Project Summary Lewy body diseases (LBDs) are highly heterogeneous neurodegenerative disorders including Parkinson's disease (PD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB). LBDs are characterized by the abnormal aggregation of the protein α-synuclein in neuronal cell bodies (Lewy Body) and neurites which are currently considered to be the common cause of the diseases. Lewy Body deposition starts in the caudal brainstem of PD but in the neocortex of DLB cases. The regional differences of initial α-synuclein deposition correlate with neuronal loss in the corresponding regions - dopamine neurons in the substantia nigra (SN) and neurons of unknown identity in the neocortex, and the unique clinical manifestations with a predominant motor symptom in PD whereas early dementia in DLB. Why some particular neurons and brain regions are affected at the disease onset, whereas the neighboring cells and regions not? This is a fundamental question in the field of neurodegenerative diseases that this proposal will address via novel genomics technologies and bioinformatics tools. In an initial pilot study, using single nucleus RNA-sequencing (snRNA-seq) analyses, we identified a novel disease-associated astrocyte (DAA) subpopulation and demonstrated that DAA contributed to increased inflammation, amyloid pathology, and neurodegenerative disease pathogenesis whereas parenchymal astrocytes had compromised functionality in both AD and PD brains. Additionally, we identified three microglia subpopulations that were similar to but with marker gene expression profiles distinct from the conventional resting (M0), M1, and M2 activated microglia. We observed deficient microglia functionality shared across all microglia subpopulations and uniquely up-regulated inflammatory pathways in PD suggesting common and PD-specific mechanisms of neurodegeneration. These data provide us with an exclusive opportunity to analyze the relationships between these glia subpopulations and selective regional and neuronal vulnerability in different diseases. In Aim 1, we will identify vulnerable neuronal types in the frontal cortex (FC) and SN of patients with PD, PDD, and DLB. In Aim 2, we will test the hypothesis that astrocyte/microglia dysfunction underlies the mechanism of the selective regional vulnerability of LBD. In Aim 3, we will test the hypothesis that dysregulated interactions between neurons and astrocytes/microglia underlie the mechanism of the selective neuronal vulnerability of LBD. Our study will provide deep insights into the molecular mechanisms of selective neuronal and regional vulnerability in LBDs. Besides, our study will provide molecular biomarkers and tools for neuron cell-type-specific protection and targeted astrocyte/microglia subpopulation isolation and manipulation. Furthermore, our study will provide molecular biomarkers for distinguishing PD, PDD, and DLB, which is very important but a considerable challenge today.