique Pattern of Retinal Degeneration. Front Mol Neurosci 14: 693967. PubMed ID: 34290587

Dehydrodolichyl diphosphate synthase (DHDDS) is a ubiquitously expressed enzyme that catalyzes cis-prenyl chain elongation to produce the poly-prenyl backbone of dolichol. Individuals who have biallelic missense mutations in the DHDDS gene are presented with non-syndromic retinitis pigmentosa with unknown underlying mechanism. This study used the Drosophila model to compromise DHDDS ortholog gene (CG10778) in order to look for cellular and molecular mechanisms that, when defective, might be responsible for this retinal disease. The Gal4/UAS system was used to suppress the expression of CG10778 via RNAi-mediated-knockdown in various tissues. Targeted knockdown of CG10778-mRNA in the early embryo using the actin promoter or in the developing wings using the nub promoter resulted in lethality, or wings loss, respectively. Targeted expression of CG10778-RNAi using the glass multiple reporter (GMR)-Gal4 driver (GMR-DHDDS-RNAi) in the larva eye disc and pupal retina resulted in a complex phenotype: (a) TEM retinal sections revealed a unique pattern of retinal-degeneration, where photoreceptors R2 and R5 exhibited a nearly normal structure of their signaling-compartment (rhabdomere), but only at the region of the nucleus, while all other photoreceptors showed retinal degeneration at all regions. (b) Western blot analysis revealed a drastic reduction in rhodopsin levels in GMR-DHDDS-RNAi-flies and TEM sections showed an abnormal accumulation of endoplasmic reticulum (ER). To conclude, compromising DHDDS in the developing retina, while allowing formation of the retina, resulted in a unique pattern of retinal degeneration, characterized by a dramatic reduction in rhodopsin protein level and an abnormal accumulation of ER membranes in the photoreceptors cells, thus indicating that DHDDS is essential for normal retinal formation (Minke, 2021).
Stankovic, D., Claudius, A. K., Schertel, T., Bresser, T. and Uhlirova, M. (2020). Drosophila model to study Retinitis pigmentosa pathology associated with mutations in the core splicing factor Prp8. Dis Model Mech. PubMed ID: 32424050

Retinitis pigmentosa (RP) represents genetically heterogeneous and clinically variable disease characterized by progressive degeneration of photoreceptors resulting in a gradual loss of vision. The autosomal dominant RP type 13 (RP13) has been linked to the malfunction of PRPF8, the essential component of the spliceosome. Over twenty different RP-associated PRPF8 mutations have been identified in human patients. However, the cellular and molecular consequences of their expression in vivo in specific tissue contexts remain largely unknown. This study establish the Drosophila melanogaster model for RP13 by introducing the nine distinct RP mutations into the fly Prp8 ortholog and expressing these mutant proteins in precise spatiotemporal patterns using the Gal4/UAS system. All nine RP-Prp8 mutations negatively impacted developmental timing, albeit to a different extent, when expressed in the endocrine cells producing the primary insect molting hormone. In the developing eye primordium, uncommitted epithelial precursors rather than differentiated photoreceptors appeared sensitive to Prp8 malfunction. Expression of the two most pathogenic variants, Prp8(S>F) and Prp8(H>R), induced apoptosis causing alterations to the adult eye morphology. The affected tissue mounted stress and cytoprotective responses, while genetic programs underlying neuronal function were attenuated. Importantly, the penetrance and expressivity increased under prp8 heterozygosity. In contrast, blocking apoptosis alleviated cell loss but not the redox imbalance. Remarkably, the pathogenicity of the RP-Prp8 mutations in the Drosophila correlates with the severity of clinical phenotypes in patients carrying the equivalent mutations highlighting the suitability of the Drosophila model for in-depth functional studies of the mechanisms underlying RP13 etiology.

Park, J. E., Tron, T. X. T., Park, N., Yeom, J., Kim, K. and Kang, M. J. (2020). The Function of Drosophila USP14 in Endoplasmic Reticulum Stress and Retinal Degeneration in a Model for Autosomal Dominant Retinitis Pigmentosa. Biology (Basel) 9(10). PubMed ID: 33053617

Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. This study analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, it was observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress (Park, 2020).
Westin, I. M., Jonsson, F., Osterman, L., Holmberg, M., Burstedt, M. and Golovleva, I. (2021). EYS mutations and implementation of minigene assay for variant classification in EYS-associated retinitis pigmentosa in northern Sweden. Sci Rep 11(1): 7696. PubMed ID: 33833316

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations. The ortholog of Drosophila eyes shut/spacemaker, EYS on chromosome 6q12 is a major genetic cause of recessive RP worldwide, with prevalence of 5 to 30%. In this study, by using targeted NGS, MLPA and Sanger sequencing, the EYS gene was identifed as one of the most common genetic cause of autosomal recessive RP in northern Sweden accounting for at least 16%. The most frequent pathogenic variant was c.8648_8655del that in some patients was identified in cis with c.1155T>A, indicating Finnish ancestry. Two novel EYS variants, c.2992_2992+6delinsTG and c.3877+1G>A caused exon skipping in human embryonic kidney cells, HEK293T and in retinal pigment epithelium cells, ARPE-19 demonstrating that in vitro minigene assay is a straightforward tool for the analysis of intronic variants. It is concluded, that whenever it is possible, functional testing is of great value for classification of intronic EYS variants and the following molecular testing of family members, their genetic counselling, and inclusion of RP patients to future treatment studies (Westin, 2021).
Hebbar, S., Lehmann, M., Behrens, S., Halsig, C., Leng, W., Yuan, M., Winkler, S. and Knust, E. (2021). Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila. Biol Open 10(1). PubMed ID: 33495354

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, mutations were induced in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented in this study will also contribute to further unravel molecular mechanisms underlying the human disease (Hebbar, 2021).

Retinitis pigmentosa is a clinically heterogeneous group of retinal dystrophies, which affects more than one million people worldwide. It often starts with night blindness in early childhood, continues with the loss of the peripheral visual field (tunnel vision), and progresses to complete blindness in later life due to gradual degeneration of photoreceptor cells (PRCs). RP is a genetically heterogeneous disease and can be inherited as autosomal dominant (adRP), autosomal recessive (arRP) or X-linked (xlRP) disease. So far >90 genes have been identified that are causally related to non-syndromic RP. Affected genes are functionally diverse. Some of them are expressed specifically in PRCs and encode, among others, transcription factors (e.g., CRX, an otx-like photoreceptor homeobox gene), components of the light-induced signalling cascade, including the visual pigment rhodopsin (Rho/RHO in Drosophila/human), or genes controlling vitamin A metabolism (e.g., RLBP-1, encoding Retinaldehyde-binding protein). Other genes are associated with a more general control of cellular homeostasis, for example genes involved in trafficking or cell polarity (e.g. CRB1). Interestingly, the second-largest group of genes causing adRP, comprising 7 of 25 genes known, encodes regulators of the splicing machinery. So far, mutations in five pre-mRNA processing factor (PRPF) genes, PRPF3, PRPF4, PRPF6, PRPF8 and PRPF31, have been linked to adRP, namely RP18, RP70, RP60, RP13 and RP11, respectively. Pim1-associated protein (PAP1) and small nuclear ribonuclearprotein-200 (SNRNP200), two genes also involved in splicing, have been suggested to be associated with RP9 and RP33, respectively. The five PRPF genes encode components regulating the assembly of the U4/U6.U5 tri-snRNP, a major module of the pre-mRNA spliceosome machinery. Several hypotheses have been put forward to explain why mutations in ubiquitously expressed components of the general splicing machinery show a dominant phenotype only in the retina. One hypothesis suggests that PRCs with only half the copy number of a gene encoding a general splicing component cannot cope with the elevated demand of RNA-/protein synthesis required to maintain the exceptionally high metabolic rate of PRCs in comparison to other tissues. Hence, halving their gene dose eventually results in apoptosis. Although this model is currently favoured, other mechanisms, such as impaired splicing of PRC-specific mRNAs or toxic effects caused by accumulation of mutant proteins have been discussed and may contribute to the disease phenotype. More recent data obtained from retinal organoids established from RP11 patients showed that removing one copy of PRPF31 affects the splicing machinery specifically in retinal and retinal pigment epithelial (RPE) cells, but not in patient-derived fibroblasts or iPS cells (Hebbar, 2021).

The observation that all adRP-associated genes involved in splicing are highly conserved from yeast to human allows use of model organisms to unravel the genetic and cell biological functions of these genes in order to obtain mechanistic insight into the origin of the diseases. In the case of RP11, the disease caused by mutations in PRPF31, three mouse models have been generated by knock-in and knock-out approaches. Unexpectedly, mice heterozygous mutant for a null allele or a point mutation that recapitulates a mutation in the corresponding human gene did not show any sign of retinal degeneration in 12- and 18-month-old mice, respectively. Further analyses revealed that the retinal pigment epithelium, rather than the PRCs, is the primary tissue affected in Prpf31 heterozygous mice. Other data show that homozygous PRPF31 mice are not viable. Morpholino-induced knockdown of zebrafish Prpf31 results in strong defects in PRC morphogenesis and survival. Defects induced by retina-specific expression of zebrafish Prpf31 constructs that encode proteins with the same mutations as those mapped in RP11 patients (called AD5 and SP117) were explained to occur by either haplo-insufficiency or by a dominant-negative effect of the mutant protein. In Drosophila, no mutations in the orthologue Prp31 have been identified so far. RNAi-mediated knockdown of Prp31 in the Drosophila eye has been shown to result in abnormal eye development, ranging from smaller eyes to complete absence of the eye, including loss of PRCs and pigment cells (Hebbar, 2021).

In order to get better insights into the mechanisms by which Prp31 prevents retinal degeneration this study aimed to establish a meaningful Drosophila model for RP11-associated retinal degeneration. Therefore two mutant alleles were isolated of Prp31, Prp31P17 and Prp31P18, which carry missense mutations affecting conserved amino acids. Flies heterozygous for either of these mutations are viable and develop normally. Strikingly, when exposed to constant light, these mutant flies undergo retinal degeneration, thus mimicking the disease phenotype of RP11 patients. Degeneration of mutant PRCs is associated with accumulation and abnormal distribution of the visual pigment rhodopsin, Rh1, in PRCs. Reduction of dietary vitamin A, a precursor of the chromophore 11-cis-3-hydroxyretinal, which binds to opsin to generate the functional rhodopsin, mitigates both aspects of the mutant phenotype, rhodopsin accumulation and retinal degeneration. From this it is concluded that Rh1 accumulation and/or misdistribution reflect a degeneration-prone condition in the Prp31 mutant retina (Hebbar, 2021).

The results reveal that mutations in the Drosophila orthologue Prp31 induce PRC degeneration under light stress, thus mimicking features of RP11-associated symptoms. Similar to those in human, mutations in Drosophila Prp31 are haplo-insufficient and lead to retinal degeneration when heterozygous. This is in stark contrast to mice heterozygous for Prpf31, which did not show any signs of PRC degeneration, but rather late-onset defects in the retinal pigment epithelium (Hebbar, 2021).

