Project Summary Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS) are irreversible and currently incurable neurodegenerative diseases with more than 65,000 new cases in the USA each year. Their core motor symptoms are respectively caused by dysfunction and death of dopaminergic neurons within the substantia nigra and motor neurons in the cortex, brainstem, and spinal cord. However, non-cell-autonomous contributions to disease progression are widely recognized and include cerebrovascular (CV) dysfunction. The CV is formed by several highly specialized cell populations, including brain endothelial cells (BECs), mural cells, fibroblasts, and glia. Given the CV’s critical role in regulating biomolecule transport into and out of the brain, blood flow, and responses to physical or chemical stress, understanding the molecular underpinnings of early CV changes during PD and ALS may be critical to develop disease-modifying treatments. Prior work indicates that CV changes can occur during the progression of PD and ALS, including leakage of the blood-brain barrier (BBB), angiogenesis, dysfunctional efflux activity, dysregulated blood flow, and increased immune cell trafficking. However, findings from brain imaging (MRI) and histological analysis are not inclusive of all CV functions nor able to identify transcriptional regulators, while studies using animal models are not representative of sporadic human disease which accounts for ~90% of PD and ALS cases. In this proposal, I will characterize cerebrovascular dysfunction during sporadic PD and ALS with cell type-specificity and whole genome-resolution from post-mortem tissue, and will benchmark the degree to which this dysfunction is recapitulated by iPSC-derived in vitro models. This work is grounded in recent application of blood-vessel enrichment (BVE) and single nucleus RNA sequencing (snRNA-seq) approaches to profile gene expression of CV cells, and the development of transcription factor overexpression-based differentiation of BECs from induced pluripotent stem cells (iPSCs). In Aim 1A, I will conduct snRNA-seq on blood vessel enriched substantia nigra from post-mortem PD patients and age-matched healthy controls, and will then validate cell type-specific dysfunction using immunofluorescence and in situ hybridization studies. In Aim 1B, I will differentiate BECs from PD patient iPSCs and age-matched healthy controls and then conduct snRNA-seq to determine how post-mortem hallmarks of dysfunction are reflected in vitro. In Aim 2, I will take a similar approach by conducting snRNA-seq on ALS patients blood vessel enriched motor cortex and iPSC-derived BECs compared to healthy age-matched post-mortem tissue and iPSC controls. By characterizing CV gene expression using cutting-edge single nucleus profiling of PD and ALS post- mortem tissue and iPSC-derived models, this proposal will identity previously unrecognized mechanisms of CV dysfunction and serve as a critical launchpad for future...