Dimitri Krainc - US grants

DESCRIPTION: (Adapted From The Applicant's Abstract): The goal of this project is to elucidate the function of huntingtin protein, mutation of which underlies Huntington's disease (HD). These mutations are expansions of a polyglutamine repeat motif (coded by CAG trinucleotide repeats in the mRNA) within the protein, making HD one of several known "glutamine repeat disorders". In this group of neurodegenerative diseases, an expansion of a polyglutamine repeat in a number of different proteins confers the various disease phenotypes. However, in spite of the relatively restricted patterns of differential cell death in HD and the other disorders, the gene products are widely distributed in both central nervous system and peripheral tissues, and both their normal functions and mechanisms of disease are largely unknown. This study will examine the function of huntingtin through three specific aims. First, analysis of the interaction between huntingtin and Sp1 will be performed. In our preliminary work, using the yeast two-hybrid system we showed that the N-terminus portion of huntingtin specifically interacts with transcription factor Spl. In this study the effect of expansions of the polyglutamine tract on the strength of Sp1-huntingtin interaction will be assessed. The domains of interaction will be studied by deletional analysis of the respective cDNA's. The effect of huntingtin protein with variable glutamine repeats on transcription will then be determined. Our preliminary results showed that huntingtin and Sp1 synergistically activate the promoter of NMDAR1 subtype of glutamate receptor. To examine the promoter regions involved in Sp1/huntingtin mediated regulation, a deletion analysis and in-vitro mutagenesis of promoter elements and proteins will be employed. Finally, we will attempt to generate a huntingtin dominant-negative construct to alter the activity of NMDAR1 promoter.

Huntington's Disease (HD) is dominantly inherited neurodegenerative disorder manifested by psychiatric, cognitive and motor symptoms typically starting in mid life and progressing toward death. HD is caused by an expansion of a polyglutamine tract in tile huntingtin protein. The number of diseases caused by polyglutamine expansions continues to, grow and a common mechanism could underlie these disorders. One hypothesis suggests that acting within the nucleus, polyglutamine expansion results in aberrant interactions with nuclear proteins, thereby leading to transcriptional dysregulation. Gene expression arrays on DNA microchips have showed that the scope of transcriptional changes in transgenic HD mice involves different groups of genes, including neurotransmitter receptors. Interestingly, when the known regulatory sequences of these genes were examined it became apparent that they contained binding sites for transcription factor Spl, suggesting that huntingtin may interfere with Spl-mediated transcription. Our preliminary data demonstrate that both normal and mutant huntingtin interact with Spl, whereas only mutant huntingtin significantly inhibits Splmediated transcription of dopamine receptors. In addition, we showed that Spl and its coactivators play a critical role in huntingtin-induced neuronal toxicity. In this work we propose to use our model to accomplish the following Specific Aims: 1) Characterize the specific components of transcriptional machinery required by normal or mutant huntingtin to regulate Spl-mediated expression of dopamine receptors in HD. 2) Examine activity and expression levels of Spl and related factors in transgenic and human HI) brain 3) Determine whether interference by drugs such as mithramycin and HDAC inhibitors with ,Spl-mediated gene transcription may protect against mutant huntingtin. Completion of these aims should reveal new insights into general mechanisms of neurodegeneration and lead to the identification of potential molecular targets for new HI) therapies.