By using three different genetic approaches this study provides convincing evidence that the knockdown of Prp31 is the cause of the retinal degeneration observed. (1) The two Prp31 alleles induced by TILLING (Prp31P17 and Prp31P18) carry missense mutations in conserved amino acids of the coding region, which are predicted to be damaging. (2) Flies heterozygous for any of three deletions, which completely remove the Prp31 locus, exhibit comparable phenotypes as flies heterozygous for Prp31 point mutations. (3) RNAi-mediated knockdown of Prp31 results in light-induced retinal degeneration. The results obtained suggest that the two missense mutations mapped in Prp31P17 and Prp31P18 are strong hypomorphic alleles. First, the two Drosophila alleles characterised in this study are hemizygous (Prp31/deficiency) and homozygous (in the case of Prp31P18) viable and fertile. Second, mutations in the two established Prp31 fly lines are missense mutations, one located N-terminal to the NOSIC domain in Prp31P17 (G90R) and the other in the Nop domain in Prp31P18 (P277L), which most likely result in a reduced function of the respective proteins. Whether protein levels are also decreased cannot be answered due to the lack of specific antibodies. The mutated amino acid residue in Drosophila Prp31P18 (P277L) lies within the snoRNA binding domain (NOP domain. Interestingly, many point mutations in human PRPF31, which are linked to RP11, have been mapped to the Nop domain. Similar as in yeast, the Nop domain in human PRPF31 is involved in an essential step in the formation of the U4/U6-U5 tri-snRNP by building a complex of the U4 snRNA and a 15.5K protein, thus stabilising the U4/U6 snRNA junction. The mutated proline in Drosophila Prp31P18 precedes a histidine (H278), which corresponds to amino acid H270 in the human protein. Mutations in H270 in the Nop domain of human PRPF31 result in a reduced affinity of PRPF31 to the complex formed by a stem-loop structure of the U4 snRNA and the 15.5K protein. Therefore, it is tempting to speculate that the Drosophila P277L mutation could similarly weaken, but not abolish the corresponding interaction of the mutant Prp31 protein with the U4/U6 complex. Further experiments are required to determine the functional consequences of the molecular lesions identified in Drosophila Prp31 (Hebbar, 2021).

It was noticed that the retinal phenotype observed upon reduction of Prp31 is more variable than that observed upon loss of crb. This could be due to the fact that all Prp31 conditions analysed represent hypomorphic conditions, possibly retaining some residual protein function(s). However, the expressivity of the mutant phenotype is not increased in Prp31/deficiency flies (carrying only one mutant copy) in comparison to that of Prp31/+ flies, which carry one mutant and one wild-type allele. Interestingly, human RP11 patients heterozygous for mutations in Prpf31 show an unusually high degree of phenotypic non-penetrance and can even be asymptomatic. Various causes have been uncovered to explain this feature. These include a highly variable expression level of the remaining wild-type Prpf31 allele, possibly due to changes in the expression levels of trans-acting regulators. In addition, mutant PRPF31 proteins can form cytoplasmic aggregates in RPE cells, thus reducing the amount of protein entering the nucleus, or can impair overall transcription or splicing, as described in Prpf31 zebrafish models. Finally, mutations in unlinked genes have been suggested to modify the disease severity of patients (Hebbar, 2021).

Not only in flies, but also in human, mutations in PRPF31 affect only the retina, despite the importance of this splicing regulator in all cells. Recently published data show that impaired PRPF31 function can affect the splicing of target genes in a cell-type specific manner. Strikingly, retinal cells isolated from RP11 patient-derived retinal organoids exhibit mis-splicing of genes that encode components of the splicing machinery itself. This was not observed in fibroblasts or iPS cells derived from the same patients. Similar results were obtained from the retina and the RPE of Prpf31/+ mice. Mutant RPE cells additionally revealed splicing defects in transcripts of genes with functions in ciliogenesis, cell polarity and cellular adhesion, which could explain the previously described RPE defects in these mice (Hebbar, 2021).

In the retina of flies lacking one functional copy of Prp31, PRCs showed increased levels of Rh1, both in the rhabdomeres and in the cytoplasm, as revealed by immunostaining and confirmed by western blot analysis. However, increased Rh1 levels did not affect rhabdomere size or structure. This is in contrast to results obtained in the mouse, where transgenic overexpression of either wild-type bovine or human rhodopsin induced an increase in outer segment volume of rod PRCs. In several other Drosophila mutants, accumulation of Rh1 in endocytic compartments has been suggested to cause retinal degeneration due to its toxicity. For example, dominant mutations in Drosophila ninaE result in ER accumulation of misfolded Rh1 due to impaired protein maturation. This, in turn, causes an overproduction of ER cisternae and induces the unfolded protein response (UPR), which eventually leads to apoptosis of PRCs, both in flies and in mammals (Hebbar, 2021).

Interestingly, mis-localisation of rhodopsin in human PRCs to sites other than the outer segment is a common characteristic of adRP induced by mutations in rhodopsin and is considered to contribute to the pathological severity. The current data suggest that increased accumulation of rhodopsin contributes to degeneration in Prp31 mutant retinas. Reduction of Rh1 by depletion of dietary carotenoid not only obliterated increased Rh1 immunoreactivity and opsin retention in perinuclear compartments in Prp31 mutants, but also reduced the degree of PRC degeneration. However, whether increased Rh1 accumulation in the rhabdomere or in the cytoplasm contributes to light-dependent PRC degeneration of Prp31 mutant flies needs to be explored in the future (Hebbar, 2021).

The data further suggest that Prp31 regulates, directly or indirectly, Rh1 levels at a posttranscriptional level, since no increase of RNA levels was detected in heads of Prp31/+ flies. This result is different from that obtained in primary murine retinal cell cultures, where expression of a mutant Prpf31 gene reduced rhodopsin expression, as a result of impaired splicing of the rhodopsin pre-mRNA. Similarly, siRNA-mediated knockdown of PRPF31 function in human organotypic retinal flat-mount cultures (HORFC) reduced mRNAs encoding genes involved in phototransduction and photoreceptor structure, including rhodopsin. Interestingly, the Prp31 mutants described in this study show increased mRNA levels of an evolutionary conserved actin monomer binding protein called twinfilin (twf), which inhibits actin polymerisation. Knockdown of twf results in excessive cytoplasmic Rh1 staining, suggesting defects in its trafficking. In Prp31 mutants, an increase in rhabdomeric Rh1 was observed as well as increased twf mRNA. From this correlation it is hypothesised that upregulation of twf mRNA in Prp31 might be in part responsible for at least the rhabdomeric Rh1 accumulation. Rh1 also accumulates in the cytoplasm of Prp31 mutant PRCs. The current data exclude the role of Rab11-mediated targeting of Rh1 in this accumulation. Now, it remains to be determined if the deregulation of other trafficking routes or the upregulation of twf contributes to the increased Rh1 in the cytoplasm. In the future, it may be interesting to explore the link between increased Rh1 levels as observed in Drosophila Prp31 mutants, increased mRNA levels of twinfilin and impaired Rh1 trafficking. Additionally, a detailed transcriptome analysis should elucidate possible defects in transcription and/or splicing of target genes, thus also allowing a better understanding of the aetiology of the human disease (Hebbar, 2021).

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Amstutz, J., Khalifa, A., Palu, R. and Jahan, K. (2020). Cluster-Based Analysis of Retinitis Pigmentosa Modifiers Using Drosophila Eye Size and Gene Expression Data. Genes (Basel) 13(2). PubMed ID: 35205430

Abstract
The goal of this research is to computationally identify candidate modifiers for retinitis pigmentosa (RP), a group of rare genetic disorders that trigger the cellular degeneration of retinal tissue. RP being subject to phenotypic variation complicates diagnosis and treatment of the disease. In a previous study, modifiers of RP were identified by an association between genetic variation in the DNA sequence and variation in eye size in a well-characterized Drosophila model of RP. This study instead focusses on RNA expression data to identify candidate modifier genes whose expression is correlated with phenotypic variation in eye size. The proposed approach uses the K-Means algorithm to cluster 171 Drosophila strains based on their expression profiles for 18,140 genes in adult females. This algorithm is designed to investigate the correlation between Drosophila eye size and genetic expression and gather suspect genes from clusters with abnormally large or small eyes. The clustering algorithm was implemented using the R scripting language and successfully identified 10 suspected candidate modifiers for RP. This analysis was followed by a validation study that tested seven candidate modifiers and found that the loss of five of them significantly altered the degeneration phenotype and thus can be labeled as a bona fide modifier of disease.

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Zhao, N., Li, N. and Wang, T. (2023). PERK prevents rhodopsin degradation during retinitis pigmentosa by inhibiting IRE1-induced autophagy. J Cell Biol 222(5). PubMed ID: 37022709

Abstract

Chronic endoplasmic reticulum (ER) stress is the underlying cause of many degenerative diseases, including autosomal dominant retinitis pigmentosa (adRP). In adRP, mutant rhodopsins accumulate and cause ER stress. This destabilizes wild-type rhodopsin and triggers photoreceptor cell degeneration. To reveal the mechanisms by which these mutant rhodopsins exert their dominant-negative effects, this study established an in vivo fluorescence reporter system to monitor mutant and wild-type rhodopsin in Drosophila. By performing a genome-wide genetic screen, PERK signaling was found to play a key role in maintaining rhodopsin homeostasis by attenuating IRE1 activities. Degradation of wild-type rhodopsin is mediated by selective autophagy of ER, which is induced by uncontrolled IRE1/XBP1 signaling and insufficient proteasome activities. Moreover, upregulation of PERK signaling prevents autophagy and suppresses retinal degeneration in the adRP model. These findings establish a pathological role for autophagy in this neurodegenerative condition and indicate that promoting PERK activity could be used to treat ER stress-related neuropathies, including adRP.

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Ribosomopathies

Baral, S. S., Lieux, M. E. and DiMario, P. J. (2020). Nucleolar stress in Drosophila neuroblasts, a model for human ribosomopathies. Biol Open. PubMed ID:
32184230

Different stem cells or progenitor cells display variable threshold requirements for functional ribosomes. This is particularly true for several human ribosomopathies in which select embryonic neural crest cells or adult bone marrow stem cells, but not others, show lethality due to failures in ribosome biogenesis or function (now known as nucleolar stress). To determine if various Drosophila neuroblasts display differential sensitivities to nucleolar stress, CRISPR-Cas9 was used to disrupt the Nopp140 gene that encodes two splice variant ribosome biogenesis factors (RBFs). Disruption of Nopp140 induced nucleolar stress that arrested larvae in the second instar stage. While the majority of larval neuroblasts arrested development, the Mushroom Body (MB) neuroblasts continued to proliferate as shown by their maintenance of deadpan, a neuroblast-specific transcription factor, and by their continued EdU incorporation. MB neuroblasts in wild type larvae appeared to contain more fibrillarin and Nopp140 in their nucleoli as compared to other neuroblasts, indicating that MB neuroblasts stockpile RBFs as they proliferate in late embryogenesis while other neuroblasts normally enter quiescence. A greater abundance of Nopp140 encoded by maternal transcripts in Nopp140-/- MB neuroblasts of 1-2 day old larvae likely rendered these cells more resilient to nucleolar stress (Baral, 2020).

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Sarcopenia

Hunt, L. C., Schadeberg, B., Stover, J., Haugen, B., Pagala, V., Wang, Y. D., Puglise, J., Barton, E. R., Peng, J. and Demontis, F. (2021) (2021). Antagonistic control of myofiber size and muscle protein quality control by the ubiquitin ligase UBR4 during aging. Nat Commun 12(1): 1418. PubMed ID: 33658508

Abstract

Sarcopenia is a degenerative condition that consists in age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. This study found that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that UBR4 increases the proteolytic activity of the proteasome. Importantly, muscle-specific UBR4 loss rescues age-associated myofiber atrophy in mice. However, UBR4 loss reduces the muscle specific force and accelerates the decline in muscle protein quality that occurs with aging in mice. Similarly, hypertrophic signaling induced via muscle-specific loss of UBR4/poe and of ESCRT members (HGS/Hrs, STAM, USP8) that degrade ubiquitinated membrane proteins compromises muscle function and shortens lifespan in Drosophila by reducing protein quality control. Altogether, these findings indicate that these ubiquitin ligases antithetically regulate myofiber size and muscle protein quality control (Hunt, 2021).

