Transcriptional regulation of neuroprotective microglia subtypes in health and disease

项目摘要 这项建议提出了一种小胶质细胞功能状态由转录因子调节的新方法 PU.1在淀粉样蛋白病理背景下及其对阿尔茨海默氏病神经保护的潜在贡献 疾病(AD)。PU.1的表达水平对指导对髓系与淋巴系的承诺至关重要 并富含小胶质细胞。PU.1还决定巨噬细胞的炎症激活状态 通过启动顺式调控区域，在刺激下诱导适当的基因表达。最近，全基因组 关联研究(Gwas)发现了SPI1(编码PU.1)的一个变异，它调节AD的风险。不像 其他变体，Spi1变体是独特的，因为它导致PU.1表达减少，并具有保护性 对AD风险的影响。在AD的5xFAD淀粉样蛋白小鼠模型中，我观察到了异质蛋白的表达 PU1在小胶质细胞中的表达。最近的研究支持这样一种观点，即特定的小胶质细胞激活状态具有独特的影响 阿尔茨海默病的发病机制。疾病相关的小胶质细胞(DAM)被认为在AD中起到保护作用 对淀粉样斑块反应强烈，而炎症性人群可能在 疾病。我推测PU1在小胶质细胞中的低表达诱导了神经保护功能状态。 抗AD的小胶质细胞。我们建立了增加(Spi1cOE)或减少(Spi1cKD)的小鼠遗传模型 PU1在小胶质细胞中特异表达，并与5xFAD淀粉样病模型交叉。核糖体 对这些小胶质细胞的分析显示，大量转录发生了变化。要了解PU.1的表达方式 水平调节小胶质细胞的激活状态在淀粉样蛋白病理过程中，我进行了单核RNA测序。 这些新数据表明，5xFAD中的Spi1cKD有利于与神经保护相关的小胶质细胞状态(DAM)。 同时减轻处于毒性状态(炎症)的细胞比例。知道这些小胶质细胞在 与淀粉样斑块的关系将增强我们对这种状态在哪里发生以及在哪里发生的理解 这些小胶质细胞可能起保护作用，也可能起有害作用。在我的第一个目标中，我将使用多路传输的错误稳健 应用原位杂交技术(MERFISH)对DAM和炎症激活状态的小胶质细胞进行空间定位 5xFAD中的Spi1cKD或Spi1cOE。除了这些转录变化外，我们还显示出 Spi1cKD 5xFAD动物的补体蛋白C1qa。我们进一步发现，Spi1cKD小胶质细胞阻止了 突触丢失是人类阿尔茨海默病的特征，并在5xFAD模型中概括。事实证明， 在阿尔茨海默病中，补体的病理性激活与炎症激活一起发生，并导致突触丢失。 因此，我建议在体外评估不同PU.1表达水平的小胶质细胞产生C1qA的能力 以及C1qa在5xFAD小鼠体内与Spi1cKD和Spi1cOE小胶质细胞的突触共定位。

Project Summary This proposal addresses a novel way in which microglia functional states are regulated by the transcription factor PU.1 in the context of amyloid pathology and its potential contribution to neuroprotection against Alzheimer’s disease (AD). The expression level of PU.1 is crucial for instructing commitment to myeloid versus lymphoid lineages and is enriched in microglia. PU.1 also determines the inflammatory activation state of macrophages by priming cis-regulatory regions to elicit appropriate gene expression upon stimulation. Recently, genome wide association studies (GWAS) have identified a variant in Spi1 (encoding PU.1) that modulates AD risk. Unlike other variants, the Spi1 variant is unique in that it leads to a reduction in PU.1 expression and has a protective effect against AD risk. In a 5xFAD amyloid mouse model of AD, I observed heterogeneous protein expression of PU.1 in microglia. Recent work supports the idea that specific microglia activation states have unique impacts on AD pathogenesis. The Disease Associated Microglia (DAMs) are thought to serve a protective role in AD by robustly responding to amyloid plaques whereas inflammatory populations potentially play a harmful role in disease. I hypothesize that low PU.1 expression in microglia induces neuroprotective functional states in microglia against AD. We generated genetic mouse models that increase (Spi1cOE) or decrease (Spi1cKD) PU.1 expression specifically in microglia and crossed them to the 5xFAD amyloid disease model. Ribosomal profiling of these microglia revealed significant bulk transcriptional changes. To understand how PU.1 expression level regulates microglia activation states during amyloid pathology, I performed single nuclei RNA-sequencing. These new data reveal that Spi1cKD in 5xFAD favors microglia states associated with neuroprotection (DAM) while attenuating the proportion of cells in toxic states (inflammatory). Knowing the location of these microglia in relation to amyloid plaques will enhance our understanding of where engagement of such states occur and where these microglia may be exerting protective or harmful effects. In my first aim, I will use multiplexed error-robust in situ hybridization (MERFISH) to spatially identify microglia in DAM and inflammatory activation states with Spi1cKD or Spi1cOE in 5xFAD. In addition to these transcriptional changes, we showed a reduction in the complement protein C1qa in Spi1cKD 5xFAD animals. We further found that Spi1cKD microglia prevented synapse loss that is characteristic in human AD and recapitulated in the 5xFAD model. It has been shown that pathological activation of complement occurs with inflammatory activation and contributes to synapse loss in AD. Therefore, I propose to assess the production of C1qa by microglia with differential PU.1 expression level in vitro and synaptic co-localization of C1qa in 5xFAD mice with Spi1cKD and Spi1cOE microglia.