Leo Pallanck - US grants

IBN 97-34125 PALLANCK. This is a CAREER award that has both a research and educational component. In his research, Dr. Pallanck will investigate the mechanisms of regulated release of neurotransmitters at neuronal synapses. He will use the powerful genetic and molecular approaches to available in the fruit fly Drosophila to define cellular and molecular mechanisms involved in this process. Neurons communicate with each other and with targets such as muscle cells with neurotransmitter signaling molecules, which are released when small vesicles inside the synaptic terminal fuse with the outer membrane of the cell. Recent biochemical work has identified candidate molecules that are involved in recruiting vesicles to the cell membrane at precise locations, promoting fusion, and allowing the vesicle to reform when cell membrane is pinched off towards the inside of the cell. Dr. Pallanck's research will ask which of these steps are carried out by which of the previously identified molecules, and whether there are components of the release apparatus that remain unidentified by the previous biochemical studies. In previous work, Dr. Pallanck and his collaborators has identified in Drosophila two different forms of one of the key candidate release molecules, N-ethylmaleimide-Sensitive Fusion protein (NSF). One goal of the current research is to test the hypothesis that multiple NSF proteins are involved in the targeting, fusion, and/or recycling of synaptic vesicles. Molecular, electrophysiological and anatomical studies as the electron microscope level will all be employed in these studies. To address the second goal, Dr. Pallanck's laboratory will carry out classical genetic screens to identify novel components of the vesicular release apparatus. The properties of neurotransmitter release appear to be similar at synapses in all animals, and to share mechanisms with the vesicular release of other substances such as hormones. Thus, the results from this study will contribute to a general understanding of this process.
Dr. Pallanck's educational objective is to incorporate a research perspective into the undergraduate genetics curriculum at his university. He will be teaching the core genetics course taken by most undergraduate biology majors, and will use this forum to emphasize the experimental basis of scientific knowledge and to introduce students to current research opportunities in his department. He will also serve as undergraduate research coordinator, and will develop an undergraduate research seminar course. Through these three avenues, Dr. Pallanck will be instrumental in preparing undergraduate students for a career in research at a critical stage in their educational development.

DESCRIPTION (Provided by Applicant): Parkinson's disease is a prevalent neurodegenerative disorder characterized by tremors, rigidity, and bradykinesia. These symptoms are thought to arise from the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Recently, mutations of the parkin gene, which encodes a ubiquitin-protein ligase, were found to underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinson's disease (AR-JP). While this advance provides clues to the mechanism responsible for pathology in AR-JP, the cellular targets of the parkin ubiquitin-protein ligase activity and the specific biochemical pathways affected by parkin mutations remain largely unknown. To address these issues, the objectives of this proposal are to create a Drosophila melanogaster model of AR-JP through mutational analysis of a Drosophila parkin ortholog, and to use this fly AR-JP model to investigate the molecular mechanisms of neuronal dysfunction underlying parkin deficiency. Two main hypotheses will be explored in this proposal: (a) parkin sequesters alpha-synuclein protein into Lewy bodies and this function represents a cellular mechanism of alpha-synuclein detoxification; (b) neurodegeneration triggered by parkin mutations results from accumulation of parkin substrate(s). To accomplish the objectives of this proposal, the following specific aims will be pursued: (1) Generate and characterize Drosophila parkin (D-parkin) mutants; (2) Determine whether altered D-parkin expression affects the time course and extent of alpha-synuclein induced neurodegeneration and Lewy body formation; (3) Identify modifiers of a D-parkin mutant phenotype; (4) Isolate D-parkin-binding components and investigate structure-function relationships in D-parkin. Results from this work should clarify the relationship between parkin dysfunction and neurodegeneration, and possibly reveal strategies for treatment of Parkinson's disease.

