Using human iPSC-derived microglia to understand the biological function of Alzheimer's risk variants

使用人类 iPSC 衍生的小胶质细胞了解阿尔茨海默病风险变异的生物学功能

• 批准号: 10470313
• 负责人: Sarah M Neuner
• 金额: $ 6.76万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-29 至 2023-09-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470313
• 关键词: Alleles; Alzheimer' s Disease; Alzheimer' s disease risk; Amyloid; Amyloid deposition; Apoptotic; Behavior; Binding; Biological; Biological Assay; Biological Process; Brain; Cell Nucleus; Cell Transplantation; Cell physiology; Cells; Cellular Morphology; Chromatin; Chromosome 11; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive; Computer Analysis; Data; Dementia; Development; Disease; Ectoderm; Elderly; Elements; Embryo; Enhancers; Epigenetic Process; Etiology; Exposure to; Foundations; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Genomic Segment; Genomic approach; Genomics; Global Change; Goals; Histones; Human; Immunization; Immunohistochemistry; In Vitro; Inflammatory; Injections; Knock-out; Lead; Maintenance; Measures; Methods; Microglia; Minor; Morphology; Mus; Myelogenous; Myeloid Cells; Nucleotides; Outcome; PRC1 Protein; Pathway interactions; Patients; Phagocytes; Phenotype; Physiology; Play; Public Health; Regulation; Research Personnel; Research Training; Role; Scientist; Statistical Study; Stimulus; Techniques; Testing; Translating; Transplantation; Validation; Variant; Work; XCL1 gene; behavior in vitro; causal variant; cell type; differentiation protocol; disorder risk; functional genomics; genetic variant; genome wide association study; genomic locus; human data; in vivo; induced pluripotent stem cell; innovation; interest; macrophage; member; methylome; monocyte; novel; precise genome editing; precursor cell; response; risk variant; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

Project Summary 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.

项目总结