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the function of huntingtin protein, mutation of which underlies Huntington's disease (HD). These mutations are expansions of a polyglutamine repeat motif (coded by CAG trinucleotide repeats in the mRNA) within the protein, making HD one of several known "glutamine repeat disorders". In this group of neurodegenerative diseases, an expansion of a polyglutamine repeat in a number of different proteins confers the various disease phenotypes. However, in spite of the relatively restricted patterns of differential cell death in HD and the other disorders, the gene products are widely distributed in both central nervous system and peripheral tissues, and both their normal functions and mechanisms of disease are largely unknown. Transcriptional dysregulation and loss of function of transcriptional co-activator proteins have been implicated in the pathogenesis of these diseases. Gene expression arrays on DNA microchips have recently been used to examine the scope of transcriptional changes in transgenic HD mice. The results showed that the genes whose expression levels were altered were part of several different molecular systems, suggesting that mutant huntingtin may affect common transcriptional mechanisms. Our preliminary data demonstrates that mutant huntingtin interacts with components of TFIID complex to inhibit transcription of target genes. In this work we will use cultured striatal cells and transgenic HD mice and postmortem human HD brain samples to identify mechanisms by which mutant huntingtin could trigger neuronal cell death by interfering with the function of TFIID. We will characterize the pathways leading to tissue-restricted transcriptional dysregulation in HD, focusing on the earliest changes in gene expression triggered by mutant huntingtin. Completion of these aims should reveal new insights into general mechanisms of polyglutamine-induced neurodegeneration and lead to identification of new molecular targets for therapies in HD.

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the function of huntingtin protein, mutation of which underlies Huntington's disease (HD). These mutations are expansions of a polyglutamine repeat motif (coded by CAG trinucleotide repeats in the mRNA) within the protein, making HD one of several known "glutamine repeat disorders". In this group of neurodegenerative diseases, an expansion of a polyglutamine repeat in a number of different proteins confers the various disease phenotypes. However, in spite of the relatively restricted patterns of differential cell death in HD and the other disorders, the gene products are widely distributed in both central nervous system and peripheral tissues, and both their normal functions and mechanisms of disease are largely unknown. Transcriptional dysregulation and loss of function of transcriptional co-activator proteins have been implicated in the pathogenesis of these diseases. Gene expression arrays on DNA microchips have recently been used to examine the scope of transcriptional changes in transgenic HD mice. The results showed that the genes whose expression levels were altered were part of several different molecular systems, suggesting that mutant huntingtin may affect common transcriptional mechanisms. Our preliminary data demonstrates that mutant huntingtin interacts with components of TFIID complex to inhibit transcription of target genes. In this work we will use cultured striatal cells and transgenic HD mice and postmortem human HD brain samples to identify mechanisms by which mutant huntingtin could trigger neuronal cell death by interfering with the function of TFIID. We will characterize the pathways leading to tissue-restricted transcriptional dysregulation in HD, focusing on the earliest changes in gene expression triggered by mutant huntingtin. Completion of these aims should reveal new insights into general mechanisms of polyglutamine-induced neurodegeneration and lead to identification of new molecular targets for therapies in HD.

Huntington's Disease (HD) is dominantly inherited neurodegenerative disorder manifested by psychiatric, cognitive and motor symptoms typically starting in mid life and progressing toward death. HD is caused by an expansion of a polyglutamine tract in tile huntingtin protein. The number of diseases caused by polyglutamine expansions continues to, grow and a common mechanism could underlie these disorders. One hypothesis suggests that acting within the nucleus, polyglutamine expansion results in aberrant interactions with nuclear proteins, thereby leading to transcriptional dysregulation. Gene expression arrays on DNA microchips have showed that the scope of transcriptional changes in transgenic HD mice involves different groups of genes, including neurotransmitter receptors. Interestingly, when the known regulatory sequences of these genes were examined it became apparent that they contained binding sites for transcription factor Spl, suggesting that huntingtin may interfere with Spl-mediated transcription. Our preliminary data demonstrate that both normal and mutant huntingtin interact with Spl, whereas only mutant huntingtin significantly inhibits Splmediated transcription of dopamine receptors. In addition, we showed that Spl and its coactivators play a critical role in huntingtin-induced neuronal toxicity. In this work we propose to use our model to accomplish the following Specific Aims: 1) Characterize the specific components of transcriptional machinery required by normal or mutant huntingtin to regulate Spl-mediated expression of dopamine receptors in HD. 2) Examine activity and expression levels of Spl and related factors in transgenic and human HI) brain 3) Determine whether interference by drugs such as mithramycin and HDAC inhibitors with ,Spl-mediated gene transcription may protect against mutant huntingtin. Completion of these aims should reveal new insights into general mechanisms of neurodegeneration and lead to the identification of potential molecular targets for new HI) therapies.

