Chromatin accessibility mapping for functional GWAS risk variants of neuropsychiatric and neurodegenerative disorders in hiPSC-derived neural cells

Despite genome-wide association studies (GWAS) of late-onset Alzheimer's disease (LOAD) have identified 75 genetic risk loci 2-7, the disease causal mechanism underlying most GWAS risk loci remains largely unknown. A major challenge is how to identify a putative causal disease variant among multiple risk variants at individual GWAS risk locus and functionally link the risk allele to LOAD-relevant cellular phenotypes. Leveraging our recent approach for identifying functional noncoding GWAS risk variants that show allele-specific open chromatin (ASoC), we systematically identified putative causal LOAD risk variants in human iPSC-derived neurons and glia cells, and established a causal link between PICALM risk allele to unappreciated microglia (MG)-specific role of PICALM in lipid droplet (LD) accumulation and phagocytosis. Our ASoC mapping identified functional risk variants for 26 AD risk loci, most of which are MG-specific. At the PICALM locus, the LOAD risk allele of the ASoC SNP rs10792832 altered chromatin accessibility to PU.1 binding and reduced PICALM expression in MG, thereby leading to impaired phagocytosis of Amyloid beta (Aß) and myelin debris. Interestingly, transcriptomic profiling of MG carrying PICALM risk allele not only supported abnormal phagocytosis but also revealed enrichment of gene pathways related to cholesterol synthesis and LD formation. Further genetic and pharmacological perturbations in MG established the causal link of the PICALM risk allele with PICALM reduction, LD accumulation and phagocytosis deficits. Our work elucidates an MG-specific role of PICALM in regulating lipid/cholesterol accumulation that is potentially linked to AD pathophysiology, providing a functional neurobiological basis for developing novel clinical interventions. Overall design: We systematically identified putative causal LOAD risk variants in human iPSC-derived neurons and glia cells and established a causal link between the PICALM risk allele and the unappreciated microglia (MG)- specific role of PICALM in lipid droplet (LD) accumulation and phagocytosis. Our ASoC mapping identified functional risk variants for 26 AD risk loci, most of which are MG-specific.

尽管针对晚发性阿尔茨海默病（late-onset Alzheimer's disease, LOAD）的全基因组关联研究（genome-wide association studies, GWAS）已在2-7项研究中鉴定出75个遗传风险位点，但绝大多数GWAS风险位点背后的疾病致病机制仍基本未知。一项核心挑战在于，如何在单个GWAS风险位点的多个风险变异中鉴定出推定的致病疾病变异，并将风险等位基因与LOAD相关的细胞表型建立功能关联。本研究依托我们近期开发的用于识别携带等位基因特异性开放染色质（allele-specific open chromatin, ASoC）特征的功能性非编码GWAS风险变异的方法，系统地在人类诱导多能干细胞（induced pluripotent stem cell, iPSC）分化的神经元和神经胶质细胞中鉴定出推定的LOAD风险变异，并建立了PICALM风险等位基因与此前未被认知的小胶质细胞（microglia, MG）中PICALM在脂滴（lipid droplet, LD）积累及吞噬作用中的特异性功能之间的因果关联。我们通过ASoC定位分析，鉴定出26个AD风险位点的功能性风险变异，其中绝大多数为小胶质细胞特异性变异。在PICALM位点，LOAD风险相关的ASoC单核苷酸多态性（single nucleotide polymorphism, SNP）rs10792832改变了PU.1结合的染色质可及性，并降低了小胶质细胞中PICALM的表达，进而导致β淀粉样蛋白（Amyloid beta, Aβ）与髓鞘碎片的吞噬作用受损。值得注意的是，对携带PICALM风险等位基因的小胶质细胞进行转录组谱分析，不仅证实了吞噬作用异常，还揭示了与胆固醇合成及脂滴形成相关的基因通路富集。后续在小胶质细胞中开展的遗传学与药理学扰动实验，进一步证实了PICALM风险等位基因与PICALM表达降低、脂滴积累及吞噬功能缺陷之间的因果关联。本研究阐明了PICALM在小胶质细胞中调控脂质/胆固醇积累的特异性功能，这一功能或与AD的病理生理过程相关，为开发新型临床干预手段提供了功能性神经生物学依据。总体实验设计：我们系统地在人类诱导多能干细胞分化的神经元和神经胶质细胞中鉴定出推定的LOAD风险变异，并建立了PICALM风险等位基因与此前未被认知的小胶质细胞中PICALM在脂滴积累及吞噬作用中的特异性功能之间的因果关联。我们的ASoC定位分析鉴定出26个AD风险位点的功能性风险变异，其中绝大多数为小胶质细胞特异性变异。