Neurodegeneration In Drosophila Research Articles

The Zn2+ transporter ZIP7 enhances endoplasmic-reticulum-associated protein degradation and prevents neurodegeneration in Drosophila.

Proteotoxic stress drives numerous degenerative diseases. Cells initially adapt to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated protein degradation (ERAD). However, persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. The ER-localized Zn2+ transporter ZIP7 is conserved from plants to humans and required for intestinal self-renewal, Notch signaling, cell motility, and survival. However, a unifying mechanism underlying these diverse phenotypes was unknown. In studying Drosophila border cell migration, we discovered that ZIP7-mediated Zn2+ transport enhances the obligatory deubiquitination of proteins by the Rpn11 Zn2+ metalloproteinase in the proteasome lid. In human cells, ZIP7 and Zn2+ are limiting for deubiquitination. In a Drosophila model of neurodegeneration caused by misfolded rhodopsin (Rh1), ZIP7 overexpression degrades misfolded Rh1 and rescues photoreceptor viability and fly vision. Thus, ZIP7-mediated Zn2+ transport is a previously unknown, rate-limiting step for ERAD invivo with therapeutic potential in protein misfolding diseases.

First person – Akanksha Onkar

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Akanksha Onkar is first author on ‘ Increase in brain glycogen levels ameliorates Huntington's disease phenotype and rescues neurodegeneration in Drosophila’, published in DMM. Akanksha conducted the research described in this article while a Ph.D. scholar in Dr S. Ganesh's lab at Indian Institute of Technology Kanpur, Kanpur, India. She is now a postdoctoral researcher in the lab of Dr Adrian Erlebacher at University of California San Francisco, investigating the moonlighting roles of carbohydrates in normal physiology and disease states.

Increase in brain glycogen levels ameliorates Huntington's disease phenotype and rescues neurodegeneration in Drosophila.

Under normal physiological conditions, the mammalian brain contains very little glycogen, most of which is stored in astrocytes. However, the aging brain and the subareas of the brain in patients with neurodegenerative disorders tend to accumulate glycogen, the cause and significance of which remain largely unexplored. Using cellular models, we have recently demonstrated a neuroprotective role for neuronal glycogen and glycogen synthase in the context of Huntington's disease. To gain insight into the role of brain glycogen in regulating proteotoxicity, we utilized a Drosophila model of Huntington's disease, in which glycogen synthase is either knocked down or expressed ectopically. Enhancing glycogen synthesis in the brains of flies with Huntington's disease decreased mutant Huntingtin aggregation and reduced oxidative stress by activating auto-lysosomal functions. Further, overexpression of glycogen synthase in the brain rescues photoreceptor degeneration, improves locomotor deficits and increases fitness traits in this Huntington's disease model. We, thus, provide in vivo evidence for the neuroprotective functions of glycogen synthase and glycogen in neurodegenerative conditions, and their role in the neuronal autophagy process.

Expanded polyQ aggregates interact with sarco-endoplasmic reticulum calcium ATPase and Drosophila inhibitor of apoptosis protein1 to regulate polyQ mediated neurodegeneration in Drosophila

Polyglutamine (polyQ) induced neurodegeneration is one of the leading causes of progressive neurodegenerative disorders characterized clinically by deteriorating movement defects, psychiatric disability, and dementia. Calcium [Ca2+] homeostasis, which is essential for the functioning of neuronal cells, is disrupted under these pathological conditions. In this paper, we simulated Huntington's disease phenotype in the neuronal cells of the Drosophila eye and identified [Ca2+] pump, sarco-endoplasmic reticulum calcium ATPase (SERCA), as one of the genetic modifiers of the neurodegenerative phenotype. This paper shows genetic and molecular interaction between polyglutamine (polyQ) aggregates, SERCA and DIAP1. We present evidence that polyQ aggregates interact with SERCA and alter its dynamics, resulting in a decrease in cytosolic [Ca2+] and an increase in ER [Ca2+], and thus toxicity. Downregulating SERCA lowers the enhanced calcium levels in the ER and rescues, morphological and functional defects caused due to expanded polyQ repeats. Cell proliferation markers such as Yorkie (Yki), Scalloped (Sd), and phosphatidylinositol 3 kinases/protein kinase B (PI3K/Akt), also respond to varying levels of calcium due to genetic manipulations, adding to the amelioration of degeneration. These results imply that neurodegeneration due to expanded polyQ repeats is sensitive to SERCA activity, and its manipulation can be an important step toward its therapeutic measures.

Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster

A-kinase anchoring protein (AKAP) comprises a family of scaffold proteins, which decides the subcellular localisation of a combination of signalling molecules. Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. We have earlier reported that Spoon suppresses ribonuclear foci formed by trinucleotide repeat expanded transcripts associated with Spinocerebellar Ataxia 8 neurodegeneration in Drosophila. However, the role of Spoonbill in cellular signalling was unexplored. In this report, we have unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. We have shown here that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that the spoon interacts with components of the JNK pathway, TGF-β activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, we found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. Hence, we propose that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson’s Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis.