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Schizophrenia

Nagy, S., Maurer, G. W., Hentze, J. L., Rose, M., Werge, T. M. and Rewitz, K. (2018). AMPK signaling linked to the schizophrenia-associated 1q21.1 deletion is required for neuronal and sleep maintenance. PLoS Genet 14(12): e1007623. PubMed ID:
30566533

Abstract

The human 1q21.1 deletion of ten genes is associated with increased risk of schizophrenia. This deletion involves the beta-subunit of the AMP-activated protein kinase (AMPK) complex, a key energy sensor in the cell. Although neurons have a high demand for energy and low capacity to store nutrients, the role of AMPK in neuronal physiology is poorly defined. This study shows that AMPK is important in the nervous system for maintaining neuronal integrity and for stress survival and longevity in Drosophila. To understand the impact of this signaling system on behavior and its potential contribution to the 1q21.1 deletion syndrome, this study focused on sleep, an important role of which is proposed to be the reestablishment of neuronal energy levels that are diminished during energy-demanding wakefulness. Sleep disturbances are one of the most common problems affecting individuals with psychiatric disorders. This study shows that AMPK is required for maintenance of proper sleep architecture and for sleep recovery following sleep deprivation. Neuronal AMPKbeta loss specifically leads to sleep fragmentation and causes dysregulation of genes believed to play a role in sleep homeostasis. These data also suggest that AMPKbeta loss may contribute to the increased risk of developing mental disorders and sleep disturbances associated with the human 1q21.1 deletion (Nagy, 2018).

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Hidalgo, S., Castro, C., Zarate, R. V., Valderrama, B. P., Hodge, J. J. L. and Campusano, J. M. (2020). The behavioral and neurochemical characterization of a Drosophila dysbindin mutant supports the contribution of serotonin to schizophrenia negative symptoms. Neurochem Int 138: 104753. PubMed ID: 32416114

Abstract

Mutations in the dystrobrevin binding protein 1 (DTNBP1) gene that encodes for the dysbindin-1 protein, are associated with a higher risk for schizophrenia. Interestingly, individuals carrying high-risk alleles in this gene have been associated with an increased incidence of negative symptoms for the disease, which include anhedonia, avolition and social withdrawal. This study evaluated behavioral and neurochemical changes in a hypomorphic Drosophila mutant for the orthologue of human Dysbindin-1, dysb1. Mutant dysb1 flies exhibit altered social space parameters, suggesting asocial behavior, accompanied by reduced olfactory performance. Moreover, dysb1 mutant flies show poor performance in basal and startle-induced locomotor activity. This study also reports a reduction in serotonin brain levels and changes in the expression of the Drosophila serotonin transporter (dSERT) in dysb1 flies. The data show that the serotonin-releasing amphetamine derivative 4-methylthioamphetamine (4-MTA) modulates social spacing and locomotion in control flies, suggesting that serotonergic circuits modulate these behaviors. 4-MTA was unable to modify the behavioral deficiencies in mutant flies, which is consistent with the idea that the efficiency of pharmacological agents acting at dSERT depends on functional serotonergic circuits. Thus, these data show that the dysb1 mutant exhibits behavioral deficits that mirror some aspects of the endophenotypes associated with the negative symptoms of schizophrenia. It is argued that at least part of the behavioral aspects associated with these symptoms could be explained by a serotonergic deficit. The dysb1 mutant presents an opportunity to study the molecular underpinnings of schizophrenia negative symptoms and reveals new potential targets for treatment of the disease (Hidalgo, 2020).

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Shwachman-Diamond syndrome

Takai, A., Chiyonobu, T., Ueoka, I., Tanaka, R., Tozawa, T., Yoshida, H., Morimoto, M., Hosoi, H. and Yamaguchi, M. (2020). A novel Drosophila model for neurodevelopmental disorders associated with Shwachman-Diamond syndrome. Neurosci Lett 739: 135449. PubMed ID: 33115644

Abstract

Genetic defects in ribosome biogenesis result in a group of diseases called ribosomopathies. Patients with ribosomopathies manifest multiorgan phenotypes, including neurological impairments. A well-characterized ribosomopathy, Shwachman-Diamond syndrome (SDS), is mainly associated with loss-of-function mutations in the causal gene SBDS. Children with SDS have neurodevelopmental disorders; however, the neurological consequences of SBDS dysfunction remain poorly defined. This study investigated the phenotype of Drosophila melanogaster following knockdown of CG8549, the Drosophila ortholog of human SBDS, to provide evidence for the neurological consequences of reduction in physiological SBDS functions. The pan-neuron-specific knockdown of CG8549 was associated with locomotive disabilities, mechanically induced seizures, hyperactivity, learning impairments, and anatomical defects in presynaptic terminals. These results provide the first evidence of a direct link between a reduction in physiological SBDS function and neurological impairments (Takai, 2020).

Tan, S., Kermasson, L., Hilcenko, C., Kargas, V., Traynor, D., Boukerrou, A. Z., Escudero-Urquijo, N., Faille, A., Bertrand, A., Rossmann, M., Goyenechea, B., Jin, L., Moreil, J., Alibeu, O., Beaupain, B., Bole-Feysot, C., Fumagalli, S., Kaltenbach, S., Martignoles, J. A., Masson, C., Nitschke, P., Parisot, M., Pouliet, A., Radford-Weiss, I., Tores, F., de Villartay, J. P., Zarhrate, M., Koh, A. L., Phua, K. B., Reversade, B., Bond, P. J., Bellanne-Chantelot, C., Callebaut, I., Delhommeau, F., Donadieu, J., Warren, A. J. and Revy, P. (2021). Somatic genetic rescue of a germline ribosome assembly defect. Nat Commun 12(1): 5044. PubMed ID: 34413298

Abstract

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. This study established that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 (see Drosophila eIF6) from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. This study identified diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. These data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS (Tan, 2021).

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16p12.1 deletion

Pizzo, L., Lasser, M., Yusuff, T., Jensen, M., Ingraham, P., Huber, E., Singh, M. D., Monahan, C., Iyer, J., Desai, I., Karthikeyan, S., Gould, D. J., Yennawar, S., Weiner, A. T., Pounraja, V. K., Krishnan, A., Rolls, M. M., Lowery, L. A. and Girirajan, S.. (2021). Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis. PLoS Genet 17(4): e1009112. PubMed ID: 33819264
Summary:

Abstract

Previous work identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. It was hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, this study examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. Knockdown of 16p12.1 homologs affected multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. These results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation.

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Skin cancer and nucleotide excision repair

Yang, Y., He, S., Wang, Q., Li, F., Kwak, M. J., Chen, S., O'Connell, D., Zhang, T., Pirooz, S. D., Jeon, Y., Chimge, N. O., Frenkel, B., Choi, Y., Aldrovandi, G. M., Oh, B. H., Yuan, Z. and Liang, C. (2016). Autophagic UVRAG promotes UV-induced photolesion repair by activation of the CRL4(DDB2) E3 ligase. Mol Cell 62: 507-519. PubMed ID:
27203177

Abstract

UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. This study shows that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. The importance of UVRAG for UV-damage tolerance was confirmed using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. These results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition (Yang, 2016).

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Snyder-Robinson Syndrome

Stewart, T. R. M., Foley, J. R., Holbert, C. E., Khomutov, M. A., Rastkari, N., Tao, X., Khomutov, A. R., Zhai, R. G. and Casero, R. A. (2023). Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome: mechanism of action and therapeutic potential. bioRxiv. PubMed ID: 37034775

Abstract
Snyder-Robinson Syndrome (SRS) is caused by mutations in the spermine synthase (SMS) gene, the enzyme product of which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonic musculature, and seizures, along with other more variable symptoms. Currently, medical management focuses on treating these symptoms without addressing the underlying molecular cause of the disease. Reduced SMS catalytic activity in cells of SRS patients causes the accumulation of spermidine, while spermine levels are reduced. The resulting exaggeration in spermidine-to-spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity in the patient. These studies aim to pharmacologically manipulate polyamine metabolism to correct this polyamine imbalance and investigate the potential of this approach as a therapeutic strategy for affected individuals. This study reports the use of difluoromethylornithine (DFMO; eflornithine), an FDA-approved inhibitor of polyamine biosynthesis, in re-establishing normal spermidine-to-spermine ratios in SRS patient cells. Through mechanistic studies, it was demonstrated that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of existing spermidine into spermine in cell lines with hypomorphic variants of SMS. Further, DFMO treatment induces a compensatory uptake of exogenous polyamines, including spermine and spermine mimetics, cooperatively reducing spermidine and increasing spermine levels. In a Drosophila SRS model characterized by reduced lifespan, adding DFMO to the feed extended lifespan. As nearly all known SRS patient mutations are hypomorphic, these studies form a foundation for future translational studies with significant therapeutic potential.

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Spliceosomopathy

Ayers, K. L., Eggers, S., ..., BenZeev, B. and Sinclair, A. H. (2023). Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects. Nat Commun 14(1): 3403. PubMed ID:
37296101

Abstract
Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Thus study identified recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 (Rnp4F) reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which is tentatively proposed to be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). These findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

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Spondyloarthritis

Grandon, B., Rincheval-Arnold, A., Jah, N., Corsi, J. M., Araujo, L. M., Glatigny, S., Prevost, E., Roche, D., Chiocchia, G., Guenal, I., Gaumer, S. and Breban, M. (2019). HLA-B27 alters BMP/TGFbeta signalling in Drosophila, revealing putative pathogenic mechanism for spondyloarthritis. Ann Rheum Dis. PubMed ID:
31563893

Abstract

The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. Transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, were generated alone or in combination with human beta2-microglobulin (hbeta2m), under tissue-specific drivers. Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hbeta2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated Mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor beta (TGFbeta). It is concluded that antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFbeta/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development (Grandon, 2019).

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Spinal muscular atrophy

Borg, R. M., Fenech Salerno, B., Vassallo, N., Bordonne, R. and Cauchi, R. J. (2016). Disruption of snRNP biogenesis factors Tgs1 and pICln induces phenotypes that mirror aspects of SMN-Gemins complex perturbation in Drosophila, providing new insights into spinal muscular atrophy. Neurobiol Dis 94: 245-258. PubMed ID:
27388936

Abstract

The neuromuscular disorder, spinal muscular atrophy (SMA), results from insufficient levels of the survival motor neuron (SMN; see Drosophila Smn) protein. Together with Gemins 2-8 and Unrip, SMN forms the large macromolecular SMN-Gemins complex, which is known to be indispensable for chaperoning the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). It remains unclear whether disruption of this function is responsible for the selective neuromuscular degeneration in SMA. This present study shows that loss of wing morphogenesis defect (wmd), the Drosophila Unrip orthologue, has a negative impact on the motor system. However, due to lack of a functional relationship between wmd/Unrip and Gemin3, it is likely that Unrip joined the SMN-Gemins complex only recently in evolution. Second, disruption of either Tgs1 or pICln, two cardinal players in snRNP biogenesis, results in viability and motor phenotypes that closely resemble those previously uncovered on loss of the constituent members of the SMN-Gemins complex. Interestingly, overexpression of both factors leads to motor dysfunction in Drosophila, a situation analogous to that of Gemin2. Toxicity is conserved in the yeast S. pombe where pICln overexpression induces a surplus of Sm proteins in the cytoplasm, indicating that a block in snRNP biogenesis is partly responsible for this phenotype. Importantly, this study shows a strong functional relationship and a physical interaction between Gemin3 and either Tgs1 or pICln. It is proposed that snRNP biogenesis is the pathway connecting the SMN-Gemins complex to a functional neuromuscular system, and its disturbance most likely leads to the motor dysfunction that is typical in SMA (Borg, 2016).

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Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022). Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Summary:

Increasing evidence points to the involvement of cell types other than motor neurons in both ALS and SMA, the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. This study asked whether the Smn protein, the causative factor for SMA, is required selectively in glia. Loss of Smn function in glia during development was shown to reduce survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology. In contrast, gain of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal snRNPs. Glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability. These findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.