DESCRIPTION (provided by applicant): Parkinson's disease is a prevalent neurodegenerative disorder characterized by tremors, rigidity, and bradykinesia. These symptoms arise from the degeneration of dopaminergic neurons in the substantia nigra. The cellular and molecular mechanisms responsible for neurodegeneration in Parkinson's disease remain poorly understood, although genetic and environmental factors both appear to play contributing roles. Recently, loss-of-function mutations in DJ-1, a gene of unknown function, were found to be responsible for an autosomal recessive form of Parkinson's disease. To explore the normal biological function of DJ-1, and the mechanism by which loss of DJ-1 function results in neurodegeneration, we propose to subject a pair of highly conserved Drosophila DJ-1 homologs (designated DJ-1a and DJ-1b) to mutational analysis. DJ-1a and DJ-1b function will be perturbed using P element mutagenesis, gene-targeting and double stranded RNA interference methods. The phenotypes resulting from perturbation of these genes will be fully characterized, including an analysis of dopaminergic neuron integrity. Additionally, we will characterize the global gene expression changes resulting from loss of DJ-1a and DJ-1b function and initiate screens for genetic modifiers of the DJ-1a and DJ-1b phenotypes to elucidate the biochemical pathways in which these genes function. This work should clarify the normal cellular role of DJ-1 and provide a foundation for further hypothesis-driven investigation of DJ-1 function.

[unreadable] DESCRIPTION (provided by applicant): Loss-of-function mutations of the parkin gene, which encodes a ubiquitin-protein ligase, are a major cause of early-onset Parkinson's disease, and increasing evidence suggests that parkin dysfunction may also play a role in late-onset typical Parkinson's disease. To explore the biological role of parkin, and the mechanism by which loss of parkin function results in neurodegeneration, we recently created a Drosophila model of Parkinson's disease through mutational inactivation of a highly-conserved Drosophila parkin ortholog. Drosophila parkin mutants are semi-viable and display a late developmental defect in spermatid formation, widespread apoptotic degeneration of flight muscle, and degeneration of a subset of dopamine neurons in the central nervous system. Mitochondrial pathology is a prominent and early characteristic of tissue degeneration in parkin mutants and a significant fraction of Parkin localizes to mitochondria. From these findings we hypothesize that Parkin promotes mitochondrial integrity by directly ubiquitinating particular mitochondrial targets. To identify the substrates of Parkin that mediate mitochondrial and tissue integrity we propose to use in vivo proteomic approaches to identify Parkin-binding components and proteins displaying reduced ubiquitination in parkin mutants. An advantage of using Drosophila for these studies is that findings from our proteomic studies can be rapidly validated in tissues that require Parkin for viability, including dopamine neurons. This work should clarify the biological role of Parkin and will serve as a foundation for future hypothesis-driven investigation of parkin pathogenesis. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common and highly debilitating movement disorder caused by the degeneration of dopaminergic neurons in the midbrain. The molecular mechanisms responsible for PD pathogenesis are poorly understood, and there are currently no preventative treatments for this disorder. Epidemiological studies aimed at the identification of environmental factors that influence the incidence of PD have provided overwhelming data in support of a substantially reduced risk of PD among coffee and tobacco users. While there are several possible interpretations of these findings, we hypothesize that tobacco and coffee contain chemicals that confer neuroprotection. To test this hypothesis we have been using two different Drosophila models of PD. Specifically, the two PD models that we have been using in our work include a fly strain bearing a null allele of the parkin gene, and a transgenic fly strain overexpressing the human 1-synuclein protein. Both of our fly models exhibit multiple characteristics of PD, most notably, selective degeneration of dopaminergic neurons in the central nervous system. In preliminary studies we have used our fly PD models to test the neuroprotective potential of tobacco and coffee extracts and to compare the neuroprotective effects of these extracts to nicotine