Downregulation of TOP2 modulates neurodegeneration caused by GGGGCC expanded repeats

GGGGCC repeats in a non-coding region of the C9orf72 gene have been identified as a major genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We previously showed that the GGGGCC expanded repeats alone were sufficient to cause neurodegeneration in Drosophila. Recent evidence indicates that GGGGCC expanded repeats can modify various gene transcriptomes. To determine the role of these genes in GGGGCC-mediated neurotoxicity, we screened an established Drosophila model expressing GGGGCC expanded repeats in this study. Our results showed that knockdown of the DNA topoisomerase II (Top2) gene can specifically modulate GGGGCC-associated neurodegeneration of the eye. Furthermore, chemical inhibition of Top2 or siRNA-induced Top2 downregulation could alleviate the GGGGCC-mediated neurotoxicity in Drosophila assessed by eye neurodegeneration and locomotion impairment. By contrast, upregulated Top2 levels were detected in Drosophila strains, and moreover, TOP2A level was also upregulated in Neuro-2a cells expressing GGGGCC expanded repeats, as well as in the brains of Sod1G93A model mice. This indicated that elevated levels of TOP2A may be involved in a pathway common to the pathophysiology of distinct ALS forms. Moreover, through RNA-sequencing, a total of 67 genes, involved in the pathways of intracellular signaling cascades, peripheral nervous system development, and others, were identified as potential targets of TOP2A to modulate GGGGCC-mediated neurodegeneration.

Harnessing the paradoxical phenotypes of APOE ɛ2 and APOE ɛ4 to identify genetic modifiers in Alzheimer's disease.

The strongest genetic risk factor for idiopathic late‐onset Alzheimer's disease (LOAD) is apolipoprotein E (APOE) ɛ4, while the APOE ɛ2 allele is protective. However, there are paradoxical APOE ɛ4 carriers who remain disease‐free and APOE ɛ2 carriers with LOAD. We compared exomes of healthy APOE ɛ4 carriers and APOE ɛ2 Alzheimer's disease (AD) patients, prioritizing coding variants based on their predicted functional impact, and identified 216 genes with differential mutational load between these two populations. These candidate genes were significantly dysregulated in LOAD brains, and many modulated tau‐ or β42‐induced neurodegeneration in Drosophila. Variants in these genes were associated with AD risk, even in APOE ɛ3 homozygotes, showing robust predictive power for risk stratification. Network analyses revealed involvement of candidate genes in brain cell type‐specific pathways including synaptic biology, dendritic spine pruning and inflammation. These potential modifiers of LOAD may constitute novel biomarkers, provide potential therapeutic intervention avenues, and support applying this approach as larger whole exome sequencing cohorts become available.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

Daily blue-light exposure shortens lifespan and causes brain neurodegeneration in Drosophila

Light is necessary for life, but prolonged exposure to artificial light is a matter of increasing health concern. Humans are exposed to increased amounts of light in the blue spectrum produced by light-emitting diodes (LEDs), which can interfere with normal sleep cycles. The LED technologies are relatively new; therefore, the long-term effects of exposure to blue light across the lifespan are not understood. We investigated the effects of light in the model organism, Drosophila melanogaster, and determined that flies maintained in daily cycles of 12-h blue LED and 12-h darkness had significantly reduced longevity compared with flies maintained in constant darkness or in white light with blue wavelengths blocked. Exposure of adult flies to 12 h of blue light per day accelerated aging phenotypes causing damage to retinal cells, brain neurodegeneration, and impaired locomotion. We report that brain damage and locomotor impairments do not depend on the degeneration in the retina, as these phenotypes were evident under blue light in flies with genetically ablated eyes. Blue light induces expression of stress-responsive genes in old flies but not in young, suggesting that cumulative light exposure acts as a stressor during aging. We also determined that several known blue-light-sensitive proteins are not acting in pathways mediating detrimental light effects. Our study reveals the unexpected effects of blue light on fly brain and establishes Drosophila as a model in which to investigate long-term effects of blue light at the cellular and organismal level.

Planar cell polarity gene Fuz triggers apoptosis in neurodegenerative disease models.

Planar cell polarity (PCP) describes a cell-cell communication process through which individual cells coordinate and align within the plane of a tissue. In this study, we show 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 We show that 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 α-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.

