Determining the Interacting Effects of GBA, SNCA, and APOE on a-Synuclein Pathology Severity in Dementia with Lewy Bodies and Parkinson's Disease - Project Summary Synucleinopathies, including dementia with Lewy bodies (DLB) and Parkinson’s disease (PD), are a growing health crisis, affecting over 1 million people in the United States and an estimated 10 million people globally. Synucleinopathies are caused by accumulation of the protein α-synuclein (α-syn) into unnatural fibrils in the brain and can manifest with motor symptoms, cognitive symptoms, or a combination of the two. There are currently no effective treatments to cure or prevent synucleinopathies, in large part because there is no clear cause for disease. There are many identified genetic and environmental risk factors associated with synucleinopathies, but the context in which these factors lead to disease is still unknown. In addition to direct mutations to SNCA, the gene that encodes α-syn, mutations to GBA and APOE are well established risk factors and have been shown to increase α-syn aggregation. However, none of these risk factors are great predictors of disease on their own, suggesting unknown genetic and environmental interactions likely influence the initiation, severity, and clinical outcomes of α-syn pathology. In this proposal, I focus on how both cell type and genotype interact with each other to modulate α-syn pathology severity. I hypothesize that α-syn pathology is precipitated by epistatic interactions between genetic factors that disrupt homeostasis in different cell types and leaves dopaminergic neurons vulnerable to deleterious α-syn pathology and neurodegeneration. I will investigate this hypothesis using two independent, complementary approaches to dissect the cellular and molecular mechanisms that modulate α-syn phenotypes. First, I will couple GBA knockdown with SNCA-triplication, wild-type, and knockout cell lines. There is a clear link between GBA mutations and α-syn aggregation, but how these proteins connect is still unknown. I will use high-content imaging with genetically encoded fluorescent markers to quantify lysosomal and mitochondrial dynamics in live cells. This will reveal how GBA responds to different α-syn dosages and how that affects cellular homeostasis. Second, I will leverage isogenic lines containing APOE allelic variants and GBA knockdown. GBA and APOE are involved in lipid metabolism, suggesting a putative link between lipid metabolism disruption and α-syn pathology, although there is no published connection between the two proteins. I will use next generation genomic sequencing and proteomic approaches to determine the interacting effects between SNCA, GBA, and APOE allelic variants on α-syn pathology severity. Completion of this aim will uncover whether GBA and APOE are acting through converging or independent pathways, expanding our knowledge of the network that contributes to disease pathology. By coupling multiple genetic risk factors into a multi-cell type model, I will pioneer new technology and approaches to dissect epistatic mechanisms that influence α-syn pathology. These insights will guide us in developing new therapeutic approaches, enabling earlier detection, treatment, and prevention of synucleinopathies.