Joseph R. Mazzulli, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Protein accumulation is a soundly documented feature of all age-related neurodegenerative disorders, however the initiating events that lead to their formation, as well as their relationship to disease, remains unknown. Parkinson's disease (PD) is characterized by the conversion of a normally soluble synaptic protein called a-synuclein into insoluble amyloid fibrils that comprise Lewy body inclusions within Parkinson's brain. Our recent data indicated that disruption of cellular degradation capacity through mutations in the lysosomal gene GBA1 contribute to the aggregation of a-synuclein. This suggested that disruption of lysosomal function contributes to the formation of Lewy bodies. Interestingly, we found that when a-synuclein accumulates, it can in turn feedback to inhibit the lysosomal system, thus causing a self-propagating cycle that promotes amyloid formation and growth within neurons. Our preliminary data indicate that a-synuclein inhibits the trafficking of hydrolases and prevents them from reaching the lysosomal compartment; however the molecular mechanism is not known. Experiments outlined in this application aim to delineate how a-syn disrupts lysosomes using cell lines, PD patient-derived induced pluripotent stem cell models, transgenic mice, and PD brain. Our goals are to 1) define the relationship between distinct a-syn aggregated assemblies and lysosomal dysfunction / neurotoxicity, 2) determine how a-syn affects protein trafficking of lysosomal hydrolases, 3) discover new rescue pathways in PD centered around promoting hydrolase folding and trafficking to the lysosome. These studies will provide new insight into the mechanism of how amyloid aggregates disrupt cellular processes, and identify novel therapeutic pathways for synucleinopathies centered on enhancement of the lysosomal clearance pathway.

Disruptions in protein homeostasis (proteostasis) have long been implicated as a mechanism that leads to neurodegeneration, however global changes that occur to the proteome have not been explored in human midbrain neurons. a-Synuclein, the protein that accumulates within defining histopathological inclusions called Lewy bodies, disrupts essential cellular pathways linked to proteostasis such as protein trafficking and lysosomal degradation pathways. Our previous studies using familial PD neuronal cultures harboring a triplication in the a-syn gene (SNCA trp) indicates that lysosomal proteolysis and protein trafficking are disrupted. Here, we propose to study the downstream consequence of a-syn accumulation and its global effects on the proteome using induced pluripotent stem cell (iPSC)-derived human midbrain models and PD brain. We hypothesize that a-syn accumulation leads to a selective accumulation and aggregation of a metastable sub-proteome with essential cellular functions. Aggregation of these critical proteins leads to loss-of-function and subsequent neurodegeneration. These studies are exploratory and we anticipate will generate new hypotheses for how human neurons handle the burden of protein accumulation in disease. They may contribute to our understanding of how a-syn accumulation leads to neurodegeneration, and identify novel pathways for therapeutic intervention. From a basic science perspective, these studies will help us to understand how human neurons respond to aggregation-induced stress through remodeling of the proteome.

Parkinson's disease (PD) is characterized by the presence of Lewy body inclusions in the nervous system comprised of insoluble alpha-synuclein (a-syn). The mechanisms that dictate the conversion of a-syn from its physiological state into pathogenic aggregates are not completely understood. Genetic studies have provided clues into the etiology of PD and causative factors that might promote a-syn inclusions. Among these, mutations in the lysosomal system have emerged as key risk factors for PD. Since lysosomes can degrade physiological a-syn, loss-of-lysosomal function may promote a-syn accumulation and drive aggregation. Mutations in lysosomal GBA1 have been identified as the strongest risk factor in PD, and are also highly associated with early onset dementia in PD patients. GBA1 mutations are also strong risk factors for a related synucleinopathy, Dementia with Lewy bodies (odds ratio=8.28), that often involves the co-aggregation of other disease-linked proteins linked to Alzheimer's disease such as tau and A?. GBA1 encodes lysosomal beta- Glucocerebrosidase (GCase), which degrades glucosylceramide (GluCer) in lysosomes. Homozygous GBA1 mutations cause the rare lysosomal storage disorder, Gaucher's disease (GD), which is also characterized by Lewy body inclusions in the nervous system. All of the GD or PD associated mutations identified to date have been well characterized and cause loss-of-enzymatic function. Additionally, GluCer substrate accumulation has been documented in patient-derived tissues and iPS neurons of both diseases. Our previous studies have indicated that GluCer directly converts physiological a-syn conformers into pathogenic assembly-state species in GD iPSn models and patient derived midbrain neurons. Here, we propose to study the mechanism of GluCer-induced aggregation by examining the how aggregates propagate and persist in human iPSn models. Pathogenic a-syn oligomers persist for hundreds of days in patient-derived iPS neurons, and our studies will examine potential mechanisms to explain this phenomenon. We will determine if the unique conformational information of GluCer-induced oligomers can be replicated by interaction and conversion of physiological conformers, their ability to cross-seed aggregates with tau or A?, neuron-to-neuron dissemination, and transmission of metabolic dysfunction. It is well known that PD and other synucleinopathies demonstrate remarkable heterogeneity for which there is no mechanistic explanation. Our studies will begin to examine this process by testing the cross-seeding potential of GluCer-induce a-syn oligomers. We plan to study these processes in human iPS midbrain models, established cell lines, and mouse models of GD. We will relate GluCer induced changes in pathological a-syn to comorbid pathologies and neuronal toxicity in vitro and in vivo. If successful, these studies will provide novel insight into the mechanisms of GBA1-associated PD, they may help to explain the high frequency of dementia and disease heterogeneity in PD patients, and may help to provide novel therapeutics to eliminate pathogenic propagation induced by glycosphingolipids.

