Probing Heterogeneity of Alzheimer's Disease Using iPSCs

项目总结 人们越来越重视了解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β蛋白的斑块和 含有tau的神经纤维缠结。小胶质细胞被认为在疾病过程中起着各种关键作用。 进展包括突触吞噬、细胞因子释放和吞噬淀粉样β蛋白(Aβ)。此外， 最近的GWAS研究表明，先天免疫过程与负荷有关，支持了 了解阿尔茨海默病风险和进展的神经免疫学过程。所有最近的大型- GWA量表已经在INPP5D基因座发现了与AD显著相关的SNPs。INPP5D 在人类成年大脑中，表达主要限于小胶质细胞，我们已经证实了之前的 发现阿尔茨海默病患者脑内INPP5D的RNA水平升高。然而，通过定量Western blotting，我们 表明阿尔茨海默病患者脑内全长、水溶性INPP5D蛋白水平降低。学习泛函 INPP5D减少的后果，我们同时使用药物抑制和CRISPR-Cas9基因组 降低人iPSC来源的小胶质细胞中INPP5D活性的工程。通过无偏见的RNA和蛋白质组学 分析和一系列药理操作，我们证明了INPP5D活性的降低 诱导免疫信号的变化，更具体地说，是激活炎症小体。这些研究 提出了重要的问题，我们打算在这项关于#年INPP5D组成的提案中解决这些问题 阿尔茨海默病脑内小胶质细胞(Aim 1)，连接INPP5D和炎症体的分子机制(S) 激活(目标2)，以及INPP5D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(目标3)。在目标1中，我们将使用定量免疫染色，顺序 提取、免疫印迹和酶联免疫吸附试验检测INPP5D水平和炎症体标志物 在大量人脑样本和IPSC来源的小胶质细胞培养中激活。AIM 2审问 可能将INPP5D活性与炎症小体激活联系起来的候选通路 分析。其中第一个涉及PLA2G7的上调，第二个是通过清道夫的失调 第三种是通过破坏溶酶体的功能。最后，在目标3中，我们使用了 IPSC来源的神经元、星形胶质细胞和小胶质细胞以及条件性INPP5D基因敲除小鼠模型的确定 小胶质细胞中INPP5D水平降低对神经元和星形胶质细胞生物学的影响 与广告相关的环境。总而言之，这些研究将为我们理解 INPP5D在健康和疾病中对小胶质细胞的基础作用，炎性小体激活的调节 人小胶质细胞，以及小胶质细胞亚溶炎性小体激活对神经元和 星形细胞生物学。

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.