Nazo, the Drosophila homolog of the NBIA-mutated protein-c19orf12, is required for triglyceride homeostasis.

Lipid dyshomeostasis has been implicated in a variety of diseases ranging from obesity to neurodegenerative disorders such as Neurodegeneration with Brain Iron Accumulation (NBIA). Here, we uncover the physiological role of Nazo, the Drosophila melanogaster homolog of the NBIA-mutated protein-c19orf12, whose function has been elusive. Ablation of Drosophila c19orf12 homologs leads to dysregulation of multiple lipid metabolism genes. nazo mutants exhibit markedly reduced gut lipid droplet and whole-body triglyceride contents. Consequently, they are sensitive to starvation and oxidative stress. Nazo is required for maintaining normal levels of Perilipin-2, an inhibitor of the lipase-Brummer. Concurrent knockdown of Brummer or overexpression of Perilipin-2 rescues the nazo phenotype, suggesting that this defect, at least in part, may arise from diminished Perilipin-2 on lipid droplets leading to aberrant Brummer-mediated lipolysis. Our findings potentially provide novel insights into the role of c19orf12 as a possible link between lipid dyshomeostasis and neurodegeneration, particularly in the context of NBIA.

Tissue-restricted inhibition of mTOR using chemical genetics.

Mammalian target of rapamycin (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 Saccharomyces 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.

Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles.

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.

Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression.

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.

Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ.

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.

Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles.

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.

Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

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.

PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix.

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.

Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo.

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.

Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants.

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.

Topograph, a software platform for precursor enrichment corrected global protein turnover measurements.

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.

A Drosophila model for mechanistic investigation of tau protein spread.

Brain protein aggregates are a hallmark of neurodegenerative disease. Previous work indicates that specific protein components of these aggregates are toxic, including tau in Alzheimer's disease and related tauopathies. Increasing evidence also indicates that these toxic proteins traffic between cells in a prion-like fashion, thereby spreading pathology from one brain region to another. However, the mechanisms involved in trafficking are poorly understood. We therefore developed a transgenic Drosophila model to facilitate rapid evaluation of candidate tau trafficking modifiers. Our model uses the bipartite Q system to drive co-expression of tau and GFP in the fly eye. We find age-dependent tau spread into the brain, represented by detection of tau, but not GFP in the brain. We also found that tau trafficking was attenuated upon inhibition of the endocytic factor dynamin or the kinase glycogen synthase kinase-3ß ( GSK-3ß ). Further work revealed that dynamin promotes tau uptake in recipient tissues, whereas GSK-3ß appears to promote tau spread via direct phosphorylation of tau. Our robust and flexible system will promote the identification of tau trafficking components involved in the pathogenesis of neurodegenerative diseases. SUMMARY STATEMENT: The trafficking of toxic proteins in neurodegenerative disease is well-known but poorly understood. Our model will allow rapid and new insight into molecular mechanisms underlying this process.

Glucocerebrosidase deficiency leads to neuropathology via cellular immune activation.

Mutations in GBA (glucosylceramidase beta), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the strongest genetic risk factor for the neurodegenerative disorders Parkinson's disease (PD) and Lewy body dementia. Recent work has suggested that neuroinflammation may be an important factor in the risk conferred by GBA mutations. We therefore systematically tested the contributions of immune-related genes to neuropathology in a Drosophila model of GCase deficiency. We identified target immune factors via RNA-Seq and proteomics on heads from GCase-deficient flies, which revealed both increased abundance of humoral factors and increased macrophage activation. We then manipulated the identified immune factors and measured their effect on head protein aggregates, a hallmark of neurodegenerative disease. Genetic ablation of humoral (secreted) immune factors did not suppress the development of protein aggregation. By contrast, re-expressing Gba1b in activated macrophages suppressed head protein aggregation in Gba1b mutants and rescued their lifespan and behavioral deficits. Moreover, reducing the GCase substrate glucosylceramide in activated macrophages also ameliorated Gba1b mutant phenotypes. Taken together, our findings show that glucosylceramide accumulation due to GCase deficiency leads to macrophage activation, which in turn promotes the development of neuropathology.

Drosophila NPC1b promotes an early step in sterol absorption from the midgut epithelium.

