The InteractiveFly: Drosophila as a Model for Human Diseases


Miscellaneous Drosophila Disease Models

Miscellaneous Drosophila Disease Models

Adrenoleukodystrophy

Adult-onset inherited myopathy

Alcoholism
Ataxia-telangiectasia

Berardinelli-Seip congenital lipodystrophy type 2

Cardiac lipotoxicity

Charcot-Marie-Tooth Disease

Colorectal cancer

Congenital disorders of glycosylation

Epilepsy and seizure disorders

Episodic ataxia

Freeman Sheldon Syndrome, a skeletal muscle contracture syndromes
Friedrich's ataxia, a recessive neurodegeneration disorder

Galactosemia

Glioblastoma

Human papillomavirus E6-induced malignancy

Hypertrophic cardiomyopathy
Inclusion body myopathy type 3

Insomnia

Intellectual Disability associated with WAC

Kohlschutter-Tonz syndrome

Laminopathy

Leigh Syndrome

Lysosomal storage disease

Mitochondrial disease

Myeloproliferative neoplasms

Myopathy

Myotonic Dystrophy

Nephrotic Syndrome

N-Glycanase 1 deficiency

Nonsyndromic X-linked mental retardation

Obesity

Peroxisome biogenesis disorders

Perry syndrome

Polyglutamine (polyQ) disorders

Prion disease

Retinitis pigmentosa

Schizophrenia

Skin cancer and nucleotide excision repair

Spinal muscular atrophy

Spinocerebellar ataxia

Squamous cell carcinoma

Wolfram syndrome
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

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

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

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).

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

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

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

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).

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Ataxia-telangiectasia

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).

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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

Abstract

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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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

Abstract

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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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.

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).

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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).

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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.

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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). 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

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). 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 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).

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Episodic Ataxia

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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Friedrich's ataxia, a recessive neurodegeneration disorder

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).

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Friedrich's ataxia, a recessive neurodegeneration disorder

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.


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).

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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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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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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. However, 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

The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production

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).

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Inclusion body myopathy type 3

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 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects. Dis Model Mech [Epub ahead of print]. 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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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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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).

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).

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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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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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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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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).

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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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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).

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Myotonic Dystrophy

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).


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. (2018). (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).

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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).

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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).

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Nonsyndromic X-linked mental retardation

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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Obesity

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). 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). 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. (2019). Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption. Nat Commun 10(1): 2700. PubMed ID: 31221967

Abstract

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). 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). 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

Dynamic mitochondrial responses to a high-fat diet in Drosophila melanogaster

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). 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. (2019). Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption. Nat Commun 10(1): 2700. PubMed ID: 31221967

Abstract

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).

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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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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).

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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).

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).

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Spinocerebellar ataxia

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).

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).

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).

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

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

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

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).

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

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).

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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).

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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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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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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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Spinocerebellar Ataxia

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).


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).

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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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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.

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Date revised: 15 April 2018

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