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Raimer, A. C., Singh, S. S., Edula, M. R., Paris-Davila, T., Vandadi, V., Spring, A. M. and Matera, A. G. (2020). Temperature-sensitive spinal muscular atrophy-causing point mutations lead to SMN instability, locomotor defects and premature lethality in Drosophila. Dis Model Mech 13(5). PubMed ID: 32501283

Abstract

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. This study shows that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function and adult longevity. These disease-related phenotypes are domain specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Owing to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, it was found that only baseline levels of SMN are required during larval stages, whereas high levels of the protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Taken together, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at crucial developmental time points that are conserved from Drosophila to humans.

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Cacciottolo, R., Ciantar, J., Lanfranco, M., Borg, R. M., Vassallo, N., Bordonne, R. and Cauchi, R. J. (2017). SMN complex member Gemin3 self-interacts and has a functional relationship with ALS-linked proteins TDP-43, FUS and Sod1. Sci Rep 9(1): 18666. PubMed ID: 31822699

Abstract

The predominant motor neuron disease in infants and adults is spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. SMA is caused by insufficient levels of the Survival Motor Neuron (SMN) protein, which operates as part of the multiprotein SMN complex that includes the DEAD-box RNA helicase Gemin3/DDX20/DP103. C9orf72, SOD1, TDP-43 and FUS are ranked as the four major genes causing familial ALS. Accumulating evidence has revealed a surprising molecular overlap between SMA and ALS. This study asked the question of whether Drosophila can also be exploited to study shared pathogenic pathways. Focusing on motor behaviour, muscle mass and survival, disruption of either TBPH/TDP-43 or Caz/FUS was found to enhance defects associated with Gemin3 loss-of-function. Gemin3-associated neuromuscular junction overgrowth was however suppressed. Sod1 depletion had a modifying effect in late adulthood. This study also showed that Gemin3 self-interacts and Gem3(DeltaN), a helicase domain deletion mutant, retains the ability to interact with its wild-type counterpart. Importantly, mutant:wild-type dimers are favoured more than wild-type:wild-type dimers. In addition to reinforcing the link between SMA and ALS, further exploration of mechanistic overlaps is now possible in a genetically tractable model organism. Notably, Gemin3 can be elevated to a candidate for modifying motor neuron degeneration (Cacciottolo, 2017).
Li, L., Ding, Z., Pang, T. L., Zhang, B., Li, C. H., Liang, A. M., Wang, Y. R., Zhou, Y., Fan, Y. J. and Xu, Y. Z. (2017). Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila. Nat Commun 11(1): 5608. PubMed ID: 33154379

Abstract

The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. By precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, this study reports that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, a large set of minor spliceosome-sensitive splicing events was identified and it was demonstrated that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals (Li, 2020).

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Squamous cell carcinoma

Fu, W., Sun, J., Huang, G., Liu, J. C., Kaufman, A., Ryan, R. J., Ramanathan, S. Y., Venkatesh, T. and Singh, B. (2016). Squamous cell carcinoma related oncogene (SCCRO) family members regulate cell growth and proliferation through their cooperative and antagonistic effects on Cullin neddylation. J Biol Chem 291(12): 6200-17. PubMed ID:
26792857

Abstract

SCCRO (squamous cell carcinoma related oncogene; a.k.a. DCUN1D1) is a highly conserved gene that functions as an E3 in neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO-/- mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led to an assessment of whether compensation by SCCRO's paralogues rescues lethality in SCCRO-/- mice. Using murine and Drosophila models, the in vivo activities of SCCRO and its paralogues were assessed in cullin neddylation (see Drosophila Cullin1). SCCRO family members were found to have overlapping and antagonistic activity that regulates neddylation and cell proliferation activities in vivo. In flies, both dSCCRO (CG7427) and dSCCRO3 (CG13322) promote neddylation and cell proliferation, whereas dSCCRO4 (CG6597) negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones, with a net decrease in neddylation activity. dSCCRO and, to a lesser extent, dSCCRO3 rescue the neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promotes and human SCCRO3 inhibits neddylation and proliferation when expressed in flies. These findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate neddylation and cell proliferation.

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Steatosis


Zhao, W., Zhang, Y., Lin, S., Li, Y., Zhu, A. J., Shi, H. and Liu, M. (2023). Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle. J Cachexia Sarcopenia Muscle. PubMed ID:
37057345

Abstract

Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. Perilipin 2 (Plin2), a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, this study used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, proteins were purified from HEK293 cells, and in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays were reconstituted to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. As a lipolysis inhibitor, Plin2 was significantly elevated in liver and muscle in patients with steatosis. Consistently, it was found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. It was further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. This study has identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. This work may provide new strategies for the prevention and treatment of steatosis (Zhao, 2023).

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Tango2 Deficiency

Asadi, P., Milev, M. P., Saint-Dic, D., Gamberi, C. and Sacher, M. (2022). Vitamin B5, a coenzyme A precursor, rescues TANGO2 deficiency disease-associated defects in Drosophila and human cells. J Inherit Metab Dis. PubMed ID:
36502486

Abstract

Mutations in the Transport and Golgi Organization 2 (TANGO2) gene are associated with intellectual deficit, neurodevelopmental delay and regression. Individuals can also present with an acute metabolic crisis that includes rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, the latter of which are potentially lethal. While preventing metabolic crises has the potential to reduce mortality, no treatments currently exist for this condition. The function of TANGO2 remains unknown but is suspected to be involved in some aspect of lipid metabolism. This study describes a model of TANGO2-related disease in the fruit fly Drosophila melanogaster that recapitulates crucial disease traits. Pairing a new fly model with human cells, the effects were examined of vitamin B5, a coenzyme A (CoA) precursor, on alleviating the cellular and organismal defects associated with TANGO2 deficiency. It was demonstrated that vitamin B5 specifically improves multiple defects associated with TANGO2 loss-of-function in Drosophila and rescues membrane trafficking defects in human cells. A partial rescue was observed of one of the fly defects by vitamin B3, though to a lesser extent than vitamin B5. These data suggest that a B complex supplement containing vitamin B5/pantothenate may have therapeutic benefits in individuals with TANGO2-deficiency disease. Possible mechanisms for the rescue are discussed that may include restoration of lipid homeostasis (Asadi, 2022).

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Tauopathy

Nisha and Sarkar, S. (2021). Downregulation of glob1 suppresses pathogenesis of human neuronal tauopathies in Drosophila by regulating tau phosphorylation and ROS generation. Neurochem Int 146: 105040. PubMed ID:
33865914

Abstract

Human tauopathies represent a group of neurodegenerative disorders, characterized by abnormal hyperphosphorylation and aggregation of tau protein, which ultimately cause neurodegeneration. The aberrant tau hyperphosphorylation is mostly attributed to the kinases/phosphatases imbalance, which is majorly contributed by the generation of reactive oxygen species (ROS). Globin(s) represent a well-conserved group of proteins which are involved in O(2) management, regulation of cellular ROS in different cell types. Similarly, Drosophila globin1 (a homologue of human globin) with its known roles in oxygen management and development of nervous system exhibits striking similarities with the mammalian neuroglobin. Several recent evidences support the hypothesis that neuroglobins are associated with Alzheimer's disease pathogenesis. It is noted that targeted expression of human-tau induces the cellular level of Glob1 protein in Drosophila tauopathy models. Subsequently, RNAi mediated restored level of Glob1 restricts the pathogenic effect of human-tau by minimizing its hyperphosphorylation via GSK-3β/p-Akt and p-JNK pathways. In addition, it also activates the Nrf2-keap1-ARE cascade to stabilize the tau-mediated increased level of ROS. These two parallel cellular events provide a significant rescue against human tau-mediated neurotoxicity in the fly models. This study reports a direct involvement of an oxygen sensing globin gene in tau etiology. In view of the fact that human genome encodes for the multiple Globin proteins including a nervous system specific neuroglobin; and therefore, these findings may pave the way to investigate if the conserved oxygen sensing globin gene(s) can be exploited in devising novel therapeutic strategies against tauopathies (Nisha, 2021).

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Traumatic Brain Injury

Blommer, J., Fischer, M. C., Olszewski, A. R., Katzenberger, R. J., Ganetzky, B. and Wassarman, D. A. (2021). Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARgamma ortholog Eip75B. PLoS One 16(10): e0258873. PubMed ID:
34699541

Abstract

Traumatic brain injury (TBI) is a common neurological disorder whose outcomes vary widely depending on a variety of environmental factors, including diet. Using a Drosophila melanogaster TBI model that reproduces key aspects of TBI in humans, this study previously found that the diet consumed immediately following a primary brain injury has a substantial effect on the incidence of mortality within 24 h (early mortality). Flies that receive equivalent primary injuries have a higher incidence of early mortality when fed high-carbohydrate diets versus water. This study reports that flies fed high-fat ketogenic diet (KD) following TBI exhibited early mortality that was equivalent to that of flies fed water and that flies protected from early mortality by KD continued to show survival benefits weeks later. KD also has beneficial effects in mammalian TBI models, indicating that the mechanism of action of KD is evolutionarily conserved. To probe the mechanism, this study examined the effect of KD in flies mutant for Eip75B, an ortholog of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) that contributes to the mechanism of action of KD and has neuroprotective effects in mammalian TBI models. The incidence of early mortality of Eip75B mutant flies was found to be higher when they were fed KD than when they were fed water following TBI. These data indicate that Eip75B/PPARγ is necessary for the beneficial effects of KD following TBI. In summary, this work provides the first evidence that KD activates PPARγ to reduce deleterious outcomes of TBI and it demonstrates the utility of the fly TBI model for dissecting molecular pathways that contribute to heterogeneity in TBI outcomes (Blommer, 2021).

van Alphen, B., Stewart, S., Iwanaszko, M., Xu, F., Li, K., Rozenfeld, S., Ramakrishnan, A., Itoh, T. Q., Sisobhan, S., Qin, Z., Lear, B. C. and Allada, R. (2022). Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila. PLoS Biol 20(1): e3001456. PubMed ID: 35081110

Abstract
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, this study developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, TBI-dependent behavior and lethality were examined in mutants of the master immune regulator NF-κB, important for AMP induction: while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

This study has developed a straightforward and reproducible Drosophila model for closed head TBI where precisely controlled strikes are delivered to the head of individually restrained, unanesthetized flies. This TBI paradigm is validated by recapitulating many of the phenotypes observed in mammalian TBI models, including increased mortality, increased neuronal cell death, impaired motor control, decreased/fragmented sleep, and hundreds of TBI-induced changes to the transcriptome, including the activation of many AMPs, indicating a strong activation of the immune response. These results set the stage to leverage Drosophila genetic tools to investigate the role of the immune response as well as novel pathways in TBI pathology (van Alphen, 2022).

The single fly paradigm is a more valid Drosophila model for TBI that circumvents the lack of specificity of currently available models or the use of anesthesia. Both previous assays induce TBI by subjecting the whole fly to trauma, which makes it hard to distinguish whether observed phenotypes are a due to TBI or a consequence of internal injuries. A recently published method uses a pneumatic device to strike an anesthetized fly's head. This method is an improvement of earlier assays and results in increased mortality in a stimulus strength-dependent manner. However, it only shows a modest reduction in locomotor activity, without demonstrating any other TBI-related phenotypes such as neuronal cell death or immune activation. The dependence on CO2 anesthesia further impairs the usefulness of this assay, as prolonged behavioral impairments in Drosophila occur even after brief exposure to CO2 anesthesia. Additionally, anesthetics that are administered either during or shortly after TBI induction can offer neuroprotective effects and alter cognitive, motor, and histological outcomes in mammalian models of TBI as well as affecting mortality in a whole body injury model in flies. The Drosophila model allows study of how TBI affects behavior and gene expression without the confounding effects of anesthesia, making it a more valid model for TBI that occurs under natural conditions (van Alphen, 2022).