and caffeine, the suspected neuroprotective agents of tobacco and coffee, respectively. Our studies indicate that coffee and tobacco are indeed neuroprotective in both of our fly models. However, nicotine and caffeine alone do not appear to confer neuroprotection, suggesting that the neuroprotective components of tobacco and coffee are novel chemical species. There are two major aims of the current application: the first aim involves identification and structural characterization of the neuroprotective components of tobacco and coffee; the second aim involves experiments to define the mechanism by which tobacco and coffee confer neuroprotection. Drosophila is ideal for these studies because the short generation time, powerful genetic tools, and evolutionary conservation of neurodegenerative disease mechanisms in this organism will allow us to quickly and efficiently explore the potential of these neuroprotective agents in a fashion that should be pertinent to humans. Our studies could ultimately lead to the development of preventative treatment strategies for PD. PUBLIC HEALTH RELEVANCE: People who smoke or drink coffee develop Parkinson's disease (PD) at a significantly lower frequency than the general public. These findings lead us to hypothesize that tobacco and coffee contain chemicals that confer protection from developing PD. We will use two different fly models of PD to test this hypothesis and to identify the specific chemical agents in tobacco and coffee that are responsible for protection. Our studies could ultimately lead to the development of preventative treatment strategies for PD. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mitochondrial dysfunction is a common feature of Parkinson's disease, but our knowledge of the underlying molecular mechanisms is rudimentary. Recently, a genetic pathway that influences mitochondrial integrity has emerged from studies of the PINK1 and parkin genes, loss-of-function mutations of which are responsible for the majority of early-onset recessive forms of parkinsonism. The PINK1 gene encodes a serine/threonine kinase that localizes to the mitochondrial inner membrane space and to the cytoplasm, whereas parkin encodes a ubiquitin-protein ligase that localizes broadly throughout the cell, including the cytoplasm and mitochondria. Mutational analyses of highly-conserved Drosophila orthologs of PINK1 and parkin indicate that PINK1 acts upstream of Parkin in a common pathway that promotes mitochondrial integrity in a subset of tissues, including indirect flight muscle and dopaminergic neurons in the central nervous system. Our recent work indicates that the PINK1/Parkin pathway influences mitochondrial and tissue integrity by promoting mitochondrial fission. We hypothesize from this and other findings that the PINK1/Parkin pathway promotes mitochondrial fission by phosphorylating and/or ubiquitinating particular components of the mitochondrial morphogenesis machinery. We further hypothesize that the cell death accompanying reduced mitochondrial fission in parkin and PINK1 mutants derives from inefficient delivery of damaged mitochondria to autophagosomes, defective trafficking of enlarged (fused) mitochondria to presynaptic nerve terminals, and/or excessive reactive oxygen species production by enlarged mitochondria. Two specific aims are proposed to address these hypotheses: the first aim will test whether the PINK1/Parkin pathway activates mitochondrial fission or inhibits mitochondrial fusion by using established mitochondrial fusion and fission assay systems, and by testing candidate substrates of the PINK1/Parkin pathway. The second aim involves genetic, molecular and cell biological experiments to distinguish between specific models by which impaired mitochondrial fission in PINK1 and parkin mutants influences tissue integrity. From these experiments, we hope to advance our long-term goal of elucidating the mechanisms underlying mitochondrial dysfunction and selective cell death in Parkinson's disease. PUBLIC HEALTH RELEVANCE: Loss-of-function mutations in the parkin or PTEN-induced kinase 1 (PINK1) genes are collectively the most frequent cause of autosomal recessive familial Parkinson's disease. Our proposal aims to understand the biological roles of Parkin and PINK1 and how their mutational inactivation results in neuronal loss. Insight from our studies of PINK1 and Parkin are likely to be relevant to more common idiopathic forms of Parkinson's disease and this insight could lead to the development of preventative treatments for this debilitating disorder.