Abstract Synucleinopathies, such as Parkinson's disease (PD), are a group of incurable neurodegenerative diseases characterized by the accumulation of aggregated alpha-synuclein (a-syn). Disturbances in protein trafficking and cellular degradation pathways, such as lysosomes, have been implicated in the pathogenesis of these disorders. The strongest genetic risk factors for PD are loss-of-function mutations in lysosomal glucocerebrosidase (GCase), that degrades glucosylceramide (GluCer). Our previous work showed that loss of GCase activity and GluCer accumulation directly induces a-syn aggregation and cell death in neurons. This indicates that lysosomal dysfunction plays an important role in PD pathogenesis, and that enhancing lysosomal function may reduce a- syn aggregation. We also showed that a-syn, when aggregated at the cell body of neurons, inhibits the trafficking of lysosomal hydrolases between the endoplasmic reticulum (ER) and the Golgi, thereby reducing lysosomal function. Therefore, methods to enhance ER-Golgi trafficking may improve lysosomal hydrolase targeting and function, and enhance the clearance of pathological a-syn aggregates. However no therapeutic targets exist that are capable of enhancing trafficking or lysosomal activity. We propose to explore the mechanism of a potential novel therapeutic target called ykt6 that functions as a Soluble NSF Attachment protein REceptor (SNARE) to enhance protein trafficking between the ER and the Golgi, therefore improving the maturation of lysosomal enzymes and hydrolase activity. Previous work has shown that cognate SNARE binding of ykt6 is regulated by farnesylation, and we will examine how farnesylation of ykt6 influences its ability to control ER-Golgi trafficking using PD patient-derived neurons from induced pluripotent stem cell (iPSC) models and isogenic corrected controls. Preliminary data indicate that inhibiting the farnesylation of ykt6 enhances the trafficking of lysosomal hydrolases and improves lysosomal function. Furthermore, pharmacological inhibition of farnesyltransferase using a brain-penetrant, clinically validated small-molecule inhibitor can reduce pathological, insoluble a-syn in cell lines in a ykt6-dependent manner. We propose to examine how the farnesyl-mediated regulatory mechanism of ykt6 function is related to its ability to enhance lysosomal function and reduce pathological a-syn. As these studies are exploratory, they are aimed to assess the future potential of ykt6 as a therapeutic target for the synucleinopathies by focusing on the mechanism that regulates its ER-Golgi trafficking activity. We expect these studies to lead to novel hypothesis for regulation of ER-Golgi trafficking and lysosomal activity, and possibly other vesicular trafficking pathways in the cell, that may be examined in future proposals. There are currently no known methods to enhance hydrolase trafficking and lysosomal function, and our studies may help to validate ykt6 as a druggable target to enhance these processes and reduce pathological forms of a-syn. Our studies may be rapidly translated into therapies since farnesyltransferase inhibitors are well-developed in the clinic as cancer therapies, leading to the possibility of repurposing these agents for the treatment of PD.

