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

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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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Abstract

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

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

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

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

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

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

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

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

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

Abstract

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

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

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

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

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

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

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

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

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

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

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

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

Abstract

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

Abstract

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

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

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

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

Abstract

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

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

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

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

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

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

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

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

Abstract

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

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

Abstract

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

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

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

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

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

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

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

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

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

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

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

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

Abstract

Summary:

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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

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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

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).

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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).

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

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

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

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

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

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

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

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

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

In order to get better insights into the mechanisms by which Prp31 prevents retinal degeneration this study aimed to establish a meaningful Drosophila model for RP11-associated retinal degeneration. Therefore two mutant alleles were isolated of Prp31, Prp31^P17 and Prp31^P18, which carry missense mutations affecting conserved amino acids. Flies heterozygous for either of these mutations are viable and develop normally. Strikingly, when exposed to constant light, these mutant flies undergo retinal degeneration, thus mimicking the disease phenotype of RP11 patients. Degeneration of mutant PRCs is associated with accumulation and abnormal distribution of the visual pigment rhodopsin, Rh1, in PRCs. Reduction of dietary vitamin A, a precursor of the chromophore 11-cis-3-hydroxyretinal, which binds to opsin to generate the functional rhodopsin, mitigates both aspects of the mutant phenotype, rhodopsin accumulation and retinal degeneration. From this it is concluded that Rh1 accumulation and/or misdistribution reflect a degeneration-prone condition in the Prp31 mutant retina (Hebbar, 2021).

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

By using three different genetic approaches this study provides convincing evidence that the knockdown of Prp31 is the cause of the retinal degeneration observed. (1) The two Prp31 alleles induced by TILLING (Prp31^P17 and Prp31^P18) carry missense mutations in conserved amino acids of the coding region, which are predicted to be damaging. (2) Flies heterozygous for any of three deletions, which completely remove the Prp31 locus, exhibit comparable phenotypes as flies heterozygous for Prp31 point mutations. (3) RNAi-mediated knockdown of Prp31 results in light-induced retinal degeneration. The results obtained suggest that the two missense mutations mapped in Prp31^P17 and Prp31^P18 are strong hypomorphic alleles. First, the two Drosophila alleles characterised in this study are hemizygous (Prp31/deficiency) and homozygous (in the case of Prp31^P18) viable and fertile. Second, mutations in the two established Prp31 fly lines are missense mutations, one located N-terminal to the NOSIC domain in Prp31^P17 (G90R) and the other in the Nop domain in Prp31^P18 (P277L), which most likely result in a reduced function of the respective proteins. Whether protein levels are also decreased cannot be answered due to the lack of specific antibodies. The mutated amino acid residue in Drosophila Prp31^P18 (P277L) lies within the snoRNA binding domain (NOP domain. Interestingly, many point mutations in human PRPF31, which are linked to RP11, have been mapped to the Nop domain. Similar as in yeast, the Nop domain in human PRPF31 is involved in an essential step in the formation of the U4/U6-U5 tri-snRNP by building a complex of the U4 snRNA and a 15.5K protein, thus stabilising the U4/U6 snRNA junction. The mutated proline in Drosophila Prp31^P18 precedes a histidine (H278), which corresponds to amino acid H270 in the human protein. Mutations in H270 in the Nop domain of human PRPF31 result in a reduced affinity of PRPF31 to the complex formed by a stem-loop structure of the U4 snRNA and the 15.5K protein. Therefore, it is tempting to speculate that the Drosophila P277L mutation could similarly weaken, but not abolish the corresponding interaction of the mutant Prp31 protein with the U4/U6 complex. Further experiments are required to determine the functional consequences of the molecular lesions identified in Drosophila Prp31 (Hebbar, 2021).

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

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

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

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

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

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

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

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

Abstract

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

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

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

Abstract

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

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Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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Abstract

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

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

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

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

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Abstract

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

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Abstract

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

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

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Abstract

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

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Abstract

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

Abstract

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

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Abstract

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

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Abstract

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

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Abstract

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

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Abstract

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

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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Abstract

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

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

Abstract

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

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

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

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

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Abstract

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

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

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Abstract

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

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Abstract

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

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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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Abstract

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

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

Abstract

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

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

Abstract

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

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

Abstract

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

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

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Abstract

Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to were generated demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, RNA-seq was used on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. This study also demonstrated that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, it was shown that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which was subsequently shown to contribute towards the proliferation phenotype in Drosophila. This work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth (Towler, 2020).