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mTOR is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product Rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a Rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this Rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in S. cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a Rapamycin-induced developmental delay in Drosophila.Significance StatementmTOR plays a number of critical organismal roles, including in cell growth, development, immunity and aging, but dissecting the tissue-specific influences of mTOR has proven challenging. This work describes a simple system for identifying the specific tissues and cells responsible for the diverse functions of mTOR, and we show that our system can be used in organisms ranging from yeast to humans.

The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. We tested this hypothesis using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, we investigated whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover. We found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, our findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a system collapse.

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson’s disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. Accordingly, we hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. We found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread., Author summary Parkinson’s disease (PD) is a common neurodegenerative disease characterized by abnormal clumps of proteins (aggregates) within the brain and other tissues which can lead to cellular dysfunction and death. Mutations in the gene GBA, which encodes glucocerebrosidase (GCase), are the strongest genetic risk factor for PD, and are associated with faster disease progression. GCase-deficient mutant flies display features suggestive of PD including increased protein aggregation in brain and muscle. We found that restoring GCase protein in the muscle of mutant flies reduced protein aggregation in muscle and the brain, suggesting a mechanism involving interaction between tissues. Previous work indicated that GBA influences extracellular vesicles (EVs)–small membrane-bound structures released by cells to communicate and/or transport cargo from cell to cell. Here, we found increased aggregated proteins within EVs of mutant flies, which was reduced by restoring GCase in muscle. In addition, we found GCase within the EVs, possibly explaining how GCase in one tissue such as muscle could reduce protein aggregation in a distant tissue like the brain. Our findings suggest that GCase influences proteins within EVs, affecting the spread of protein aggregation. This may be important to understanding PD progression and could uncover new targets to slow neurodegeneration.

The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. Our work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2., Author summary Mitochondria produce virtually all of the cellular energy through the actions of the respiratory chain (RC) complexes. However, mitochondria also present a quality control challenge for the eukaryotic cell. In particular, biogenesis of the RC complexes depends on the coordinated expression of nuclear and mitochondrially encoded subunits and an imbalance in this process can cause protein aggregation. Moreover, the RC complexes produce highly damaging reactive oxygen species as a side product of their activity. The Mitochondrial AAA+ family of proteases are believed to provide the first line of defense against these insults. The importance of this protease family is best exemplified by the severe neurodegenerative diseases that are caused by mutations in their respective genes. To better understand the biological roles of the AAA+ proteases, and the physiological consequences of their inactivation we used a genetic approach in Drosophila to study the Afg3l2 AAA+ protease. Partial inactivation of the AFG3L2 gene resulted in mitochondrial functional deficits, shortened lifespan, and neurodegeneration. Unexpectedly, we found that severely diminished mitochondrial gene expression, including transcription, and translation, primarily accounts for these phenotypes. The defects in gene expression appear to be caused by a failure in mitochondrial ribosome maturation and assembly, and possibly, sequestration of regulatory factors into inactive aggregates. Our work indicates Afg3l2 plays critical roles in degrading unfolded mitochondrial proteins and regulating mitochondrial gene expression, and that strategies to address these matters could be therapeutic in the diseases caused by mutations in the AFG3L2 gene.

Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. We tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Likewise, Gba1b mutants did not have the marked impairment of mitochondrial protein turnover seen in mitophagy-deficient parkin mutants. Proteasome activity, microautophagy, and endocytic degradation also appeared unaffected in Gba1b mutants. However, we found striking changes in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. These changes were specific to Gba1b mutants and did not represent an acceleration of normal aging. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, our findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles., Author summary Mutations in the GBA gene, which encodes the enzyme glucocerebrosidase, are common and increase the risk of Parkinson disease. A widely accepted explanation for the increased risk is that the fatty substance normally broken down by glucocerebrosidase builds up in the lysosome, which is the cell’s recycling center, until the cell can no longer get rid of damaged parts. At that point, proteins that should be destroyed in the lysosome form large clumps (aggregates) throughout the cell. We used mutant fruit flies without glucocerebrosidase to test this theory, and we were surprised to see no evidence that the lysosome was failing. The destruction of proteins usually recycled by the lysosome was not slowed down in the mutant flies. Instead, we saw evidence that the mutants’ cells might be producing too many extracellular vesicles, tiny spheres that transport cargo and messages from cell to cell. Some researchers have also suggested that extracellular vesicles carry the protein aggregates that spread between cells as Parkinson disease get worse. Our study supports this idea. It suggests that increased spread of aggregates through extracellular vesicles, rather than failure of the lysosome, might explain why GBA mutations increase the risk of neurodegenerative disease.