DESCRIPTION (provided by applicant): Over the past several years, genetic and cell biological studies of the Parkinson's disease- related factors PINK1 and Parkin have begun to delineate the mechanisms by which damaged mitochondria are selectively detected and degraded. This work has led to the model that PINK1, a mitochondrially targeted serine/threonine kinase, is selectively stabilized on the surface of damaged mitochondria where it recruits Parkin, a cytosolic E3 ubiquitin ligase. Parkin then ubiquitinates particular mitochondrial proteins to isolate the damaged mitochondria, and to promote their eventual degradation through autophagy. While this work has tremendously advanced our understanding of the mechanisms by which damaged mitochondria are detected and degraded, many critical questions remain unanswered. In particular, the factors that regulate the stability, localization and activity of PINK1 and Parkin are poorly understood. Additionally, while some of the Parkin substrates required to isolate damaged mitochondria are known, the Parkin substrates required for the subsequent autophagic degradation of these mitochondria are unknown. Finally, whether the PINK1-Parkin pathway also acts in an autophagy-independent manner to influence mitochondrial integrity is unclear. To address these and other matters, we are performing a comprehensive genetic screen in Drosophila to identify novel components of the PINK1-Parkin pathway. The goal of our proposal is to determine how some of the factors from our screen influence this pathway. Specifically, we will pursue four Aims. First, we will test the hypothesis that three key mitochondrial biogenesis-promoting factors identified in our screen are important downstream targets of regulation by PINK1 and Parkin. Second, we will test the hypothesis that two deubiquitinating enzymes identified in our screen influence the PINK1-Parkin pathway by acting directly on PINK1, Parkin, or the Parkin substrates mitofusin, miro, or PARIS. Third, we will test the model that two mitochondrial proteases identified in our screen influence the activit of PINK1 by promoting its delivery to the matrix for degradation. Fourth, we will use a novel proteomic assay of protein turnover, and simple cell biological assays to categorize the remaining modifiers in our collection. Insight from our studies should be directly relevant to the etiology and treatment of Parkinson's disease, as well as the many other diseases in which mitochondrial dysfunction is implicated.

DESCRIPTION (provided by applicant): Mitochondrial DNA (mtDNA) mutations cause a number of severe maternally transmitted diseases, and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases of the elderly. These mtDNA mutations often coexist with normal mtDNA, a condition known as heteroplasmy. The ratio of mutated to wild-type mtDNA plays a crucial role in the pathogenesis of heteroplasmic disorders, but the mechanisms that influence this ratio are largely unknown. The long-term objective of our work is to define the cellular mechanisms that govern the frequency of deleterious mtDNA mutations in heteroplasmic somatic tissues. Recent work on the Parkinson's disease-related factors PINK1 and Parkin has revealed that these factors are components of a mitochondrial quality control system (MQCS) that can detect dysfunctional mitochondria, and, in collaboration with other cellular factors, promote their autophagic degradation. We hypothesize that the MQCS acts to reduce the frequency of deleterious heteroplasmic mtDNA mutations by detecting dysfunctional mitochondria that bear mutant DNA and targeting them for degradation. To test this hypothesis, we propose to use the model organism Drosophila melanogaster to pursue three aims. The first aim will examine the influence of genetic alterations of the MQCS on the phenotypes of a Drosophila strain with an increased mtDNA mutation frequency. The second aim will examine the influence of genetic perturbations of the MQCS on the mtDNA mutation frequency using a novel next-generation sequencing method. Finally, the third aim will use a recently developed in vivo assay of mitochondrial turnover to test whether mtDNA mutations promote mitochondrial turnover, and whether genetic perturbations of the MQCS influence the effects of mtDNA mutations on turnover. Our studies will contribute to an understanding of the molecular mechanisms that influence heteroplasmy, and this knowledge could ultimately lead to the development of treatments for diseases caused by mtDNA mutations.