Green tea infusion alleviates neurodegeneration induced by mutant Huntingtin in Drosophila

Objectives: Green tea infusion contains a complex mixture of polyphenolic compounds that were shown to provide health benefits. It was previously demonstrated that (-)-epigallocatechin-3-gallate, one of the major polyphenols present in green tea, has a suppressing effect on various aspects of pathogenesis in models of Huntington’s disease (HD), an inherited neurodegenerative disorder. In this study, we aimed to investigate, whether green tea infusion prepared as for human consumption has similar positive effects.Methods: We used a transgenic Drosophila model of HD to study the effects of green tea on mutant Huntingtin induced phenotypes. We tested the effects of green tea infusion on mutant Huntingtin induced neurodegeneration, impaired motor performance, reduced viability and lifespan by pseudopupil assay, climbing assay, eclosion and survival tests, respectively. We used immunoblots to measure Huntingtin protein levels and tested generic health benefits of green tea by longevity analysis.Results: We found that green tea supplementation reduced mutant Huntingtin induced neurodegeneration in Drosophila and positively impacted the longevity of mutant Huntingtin expressing flies. However, green tea did not rescue reduced viability of Drosophila expressing mutant Huntingtin or increased longevity of wild-type fruit flies.Discussion: Our results indicate that green tea consumption might have a modest positive effect on symptoms of HD.

Pathways to neurodegeneration: lessons learnt from unbiased genetic screens in Drosophila

Neurodegenerative diseases are a complex set of disorders that are known to be caused by environmental as well as genetic factors. In the recent past, mutations in a large number of genes have been identified that are linked to several neurodegenerative diseases. The pathogenic mechanisms in most of these disorders are unknown. Recently, studies of genes that are linked to neurodegeneration in Drosophila, the fruit flies, have contributed significantly to our understanding of mechanisms of neuroprotection and degeneration. In this review, we focus on forward genetic screens in Drosophila that helped in identification of novel genes and pathogenic mechanisms linked to neurodegeneration. We also discuss identification of four novel pathways that contribute to neurodegeneration upon mitochondrial dysfunction.

Ataxin-2 Is Droppin’ Some Knowledge

Ataxin-2 is an RNA-binding protein involved in translation regulation and mutated in neurodegenerative diseases, including ALS. In this issue of Neuron, Bakthavachalu etal. (2018) demonstrate that higher-order ataxin-2 RNA/protein assemblies are necessary for both translation-dependent learning and ALS-associated neurodegeneration in Drosophila.

RhoGAPp190: A potential player in tbph-mediated neurodegeneration in Drosophila.

TDP-43 is an ubiquitous and highly conserved ribonucleoprotein involved in several cellular processes including pre-mRNA splicing, transcription, mRNA stability and transport. Notwithstanding the evidence of TDP-43 involvement in the pathogenesis of different neurodegenerative disorders (i.e. ALS and FTLD), the underlying mechanisms are still unclear. Given the high degree of functional similarity between the human and fly orthologs of TDP-43, Drosophila melanogaster is a simple and useful model to study the pathophysiological role of this protein in vivo. It has been demonstrated that the depletion of the TDP-43 fly ortholog (tbph) induces deficient locomotive behaviors and reduces life span and anatomical defects at the neuromuscular junction. In this study, using the known binding specificity of TDP-43/tbph for (UG) repeated sequences, we performed a bioinformatic screening for fly genes with at least 6 (TG) repeats in a row within the 3'-UTR regions in order to identify the genes that might be regulated by this factor. Among these genes, we were able to identify RhoGAPp190 as a potential target of the tbph-mediated neurodegeneration. RhoGAPp190 is a negative regulator of Drosophila RhoA, a GTPase protein implicated in the fine modulation of critical cellular processes including axon branch stability and motor axon defasciculation at muscle level and cognitive processes. We were able to demonstrate that the RhoGAPp190 expression is upregulated in a tbph-null fly model, providing evidence that this deregulation is associated to tbph silencing. Our results introduce RhoGAPp190 as a novel potential mediator in the complex scenario of events resulting from in vivo tbph loss-of-function.

Neuroprotective action of 4-Hydroxyisophthalic acid against paraquat-induced motor impairment involves amelioration of mitochondrial damage and neurodegeneration in Drosophila

Neurodegenerative disorders including Parkinson’s disease (PD) are believed to be caused by oxidative stress and mitochondrial dysfunction. Exposure to environmental agents such as pesticides has been implicated in the etiology of sporadic PD. Paraquat (PQ), a widely used herbicide, induces PD symptoms in laboratory animals including Drosophila. PQ acts as a free radical generator and induces oxidative damage, which is implicated in neuronal cell death. Drosophila model of PQ-induced PD offers a convenient tool for mechanistic studies and, to assess the neuroprotective potential of natural antioxidants. We have investigated the neuroprotective potential of 4-Hydroxyisophthalic acid (DHA-I), a novel bioactive molecule from the roots of Decalepis hamiltonii, against PQ-induced locomotor impairment and neurodegeneration in Drosophila melanogaster. Our study shows that PQ treatment results in movement disorder associated with oxidative stress-mediated mitochondrial damage and neurodegeneration in the brain as evident by ultrastructural observations. Treatment with DHA-I markedly attenuated locomotor deficits, oxidative stress, mitochondrial damage, and neurodegenerative changes induced by PQ in Drosophila. Our results show that DHA-I could be a promising natural antioxidant and a neuroprotective molecule targeting oxidative stress-mediated mitochondrial dysfunction with therapeutic potential for neurodegenerative disorders.