[unreadable] DESCRIPTION (provided by applicant): The goal of this project is to elucidate the function of huntingtin protein, mutation of which underlies Huntingtin's disease (HD). These mutations are expansions of a polyglutamine repeat motif (coded by CAG trinucleotide repeats in the mRNA) within the protein, making HD one of several known "glutamine repeat disorders". In this group of neurodegenerative diseases, an expansion of a polyglutamine repeat in a number of different proteins confers the various disease phenotypes. Transcriptional dysregulation and abnormalities in energy metabolism have been implicated in the pathogenesis of HD. Here we hypothesize that these two processes may be functionally linked in HD. Our preliminary data demonstrates that mutant huntingtin inhibits transcription of coactivator PGC-1a, a major regulator of mitochondrial biogenesis and cellular respiration in several cell types. Increased PGC-1a levels in different tissues lead to enhanced mitochondrial electron transport that enable cells to meet raising energy demands. In this work, we propose to analyze the transcriptional mechanisms by which mutant huntingtin interferes with the function of PGC-1a. Transgenic mice expressing PGC-1a in the brain will be generated and crossed with HD mice to determine whether gain-of-function of PGC-1a alters survival and neuropathology in HD transgenic mice. In addition, mechanisms that lead to modulation of PGC-1a activity in HD will be studied. Upstream regulators controlling PGC-1a transcription in HD cells will be identified to determine whether activation of PGC-1a leads to correction of energy deficits in HD. Target genes of PGC-1a and markers of mitochondrial function will be analyzed in HD cell and tissues. Completion of these aims should reveal new insights into general mechanisms of polyglutamine-induced neurodegeneration and lead to identification of new molecular targets for therapies in HD. [unreadable] PUBLIC HEALTH RELEVANCE: The goal of this project is to elucidate neuroprotective mechanisms in Huntington's disease (HD). We have evidence that disruptions of energy metabolism and deregulation of gene transcription are functionally linked in HD. Our preliminary data suggest that mutant huntingtin represses transcriptional regulation of peroxisome proliferator activator receptor coactivator 1 alpha (PGC-1a). Recent studies have implicated PGC-1a as a major regulator of mitochondrial biogenesis and cellular respiration in several cell types, suggesting that disruption of PGC-1a expression may lead to cellular dysfunction and cell death. We propose a study to search for activators and modulators of PGC-1a expression and activity in HD. Since PGC-1alpha has been implicated in various disorders, including Parkinson's disease, such regulators of PGC-1a function may prove valuable for development of neuroprotective therapies for HD and other neurodegenerative disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Accumulation and aggregation of disease-causing proteins is a hallmark of several neurodegenerative disorders such as Parkinson's, Alzheimer's and Huntington's disease. One of the main goals of research in neurodegenerative disorders has been to improve clearance of these accumulated proteins by selectively activating degradation machinery such as autophagy. Current approaches to modulate autophagy result in global and non-specific activation of autophagic and other cellular pathways. Using an example of Huntington's disease, we reported that selective clearance can be achieved by posttranslational modification of the mutant htt by acetylation at lysine residue 444 (K444). Increased acetylation at K444 facilitates trafficking of mutant Htt into autophagosomes, significantly improves clearance of the mutant protein by macroautophagy and reverses the toxic effects of mutant huntingtin in vitro and in vivo. These preliminary studies suggest a critical role for acetylation in the control of regulated clearance of mutant huntingtin. Here we propose to examine the molecular pathways of such acetylation-mediated clearance of mutant htt. In Aim1 we propose to validate the role of mutant htt acetylation in vivo by generating a knockin mouse model expressing acetylation-resistant full-length mutant htt to perform biochemical, neuropathological and behavioral studies. Aim 2 will examine the interactions of autophagy machinery with acetylated mutant huntingtin. The cargo recognition process of proteins destined for the autophagosome is not well understood. We propose to assess interactions of acetyl-htt with the autophagy machinery, specifically the proteins involved in shuttling protein cargo to the autophagosome. In Aim 3 we will examine the role of HDAC inhibitors in promoting clearance of mutant huntingtin. Using cellular models systems, we will examine the relative contribution of various HDACs to clearance of acetylated mutant Htt. In summary, the proposed study will examine how cells regulate mutant Htt levels, a critical risk factor for HD. Identifying the mechanisms that control mutant Htt levels could lead to novel therapeutic strategies by directly targeting mutant Htt. Understanding the molecular mechanisms involved in the recognition and selective autophagic clearance of mutant Htt may facilitate studies of other disease-causing proteins that accumulate in various neurodegenerative disorders.