? DESCRIPTION (provided by applicant): Gaucher's disease (GD), the most common lysosomal storage disorder, is caused by recessively inherited mutations in the glucosidase, beta, acid (GBA1) gene, which encodes glucocerebrosidase, a lysosomal enzyme that catalyzes the breakdown of the sphingolipid glucosylceramide. GD encompasses a wide spectrum of clinical symptoms, including severe and untreatable neurological abnormalities. Over the past ten years it has become increasingly clear that patients with GD also have a dramatically elevated risk of Parkinson's disease (PD), a common movement disorder characterized by the death of dopaminergic neurons in the midbrain and the accumulation of intracellular neuronal protein aggregates known as Lewy bodies. More recently, this finding has led to the discovery that heterozygous mutations in GBA1 are the most common genetic association with PD and Lewy body dementia (LBD), possibly accounting for 7-10% of these diseases. The mechanisms by which GBA1 mutations cause neurological symptoms of GD, and increase the risk of PD and LBD, are poorly understood. We hypothesize that the neurological symptoms of GD derive from the lysosomal accumulation of glucosylceramide, and a consequent defect in the degradative capacity of the lysosome. We further hypothesize that mutations in GBA1 increase the risk for PD and LBD by impairing the lysosomal degradation of ?-synuclein protein, a major component of the Lewy body aggregates that define both of these diseases. Finally, we hypothesize that genetic modifiers largely account for the poor genotype-phenotype correlation observed in GD and the low penetrance of GBA1 mutations in PD and LBD. To explore these hypotheses, we have created a Drosophila model of glucocerebrosidase deficiency that exhibits shortened lifespan, locomotor and memory deficits, neurodegeneration, and accumulation of protein aggregates. We propose to use our Drosophila model of glucocerebrosidase deficiency, as well as an existing mouse model of glucocerebrosidase deficiency, to pursue three aims. First, we will test the hypothesis that lysosomal glucosylceramide accumulation is responsible for the phenotypes of our Drosophila model of glucocerebrosidase deficiency. Second, we will test the hypothesis that glucocerebrosidase deficiency impairs lysosomal protein degradation, including the degradation of ?-synuclein. Third, we will conduct a genetic screen to identify novel modifiers of the phenotypes caused by glucocerebrosidase deficiency. Our work will contribute to a mechanistic understanding of GD, PD and LBD, and this insight will facilitate risk assessment in the clinic and provide a foundation for the development of treatments.

? DESCRIPTION (provided by applicant): Recent studies of the Parkinson's disease factors PINK1 and Parkin indicate that they play a critical role in the degradation of damaged mitochondria through a mitochondrial selective form of autophagy, termed mitophagy. This advance has fostered a greater appreciation of the importance of mitochondrial quality control to human health, and has led to a dramatic increase in research on mitophagy. However, mitophagy is only one of several mechanisms of mitochondrial quality control, and our recent work indicates that mitophagy accounts for less than half of all mitochondrial protein degradation in the model organism Drosophila. This unexpected finding suggests that non- mitophagic degradative processes are primarily responsible for mitochondrial protein quality control. We hypothesize from this and other findings that mitochondrial-resident proteases account for the majority of the protein degradative quality control that occurs in mitochondria. While genetic studies of the mitochondrial ATPase Associated with diverse cellular Activities (AAA+) family of proteases in yeast suggest that they play an important role in mitochondrial protein quality control, few of their substrates are known. Furthermore, the metazoan counterparts of these proteases have been little studied, despite the fact that mutations in the genes encoding several of them cause human disease. We propose to identify in vivo substrates of the four metazoan AAA+ mitochondrial proteases in Drosophila by using a stable isotope proteomic method to compare the half-lives of mitochondrial proteins in WT flies with those in flies that bear genetic perturbations targeting these proteases. Moreover, we will test whether these proteases degrade mitochondrial proteins that are damaged by two oft-cited mitochondrial stresses: oxidation and protein misfolding. Our work will advance the basic understanding of mitochondrial quality control by defining the biological roles of these proteases, and will provide a foundation to study the mechanisms by which mutations in the genes encoding these proteases cause disease.