Discovery and functional prioritization of Parkinson\u2019s disease candidate genes from large-scale whole exome sequencing

BackgroundWhole-exome sequencing (WES) has been successful in identifying genes that cause familial Parkinson’s disease (PD). However, until now this approach has not been deployed to study large cohorts of unrelated participants. To discover rare PD susceptibility variants, we performed WES in 1148 unrelated cases and 503 control participants. Candidate genes were subsequently validated for functions relevant to PD based on parallel RNA-interference (RNAi) screens in human cell culture and Drosophila and C. elegans models.ResultsAssuming autosomal recessive inheritance, we identify 27 genes that have homozygous or compound heterozygous loss-of-function variants in PD cases. Definitive replication and confirmation of these findings were hindered by potential heterogeneity and by the rarity of the implicated alleles. We therefore looked for potential genetic interactions with established PD mechanisms. Following RNAi-mediated knockdown, 15 of the genes modulated mitochondrial dynamics in human neuronal cultures and four candidates enhanced α-synuclein-induced neurodegeneration in Drosophila. Based on complementary analyses in independent human datasets, five functionally validated genes—GPATCH2L, UHRF1BP1L, PTPRH, ARSB, and VPS13C—also showed evidence consistent with genetic replication.ConclusionsBy integrating human genetic and functional evidence, we identify several PD susceptibility gene candidates for further investigation. Our approach highlights a powerful experimental strategy with broad applicability for future studies of disorders with complex genetic etiologies.

Dietary Iron Oxide Nanoparticles Delay Aging and Ameliorate Neurodegeneration in Drosophila.

Dietary iron oxide nanoparticles are shown to ameliorate neurodegeneration in a Drosophelia Alzheimer's disease model. Iron oxide nanoparticles can mimic catalase and can decompose reactive oxygen species (ROS). This has potential therapeutic uses for aging, metabolic disorders, and neurodegenerative diseases, in which increased production of ROS is closely implicated.

Classification of Parkinson's Disease Genotypes in Drosophila Using Spatiotemporal Profiling of Vision.

Electrophysiological studies indicate altered contrast processing in some Parkinson’s Disease (PD) patients. We recently demonstrated that vision is altered in Drosophila PD models and hypothesised that different types of genetic and idiopathic PD may affect dopaminergic visual signalling pathways differently. Here we asked whether visual responses in Drosophila could be used to identify PD mutations. To mimic a clinical setting a range of flies was used. Young flies from four control lines were compared to three early-onset PD mutations (PINK1, DJ-1α and DJ-1β), and to two other neurodegenerative mutations, one in the fly LRRK2 orthologue (dLRRK) the other in eggroll, a model of general neurodegeneration in Drosophila. Stimuli were contrast reversing gratings spanning 64 spatiotemporal frequency combinations. We recorded the steady-state visually-evoked response amplitude across all combinations. We found that the pattern of neuronal responses differed between genotypes. Wild-type and early-onset PD flies formed separate clusters; the late-onset mutation is an outlier. Neuronal responses in early-onset PD flies were stronger than in wild-types. Multivariate pattern analysis grouped flies by PD/non-PD genotype with an accuracy >85%. We propose that machine learning algorithms may be useful in increasing the diagnostic specificity of human electrophysiological measurements in both animal models and PD patients.

Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Aβ(42)-induced locomotor decline in Drosophila.

The negative-geotaxis climbing assay is used to efficiently study aging and neurodegeneration in Drosophila. To make it suitable for large-scale study, a method called the rapid iterative negative geotaxis (RING) assay has been established by simultaneously photographing the climbing of multiple groups of flies when they are manually tapped down in test tubes. Here, we automated the assay by using a well-controlled electric motor to drive the tapping, and a homemade program to analyze the climbing height of flies. Using the automated RING (aRING) assay, we found that the climbing ability of a strain of wild-type flies, males in particular, declined rapidly before day 21 after eclosion, but slowly from day 21 to 35. We also found that the expression of arctic mutant Aβ42 accelerated the age-dependent decline in the climbing ability of flies. Moreover, using aRING, we examined the effect of third chromosome deficiencies on the accelerated locomotor decline in Aβ42-expressing flies, and isolated 7 suppressors and 15 enhancers.