SUMMARY Lysosomal dysfunction has been increasingly implicated in the development of neurodegenerative diseases including Parkinson's disease (PD). Mutations in GBA1 encoding ?-glucocerebrosidase (GCase) cause Gaucher disease (GD), the most prevalent lysosomal storage disorder and represent an important genetic risk factor for synucleinopathies including PD and dementia with Lewy Bodies (DLB). A significant reduction in GCase activity has been also reported in brains of sporadic PD patients, suggesting that reduced GCase activity may represent a common feature of PD pathogenesis. Genetic linkage with PD and DLB has been also demonstrated for lysosomal membrane protein LIMP-2, the lysosomal trafficking receptor for GCase and GD modifier. Mutations in SCARB2 encoding LIMP-2 itself are disease-causing for a rare form of progressive myoclonic epilepsy associated with renal failure (AMRF). Our previous data showed that loss of function of LIMP-2 results in mistrafficking and lysosomal depletion of GCase and is also associated with a PD-like pathology in mice. We have also demonstrated that by elevating the levels of LIMP-2, lysosomal GCase activity was enhanced and ?-syn levels reduced, suggesting that both proteins are functionally linked in the regulation of lysosomal function and ?-syn metabolism. Therefore, we hypothesize that it will be critically important to consider LIMP-2-GCase interaction in the development of activators of GCase as potential therapeutics for PD and related synucleinopathies. To test this hypothesis, we propose to further examine the contribution of LIMP-2 in the pathogenesis of synucleinopathies by providing molecular insights into the formation and trafficking of the LIMP-2/GCase complex, and its role in normal and diseased human neurons. Aim 1 will examine the assembly and stoichiometry of the wild type LIMP-2/GCase complex in live cells by using photoactivatable amino acids, pulse chase and immunoprecipitations experiments. These studies will be extended to GCase and LIMP-2 patient-linked mutations to determine their impact on the formation of the LIMP-2/GCase complex. Aim 2 will examine time-dependent phenotypes in LIMP-2- and GCase-deficient human midbrain neurons. We will generate iPSC-derived midbrain neurons from LIMP-2 patient fibroblasts to examine subcellular localization of GCase and GCase/LIMP-2 complex, lysosomal proteolysis and morphology, accumulation of lipid substrates and ?-synuclein in a time-dependent manner. In Aim 3 we will perform neuropathological characterization of a mouse model expressing LIMP-2 that is deficient in binding to GCase. To directly examine if the neurological phenotypes we previously described in LIMP-2 knockout mice result from depletion of lysosomal GCase, we generated a mouse model expressing LIMP-2-Y163D mutant that cannot bind GCase. These mice will be analyzed for ?-syn and lipid accumulation, lysosomal dysfunction, inflammation and neurotoxicity. These experiments will also establish whether the neuropathology mediated by LIMP-2 deficiency may be at least in part independent of GCase.