The NPC1 family of proteins plays crucial roles in the intestinal absorption and intracellular trafficking of sterols. The Drosophila genome encodes two NPC1 homologs, one of which, NPC1a, is required for intracellular sterol trafficking in many tissues. Here we show that the other Drosophila NPC1 family member, NPC1b, is expressed in the midgut epithelium and that NPC1b is essential for growth during the early larval stages of development. NPC1b mutants are severely defective in sterol absorption, and the midgut epithelium of NPC1b mutants is deficient in sterols and sterol trafficking intermediates. By contrast, NPC1a mutants absorb sterols more efficiently than wild-type animals, and, unexpectedly, NPC1b;NPC1a double mutants absorb sterols as efficiently as wild-type animals. Together, these findings suggest that NPC1b plays an early role in sterol absorption, although sterol absorption continues at high efficiency through an NPC1a- and NPC1b-independent mechanism under conditions of impaired intracellular sterol trafficking.

Decaffeinated coffee and nicotine-free tobacco provide neuroprotection in Drosophila models of Parkinson's disease through an NRF2-dependent mechanism.

Epidemiological studies have revealed a significantly reduced risk of Parkinson's disease (PD) among coffee and tobacco users, although it is unclear whether these correlations reflect neuroprotective/symptomatic effects of these agents or preexisting differences in the brains of tobacco and coffee users. Here, we report that coffee and tobacco, but not caffeine or nicotine, are neuroprotective in fly PD models. We further report that decaffeinated coffee and nicotine-free tobacco are as neuroprotective as their caffeine and nicotine-containing counterparts and that the neuroprotective effects of decaffeinated coffee and nicotine-free tobacco are also evident in Drosophila models of Alzheimer's disease and polyglutamine disease. Finally, we report that the neuroprotective effects of decaffeinated coffee and nicotine-free tobacco require the cytoprotective transcription factor Nrf2 and that a known Nrf2 activator in coffee, cafestol, is also able to confer neuroprotection in our fly models of PD. Our findings indicate that coffee and tobacco contain Nrf2-activating compounds that may account for the reduced risk of PD among coffee and tobacco users. These compounds represent attractive candidates for therapeutic intervention in PD and perhaps other neurodegenerative diseases.

The PINK1/Parkin pathway regulates mitochondrial morphology.

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.

Slowed Protein Turnover in Aging Drosophila Reflects a Shift in Cellular Priorities.

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.

Induction of the phase II detoxification pathway suppresses neuron loss in Drosophila models of Parkinson's disease.

Alpha-synuclein aggregates are a common feature of sporadic Parkinson's disease (PD), and mutations that increase alpha-synuclein abundance confer rare heritable forms of PD. Although these findings suggest that alpha-synuclein plays a central role in the pathogenesis of this disorder, little is known of the mechanism by which alpha-synuclein promotes neuron loss or the factors that regulate alpha-synuclein toxicity. To address these matters, we tested candidate modifiers of alpha-synuclein toxicity using a Drosophila model of PD. In the current work, we focused on phase II detoxification enzymes involved in glutathione metabolism. We find that the neuronal death accompanying alpha-synuclein expression in Drosophila is 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 in Drosophila parkin mutants, a loss-of-function model of PD. Our results suggest that oxidative stress is a feature of alpha-synuclein toxicity and that induction of the phase II detoxification pathway represents a potential preventative therapy for PD.

The PINK1/Parkin pathway: a mitochondrial quality control system?

Significant insight into the mechanisms that contribute to dopaminergic neurodegeneration in Parkinson disease has been gained from the analysis of genes linked to rare heritable forms of parkinsonism such as PINK1 and parkin, loss-of-function mutations of which cause autosomal recessive parkinsonism. PINK1 encodes a mitochondrially targeted Ser/Thr kinase and parkin encodes a ubiquitin-protein ligase. Functional studies of PINK1 and Parkin in animal and cellular model systems have shown that both proteins play important roles in maintaining mitochondrial integrity. Genetic studies of PINK1 and Parkin orthologs in flies have shown that PINK1 acts upstream from Parkin in a common pathway that appears to regulate mitochondrial morphology. Mitochondrial morphology is regulated by mitochondrial fission and fusion-promoting proteins, and is important in a variety of contexts, including mitochondrial trafficking and mitochondrial quality control. In particular, mitochondrial fission appears to promote the segregation of terminally dysfunctional mitochondria for degradation in the lysosome through a process termed mitophagy. Recent work has shown that Parkin promotes the degradation of dysfunctional mitochondria in vertebrate cell culture. Here we postulate a model whereby the PINK1/Parkin pathway regulates mitochondrial dynamics in an effort to promote the turnover of damaged mitochondria.