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential, and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex I-associated neurodegeneration.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis., Author summary The energy needs of an animal cell are supplied by tiny organelles known as mitochondria. Each of the many mitochondria in a cell has a set of blueprints for making more mitochondria, known as mitochondrial DNA (mtDNA). As animals age, their mtDNA acquires irreversible defects called mutations, which accumulate and may cause aging. Cells can selectively destroy malfunctioning mitochondria, so we hypothesized that mitochondria with harmful mutations would be selectively destroyed. To test our theory, we created a fruit fly strain with a high mtDNA mutation rate. Our hypothesis predicts that, because mitochondria bearing harmful mtDNA mutations would be destroyed, we should detect primarily less harmful mutations in our strain. However, the mtDNA mutations we detected were more harmful than expected by chance. We suggest two possible explanations: First, mitochondria with harmful mtDNA mutations may be degraded less often because they generate little energy and are not damaged by toxic byproducts of energy production. Second, cells may compensate for harmful mtDNA mutations by stimulating mitochondria to multiply, creating more healthy mitochondria but also more mitochondria with harmful mtDNA mutations. Future studies will distinguish between these models and further advance our understanding of aging and aging related disease.

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity., Author Summary Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease (GD), a lysosomal storage disease that includes neurodegenerative phenotypes. Recently, mutations in GBA1 were identified as the strongest genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), which are neurodegenerative conditions characterized by intraneuronal protein aggregates containing α-synuclein. To explore how GBA1 mutations lead to neurodegeneration in GD, PD and DLB, we developed a novel invertebrate model of GBA1 insufficiency by deleting the Drosophila GBA1 homolog, dGBA1b. We found that dGBA1b mutants have multiple phenotypes consistent with neuronal dysfunction as seen in PD, DLB and GD, and dramatically increased protein aggregation that is normally cleared through an autophagic mechanism. dGBA1b mutants expressing human α-synuclein in dopaminergic neurons led to dopaminergic neuron loss and α-synuclein aggregation. However, α-synuclein expression had minimal effect on dGBA1b mutant phenotypes, suggesting that the deleterious consequences of glucocerebrosidase deficiency are independent of α-synuclein. These findings significantly contribute to our understanding of the role of GBA1 mutations in the pathogenesis of PD, DLB, and GD, and further studies using this model should elucidate mechanisms underlying these diseases.

α-Synuclein aggregates are a common feature of sporadic Parkinson's disease (PD), and mutations that increase α-synuclein abundance confer rare heritable forms of PD. Although these findings suggest that α-synuclein plays a central role in the pathogenesis of this disorder, little is known of the mechanism by which α-synuclein promotes neuron loss or the factors that regulate α-synuclein toxicity. To address these matters, we tested candidate modifiers of α-synuclein toxicity using aDrosophilamodel of PD. In the current work, we focused on phase II detoxification enzymes involved in glutathione metabolism. We find that the neuronal death accompanying α-synuclein expression inDrosophilais enhanced by loss-of-function mutations in genes that promote glutathione synthesis and glutathione conjugation. This neuronal loss can be overcome by genetic or pharmacological interventions that increase glutathione synthesis or glutathione conjugation activity. Moreover, these same pharmacological agents suppress neuron loss inDrosophila parkinmutants, a loss-of-function model of PD. Our results suggest that oxidative stress is a feature of α-synuclein toxicity and that induction of the phase II detoxification pathway represents a potential preventative therapy for PD.

