Abstract

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

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Abstract

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

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

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

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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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Abstract

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

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

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

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

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

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

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

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Abstract

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

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

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

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

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

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

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

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

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

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

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Abstract

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

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

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Abstract

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

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Abstract

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

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Abstract

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

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Abstract

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. This study performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, 60 additional prioritized candidate genes from these patients were tested for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs (Birker, 2023). Schroeder, A. M., Allahyari, M., Vogler, G., Missinato, M. A., Nielsen, T., Yu, M. S., Theis, J. L., Larsen, L. A., Goyal, P., Rosenfeld, J., Nelson, T. J., Olson, T. M., Colas, A. R., Grossfeld, P. and Bodmer, R. (2019). Model system identification of novel congenital heart disease gene candidates: focus on RPL13. Hum Mol Genet. PubMed ID: 31625562

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

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Abstract

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

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

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

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Abstract

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

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Abstract

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

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Abstract

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

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Abstract

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

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

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

Abstract

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

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

Abstract

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

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Abstract

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

Keshav, N., Ammankallu, R., Shashidhar, Paithankar, J. G., Baliga, M. S., Patil, R. K., Kudva, A. K. and Raghu, S. V. (2022) (2021). Dextran sodium sulfate alters antioxidant status in the gut affecting the survival of Drosophila melanogaster. 3 Biotech 12(10): 280. PubMed ID: 36275361

Abstract

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

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Abstract

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

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

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

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

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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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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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Abstract
e

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

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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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Abstract

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

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

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

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

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

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

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

Abstract

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

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

Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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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Abstract

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

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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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Abstract

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

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

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Abstract

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

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Abstract

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

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

Abstract

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

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Abstract

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

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

Abstract

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

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

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

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

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

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

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

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

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

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

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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).

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

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Abstract

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

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

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

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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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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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Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

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Abstract

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

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

Abstract

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

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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).

Abstract

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

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

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

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

Abstract

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

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

Abstract

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

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Abstract

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

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Abstract

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

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

Abstract

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

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Abstract

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

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

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

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Abstract

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

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

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

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

Abstract

Obesity is associated with cognitive decline. Recent observations in mice propose an adipose tissue (AT)-brain axis. This study identified 188 genes from RNA sequencing of AT in three cohorts that were associated with performance in different cognitive domains. These genes were mostly involved in synaptic function, phosphatidylinositol metabolism, the complement cascade, anti-inflammatory signaling, and vitamin metabolism. These findings were translated into the plasma metabolome. The circulating blood expression levels of most of these genes were also associated with several cognitive domains in a cohort of 816 participants. Targeted misexpression of candidate gene ortholog in the Drosophila fat body significantly altered flies memory and learning. Among them, down-regulation of the neurotransmitter release cycle-associated gene SLC18A2 improved cognitive abilities in Drosophila and in mice. Up-regulation of RIMS1 in Drosophila fat body enhanced cognitive abilities. Current results show previously unidentified connections between AT transcriptome and brain function in humans, providing unprecedented diagnostic/therapeutic targets in AT (Oliveras-Canellas, 2023). Gendron, C. M., Chakraborty, T. S., Duran, C., Dono, T. and Pletcher, S. D. (2023). Ring neurons in the Drosophila central complex act as a rheostat for sensory modulation of aging. PLoS Biol 21(6): e3002149. PubMed ID: 37310911

Abstract

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

Abstract

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

Abstract

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

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

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

Abstract

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

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

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

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

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

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

Abstract

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

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

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

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

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

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

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

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

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

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

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

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

Abstract

Summary:

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

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

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Abstract

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

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Abstract

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

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

Abstract

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

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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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Abstract

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150^Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150^Glued 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 p150^G50R in Drosophila. Dopaminergic expression of p150^G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150^G50R 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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Abstract

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

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

Abstract

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

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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).

Abstract

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. This study used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, these data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology (Thackray, 2020).
Myers, R. R., John, A., Zhang, W., Zou, W. Q., Cembran, A. and Fernandez-Funez, P. (2023). Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila. J Biol Chem 299(7): 104881. PubMed ID: 37269948

Abstract

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

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Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

Abstract

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

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Abstract

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

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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).