Neuronal Vulnerability to Lipid Droplets and Cholesterol in Alzheimer's Disease - ABSTRACT Alzheimer's disease (AD) is a devastating neurodegenerative disorder that afflicts over 6.5 million Americans. Despite decades of research on amyloid-β (Aβ) and Tau lesions, effective treatment remains out of reach. Mounting AD genome-wide association studies (GWAS) risk loci provide opportunities for understanding disease biology and developing effective treatment. Multiple AD GWAS risk genes, such as APOE, are pivotal for lipid metabolism that is essential to brain health and disease. Under stress or in the aging brain, both astrocytes and microglia show abnormal lipid metabolism and accumulation of lipid droplets (LD). Although glial LD are known to be largely neuroprotective, LD formation in human neurons and its cellular and molecular ramifications are less clear. With excitatory neurons (Ex) derived from human induced pluripotent stem cells (iPSC), we have shown that APOE4 Ex neurons co-cultured with astrocytes accumulated more LD than APOE2/3 neurons. Our single-cell transcriptomic analysis of AD brain also identified an impaired neuron- oligodendrocyte precursor cell (OPC) communication mediated by APOE and its receptor LDLR. Interestingly, neural cholesterol was recently found to be essential to OPC proliferation and remyelination. We thus hypothesize that abnormal neuronal LD and cholesterol in AD contribute to neuronal damage and impaired neuron remyelination. Leveraging our expertise on using iPSC-derived neurons and cortical organoids as cellular models for brain disorders, we will characterize the cell type vulnerability to LD/cholesterol and the corresponding epigenomic/transcriptomic changes in the context of AD risk alleles. In Aim 1, to determine the neural vulnerability to LD and its clinical relevance in AD, we will co-culture Ex and Inhibitory (Inh) neurons of a cohort of 60 patient-specific iPSC lines, and assay neural LD, free fatty acids, cholesterol, and neural morphometrics, which will then be associated with APOE alleles and non-APOE AD polygenic risk scores. These cellular phenotypes will also be correlated with patients’ longitudinal plasma lipid levels and other clinical outcomes. In Aim 2, to understand molecular mechanisms underlying neural vulnerability to LD, we will perform single-cell RNA/ATAC-sequencing on the same neural co-cultures used in Aim 1 to identify genes with chromatin accessibility and transcription correlated with cellular LD in each cell type. In Aim 3, to examine the effect of AD risk alleles on cholesterol-mediated neuron-OPC communication and myelination, we will first use CRISPR editing to generate isogenic iPSC lines carrying AD risk alleles and then differentiate them into myelinating cerebral organoids. We will examine the effects of AD risk alleles on neuronal cholesterol levels as well as the OPC proliferation and neuron myelination. This study will significantly advance our understanding of the cellular and molecular mechanisms of neuronal vulnerability to abnormal LD and cholesterol in AD.