Parkinson's disease (PD) is a common neurodegenerative disorder that displays both sporadic and inherited forms [1]. Exposure to several common environmental toxins acting through oxidative stress has been shown to be associated with PD [2]. One recently identified inherited PD gene, DJ-1, may have a role in protection from oxidative stress [3-10], thus potentially linking a genetic cause with critical environmental risk factors. To develop an animal model that would allow integrative study of genetic and environmental influences, we have generated Drosophila lacking DJ-1 function. Fly DJ-1 homologs exhibit differential expression: DJ-1β is ubiquitous, while DJ-1α is predominantly expressed in the male germline. DJ-1α and DJ-1β double knockout flies are viable, fertile, and have a normal lifespan; however, they display a striking selective sensitivity to those environmental agents, including paraquat and rotenone, linked to PD in humans. This sensitivity results primarily from loss of DJ-1β protein, which also becomes modified upon oxidative stress. These studies demonstrate that fly DJ-1 activity is selectively involved in protection from environmental oxidative insult in vivo and that the DJ-1β protein is biochemically responsive to oxidative stress. Study of these flies will provide insight into the critical interplay of genetics and environment in PD. ©2005 Elsevier Ltd. All rights reserved.

TheN-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP) are cytosolic factors that promote vesicle fusion with a target membrane in both the constitutive and regulated secretory pathways. NSF and SNAP are thought to function by catalyzing the disassembly of a SNAP receptor (SNARE) complex consisting of membrane proteins of the secretory vesicle and target membrane. Although studies of NSF function have provided strong support for this model, the precise biochemical role of SNAP remains controversial. To further explore the function of SNAP, we have used mutational and transgenic approaches inDrosophilato investigate the effect of altered SNAP dosage on neurotransmitter release and SNARE complex metabolism. Our results indicate that reduced SNAP activity results in diminished neurotransmitter release and accumulation of a neural SNARE complex. Increased SNAP dosage results in defective synapse formation and a variety of tissue morphological defects without detectably altering the abundance of neural SNARE complexes. The SNAP overexpression phenotypes are enhanced by mutations in other secretory components and are at least partially overcome by co-overexpression of NSF, suggesting that these phenotypes derive from a specific perturbation of the secretory pathway. Our results indicate that SNAP promotes neurotransmitter release and SNARE complex disassembly but inhibits secretion when present at high abundance relative to NSF.

Neuronal function depends upon the proper formation of synaptic connections and rapid communication at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromosome 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse development and function.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. Several lines of evidence strongly implicate mitochondrial dysfunction as a major causative factor in PD, although the molecular mechanisms responsible for mitochondrial dysfunction are poorly understood. Recently, loss-of-function mutations in the parkin gene, which encodes a ubiquitin-protein ligase, were found to underlie a familial form of PD known as autosomal recessive juvenile parkinsonism (AR-JP). To gain insight into the molecular mechanism responsible for selective cell death in AR-JP, we have created a Drosophila model of this disorder. Drosophila parkin null mutants exhibit reduced lifespan, locomotor defects, and male sterility. The locomotor defects derive from apoptotic cell death of muscle subsets, whereas the male sterile phenotype derives from a spermatid individualization defect at a late stage of spermatogenesis. Mitochondrial pathology is the earliest manifestation of muscle degeneration and a prominent characteristic of individualizing spermatids in parkin mutants. These results indicate that the tissue-specific phenotypes observed in Drosophila parkin mutants result from mitochondrial dysfunction and raise the possibility that similar mitochondrial impairment triggers the selective cell loss observed in AR-JP.