Neurodegenerative disorders including Lewy body Dementia (LBD) and Parkinson?s disease (PD) are characterized by aggregation of a-synuclein (a-syn), however the downstream toxic events that lead to cell death are not understood. Proteome dysfunction is a prominent feature of synucleinopathies, as indicated by genetics and pathology. To gain a comprehensive understanding of how the proteome changes in PD, we performed a quantitative proteomic study to identify proteins that aggregate in patient derived iPSC-neurons as a consequence of a-syn accumulation. By comparing iPSC neurons expressing A53T a-syn with isogenic corrected lines, we discovered a remarkable level of selectivity in the classes of proteins that aggregate. Specifically, we found that RNA binding proteins NONO and SFPQ undergo dramatic solubility shifts from detergent soluble into the insoluble state. NONO and SFPQ are multifunctional nuclear proteins that play critical roles in transcription regulation, RNA splicing, and RNA editing of genes that regulate axon guidance. They are core components of a membraneless sub-compartment in the nucleus called the paraspeckle, and contain prion- like low complexity domains that permit phase separation under physiological conditions. Paraspeckles occur in neuronal cultures and in vivo in the brain, and are thought to play key roles in regulating homeostatic stress by transiently sequestering transcription factors and RNAs to prevent translation. Once stress subsides, paraspeckles normally dissolve and gene expression returns to normal. However, we have found that NONO and SFPQ irreversibly form pathological aggregates in patient iPSC-neurons and LBD patient brain. This effect is specifically associated with a-syn accumulation, and does not occur with general cellular stress. Mechanistic studies in iPSC-neurons suggest that formation of NONO/SFPQ aggregates is associated with loss of their functions, resulting in neurite degeneration. We find that the SFPQ transcriptional target, ADAR3 that mediates RNA editing, is nearly completely depleted in patient neurons. Here, we propose to examine the mechanism of how a-syn accumulation leads to aberrant NONO/SFPQ aggregation in the nucleus and downstream pathophysiology. Given their role in RNA splicing and editing, we propose to employ both targeted and unbiased methods to identify changes in RNA editing including RNA-seq, exon-junction microarrays to examine RNA splicing, and ChIP-seq to detect changes in SFPQ transcriptional activity. These phenotypes will be correlated with distinct aggregated forms of a-syn and neurodegeneration. Finally, we will attempt to rescue established phenotypes in patient iPSC-neurons by promoting soluble, function NONO/SFPQ. Our preliminary studies have identified a novel pathogenic pathway in synucleinopathies, and we will extend these findings by examining the mechanisms of gene dysregulation and RNA processing. We will provide the first description of RNA editing and splicing changes in patient iPSCs, which may uncover novel disease mechanisms and therapeutic strategies.

Deficits in the phospholipid phosphatidylethanolamine (PE) and its metabolites, such as ethanolamine (ETA) and phosphoethanolamine, occur in the Parkinson?s disease (PD) and Alzheimer?s disease brain. We propose that these metabolic deficits likely also occur in diseases characterized by synuclein pathology such as dementia with Lewy bodies and multiple system atrophy. Whether these metabolic deficits are a cause or consequence of disease is not known. However, we do know that low levels of PE can lead to mitochondrial dysfunction, autophagy dysfunction, and the misprocessing of glycosylphosphatidylinositol-anchored proteins, and that the homeostasis of ?-syn can certainly be impacted by decrements in these processes. In fact, data from yeast and worm models of synucleinopathies, have shown that the co-occurrence of low levels of PE (due to knocking out the mitochondrial enzyme phosphatidylserine decarboxylase, PISD) and ?-syn are synthetically toxic. ETA rescues ?-syn toxicity in PISD knockout cells, because ETA stimulates the synthesis of PE via the CDP-ethanolamine pathway, which resides in the endoplasmic reticulum. The long-term goal of this proposed research is to elucidate how ?-syn modulates autophagy. To dissect the role of the PE-ETA axis in synucleinopathies and the role of ?-syn in inhibiting autophagy, our specific aims are to: (1) determine the role of PE synthesis in autophagic-lysosomal function and clearance of ?-syn in patient iPSC-neurons. We will determine if stimulating PE synthesis with ETA will rescue autophagic phenotypes in patient neurons and whether ETA decreases the accumulation of pathologic conformations of ?-syn by increasing autophagic flux. (2) Determine the mechanism by which ?-syn (and A53T) decreases the level of PISD in patient iPSC-midbrain and excitatory-cortical cells, and in SH-SY5Y cells. We propose that a deficit in PISD produces a deficit in PE with a parallel inhibition of autophagy. We will determine whether ?-syn (and A53T) blocks the import of PISD into mitochondria by disrupting mitochondrial associated membranes (MAMs) that connect the ER with mitochondria. Alternatively, ?-syn (and A53T) may trigger the release of PISD from cells via endosomes or promote the rapid degradation of the protein. These possibilities will be analyzed by electron microscopy, isolation of mitochondria with attending lipid analysis, analysis of autophagy flux, and exosome isolation. (3) Determine whether ?-syn inhibits mitochondrion-vacuole and mitochondrion-ER contacts. We will knock out genes that regulate mitochondrion-ER contacts and separately knockout genes that regulate mitochondrion- vacuole contacts in cells with and without ?-syn expression and then evaluate how disruptions in these mitochondrion-organelle contacts affect the autophagic flux of Atg8-GFP. We have already shown that ?-syn inhibits the autophagic flux of Atg8-GFP, but these experiments dig deeper and will reveal whether the ?-syn inhibits autophagy by disrupting molecular contacts between mitochondria and the ER or vacuole. The proposed experiments will give insight into why ?-syn aggregates and how it can be prevented.