Probing Heterogeneity of Alzheimer's Disease Using iPSCs

项目摘要 人们越来越关注小胶质细胞在神经退行性疾病中的作用，包括 晚发性阿尔茨海默病（LOAD），一种由富含淀粉样蛋白β的斑块的积累定义的疾病， 含有tau的神经元缠结。小胶质细胞被认为在疾病过程中发挥着各种关键作用 包括突触吞噬、细胞因子释放和β淀粉样蛋白（Aβ）吞噬的进展。此外，本发明还 最近的GWAS研究表明先天免疫过程与LOAD有关，支持了 了解AD风险和进展的神经免疫学过程。所有最近的大型- 规模GWAS已经鉴定出与AD显著相关的INPP 5D基因座的SNP。INPP5D 表达主要局限于成人大脑中的小胶质细胞， 研究发现，在AD脑中INPP 5D的RNA水平升高。然而，通过定量Western印迹，我们 显示了AD脑中全长水溶性INPP 5D蛋白水平降低。研究功能性 为了减少INPP 5D的后果，我们使用了药理学抑制和CRISPR-Cas9基因组 工程化以降低人iPSC衍生的小胶质细胞中的INPP 5D活性。通过无偏RNA和蛋白质组学 分析和一系列药理学操作，我们证明了INPP 5D活性的降低 诱导免疫信号传导的变化，更具体地，诱导炎性体的激活。这些研究 提出了一些重要的问题，我们的目标是在这一提案中解决有关宪法的INPP 5D， 阿尔茨海默氏症脑中的小胶质细胞（目的1），连接INPP 5D和炎性小体的分子机制 激活（目的2），以及INPP 5D活性丧失和炎性小体激活的功能后果。 星形胶质细胞和神经元上的小胶质细胞（aim 3）。在目标1中，我们将利用定量免疫染色， 提取和蛋白质印迹，以及ELISA，以深入探究INPP 5D水平和炎性体标志物 在大的人脑样品和iPSC衍生的小神经胶质细胞培养物的队列中进行激活。目标2询问 可能将INPP 5D活性与炎性小体激活联系起来的候选途径， 分析。其中第一个涉及PLA 2G 7的上调，第二个通过清道夫的失调， 受体，第三种通过破坏溶酶体功能。最后，在目标3中，我们利用了 iPSC衍生的神经元、星形胶质细胞和小胶质细胞以及条件性INPP 5D敲除小鼠模型，以确定 小胶质细胞中INPP 5D水平降低对神经元和星形胶质细胞生物学的影响 AD相关环境。总之，这些研究将为我们理解 INPP 5D在健康和疾病中的小胶质细胞中的基本作用， 人小胶质细胞，以及小胶质细胞中亚溶解性炎性小体激活对神经元和 星形胶质细胞生物学

PROJECT SUMMARY There is an increasing focus on understanding the role of microglia in neurodegenerative disorders including late-onset Alzheimer’s disease (LOAD), a disease defined by the accumulation of amyloid beta rich plaques and neurofibrillary tangles containing tau. Microglia are proposed to play a variety of critical roles during disease progression including synaptic engulfment, cytokine release, and phagocytosis of amyloid beta (Aβ). Further, recent GWAS studies have implicated innate immune processes in LOAD, supporting the importance of understanding the neuroimmunological processes underlying the risk and progression of AD. All recent large- scale GWAS have identified SNPs at the INPP5D locus that are significantly associated with AD. INPP5D expression is largely restricted to microglial cells in the human adult brain and we have confirmed previous findings that RNA levels of INPP5D are elevated in AD brain. However, through quantitative western blotting, we show that protein levels of full length, water-soluble INPP5D are reduced in AD brain. To study the functional consequences of reduced INPP5D, we used both pharmacological inhibition and CRISPR-Cas9 genome engineering to lower INPP5D activity in human iPSC-derived microglia. Through unbiased RNA and proteomic profiling and a series of pharmacological manipulations, we demonstrated that reduction of INPP5D activity induces changes in immune signaling and, more specifically, the activation of the inflammasome. These studies have raised important questions that we aim to address in this proposal regarding the constitution of INPP5D in microglia in the Alzheimer’s brain (aim 1), the molecular mechanism(s) linking INPP5D and inflammasome activation (aim 2), and the functional consequences of loss of INPP5D activity and inflammasome activation in microglia on astrocytes and neurons (aim 3). In aim 1, we will utilize quantitative immunostaining, sequential extraction and western blotting, and ELISA to deeply interrogate INPP5D levels and markers of inflammasome activation across a large cohort of human brain samples and iPSC-derived microglia cultures. Aim 2 interrogates candidate pathways that may link INPP5D activity with inflammasome activation that arose from our preliminary analyses. The first of these involves upregulation of PLA2G7, second through dysregulation of scavenger receptors and the third through disruption of lysosome function. Finally, in aim 3 we utilize co-culture models of iPSC-derived neurons, astrocytes and microglia and a conditional INPP5D knock out mouse model to determine the consequences of reduction of INPP5D levels in microglia on neuron and astrocyte biology in the context of AD relevant environments. Together, these studies will provide important new insights into our understanding of the fundamental role of INPP5D in microglia in health and disease, regulation of inflammasome activation in human microglia, and the consequences of sub-lytic inflammasome activation in microglia on neuron and astrocyte biology.