DESCRIPTION (provided by applicant): Mitochondrial DNA mutations cause a number of severe childhood-onset mitochondrial syndromes, and mitochondrial dysfunction is associated with common age-related diseases such as diabetes, Alzheimer's disease and Parkinson's disease. However, there are currently no curative treatments for any of these diseases. One promising avenue of therapy for mitochondrial diseases involves the use of chemical agents that can activate a cellular quality control pathway that is capable of selectively eliminating dysfunctional mitochondria. Recent work indicates that the PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin function in such a pathway. Moreover, our preliminary studies demonstrate that overexpression of PINK1 and Parkin in the fruit fly Drosophila melanogaster rescues fly models of mitochondrial disease, thus validating the therapeutic potential of this pathway. The goal of our proposal is to test whether PINK1-Parkin pathway activating compounds identified from high-throughput cell culture-based screens can also rescue fly models of mitochondrial disease. To achieve this goal, we propose two aims. First, we propose to test whether PINK1-Parkin pathway activating compounds identified from cell culture-based screens can rescue an easily assayed phenotype associated with our Drosophila models of mitochondrial disease. Second, we will test whether the most promising compounds identified in our first aim act in a PINK1 and Parkin-dependent fashion, and whether these compounds can rescue other phenotypes of our mitochondrial disease models. Our work will potentially identify compounds that can be used to treat a wide variety of human diseases.

Accumulation of brain protein aggregates is a hallmark of Alzheimer?s disease and many other neurodegenerative disorders. Brain protein aggregation was originally assumed to be a cell-autonomous phenomenon in Alzheimer?s disease, but increasing evidence indicates that non?cell-autonomous processes also play major roles by promoting the spread of toxic aggregation-prone proteins. Although the principal components of the brain protein aggregates that accumulate in Alzheimer?s disease victims have been detected in extracellular vesicles (EVs), the mechanisms underlying the spread of protein aggregates in Alzheimer?s disease are poorly understood. Recent work in our lab suggests that the GBA gene plays an important role in this process. GBA encodes the lysosomal enzyme glucocerebrosidase, which catalyzes the conversion of the sphingolipid glucosylceramide to glucose and ceramide. Mutations in GBA cause several neurodegenerative diseases characterized by the accumulation of protein aggregates in the brain. To study the mechanisms by which mutations in GBA cause disease, we created a Drosophila model of glucocerebrosidase deficiency by deleting the Drosophila GBA ortholog, GBA1b. GBA1b mutants accumulate ubiquitinated protein aggregates, show age-related neurodegeneration, have changes in the turnover and abundance of EV proteins and exhibit a six-fold elevation in EV abundance. Furthermore, ectopic expression of GBA1b in peripheral tissues such as muscle or gut rescued protein aggregation in the brain. These findings lead us to hypothesize that mutations in GBA1b result in lipidomic alterations promoting the overproduction of extracellular vesicles that spread protein aggregates from peripheral tissues to the brain and possibly between cells in the brain. To test this hypothesis and explore the underlying mechanisms, we propose three aims. First, we will test whether the non?cell-autonomous rescue of brain protein aggregation in GBA1b mutants is mediated by extracellular vesicles, and whether this pathway influences the protein aggregates seen in other common neurodegenerative diseases, including Alzheimer?s disease. Second, we will test whether GBA1b mutants promote the spread of endogenous protein aggregates, as well as those seen in Alzheimer?s and prion disease, by blocking the production of EVs in GBA1b mutants and by transplanting EVs from GBA1b mutants to WT flies. Third, we will perform proteomic, lipidomic and cell biological experiments to explore how mutations in GBA1b alter extracellular vesicle formation and composition. Given the increasing evidence of peripheral influences on the spread of brain protein aggregates in Alzheimer?s disease and other neurodegenerative disorders, we anticipate that our findings will advance our understanding of this phenomenon and also create novel opportunities for therapeutic intervention in these diseases.