The force used in this study (8.34 N) is higher than the force used in the HIT assay (2.5 N). When designing the TBI paradigm, several commercially available solenoids were tested for their ability to induce TBI, and the one that gave the best results was used. A higher force may be needed because brain damage is caused by the direct impact of the solenoid to the fly head, where the fly head moves with the solenoid rather than full body injury or compression injuries used in the other Drosophila TBI assays. Although it cannot be excluded that the neck is not damaged in this assay, cell death was observed in the central brain and significant changes in glia after TBI were observed, suggesting that TBI does occur (van Alphen, 2022).

This study also elucidate, in an unbiased manner, the genomic response to TBI. Glial cells play an important role in immune responses in both mammals and Drosophila, and changes to glial morphology and function were reported in earlier Drosophila TBI models. Until now, profiling TBI-induced changes in gene expression have either been limited to a small number of preselected genes in both mammals and Drosophila or focused on whole brain tissue rather than individual cell types. Using TRAP in combination with RNA-seq, previously reported up-regulation of Attacin-C, Diptericin-B, and Metchnikowin was validated. Additionally, an acute, broad-spectrum immune response was detected, where AMPs and stress response genes are up-regulated 24 hours after TBI. These include antibacterial, antifungal, and antiviral peptides as well as peptides from the Tot family, which are secreted as part of a stress response induced by bacteria, UV, heat, and mechanical stress. Although an increase in the heatshock protein 70 family of stress response genes was reported earlier, this study detected a significant glial up-regulation only in Hsp70BC (van Alphen, 2022).

Three days after TBI, only Attacin-C, Diptericin A, and Metchnikowin are up-regulated. Seven days after TBI, AMPs or stress response genes are not detectably up-regulated. These findings match reports in mammalian TBI models, where inflammatory gene expression spikes shortly after TBI but mostly dies down during subsequent days. Using CRISPR deletions of AMP classes, this study demonstrates that most AMPs not only protect against microbes but are also crucial in promoting survival after TBI. The exception is Defensin, as loss of this peptide increases survival, indicating that the Drosophila innate immune response to TBI can have both beneficial and detrimental effects. While loss of AMPs may render flies more susceptible to TBI, the hypothesis that AMP induction after TBI actively plays a role in mediating TBI effects is favored (van Alphen, 2022).

Besides validating the Drosophila model with the detection of a strongly up-regulated immune response, several novel genes were detected among the total of 512 different glial genes that were either up- or down-regulated after TBI. Immune and stress response only make up 157 out of 512 differentially expressed glial genes. Genes involved in proteolysis and protein folding are a prominent portion (85/512) of these differentially expressed genes, yet their role in TBI is poorly understood. These results demonstrate that there are other candidate pathways that may modulate recovery, and Drosophila can be used as a first line screen to test their in vivo function and to disentangle beneficial from detrimental responses (van Alphen, 2022).

This study has successfully applied in vivo genetics to identify in vivo pathways important for TBI. Loss of master immune regulator NF-κB results in increased mortality after TBI, yet it protects against TBI-induced impairments in sleep and motor control. These findings align with previous reports showing links between sleep and the immune response in flies where NF-κB is required to alter sleep architecture after exposure to septic or aseptic injuries. It will be of interest to determine if NF-κB is required for TBI-induced cell death. One possibility is that sleep impairments can be a side effect of melanization, an invertebrate defense mechanism that requires dopamine as melanin precursor. If dopamine is up-regulated to create more melanin, decreased sleep would be a side effect. Consistent with this hypothesis, changes were observed in fumin and pale, which likely result in increased dopamine levels (van Alphen, 2022).

However, the role of sleep after injury is complex. Two recent studies demonstrated that sleep is increased after antennal transection and facilitates Wallerian degeneration and glia-mediated clearance of axonal debris, suggesting that different types of injury have different effects on sleep. Interestingly, sleep disturbances can increase the up-regulation of immune genes. Thus, it is possible that decreased sleep after TBI contributes to survival by stimulating the immune response. Some support is found for this hypothesis in the difference in TBI-induced changes to sleep in flies that survive 7 days of TBI versus flies that die within 7 days after TBI, where the survivors sleep significantly less for 4 days post-TBI and dying flies sleep is nearly unaffected. Additionally, immune response genes are up-regulated for up to 3 days after TBI, which correlates with the observed sleep impairments. Also, the engulfment receptor Draper, which mediates Wallerian degeneration, is not up-regulated in the glial TRAP-seq data, suggesting that Wallerian degeneration, and its accompanying increase in sleep, is not part of the response to dCHI (van Alphen, 2022).

TBI results in impaired climbing behavior that persists for up to 7 days, yet impairments to sleep disappear after a few days. Recently, it was shown that TBI through head compression results in impaired memory, as quantified through courtship conditioning, indicating that TBI also results in persistent memory defects (van Alphen, 2022).

Recently, it was shown that repressing neuronal NF-κB in a mouse model of TBI increases post-TBI mortality, as in the current studies, without reducing behavioral impairments, suggesting that nonneuronal NF-κB could underlie behavioral impairments after TBI. This study demonstrates that behavioral responses to TBI (for example, sleep and geotaxis) are abolished in mutants of the transcription factor NF-κB Relish, which plays a central role in regulating stress-associated and inflammatory gene expression in both mammals and flies. Nonetheless, Relish null mutants show increased mortality after TBI, but none of the behavioral impairments observed in wild-type flies, indicating that these impairments might be a side effect of immune activation rather than direct injury. The demonstration of an in vivo role for TBI-regulated genes will be important for defining their function (van Alphen, 2022).

In summary, the dCHI assay recapitulates many of the physiological symptoms observed in mammals, demonstrating that fruit flies are a valid model to study physiological responses to TBI. Both a potent induction of immune pathways and a requirement for an immune master regulator was demonstrated in mediating TBI effects on behavior. This model now provides a platform to perform unbiased genetic screens to study how gene expression changes after TBI in unanesthetized, awake animals result in the long-term sequelae of TBI. These studies raise the possibility of rapidly identifying key genes and pathways that are neuroprotective for TBI, thereby providing a high-throughput approach that could facilitate the discovery of novel genes and therapeutics that offer better outcomes after TBI (van Alphen, 2022). Swanson, L. C., Rimkus, S. A., Ganetzky, B. and Wassarman, D. A. (2020). Loss of the Antimicrobial Peptide Mechnikowin Protects Against Traumatic Brain Injury Outcomes in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32631949

Abstract
Neuroinflammation is a major pathophysiological feature of traumatic brain injury (TBI). Early and persistent activation of innate immune response signaling pathways by primary injuries is associated with secondary cellular injuries that cause TBI outcomes to change over time. A Drosophila melanogaster model was used to investigate the role of antimicrobial peptides (AMPs) in acute and chronic outcomes of closed-head TBI. AMPs are effectors of pathogen and stress defense mechanisms mediated by the evolutionarily conserved Toll Immune-deficiency (Imd) innate immune response pathways that activate Nuclear Factor kappa B (NF-kB) transcription factors. This study analyzed the effect of null mutations in 10 of the 14 known Drosophila AMP genes on TBI outcomes. Mutation of Metchnikowin (Mtk) was unique in protecting flies from mortality within the 24 h following TBI under two diet conditions that produce different levels of mortality. In addition, Mtk mutants had reduced behavioral deficits at 24 h following TBI and increased lifespan either in the absence or presence of TBI. Using a transcriptional reporter of gene expression, it was found that TBI increased Mtk expression in the brain. Quantitative analysis of mRNA in whole flies revealed that expression of other AMPs in the Toll and Imd pathways as well as NF-κB transcription factors were not altered in Mtk mutants. Overall, these results demonstrate that Mtk plays an infection-independent role in the fly nervous system, and TBI-induced expression of Mtk in the brain activates acute and chronic secondary injury pathways that are also activated during normal aging.
Lika, J., Katzenberger, R. J., Ganetzky, B. and Wassarman, D. A. (2021). Effects of blunt force injuries in third-instar Drosophila larvae persist through metamorphosis and reduce adult lifespan. MicroPubl Biol 2021. PubMed ID: 34278243

Abstract

Blunt force injuries are a significant cause of disability and death worldwide. This study describes a Drosophila melanogaster model of blunt force injury that can be used to investigate cellular and molecular mechanisms that underlie the short-term and long-term effects of injuries sustained at a juvenile stage of development. Injuries inflicted in late third-instar larvae using the spring-based High-Impact Trauma (HIT) device robustly activated the humoral defense response process of melanization and caused larval and pupal lethality. Additionally, adults that developed from injured larvae had reduced lifespans, indicating that cellular and molecular mechanisms activated by blunt force injuries in larvae persist through metamorphosis and adult development. Previously, the HIT device has been used to investigate genetic and environmental factors underlying mechanisms that contribute to consequences of blunt force injuries incurred in adult flies. This work expands use of the HIT device to a juvenile stage of development, offering the opportunity to investigate whether the consequences of blunt force injuries involve different factors and mechanisms at different stages of development.
Scharenbrock, A. R., Katzenberger, R. J., Fischer, M. C., Ganetzky, B. and Wassarman, D. A. (2021). Beta-blockers reduce intestinal permeability and early mortality following traumatic brain injury in Drosophila. MicroPubl Biol 2021. PubMed ID: 34723144
Summary:

Abstract
Traumatic brain injury (TBI) frequently leads to non-neurological consequences such as intestinal permeability. The beta-blocker drug labetalol, which inhibits binding of catecholamine neurotransmitters to adrenergic receptors, reduces intestinal permeability in a rat TBI model. Using a Drosophila melanogaster TBI model, previous studies found a strong positive correlation between intestinal permeability and mortality within 24 hours of TBI in a standard laboratory line (w1118) and across genetically diverse inbred lines from the Drosophila Genetic Reference Panel (DGRP). This study reports that feeding injured w1118 flies the beta-blockers labetalol and metoprolol reduced intestinal permeability and mortality. Additionally, metoprolol reduced intestinal permeability when 18 DGRP fly lines were analyzed in aggregate, but neither beta-blocker affected mortality. These data indicate that the mechanism underlying disruption of the intestinal barrier by adrenergic signaling following TBI is conserved between humans and flies and that mortality following TBI in flies is not strictly dependent on disruption of the intestinal barrier. Thus, the fly TBI model is useful for shedding light on the mechanism and consequences of adrenergic signaling between the brain and intestine following TBI in humans.
Crocker, K. L., Marischuk, K., Rimkus, S. A., Zhou, H., Yin, J. C. P. and Boekhoff-Falk, G. (2021). Neurogenesis in the adult Drosophila brain. Genetics. PubMed ID: 34117750

Abstract

Neurodegenerative diseases such as Alzheimer's and Parkinson's currently affect ∼25 million people worldwide. The global incidence of traumatic brain injury (TBI) is estimated at ∼70 million/year. Both neurodegenerative diseases and TBI remain without effective treatments. This study utilized adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long term goal of identifying targets for neural regenerative therapies. This study specifically focused on neurogenesis, i.e. the generation of new cells, as opposed to the regrowth of specific subcellular structures such as axons. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a Penetrating Traumatic Brain Injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. Both new glia and new neurons were detected, along with the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within two weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. It is anticipated that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair (Crocker, 2021).
Shorey, M., Stone, M. C., Mandel, J. and Rolls, M. M. (2020). Neurons survive simultaneous injury to axons and dendrites and regrow both types of processes in vivo. Dev Biol. PubMed ID: 32687893