DESCRIPTION (provided by applicant): Sirt1, an NAD-dependent protein deacetylase has emerged as important regulator of mammalian transcription in response to cellular metabolic status and stress. SIRT1 protects from Wallerian degeneration and protects against neurodegeneration in models of Alzheimer's disease, Amyotrophic lateral sclerosis, Huntington's and Parkinson's disease. The fact that Sirt1 protects in various disease models suggests a more general function of Sirt1 in normal and abnormal neuronal function. However, normal function of Sirt1 in neurons remains largely unknown. As part of our initial effort to defin the physiological function of Sirt1 in CNS, we found that SIRT1 deacetylates TORC1 (Transducer of Regulated CREB activity) and activates CREB mediated transcription. As coactivator of CREB, TORC1 regulates transcription of a number of important genes that have been implicated in the pathogenesis of neurodegenerative disorders, such as PGC-1alpha and BDNF. TORC1 regulates neuronal activity-dependent CREB transcription and we hypothesize that Sirt1 plays a role in this process. Since TORC1 is only expressed in CNS and testis, it potentially represents a unique target of neuronal Sirt1 function. However, it is likely that Sirt1 regulates other targets in neurons, especially in light of the fact that more than forty targets of Sirt1 have been identified in non-neuronal systems. We found that Sirt1 deactylase activity was inhibited by mutant huntingtin. This inhibition presumably leads to deregulation of numerous targets of Sirt1 in HD brain. We propose to examine the role of Sirt1 and its targets in normal and HD neurons. Neuronal activity-dependent regulation gene expression will be assessed in primary neurons in the presence or absence of Sirt1. TORC1-dependent expression profiles will be compared with Sirt1 profiles. We will also examine if higher levels of Sirt1 overexpression afford dose-dependent neuroprotection in HD-like mice. Unbiased and biased studies will be performed to examine Sirt1 targets in HD pathogenesis. More generally, this work will further our understanding of the precise mechanistic link between the sirtuins and healthy brain aging, and potentiate development of drugs that delay and ameliorate neurodegenerative diseases.

? DESCRIPTION (provided by applicant): Dopamine (DA) deficiency, caused by DA neuron degeneration, underpins the devastating motor symptoms of Parkinson's disease (PD). DA neurons located in the ventral tier of the substantia nigra pars compacta (SNc), are particularly vulnerable, compared to those in the dorsal tier of the SNc or ventral tegmental area (VTA). Why these DA neurons display differential vulnerability remains enigmatic. Understanding the underlying mechanisms would shed light on degeneration as well as potential neuroprotective strategies to mitigate the disease. iPS-derived DA neurons are an important new method for modeling PD. Yet current protocols for generating DA neurons are not designed to generate specific DA subtypes, a critical requisite for modeling selective vulnerability. This gap exists because the molecular heterogeneity of midbrain DA neurons is not well understood. To elucidate the heterogeneity of DA neurons, we have recently used single cell molecular profiling, coupled with anatomical co-labeling studies, and revealed the existence of at least six distinct of DA neuron subtypes in mouse models. Here, we aim to use this knowledge to i. better understand DA neuron diversity in vivo ii. understand mechanisms that may influence the generation of DA neuron subtypes iii. derive and characterize two prominent DA neuronal subtypes, one located in the SNc and one in the VTA, from human iPS cells in a rational manner, and iv. use these DA neuron subtypes to examine selective vulnerability in the context of genetic PD mutations. In Aim1, we will examine how Wnt signaling may influence DA neuron subtype allocation. In Aim 2, having optimized the Wnt regimen, we will next use targeted gene manipulations to derive highly enriched cultures of two specific DA neuron subtypes, and then characterize those subtypes by physiological and transcriptomic approaches. Next, we will generate both DA neuron subtypes from iPS cells harboring a DJ-1 mutation and examine differential pathological effects on both, SNc as well as VTA DA neuron subtypes. In Aim 3, we will further characterize the phenotype of the two DA neuron subtypes in vivo. We will elucidate the projections, and complete transcriptomes of two murine DA neuron subtypes, taking advantage of genetically targeted mice. Information from this aim will further highlight the differences between these subtypes. Additionally, these results will feed back into Aims 1 and 2, to further optimize our DA neuron subtype derivation protocol. In sum, taking advantage of the combined expertise and extensive interactions of two labs, we propose a cohesive plan based on molecular logic, to derive distinct DA neuron subtypes from iPS cells and aim to improve modelling PD. These studies will open the future possibility of understanding the effects of a range of PD mutations on selective vulnerability.