The accumulation of damaged mitochondria has been proposed as a key factor in aging and the pathogenesis of many common age-related diseases, including Parkinson disease (PD). Recently, in vitro studies of the PD-related proteins Parkin and PINK1 have found that these factors act in a common pathway to promote the selective autophagic degradation of damaged mitochondria (mitophagy). However, whether Parkin and PINK1 promote mitophagy under normal physiological conditions in vivo is unknown. To address this question, we used a proteomic approach in Drosophila to compare the rates of mitochondrial protein turnover in parkin mutants, PINK1 mutants, and control flies. We found that parkin null mutants showed a significant overall slowing of mitochondrial protein turnover, similar to but less severe than the slowing seen in autophagy-deficient Atg7 mutants, consistent with the model that Parkin acts upstream of Atg7 to promote mitophagy. By contrast, the turnover of many mitochondrial respiratory chain (RC) subunits showed greater impairment in parkin than Atg7 mutants, and RC turnover was also selectively impaired in PINK1 mutants. Our findings show that the PINK1–Parkin pathway promotes mitophagy in vivo and, unexpectedly, also promotes selective turnover of mitochondrial RC subunits. Failure to degrade damaged RC proteins could account for the RC deficits seen in many PD patients and may play an important role in PD pathogenesis.

Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, we developed a method to purify and characterize neural subtypes of interest from the adult Drosophila brain. Using this method, we find that dopaminergic neurons from Drosophila parkin mutants accumulate enlarged, depolarized mitochondria, and that genetic perturbations that promote mitochondrial fragmentation and turnover rescue the mitochondrial depolarization and neurodegenerative phenotypes of parkin mutants. In contrast, cholinergic neurons from parkin mutants accumulate enlarged depolarized mitochondria to a lesser extent than dopaminergic neurons, suggesting that a higher rate of mitochondrial damage, or a deficiency in alternative mechanisms to repair or eliminate damaged mitochondria explains the selective vulnerability of dopaminergic neurons in Parkinson disease. Our study validates key tenets of the model that PINK1 and Parkin promote the fragmentation and turnover of depolarized mitochondria in dopaminergic neurons. Moreover, our neural purification method provides a foundation to further explore the pathogenesis of Parkinson disease, and to address other neurobiological questions requiring the analysis of defined neural cell types.

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in three weeks. Loss-of-function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 orthologue (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD / Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss-of-function results in altered bioenergetics and mitochondrial dysfunction, and suggest that pcd shares pathogenic features with neurodegenerative disorders such as Parkinson's disease.

Loss-of-function mutations in the PTEN-induced kinase 1 ( PINK1 ) or parkin genes, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin-protein ligase, respectively, result in recessive familial forms of Parkinsonism. Genetic studies in Drosophila indicate that PINK1 acts upstream of Parkin in a common pathway that influences mitochondrial integrity in a subset of tissues, including flight muscle and dopaminergic neurons. The mechanism by which PINK1 and Parkin influence mitochondrial integrity is currently unknown, although mutations in the PINK1 and parkin genes result in enlarged or swollen mitochondria, suggesting a possible regulatory role for the PINK1/Parkin pathway in mitochondrial morphology. To address this hypothesis, we examined the influence of genetic alterations affecting the machinery that governs mitochondrial morphology on the PINK1 and parkin mutant phenotypes. We report that heterozygous loss-of-function mutations of drp1 , which encodes a key mitochondrial fission-promoting component, are largely lethal in a PINK1 or parkin mutant background. Conversely, the flight muscle degeneration and mitochondrial morphological alterations that result from mutations in PINK1 and parkin are strongly suppressed by increased drp1 gene dosage and by heterozygous loss-of-function mutations affecting the mitochondrial fusion-promoting factors OPA1 and Mfn2. Finally, we find that an eye phenotype associated with increased PINK1/Parkin pathway activity is suppressed by perturbations that reduce mitochondrial fission and enhanced by perturbations that reduce mitochondrial fusion. Our studies suggest that the PINK1/Parkin pathway promotes mitochondrial fission and that the loss of mitochondrial and tissue integrity in PINK1 and parkin mutants derives from reduced mitochondrial fission.