Abstract
Neurons extend dendrites and axons to receive and send signals. If either type of process is removed, the cell cannot function. Rather than undergoing cell death, some neurons can regrow axons and dendrites. Axon and dendrite regeneration have been examined separately and require sensing the injury and reinitiating the correct growth program. Whether neurons in vivo can sense and respond to simultaneous axon and dendrite injury with polarized regeneration has not been explored. To investigate the outcome of simultaneous axon and dendrite damage, a Drosophila model system was used in which neuronal polarity, axon regeneration, and dendrite regeneration have been characterized. After removal of the axon and all but one dendrite, the remaining dendrite was converted to a process that had a long unbranched region that extended over long distances and a region where shorter branched processes were added. These observations suggested axons and dendrites could regrow at the same time. To further test the capacity of neurons to implement polarized regeneration after axon and dendrite damage, all neurites were removed from mature neurons. In this case a long unbranched neurite and short branched neurites were regrown from the stripped cell body. Moreover, the long neurite had axonal plus-end-out microtubule polarity and the shorter neurites had mixed polarity consistent with dendrite identity. The long process also accumulated endoplasmic reticulum at its tip like regenerating axons. It is concluded that neurons in vivo can respond to simultaneous axon and dendrite injury by initiating growth of a new axon and new dendrites.
Saikumar, J., Byrns, C. N., Hemphill, M., Meaney, D. F. and Bonini, N. M. (2020). Dynamic neural and glial responses of a head-specific model for traumatic brain injury in Drosophila. Proc Natl Acad Sci U S A 117(29): 17269-17277. PubMed ID: 32611818

Abstract
Traumatic brain injury (TBI) is the strongest environmental risk factor for the accelerated development of neurodegenerative diseases. There are currently no therapeutics to address this due to lack of insight into mechanisms of injury progression, which are challenging to study in mammalian models. This study has developed and extensively characterized a head-specific approach to TBI in Drosophila, a powerful genetic system that shares many conserved genes and pathways with humans. The Drosophila TBI (dTBI) device inflicts mild, moderate, or severe brain trauma by precise compression of the head using a piezoelectric actuator. Head-injured animals display features characteristic of mammalian TBI, including severity-dependent ataxia, life span reduction, and brain degeneration. Severe dTBI is associated with cognitive decline and transient glial dysfunction, and stimulates antioxidant, proteasome, and chaperone activity. Moreover, genetic or environmental augmentation of the stress response protects from severe dTBI-induced brain degeneration and life span deficits. Together, these findings present a tunable, head-specific approach for TBI in Drosophila that recapitulates mammalian injury phenotypes and underscores the ability of the stress response to mitigate TBI-induced brain degeneration.
Shah, E. J., Gurdziel, K. and Ruden, D. M. (2020). Drosophila Exhibit Divergent Sex-Based Responses in Transcription and Motor Function After Traumatic Brain Injury. Front Neurol 11: 511. PubMed ID: 32636795

Abstract
Every year, millions of people in the US suffer brain damage from mild to severe traumatic brain injuries (TBI) that result from a sudden impact to the head. Despite TBI being a leading cause of death and disability worldwide, sex differences that contribute to varied outcomes post-injury are not extensively studied and therefore, poorly understood. This study aimed to explore biological sex as a variable influencing response to TBI using Drosophila melanogaster as a model, since flies have been shown to exhibit symptoms commonly seen in other mammalian models of TBI. After inflicting TBI using the high-impact trauma device, w (1118) fly brains were isolated and gene transcription changes were assessed in male and female flies at control and 1, 2, and 4 hr after TBI. The results suggest that overall, Drosophila females show more gene transcript changes than males. Females also exhibit upregulated expression changes in immune response and mitochondrial genes across all time-points. In addition, the impact of injury on mitochondrial health and motor function was assessed in both sexes before and after injury. Although both sexes report similar changes in mitochondrial oxidation and negative geotaxis, locomotor activity appears to be more impaired in males than females. These data suggest that sex-differences not only influence the response to TBI but also contribute to varied outcomes post-injury.

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Trichohepatoenteric syndrome

Ohnuma, K., Kishita, Y., Nyuzuki, H., Kohda, M., Ohtsu, Y., Takeo, S., Asano, T., Sato-Miyata, Y., Ohtake, A., Murayama, K., Okazaki, Y. and Aigaki, T. (2020). Ski3/TTC37 deficiency associated with trichohepatoenteric syndrome causes mitochondrial dysfunction in Drosophila. FEBS Lett. PubMed ID:
32294252

Abstract

Tetratricopeptide repeat protein 37 (TTC37) is a causative gene of trichohepatoenteric syndrome (THES). However, little is known about the pathogenesis of this disease. This study characterized the phenotype of a Drosophila model in which ski3, a homolog of TTC37, is disrupted. The mutant flies are pupal lethal, and the pupal lethality is partially rescued by transgenic expression of wild-type ski3 or human TTC37. The mutant larvae show growth retardation, heart arrhythmia, triacylglycerol accumulation, and aberrant metabolism of glycolysis and the TCA cycle. Moreover, mitochondrial membrane potential and respiratory chain complex activities are significantly reduced in the mutants. These results demonstrate that ski3 deficiency causes mitochondrial dysfunction, which may underlie the pathogenesis of THES (Ohnuma, 2020).

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Triosephosphate isomerase (TPI) deficiency

Hrizo, S. L., Eicher, S. L., Myers, T. D., McGrath, I., Wodrich, A. P. K., Venkatesh, H., Manjooran, D., Swoger, S., Gagnon, K., Bruskin, M., Lebedev, M. V., Zheng, S., Vitantonio, A., Kim, S., Lamb, Z. J., Vogt, A., Ruzhnikov, M. R. Z. and Palladino, M. J. (2021). Identification of protein quality control regulators using a Drosophila model of TPI deficiency. Neurobiol Dis 152: 105299. PubMed ID:
33600953

Abstract
Triosephosphate isomerase (TPI) deficiency (Df) is a rare recessive metabolic disorder that manifests as hemolytic anemia, locomotor impairment, and progressive neurodegeneration. Drosophila with the recessive TPI (sugarkill) allele (a.k.a. sgk or M81T) exhibit progressive locomotor impairment, neuromuscular impairment and reduced longevity, modeling the human disorder. TPI(sugarkill) produces a functional protein that is degraded by the proteasome. Molecular chaperones, such as Hsp70 and Hsp90, have been shown to contribute to the regulation of TPI(sugarkill) degradation. In addition, stabilizing the mutant protein through chaperone modulation results in improved TPI deficiency phenotypes. To identify additional regulators of TPI(sugarkill) degradation, a genome-wide RNAi screen was performed that targeted known and predicted quality control proteins in the cell to identify novel factors that modulate TPI(sugarkill) turnover. Of the 430 proteins screened, 25 regulators of TPI(sugarkill) were identified. Proteins involved in co-translational protein quality control and ribosome function were isolated in the screen, suggesting that TPI(sugarkill) may undergo co-translational selection for polyubiquitination and proteasomal degradation as a nascent polypeptide. The proteins identified in this study may reveal novel pathways for the degradation of a functional, cytosolic protein by the ubiquitin proteasome system and define therapeutic pathways for TPI Df and other biomedically important diseases.

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Tuberous Sclerosis Complex

Kosillo, P., Doig, N. M., Ahmed, K. M., Agopyan-Miu, A., Wong, C. D., Conyers, L., Threlfell, S., Magill, P. J. and Bateup, H. S. (2019). Tsc1-mTORC1 signaling controls striatal dopamine release and cognitive flexibility. Nat Commun 10(1): 5426. PubMed ID:
31780742

Abstract

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 (see Drosophila Tsc1) or TSC2 (see Drosophila Gigas), which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. This study used cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. Loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor (see Drosophila Raptor). These results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC (Kosillo, 2019).

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Schizophrenia

Wakisaka, K. T., Muraoka, Y., Shimizu, J., Yamaguchi, M., Ueoka, I., Mizuta, I., Yoshida, H. and Yamaguchi, M. (2019). Drosophila Alpha-ketoglutarate-dependent dioxygenase AlkB is involved in repair from neuronal disorders induced by ultraviolet damage. Neuroreport. PubMed ID:
31503204

Abstract

AlkB family proteins are enzymes that repair alkylated DNA and RNA by oxidative demethylation. Nine homologs have been identified and characterized in mammals. ALKBH1 is conserved among metazoans including Drosophila. Although the ALKBH1 mouse homolog, Alkbh1 functions in neurogenesis, it currently remains unclear whether ALKBH1 plays a role in neuronal disorders induced by ultraviolet-induced DNA damage. This study has demonstrated that the Drosophila ALKBH1 homolog, AlkB contributed to recovery from neuronal disorders induced by ultraviolet damage. The knockdown of AlkB resulted in not only learning defects but also altered crawling behavior in Drosophila larvae after ultraviolet irradiation. A molecular analysis revealed that AlkB contributed to the repair of ultraviolet-induced DNA damage in the central nervous system of larvae. Therefore, it is proposed that ALKBH1 plays a role in the repair of ultraviolet-induced DNA damage in central nervous system. Ultraviolet-induced DNA damage is involved in the pathogenesis of xeroderma pigmentosum, and has recently been implicated in Parkinson's disease. The present results will contribute to understanding of neuronal diseases induced by ultraviolet-induced DNA damage (Wakisaka, 2019).

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Warburg Micro Syndrome

Takats, S., Levay, L., Boda, A., Toth, S., Simon-Vecsei, Z., Rubics, A., Varga, A., Lippai, M., Lorincz, P., Glatz, G. and Juhasz, G. (2020). The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I. FEBS J. PubMed ID:
32248620

Abstract

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. This work characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. These mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. This study finds that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, this study shows that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. These data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS (Takats, 2020).

Warburg micro syndrome (hereafter WMS) and Martsolf syndrome are severe and milder forms of the same hereditary neuromuscular disorder, respectively. Patients suffer from motor disorders, spastic paraplegia, cataract, and mental retardation. Both syndromes are caused by homozygous loss-of-function mutations affecting one of the RAB18, RAB3GAP1, RAB3GAP2, or TBC1D20 genes. Although dozens of disease-causing mutations were identified in these genes, how these lead to the onset of WMS is still poorly understood (Takats, 2020).

Rab18 is a member of the Rab GTPase family proteins, which are the main regulators of endomembrane trafficking. Rabs are switch-like proteins: They bind or release their effector proteins (tethers, motor adaptors, kinases) in their GTP- or GDP-bound states, respectively. The switch between GTP and GDP depends on specific regulatory proteins: guanosine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs promote the exchange of GDP for GTP; hence, they activate a given Rab. In contrast, GAPs promote GTPase activity and inactivation of their partner Rabs. The activating GEF for Rab18 is the heterodimer Rab3GAP complex, which consists of the catalytic Rab3GAP1 and noncatalytic Rab3GAP2 subunits. The Rab3GAP complex acts as a bivalent Rab regulator: In addition to its GEF function toward Rab18, it also serves as a GAP for Rab3. As WMS-associated Rab3 mutations have not been identified yet, the onset of WMS in Rab3GAP mutant patients is most likely due to the loss of Rab18 GEF function. Rab18 was found to localize to the ER and lipid droplets, but some studies showed that Rab18 is also present on the Golgi apparatus and on endosomes. Although it was previously demonstrated that Rab18 is involved in ER-lipid droplet tethering, regulation of lipid droplet dynamics, and replication of various viruses, there is no clear evidence for the involvement of these processes in the occurrence of WMS. As both Rab18 and Rab3GAP are regulators of endomembrane trafficking, it seems possible that a certain vesicle transport route is impaired in WMS patients (Takats, 2020).

