Neuronal Vulnerability to Lipid Droplets and Cholesterol in Alzheimer's Disease

摘要 阿尔茨海默病(AD)是一种毁灭性的神经退行性疾病，困扰着超过650万美国人。 尽管对淀粉样蛋白β(Aβ)和Tau损害已经研究了几十年，但有效的治疗仍然遥不可及。 越来越多的AD全基因组关联研究(GWAS)风险基因座提供了理解的机会 疾病生物学和开发有效的治疗方法。多个AD风险基因，如载脂蛋白E，是关键 对于大脑健康和疾病至关重要的脂肪代谢。在压力下或在老化的大脑中，两者 星形胶质细胞和小胶质细胞脂代谢异常，脂滴聚集。虽然 已知胶质细胞LD在很大程度上具有神经保护作用，在人类神经元及其细胞和分子中形成LD 其后果还不太清楚。人诱导多能干细胞来源的兴奋性神经元 (IPSC)，我们发现与星形胶质细胞共培养的APOE4Ex神经元积累的LD比 APOE2/3神经元。我们对AD大脑的单细胞转录分析也发现了一个受损的神经元- APOE及其受体LDLR介导的少突胶质前体细胞(OPC)通讯。有趣的是， 最近发现神经胆固醇对OPC的增殖和重新髓鞘形成是必不可少的。因此，我们 阿尔茨海默病患者神经元LD和胆固醇异常与神经元损伤和受损的关系 神经元重新髓鞘形成。利用我们在使用IPSC来源的神经元和皮质有机体方面的专业知识 脑部疾病的细胞模型，我们将描述细胞类型对LD/胆固醇的易感性以及 AD风险等位基因背景下相应的表观基因组/转录改变。在目标1中，确定 神经元对LD的易感性及其在AD中的临床意义，我们将对AD患者的Ex和抑制神经元进行共培养 60个患者特定的IPSC系的队列，并测定神经性LD、游离脂肪酸、胆固醇和神经性 形态计量学，这将与载脂蛋白E等位基因和非载脂蛋白E AD多基因风险分数相关联。 这些细胞表型也将与患者的纵向血脂水平和其他 临床结果。在目标2中，为了了解神经对LD易感性的分子机制，我们将 对AIM 1中使用的相同神经共培养物进行单细胞RNA/ATAC测序，以识别与 在每种细胞类型中，染色质可及性和转录与细胞LD相关。在目标3中，检查 AD风险等位基因对胆固醇介导的神经元-OPC通讯和髓鞘形成的影响，我们将首先使用 CRISPR编辑以生成携带AD风险等位基因的等基因IPSC株系，然后将它们区分为 有髓鞘的脑器官。我们将研究AD风险等位基因对神经元胆固醇水平的影响 以及OPC增殖和神经元髓鞘形成。这项研究将极大地促进我们对 AD神经元对异常LD和胆固醇易感性的细胞和分子机制。

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.