Mutations resulting in the expansion of a polyglutamine tract in the protein ataxin-2 give rise to the neurodegenerative disorders spinocerebellar ataxia type 2 and Parkinson's disease. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration are unknown. Here, we demonstrate that ataxin-2 and its Drosophila homolog, ATX2, assemble with polyribosomes and poly(A)-binding protein (PABP), a key regulator of mRNA translation. The assembly of ATX2 with polyribosomes is mediated independently by two distinct evolutionarily conserved regions of ATX2: an N-terminal Lsm/Lsm-associated domain (LsmAD), found in proteins that function in nuclear RNA processing and mRNA decay, and a PAM2 motif, found in proteins that interact physically with PABP. We further show that the PAM2 motif mediates a physical interaction of ATX2 with PABP in addition to promoting ATX2 assembly with polyribosomes. Our results suggest a model in which ATX2 binds mRNA directly through its Lsm/LsmAD domain and indirectly via binding PABP that is itself directly bound to mRNA. These findings, coupled with work on other ataxin-2 family members, suggest that ATX2 plays a direct role in translational regulation. Our results raise the possibility that polyglutamine expansions within ataxin-2 cause neurodegeneration by interfering with the translational regulation of particular mRNAs.

Despite the prevalence and severity of Parkinson's disease (PD), little is known about the molecular etiology of this disease, and preventative and disease-modifying therapies remain elusive. Recently, linkage studies have begun to identify single-gene mutations that are responsible for rare, heritable forms of PD, which offer an opportunity to gain insight into the molecular mechanisms of this disorder through the creation and analysis of appropriate animal models. One model system that is tractable for these studies is the fruit fly, Drosophila melanogaster. Analysis of several Drosophila models of PD has revealed some surprising insights into the pathogenesis of PD and begun to highlight potential treatment strategies.

Loss-of-function mutations of the parkin gene are a major cause of early-onset parkinsonism. To explore the mechanism by which loss of parkin function results in neurodegeneration, we are using a genetic approach in Drosophila . Here, we show that Drosophila parkin mutants display degeneration of a subset of dopaminergic (DA) neurons in the brain. The neurodegenerative phenotype of parkin mutants is enhanced by loss-of-function mutations of the glutathione S-transferase S1 ( GstS1 ) gene, which were identified in an unbiased genetic screen for genes that modify parkin phenotypes. Furthermore, overexpression of GstS1 in DA neurons suppresses neurodegeneration in parkin mutants. Given the previous evidence for altered glutathione metabolism and oxidative stress in sporadic Parkinson's disease (PD), these data suggest that the mechanism of DA neuron loss in Drosophila parkin mutants is similar to the mechanisms underlying sporadic PD. Moreover, these findings identify a potential therapeutic approach in treating PD.

Current knowledge of the molecular mechanisms of Parkinson's disease (PD) is dramatically advanced by the recent identification of genes underlying relatively rare heritable forms of this disorder. These breakthroughs may provide a window into the mechanisms underlying the more common sporadic form of PD. However, understanding of the functions of these genes and the mechanisms by which their mutational alteration results in neuronal death is limited. One approach to this problem that has great potential is classical genetic analysis in the fruit fly Drosophila melanogaster to identify the genetic pathways leading to pathology in fly models of this disease. This review describes the features that make Drosophila useful in studies of the genes implicated in PD and how these studies have begun to contribute to the understanding of the pathogenesis of this disorder. The Drosophila system provides a unique opportunity to evaluate the functions of genes implicated in PD and identify mechanisms relevant to this disorder in a relatively unbiased fashion. While significant progress is already made, PD modeling in Drosophila is still relatively new and the reagents and methodologies for conducting these studies are still in their infancy. There are currently Drosophila homologs of three genes identified from linkage studies of PD patients for which a description of the mutant phenotype is awaited.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. In this study we have used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.

Summary Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget's disease, or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.