Neurodegenerative disorders are characterized by the accumulation of misfolded aggregated proteins in neurons. Since neurons are permanently postmitotic, efficient intracellular protein degradation systems are critically important for normal neuronal function. Recent evidence suggests that disruption of lysosomal degradation pathways directly contributes to neurodegeneration in Parkinson's disease and related synucleinopathies. We have previously shown that loss of function of lysosomal ATPase PARK9 (ATP13A2) leads to zinc dyshomeostasis, lysosomal dysfunction and a-syn accumulation. In addition, we and other found that PARK9 localizes to multivesicular endosomes and regulates exosome biogenesis. Here, we propose to further analyze the physiological role of PARK9 in generation and secretion of exosomes and how loss of PARK9 function contributes to neuronal dysfunction and neurodegeneration. First, we will test the hypothesis that PARK9 plays an important role in the formation of intraluminal vesicles by recruitment of zinc-dependent FYVE proteins to early endosomes. Second, we will examine if a-syn secretion via exosomes and lysosomal exocytosis contributes to PARK9-mediated neuronal dysfunction. Finally, we will test the hypothesis that PARK9 is protective in synucleinopathies by overexpressing PARK9 in mouse models that accumulate a-synuclein. We will also examine propagation of a-synuclein in PARK9 knockout and transgenic mice. These findings will also provide further mechanistic insights into PARK9 loss of function in the context of Kufor-Rakeb syndrome as well as more general forms of syncleinopathies such as Parkinson's disease (PD), especially in terms of cell- to-cell transmission of a-syn that has been implicated in the pathogenesis of these disorders.

Summary While the cellular mechanisms involved in SN DA neurodegeneration are still not completely understood in PD, we recently identified a pathological cascade involving mitochondrial oxidant stress and oxidized DA that drives multiple downstream neurotoxic phenotypes in PD patient- derived neurons. Here, we propose to investigate the upstream mechanistic pathways leading to this oxidized DA accumulation in PD using imaging, biochemical and electrophysiological approaches in long-term cultures of patient-derived DA neurons. As recent genetic studies have identified synaptic genes linked to PD, our first aim will determine whether familial PD genes including parkin and LRRK2 interact with PD-linked synaptic genes to modulate synaptic vesicle endocytosis. In our second aim, we will analyze whether sporadic PD modeled by mitochondrial Complex I deficiency leads to defective synaptic vesicle turnover due to loss of synaptic ATP production. In both aims, our working hypothesis is that defects in synaptic vesicle endocytosis prevents efficient cytosolic DA uptake and, in the presence of mitochondrial oxidant stress, this deficit results in the accumulation of cytosolic oxidized DA and downstream pathogenic phenotypes. Finally, our third aim is to determine whether glutamatergic signaling through metabotropic glutamatergic receptors further drives mitochondrial oxidant stress and DA oxidation. Increased glutamatergic signaling arising from the STN and PPN could promote degeneration of SN DA neurons in the later stages of PD, but it is unclear whether the mechanisms present in rodent SN DA neurons are recapitulated in human neurons. Together, these aims will further inform the mechanisms studied in Projects 1 and 2, and elucidate their role in driving mitochondrial stress, oxidized DA accumulation and downstream PD pathogenesis in human DA neurons.