Autophagy is an evolutionarily conserved catabolic pathway of eukaryotic cells during which cells degrade aged, superfluous or damaged proteins or organelles through the lysosomal pathway. During the process of macroautophagy (hereafter autophagy), a specific membrane cistern called phagophore emerges in the cytoplasm; then, by sealing of its edges it engulfs cytoplasmic material into a double-membrane vesicle called autophagosome. Autophagosomes eventually fuse with lysosomes and give rise to autolysosomes in which the cargo gets degraded and the resulting organic monomers are released to fuel energy-producing or anabolic pathways. As a recycling and self-renewal process, autophagy contributes to maintaining the health of terminally differentiated cells (such as neurons or muscle fibers) and serves as an important general response to stress (e.g., starvation, presence of oxidative agents) in all cell types. Perturbation of autophagy, among others, can contribute to cancer, impaired immunity, and early aging. Additionally, there is a large body of evidence that flies, mice, and humans defective for autophagy suffer from neuromuscular disorders and progressive neurodegeneration (Takats, 2020).

The autophagic pathway converges with endocytosis (the other main lysosomal degradative pathway) at the level of lysosomes, and several Rab proteins are known to regulate both processes. It has been recently demonstrated that autophagosome–lysosome fusion is promoted by Rab2 and Rab7. While early endosomal Rab5 also enhances autolysosomal degradation, it regulates this process via promoting lysosomal maturation through regulating the phosphatidylinositol 3-phosphate (PI3P) content of endosomal membranes (Takats, 2020).

Phosphatidylinositol 3-phosphate is produced by the class III phosphatidylinositol 3-kinase (PI3K) complex. The core of the complex consists of three permanent subunits: the catalytic Vps34 lipid kinase and the two regulatory subunits, Vps15 and Atg6. The core complex can bind one from the two alternative and mutually exclusive subunits, Atg14 or UVRAG, which regulate its activity and organelle specificity. The complex consisting of the heterotrimeric core and Atg14 is also known as Vps34 Complex I, and it is considered to promote autophagosome formation. In contrast, the Vps34 Complex II that contains the heterotrimeric core and UVRAG produces PI3P on endosomes and promotes their maturation to late endosomes and lysosomes. It is important to note that further facultative subunits have been described in addition to Atg14 and UVRAG such as Atg38/NRBF2. Since their activity is still dependent on the presence of Atg14 or UVRAG, they do not define a new Vps34 complex type. Hereafter, the Atg14- or UVRAG-containing heterotetramers will be referred to as Vps34 Complex I or Vps34 Complex II, respectively. The Vps34 Complex II acts as a Rab5 effector on endosomal membranes, while emerging evidence suggests that the autophagosomal activity of Complex I is independent of Rab5. However, the possibility cannot be ruled out that Complex I acts as an effector of another Rab protein (Takats, 2020).

Although a few previous studies suggested that loss of Rab18 or the Rab3GAP complex influences autophagy, their precise role in the regulation of this process and whether it can be connected to WMS is still poorly understood. This study established a new Rab3GAP2-deficient Drosophila model for WMS and demonstrate that Rab3GAP-Rab18 module, through its interaction with the Vps34 Complex I, regulates autophagy by promoting the maturation of lysosomes/autolysosomes (Takats, 2020).

This study demonstrates that Rab3GAP2 mutant fruit flies display ataxia and may be used as an invertebrate model system for WMS. Furthermore, the climbing defect may at least in part arise due to an autophagy failure obvious in adult muscle and larval fat cells. The finding that the accumulation of p62 (refractory to sigma P) and Atg8a was not apparent in Rab3GAP2 mutant head lysates was surprising; but, it is possible that only a subset of neurons is defective for autophagy in these flies, which this study failed to detect. Although studies in mammalian WMS models are focusing on neuronal defects, the data suggest that it may be worth investigating other tissues of WMS patients as well (Takats, 2020).

For characterizing the role of the Rab3GAP-Rab18 module in autophagy, the genetically mosaic fat tissue of L3 larvae, a well-established system for autophagy analysis in Drosophila, was used. By analyzing Rab3GAP2 mutants and multiple independent RNAi lines, this study shows that the loss-of-function cells are less effective in autophagosome–lysosome fusion and are defective in autolysosome morphology and maturation. Additionally, it was found that the lack of Rab3GAP2 function causes striking perturbation of Rab7-positive late endosomes, autophagosomes, and (auto)lysosomes, but it does not affect Rab5-positive early endosomes. Thus, the data suggest that the Rab3GAP-Rab18 module can be considered as a general regulator of lysosome maturation. These results fit well with the findings of a recently published paper showing that Rab18 acts in concert with Rab7 during the lysosomal fusion events of autophagy and in the axonal transport of lysosomes. On the other hand, previous studies in C. elegans and cultured human cells suggested that the Rab3GAP subunits and Rab18 are rather involved in early steps of autophagy; however, it is important to note that these latter studies focused only on the amount and localization patterns of the autophagy markers Atg8 and p62, with no particular emphasis on ultrastructural analysis of the integrity of the lysosomal system. Electron microscopy observations concerning that numerous autophagosomes and autophagosome clusters are present in the cytoplasm of Rab3GAP2 mutant fat cells further suggest that loss of the Rab3GAP-Rab18 module causes major defects in (auto)lysosome function rather than in autophagosome formation. Of course, the possibility that the discrepancies between these studies arise due to a tissue-specific role of the Rab3GAP-Rab18 module in autophagy cannot be ruled out. The finding that Rab3GAP2 mutant adults obviously accumulate much more autophagy cargos in muscles than in their brain further corroborates this notion (Takats, 2020).

Since this study demonstrated that loss of Vps34 Complex I function results in phenotypes similar to those of inhibition of the Rab3GAP-Rab18 module and, furthermore, a physical interaction between the permanent Vps34 complex subunit Atg6 and the GTP-bound Rab18 was proven, it is proposed that Complex I is likely a novel Rab18 effector (Takats, 2020).

Vps34 Complex I is one of the most important regulators of autophagosome formation, and it also has a role in autophagosome maturation and fusion [(Diao, 2015). As matured, intact autophagosomes in Rab3GAP2 mutant cells were detected, it seems likely that Rab18 is critical for Vps34 Complex I activity following autophagosome formation. Localization of Rab3GAP subunits and Rab18 to autophagosomes also suggests that this module plays a role in later steps of autophagosome maturation: It facilitates their fusion with lysosomes and further enhances the maturation of the newly formed autolysosomes into enlarged degradative compartments (Takats, 2020).

The question of how could the autophagosome-localized Rab3GAP-Rab18 module affect vesicle maturation is yet to be answered. Based on the current results showing that Rab7 becomes dispersed in cells lacking the Rab3GAP-Rab18 module, it is suggested that the most important role of this module is to stabilize the Rab7-containing compartment. During their maturation, (auto)lysosomes undergo a series of membrane fusion events with endosomes, Golgi-derived vesicles, and autophagosomes. All these steps are mediated by Rab7 and its effectors such as the tethering factor HOPS complex or the adaptor protein PLEKHM1. As matured autophagosomes are also positive for Rab7, the autophagy-derived Rab7 proteins can be an important source for the lysosomal Rab7 pool. This scenario is even more likely in cell types such as fat cells, which show relatively low endocytic but high autophagic activity. Still, it cannot be ruled out that the Rab3GAP-Rab18 complex is also present on maturing endosomes or Golgi-derived transport vesicles that may also act as important Rab7 sources for maturing lysosomes. The precise contribution of these membrane transport pathways to maintaining the lysosomal Rab7 pool needs to be further investigated in the future (Takats, 2020).

This research highlights that the Rab3GAP-Rab18 module, in concert with the activity of the Vps34 Complex I, maintains the integrity of the Rab7-positive late endosomal/lysosomal compartment. Additionally, these findings that loss of Rab3GAP-Rab18 function perturbs autolysosome maturation and autophagic degradation shed light on a new possible cause of WMS development and open up potential novel therapeutic perspectives for this disease (Takats, 2020).

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Werner syndrome

Cassidy, D., Epiney, D., Salameh, C., Zhou, L. T., Salomon, R. N., Schirmer, A. E., McVey, M. and Bolterstein, E. (2019). Evidence for premature aging in a Drosophila model of Werner syndrome. Exp Gerontol: 110733. PubMed ID:
31518666

Abstract

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by patients' early onset of aging, increased risk of cancer and other age-related pathologies. WS is caused by mutations in WRN, a RecQ helicase that has essential roles responding to DNA damage and preventing genomic instability. While human WRN has both an exonuclease and helicase domain, Drosophila WRNexo has high genetic and functional homology to only the exonuclease domain of WRN. Like WRN-deficient human cells, Drosophila WRNexo null mutants (WRNexo(Delta)) are sensitive to replication stress, demonstrating mechanistic similarities between these two models. Compared to age-matched wild-type controls, WRNexo(Delta) flies exhibit increased physiological signs of aging, such as shorter lifespans, higher tumor incidence, muscle degeneration, reduced climbing ability, altered behavior, and reduced locomotor activity. Interestingly, these effects are more pronounced in females suggesting sex-specific differences in the role of WRNexo in aging. This and future mechanistic studies will contribute to linking faulty DNA repair mechanisms with the process of aging (Cassidy, 2019).

Fang, E. F., Hou, Y., Lautrup, S., Jensen, M. B., Yang, B., SenGupta, T., Caponio, D., Khezri, R., Demarest, T. G., Aman, Y., Figueroa, D., Morevati, M., Lee, H. J., Kato, H., Kassahun, H., Lee, J. H., Filippelli, D., Okur, M. N., Mangerich, A., Croteau, D. L., Maezawa, Y., Lyssiotis, C. A., Tao, J., Yokote, K., Rusten, T. E., Mattson, M. P., Jasper, H., Nilsen, H. and Bohr, V. A. (2019). NAD(+) augmentation restores mitophagy and limits accelerated aging in Werner syndrome. Nat Commun 10(1): 5284. PubMed ID: 31754102

Abstract

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. This study reports impaired mitophagy and depletion of NAD(+), a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD(+) biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD(+) repletion restores NAD(+) metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD(+) repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. These findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD(+) levels counteracts WS phenotypes (Fang, 2019).

Cox, R. L., Hofley, C. M., Tatapudy, P., Patel, R. K., Dayani, Y., Betcher, M. and LaRocque, J. R. (2019). Functional conservation of RecQ helicase BLM between humans and Drosophila melanogaster. Sci Rep 9(1): 17527. PubMed ID: 31772289

Abstract

RecQ helicases are a family of proteins involved in maintaining genome integrity with functions in DNA repair, recombination, and replication. The human RecQ helicase family consists of five helicases: BLM, WRN, RECQL, RECQL4, and RECQL5. Inherited mutations in RecQ helicases result in Bloom Syndrome (BLM mutation), Werner Syndrome (WRN mutation), Rothmund-Thomson Syndrome (RECQL4 mutation), and other genetic diseases, including cancer. The RecQ helicase family is evolutionarily conserved, as Drosophila melanogaster have three family members: DmBlm, DmRecQL4, and DmRecQL5 and DmWRNexo, which contains a conserved exonuclease domain. DmBlm has functional similarities to human BLM (hBLM) as mutants demonstrate increased sensitivity to ionizing radiation (IR) and a decrease in DNA double-strand break (DSB) repair. To determine the extent of functional conservation of RecQ helicases, hBLM was expressed in Drosophila using the GAL4 > UASp system to determine if GAL4 > UASp::hBLM can rescue DmBlm mutant sensitivity to IR. hBLM was able to rescue female DmBlm mutant sensitivity to IR, supporting functional conservation. This functional conservation is specific to BLM, as human GAL4 > UASp::RECQL was not able to rescue DmBlm mutant sensitivity to IR. These results demonstrate the conserved role of BLM in maintaining the genome while reinforcing the applicability of using Drosophila as a model system to study Bloom Syndrome (Cox, 2019)

Fang, E. F., Hou, Y., Lautrup, S., Jensen, M. B., Yang, B., SenGupta, T., Caponio, D., Khezri, R., Demarest, T. G., Aman, Y., Figueroa, D., Morevati, M., Lee, H. J., Kato, H., Kassahun, H., Lee, J. H., Filippelli, D., Okur, M. N., Mangerich, A., Croteau, D. L., Maezawa, Y., Lyssiotis, C. A., Tao, J., Yokote, K., Rusten, T. E., Mattson, M. P., Jasper, H., Nilsen, H. and Bohr, V. A. (2019). NAD(+) augmentation restores mitophagy and limits accelerated aging in Werner syndrome. Nat Commun 10(1): 5284. PubMed ID: 31754102

Abstract

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. This study reports impaired mitophagy and depletion of NAD(+), a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD(+) biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD(+) repletion restores NAD(+) metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD(+) repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. these findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD(+) levels counteracts WS phenotypes (Fang, 2019).