The common neurodegenerative disorders Alzheimer?s disease (AD) and Parkinson?s disease (PD) are both characterized by the accumulation and spread of protein aggregates through the nervous system. As in prion disorders, the protein aggregates that occur in AD and PD are believed to propagate by transfer of aggregate seeds to recipient cells, where the seeds serve as templates for conversion of their normally folded counterparts into aggregating forms. The spread of protein aggregates is accepted as an essential part of the pathogenesis of both AD and PD, yet none of the more than 100 loci that influence the risk of AD and PD are known to affect the spread of protein aggregates between tissues, and the underlying mechanisms are poorly understood. One reason for this gap in understanding is the scarcity of genetically tractable in vivo model systems for studying propagation and spread of aggregated tau and alpha-synuclein, two key aggregate-prone proteins in AD and PD. The goal of this pilot project grant is to develop a simple, modular, and highly tractable set of Drosophila models to study the spread of protein aggregation. First, we will create transgenic lines that will permit us to express multiple combinations of WT and mutant forms of tau and alpha-synuclein. We will then use these lines to express the WT forms of these proteins at low levels throughout the nervous system, while overexpressing WT or mutant forms of the same protein in a small subset of cells to initiate aggregation. Using epitope tags to distinguish the products of the transgenes, we will visualize and quantify the resulting spread of aggregates. This set of transgenic lines will facilitate rapid investigation of the effects of previously identified genetic risk factors and candidate cellular pathways on the spread of protein aggregates in neurodegenerative disease. Our system will thus advance understanding of the mechanisms underlying the spread of protein aggregates in AD and PD, which in turn may reveal new targets for therapeutic intervention in these common and debilitating disorders.

ABSTRACT: Mutations in the glucosylceramidase beta (GBA) gene cause the lysosomal lipid storage disorder Gaucher?s disease and are the most frequent genetic association with Parkinson?s disease and Lewy body dementia. GBA encodes glucocerebrosidase, a lysosomal enzyme that catalyzes the breakdown of the sphingolipid glucosylceramide to ceramide and glucose. To explore the mechanism by which mutations in GBA predispose to these diseases, we created a Drosophila model of glucocerebrosidase deficiency by inactivating the Drosophila GBA ortholog, Gba1b. Gba1b mutants recapitulate many of the features of these diseases, including shortened lifespan, locomotor impairment, accumulation of glucosylceramide, protein aggregation in brain and other tissues, and neurodegeneration. In recently published work, we reported the results of a proteomic study of Drosophila Gba1b mutants that revealed dramatic alterations in the abundance and turnover of extracellular vesicle (EV) proteins. Our experiments also demonstrated that these proteomic findings reflected actual changes in the composition of EVs, and that genetic perturbations targeting factors involved in the production of EVs suppressed a Gba1b mutant phenotype. In more recent unpublished work, we used RNA-Seq to compare transcript abundance in Gba1b mutants and controls. This study revealed a profound induction of the innate immune response pathway in Gba1b mutants. This induction was specific to Gba1b mutants and was further corroborated in our proteomic data, and RNAi-mediated knockdown of an innate immune pathway component partially suppressed the brain protein aggregation phenotype of Gba1b mutants. From these and other findings, we hypothesize that the production of glucosylceramide-enriched EVs by Gba1b mutants triggers an innate immune response because these EVs resemble the glucosylceramide-enriched EVs released by pathogens during infection. We further hypothesize that this innate immune response accounts for the phenotypes of Gba1b mutants. We propose two aims to address these hypotheses; the first will investigate the mechanism of immune activation, and the second will investigate the importance of immune activation to Gba1b mutant pathogenesis. Thus, the primary goal of our research proposal is to provide a foundation for further mechanistic work by asking the two most fundamental questions raised by our preliminary findings: how does innate immune activation occur in Gba1b mutants, and is it important to their phenotypes? Given the increasing evidence for neuroinflammation in neurodegenerative disorders, including those caused by GBA mutations, we anticipate that our work will have broad medical significance.