Regulation of diverse microglial phenotypes in neurodegeneration

阿尔茨海默病是一个多因素的过程，到目前为止，治疗目标一直是具有挑战性的。一个 大量文献支持小胶质细胞在阿尔茨海默病中的关键作用，小胶质细胞是大脑的天然免疫细胞 发病机制。操纵小胶质细胞的活动可能是一种易于处理的AD干预措施，但小胶质细胞在 广告是复杂的。小胶质细胞在体内平衡、衰老和疾病的大脑中协调一系列功能。 了解哪些小胶质细胞表型与阿尔茨海默病有关，以及这些表型是如何调节的 是开发治疗阿尔茨海默病的精确疗法所必需的。通过执行单核RNAseq(SnRNAseq) 从22例晚发性AD(LOAD)和年龄匹配的对照尸检队列中，每个人有超过5,000个小胶质细胞 我们已经确定了与AD显著相关的小胶质细胞簇。其中之一是特别的 富含内溶酶体网络(ELN)、DNA损伤反应通路和1型的基因 干扰素信号。虽然这些病理已经在AD中被记录下来，但它们也受到年龄的影响， 突出了研究一种老年神经退行性疾病的复杂性。相比之下，拥有 早发性家族性阿尔茨海默病(EOFAD)由基因变异驱动，在生命早期和死亡时发展为阿尔茨海默病， 与LOAD患者相比，EOFAD患者的全身和大脑老化变化较少。我们假设 对DNA损伤的应答由功能失调的ELN加剧在AD发病中独立于 年龄。在这项研究中，我们将利用我们独特的冰冻脑组织队列和人类诱导的多能性 干细胞(HiPSC)在3个靶点。我们将确定小胶质细胞分子图谱(目标1)和空间组织 (目标2)年轻至老年人群中有无阿尔茨海默病。我们将功能上测试我们的 使用HiPSC模型的假设，该模型缺乏与年龄相关的变化(目标3)。我们建议产生小胶质细胞 从冷冻皮质构建基因网络的转录、开放染色质和DNA甲基化图谱 19例携带PSEN1/PSEN2或APP致病变异的EOFAD个体样本比较 来自35名年龄匹配的25岁至60岁的未受影响的对照组和10名年龄在65岁至75岁的对照组的皮质样本 好几年了。我们将绘制与年龄相关的变化图，仅在整个成年年龄的小胶质细胞图谱中 25年-75年对照队列。我们将利用现有较旧的控件，并将SnRNAseq数据集作为另一个 比较和提名哪些小胶质细胞变化出现在AD病理、单独的年龄和 那些因AD和年龄的存在而加剧的疾病。从功能上衡量…的重要性 我们已经确定的AD表型，我们将使用HiPSC来源的小胶质细胞和神经元来检验这一假设 ELN功能障碍会导致损伤和核酸积累，从而导致神经元毒性。 这些整合的组学和体外研究评估了成人年龄和阿尔茨海默病患者的小胶质细胞 病理学将识别并从功能上测试调节小胶质细胞表型的因素，以告知有效 EOFAD和LOAD的治疗靶向。

Alzheimer disease is a multifactorial process that has thus far been challenging to therapeutically target. A large body of literature supports a key role for microglia, the innate immune cells of the brain, in AD pathogenesis. Manipulating microglial activity may be one tractable AD intervention however microglia roles in AD are complex. Microglia orchestrate a suite of functions in the homeostatic, aged and diseased brain. Understanding which of these microglial phenotypes contribute to AD and how these phenotypes are regulated is necessary to develop precise therapies for AD. By performing single nucleus RNAseq (snRNAseq) on more than 5,000 microglia per individual from a cohort of 22 late-onset AD (LOAD) and age-matched control autopsy samples we have identified microglia clusters significantly associated with AD. One of these is specifically enriched in genes in the endolysosomal network (ELN), DNA damage response pathways and Type 1 interferon signaling. While these pathologies have been documented in AD, they are also influenced by aging, highlighting the complication of studying a late age neurodegenerative disease. In contrast, individuals with early onset familial AD (EOFAD), driven by genetic variants develop AD early in life and at time of death, EOFAD individuals have fewer systemic and brain aging changes than those with LOAD. We hypothesize that response to DNA damage exacerbated by a dysfunctional ELN contributes to AD pathogenesis independent of age. In this study we will leverage our unique cohorts of frozen brain tissue and human induced pluripotent stem cell (hiPSC) in 3 Aims. We will determine microglial molecular profiles (Aim 1) and spatial organization (Aim 2) with and without AD across the spectrum of young to older adult age. We will functionally test our hypothesis using hiPSC models, which lack age related changes (Aim 3). We propose to generate microglial transcriptomic, open chromatin and DNA methylation profiles building gene networks from frozen cortical samples of 19 EOFAD individuals who carry pathogenic variants in PSEN1/PSEN2 or APP and compare them to cortical samples from 35 age-matched unaffected controls aged 25 to 60 years and 10 controls aged 65-75 years. We will map the changes associated with age alone in microglia profiles across the adult age in the 25yr-75 yr control cohort. We will leverage our existing older control and LOAD snRNAseq dataset as another comparison and nominate which microglial changes appear in the presence of AD pathology, age alone, and those that are exacerbated by the presence of both AD and age. To functionally measure the significance of the AD phenotypes we have identified we will use hiPSC derived microglia and neurons to test the hypothesis that ELN dysfunction contributes to damage and accumulation of nucleic acids contributing to neuronal toxicity. These integrated omics and in vitro studies evaluating human microglia across the adult age span and AD pathology will identify and functionally test factors regulating microglial phenotypes that can inform effective therapeutic targeting for both EOFAD and LOAD.