Epiney, D. G., Salameh, C., Cassidy, D., Zhou, L. T., Kruithof, J., Milutinovic, R., Andreani, T. S., Schirmer, A. E. and Bolterstein, E. (2021). Characterization of Stress Responses in a Drosophila Model of Werner Syndrome. Biomolecules 11(12). PubMed ID: 34944512
Summary:
As organisms age, their resistance to stress decreases while their risk of disease increases. This can be shown in patients with Werner syndrome (WS), which is a genetic disease characterized by accelerated aging along with increased risk of cancer and metabolic disease. WS is caused by mutations in WRN, a gene involved in DNA replication and repair. Recent research has shown that WRN mutations contribute to multiple hallmarks of aging including genomic instability, telomere attrition, and mitochondrial dysfunction. However, questions remain regarding the onset and effect of stress on early aging. This study used a fly model of WS (WRNexo(Δ)) to investigate stress response during different life stages; stress sensitivity was found to vary according to age and stressor. While larvae and young WRNexo(Δ) adults are not sensitive to exogenous oxidative stress, high antioxidant activity suggests high levels of endogenous oxidative stress. WRNexo(Δ) adults are sensitive to stress caused by elevated temperature and starvation suggesting abnormalities in energy storage and a possible link to metabolic dysfunction in WS patients. Higher levels of sleep were observed in aged WRNexo(Δ) adults suggesting an additional adaptive mechanism to protect against age-related stress. It is suggested that stress response in WRNexo(Δ) is multifaceted and evokes a systemic physiological response to protect against cellular damage. These data further validate WRNexo(Δ) flies as a WS model with which to study mechanisms of early aging and provide a foundation for development of treatments for WS and similar diseases.

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Wilms' Tumor

Towler, B. P., Pashler, A. L., Haime, H. J., Przybyl, K. M., Viegas, S. C., Matos, R. G., Morley, S. J., Arraiano, C. M. and Newbury, S. F. (2020). Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism. PLoS Genet 16(12): e1009297. PubMed ID:
33370287

Abstract

Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to were generated demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, RNA-seq was used on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. This study also demonstrated that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, it was shown that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which was subsequently shown to contribute towards the proliferation phenotype in Drosophila. This work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth (Towler, 2020).


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    Williams-Beuren Syndrome

    Medvedeva, A. V., Tokmatcheva, E. V., Kaminskaya, A. N., Vasileva, S. A., Nikitina, E. A., Zhuravlev, S. A., Zakharov, G. A., Zatsepina, O. G. and Savvateeva-Popova, E. V. (2021). Parent-of-origin effects on nuclear chromatin organization and behavior in a Drosophila model for Williams-Beuren Syndrome. Vavilovskii Zhurnal Genet Selektsii 25(5): 472-485. PubMed ID:
    34595370

    Abstract

    Prognosis of neuropsychiatric disorders in progeny requires consideration of individual parent-of-origin effects (POEs) relying on the nerve cell nuclear 3D chromatin architecture and impact of parent-specific miRNAs. Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting, or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMP dependent, the second relies on actin remodeling by small GTPase Rac1 - LIMK1 (LIM-kinase 1). Simple experimental model systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies. This study has developed a Drosophila model for Williams-Beuren Syndrome (WBS): a mutant agn(ts3) of the agnostic locus (X:11AB) harboring the dlimk1 gene. The agn(ts3) mutation drastically increases the frequency of ectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affects locomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between agn(ts3) and the wild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal gene origin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with the occurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat. Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-term memory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Several parameters of locomotion demonstrate heterosis. These data indicate that the agn(ts3) locus is capable of trans-regulating gene activity via POEs on the chromatin nuclear organization, thereby affecting behavior (Medvedeva, 2021).

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    Wolfram syndrome

    Sakakibara, Y., Sekiya, M., Fujisaki, N., Quan, X. and Iijima, K. M. (2018). Knockdown of wfs1, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in Drosophila. PLoS Genet 14(1): e1007196. PubMed ID:
    29357349

    Abstract

    Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic beta-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. This study investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases (Sakakibara, 2018).

    Most WS patients die prematurely in association with severe neurological disabilities as a result of brain atrophy; however, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. This study investigated the effects of knockdown of wfs1, a fly homolog of WFS1, in the nervous system on neuronal integrity during aging. The results demonstrated that wfs1 expression was induced upon aging, and neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration (Sakakibara, 2018).

    The process by which wfs1 deficiency induced behavioral deficits and neurodegeneration in the Drosophila model remained unclear. In pancreatic β cells, WFS1 deficiency causes chronic ER stress and subsequent cell death through the dysregulation of intracellular Ca2+ levels and disruption of ER homeostasis. In addition, a recent report using primary cultured neurons demonstrated that WFS1 deficiency alters Ca2+ homeostasis in the ER and promotes mitophagy, leading to alterations in mitochondrial distribution and dynamics. This study found that knockdown of wfs1 in the nervous system did not induce ER stress or mitochondrial dysfunction even in aged fly brains. Moreover, autophagy flux was not significantly affected in these flies. These results suggest that reductions in wfs1 function alone do not severely disrupt cellular functions, which directly causes neurodegeneration. Rather, wfs1 may play a protective role against various stressors associated with aging, and its deficiency increased the susceptibility to neurodegeneration in aged flies (Sakakibara, 2018).

    In support of this hypothesis, wfs1 expression was induced by aging and stressors, and neuronal knockdown of wfs1 rendered neurons vulnerable to oxidative stress-, altered neuronal excitability- or Alzheimer's associated tauopathy-induced behavioral deficits and neurodegeneration. Moreover, although knockdown of wfs1 in neurons did not induce obvious stress responses (Figs 4 and 5C), wfs1 deficiency significantly augmented oxidative stress and ER stress responses under stressed conditions (Fig 6F and 6G). These results suggest that wfs1 deficiency modifies the onset, progression and severity of neurological and neurodegenerative phenotypes caused by a combination of complex environmental, genetic, and age-associated factors (Sakakibara, 2018).

    In this study, knockdown of wfs1 in neurons and glial cells significantly exaggerated behavioral deficits and premature death, suggesting that neuronal and glial wfs1 work in concert to maintain neuronal integrity in flies. The mRNA expression levels of Eaat1 and Grd, which are fly orthologs of glial glutamate transporters (SLC1A3) and GABA-A receptor α subunit (GABRA6), respectively, were significantly altered in these fly brains. Moreover, loss of one copy of Eaat1 increased locomotor deficits induced by wfs1 deficiency, whereas feeding of riluzole, which is thought to inhibit glutamate release as well as GABA uptake, significantly suppressed premature death phenotypes in these flies. These results suggest that stresses caused by altered neuronal excitability, possibly due to an imbalance between glutamatergic and GABAergic tones, may underlie the observed behavioral deficits in flies with wfs1 deficiency (Sakakibara, 2018).

    Recent reports suggest that the neurological manifestations of WFS1 deficiency are associated with endocrine problems. In addition to the ER, the WFS1 protein localizes to secretory granules and regulates intragranular acidification, proinsulin processing, and exocytosis in pancreatic β cells, in part by interacting with the V1A subunit of H+ V-ATPase. Consistent with this, vasopressin processing is defective in some hypothalamic nuclei in the brain of WS patients, and the processing and secretion of growth and trophic factors is impaired in Wfs1-deficient mice. This study examined whether knockdown of wfs1 in neurons and glial cells alters the levels of a fly ortholog of Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) in fly brains. Western blot analyses revealed that the level of MANF was not significantly altered in fly brains. Further analysis is required to clarify the mechanism by which wfs1 functions in neurons and glial cells to maintain neuronal function and integrity in fly brains (Sakakibara, 2018).

    The wfs1 mutant and RNAi lines used in this study did not completely abolish wfs1 expression and function in flies. This indicates that some of the phenotypes induced by knockdown of wfs1 may be different from those caused by complete null mutation of wfs1. Nevertheless, this study demonstrated that wfs1 deficiency in the nervous system is sufficient to render neurons vulnerable to age-associated neurodegeneration in Drosophila. The results also highlight the importance of the functions of wfs1 in both neuronal and glial cells. Moreover, the data revealed a novel genetic interaction between wfs1 and Eaat1, and suggest that altered neural activity and vulnerability to oxidative stress underlie neurological phenotypes in wfs1 deficient flies (Sakakibara, 2018).

    In conclusion, this study provides insight into the molecular mechanisms underlying neurodegeneration in WS and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases such as Alzheimer's disease (Sakakibara, 2018).

    Hao, H., Song, L. and Zhang, L.(2023). Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis. PLoS Genet 19(7): e1010827. PubMed ID: 37399203
    Summary:

    Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

    Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

    Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

    Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

    In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

    MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

    GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

    It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

    The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

    In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

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    Zika

    Tafesh-Edwards, G., Kalukin, A., Bunnell, D., Chtarbanova, S. and Eleftherianos, I. (2023).Temperature and sex shape Zika virus pathogenicity in the adult Brat (cheesehead) brain: A Drosophila model for virus-associated neurological diseases. iScience 26(4): 106424. PubMed ID:
    37009222

    Abstract

    Severe neurological complications affecting brain growth and function have been well documented in newborn and adult patients infected by Zika virus (ZIKV), but the underlying mechanisms remain unknown. This study used a Drosophila melanogaster mutant, cheesehead (chs), with a mutation in the brain tumor (brat) locus that exhibits both aberrant continued proliferation and progressive neurodegeneration in the adult brain. This study reports that temperature variability is a key driver of ZIKV pathogenesis, thereby altering host mortality and causing motor dysfunction in a sex-dependent manner. Furthermore, it was shown that ZIKV is largely localized to the brat (chs) brain and activates the RNAi and apoptotic immune responses. These findings establish an in vivo model to study host innate immune responses and highlight the need of evaluating neurodegenerative deficits as a potential comorbidity in ZIKV-infected adults (Tafesh-Edwards 2023).

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    Zinc Deficiency

    Philipsen, M., Gu, C. and Ewing, A. G. (2020). Imaging Mass Spectrometry Shows That Zinc Deficiency Leads to Lipids Changes in Drosophila Brain Similar to Cognitive Impairing Drugs. Chembiochem. PubMed ID:
    32402134

    Abstract
    Several diseases and disorders have been suggested to be associated with zinc deficiency, especially learning and memory impairments. To have better understanding about the connection between lipid changes and cognitive impairments, this study investigated the effects of zinc chelated diet on certain brain lipids of Drosophila melanogaster using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The data revealed that there are increases in the levels of phosphatidyl choline and phosphatidylinositol in the central brains of the zinc deficient flies compared to the control flies. In contrast, the abundance of phosphatidylethanolamine in the brains of the zinc deficient flies is lower. These data are consistent with that of cognitive diminishing drugs providing insight into the biological and molecular effects of zinc deficiency on the major brain lipids and opening a new treatment target for cognitive deficit in zinc deficiency (Philipsen, 2020).

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    Date revised: 15 May 2022

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