Summary I believe that combining disease gene discovery approaches with in-depth follow-up mechanistic and functional studies is a unique aspect of my research program. Our recent discovery of ?human-specific? pathways and phenotypes (compared to mice) in midbrain DA neurons has led us to focus on patient-derived DA neurons to examine the function of PD-linked genes. By employing co-cultures of iPS-derived neurons, microglia and astrocytes, we will examine the interplay of cell-autonomous and no-cell autonomous pathways that lead to dysfunction of midbrain DA neurons in PD. Moreover, we will use innovative technology to simultaneously examine a large number of genetic variants in a pooled iPS approach that has not been possible previously. Finally, our recent discovery of direct contacts between lysosomes and mitochondrial has opened a completely new opportunity to examine inter- and intra-organellar dynamics in neurodegeneration. The R35 award would provide me the time, freedom and stability to be even more adventurous and, as always, follow the most interesting biology to have a high impact on the field.

With a rapidly aging population, neurodegenerative diseases such as Parkinson?s Disease (PD), are expected to rise to 25% by 2030, presenting a huge economical and emotional challenge to society. While most PD cases are sporadic, approximately 10-15% are familial. One of the few genes leading to early-onset PD is the recently discovered VPS13C (Vacuolar Protein Sorting 13 Homolog C). Autosomal-recessive mutations in Vps13C result in protein-truncation and loss of function, with patients demonstrating Lewy body pathology with ?-synuclein aggregation in both dopaminergic and cortical neurons. While Vps13C was recently implicated in the endolysosomal pathway in non-neuronal cells, its neuronal function and how loss of this function leads to PD in patient neurons still remains to be elucidated. Through an unbiased mass-spectrometry based screen, we recently identified Vps13C as a novel interactor of Ykt6, a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein critically involved in the endolysosomal pathway and linked to the pathobiology of ?-synuclein. Interestingly, we further found that the Ykt6 and Vps13C interaction was regulated by the phosphatase activity of Calcineurin, a master regulator of Ca2+ signaling and a key player of toxicity in several PD models. Importantly, we demonstrated that the Calcineurin-dependent phosphorylation site in Ykt6 is a critical regulatory step mediating autophagosome to lysosome fusion during autophagy, and Vps13C can further regulate autophagy. Based on strong preliminary data, the goals of this proposal are to investigate the role for Vps13C in regulating neuronal autophagy via its interaction with Ykt6 (Aim 1) and its functional dependence on Ca2+ dynamics mediated by Calcineurin activity (Aim 2) in PD. Our central hypothesis is that loss of Vps13C misregulates neuronal autophagy and Ca2+ dynamics, contributing to PD pathogenesis in patient neurons. The proposed studies will test our hypotheses using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from patients carrying VPS13C truncation mutations, as well as iPSC-human DA neuron CRISPR/Cas9-generated VPS13C knockout lines. To address this, we will implement: 1) micropatterned substrates which we have generated which allow for the culture of individually separated neurons over extended periods of time, 2) affinity purification coupled to mass spectrometry (AP-MS) analysis of Vps13C?s interactome under both basal and stressed conditions, 3) neuronal imaging of Vps13C dynamics and function using state-of- the-art imaging techniques available at Northwestern University?s Nikon Imaging Center Microscopy Core including live cell super-resolution microscopy, and 4) advanced organelle-specific Ca2+ and lipid sensor imaging techniques in PD patient-derived DA neurons to further elucidate Vps13C function. These studies will provide a mechanistic understanding of Vps13C?s role in autophagy and Ca2+ signaling in patient neurons. Moreover, the proposed research is significant as it offers novel insights into the endolysosomal role of Vps13C in relation to PD pathogenic phenotypes with the goal of potentially highlighting new therapeutic angles for PD.