Alzheimer?s disease is the most common form of dementia in the elderly, but the causal mechanisms involved in disease development remain poorly understood. Genome-wide association studies have identified several genomic regions associated with disease, but translating these into causal variants and genes remains a challenge. Initial studies have identified an enrichment for AD risk variants in active enhancers of human monocytes, macrophages, and microglia, suggesting many of these variants act by disrupting gene expression specifically in myeloid cells. This, along with myeloid-specific expression of several genes implicated in AD risk, strongly implicate these cells in the etiology of AD. Using fine mapping approaches and integrated functional data from human myeloid cells, we identified the gene embryonic ectoderm development (EED) as a strong candidate causal gene and a putative target of a myeloid cell enhancer containing an AD-associated functional variant on chromosome 11. While EED is known to function in the maintenance and placement of repressive histone mark H3K27me3 and has been implicated in the regulation of clearance behavior in mouse microglia, it remains relatively unstudied in the context of AD and its role in human microglia is unclear. The overall goal of this proposal is to directly test the hypothesis that EED plays a critical role in regulating human microglia behavior in vitro and in vivo, and to gain a better understanding of how AD-associated genomic elements may influence EED expression.
In Aim 1 I will combine CRISPR gene editing in human induced pluripotent stem cells (iPSCs), microglial differentiation protocols, novel transplantation methods involving direct injection of microglia precursor cells into the mouse brain, and functional genomics techniques including single-nuclei RNA sequencing with targeted functional assays to evaluate the role of EED in human microglia.
Aim 2 will evaluate the mechanism of action of our prioritized variant/enhancer pair and investigate the extent to which EED is the main target of these AD-associated elements. Specifically, I will use CRISPR to simultaneously delete the candidate enhancer and create isogenic lines homozygous for the major and minor alleles of the candidate variant and measure downstream changes in gene expression, chromatin accessibility, and transcription factor binding. Together, results here will confirm or refute computational predictions that our prioritized AD-associated variant impacts myeloid cell physiology by altering EED levels in human microglia cells via a cell-type specific enhancer. This work has the potential to both greatly impact public health through the validation of a novel AD risk gene and enhance our understanding of genes, pathways, and epigenetic mechanisms involved in disease development. In addition, functional validation of computational predictions derived from fine mapping will provide a blueprint for future researchers interested in translating the results of GWAS from statistical association to biological function. Overall, the research and training plan outlined here will make significant strides towards enabling me to become a successful independent scientist.
Dozens of genetic loci have been implicated in Alzheimer?s disease risk, yet the underlying causal genes, variants, and biological mechanisms involved in disease development remain unclear. This proposal combines patient-derived induced pluripotent stem cells, precise genome editing, and innovative transplantation techniques to experimentally investigate the impact of prioritized genes, variants, and enhancers on the physiology of disease-relevant microglia both in vitro and in vivo. This approach has the potential to both greatly enhance our understanding of genes and pathways causally involved in disease development and lay the foundation for future researchers interested in translating the results of genome-wide association studies from pure statistical association to biological function.
