Role of TREM2 gain-of-function mutations in modulating microglial pathology in Alzheimer's disease

项目摘要 全基因组关联研究发现了许多与免疫相关的阿尔茨海默病(AD)风险基因 回应。其中包括小胶质细胞受体CD33和TREM2。我们之前展示了击倒 CD33可减轻5XFAD小鼠的淀粉样β蛋白(Aβ)病理改变并改善其认知功能，两者均 被额外的TREM2基因敲除而被废除。5XFAD小鼠TREM2基因敲除加剧了β的病理和 神经变性，但小胶质细胞数量减少。小胶质细胞的rna-seq图谱显示与 T细胞吞噬和信号传导(IL-6、IL-8、急性时相反应)在5XFAD；TREM2-/-小鼠中下调。 5XFAD；CD33-/-小胶质细胞的差异基因表达依赖于TREM2的存在，提示 TREM2在CD33下游起作用。此外，我们还报道了TREM2的D87N和T96K变体 在基于NIMH家族的遗传数据和ADSP病例对照样本中增加AD风险，并表明 这些变异体增加了与TREM2配体的结合。在我们的初步研究中，我们发现功能增益 Trem2T96K变异体增加了不溶性Aβ42的水平，但同时减少了小胶质细胞数量和聚集 5XFAD小鼠Aβ斑块周围的小胶质细胞此外，Trem2T96K变异体减少了sTrem2的分泌 在5XFAD小鼠中。最后，T96K变异体导致细胞表面TREM2表达和分泌减少 人小胶质细胞系。在这里，我们建议1)评估TREM2功能增益突变对 AD的发病机制，2)研究这些突变对小胶质细胞激活和基因表达的影响。 以及3)识别和表征与AD相关的新的TREM2功能增益突变以及靶点 TREM2用于治疗目的。在目标1中，我们将研究函数增益Trem2T96K的影响 5XFAD小鼠Aβ基因突变、神经变性和斑块相关性神经性营养不良。我们 也将确定Trem2T96K是否影响5XFAD小鼠的认知功能。在目标2中，我们将调查 功能获得性Trem2T96K突变对小胶质细胞活化和向β募集的影响 5XFAD小鼠的斑块和对内毒素刺激的免疫反应。核糖核酸序列分析 小胶质细胞将揭示Trem2T96K对小胶质细胞基因表达特征的影响，以及它们与 与疾病相关的小胶质细胞。在目标3中，我们将分析来自NIMH家族的全基因组测序数据 Sample和NIA ADSP确定与AD相关的新TREM2突变，这些突变将被表征为其 对TREM2配体依赖性激活的影响。然后我们将介绍新的TREM2函数增益 应用CRISPR/Cas9系统诱变IPSC来源的人小胶质细胞样细胞及其相关研究 分子机制。最后，我们将预测和验证模拟TREM2激活的新型靶向药物 和/或使用计算方法来对抗TREM2功能丧失。这项提议的主要目标是 综合评价TREM2功能获得突变在AD发病机制中的作用 潜在的分子网络，这将有助于针对TREM2的AD治疗。

Project Summary Genome-wide association studies have identified many Alzheimer’s disease (AD) risk genes related to immune response. Among these are the microglial receptors CD33 and TREM2. We previously showed that knock-out of CD33 attenuated amyloid beta (Aβ) pathology and improved cognition in 5XFAD mice, both of which were abrogated by additional Trem2 knock-out. Knocking out Trem2 in 5XFAD mice exacerbated Aβ pathology and neurodegeneration but reduced microglia numbers. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are downregulated in 5XFAD;Trem2-/- mice. Differential gene expression in 5XFAD;CD33-/- microglia depended on the presence of Trem2, suggesting TREM2 acts downstream of CD33. Moreover, we reported that the D87N and T96K variants of TREM2 increased AD risk in the NIMH family-based genetic data and ADSP case-control samples and showed that these variants increased binding to TREM2 ligands. In our preliminary study, we found that the gain-of-function Trem2T96K variant increased levels of insoluble Aβ42 but reduced both microglia numbers and clustering of microglia around Aβ plaques in 5XFAD mice. Furthermore, the Trem2T96K variant reduced secretion of sTrem2 in 5XFAD mice. Finally, the T96K variant led to reduced cell surface expression and secretion of TREM2 in human microglial cell lines. Here, we propose to 1) assess the impact of TREM2 gain-of-function mutations on AD pathogenesis, 2) investigate the effects of these mutations on microglial activation and gene expression, and 3) identify and characterize novel AD-associated TREM2 gain-of-function mutations as well as target TREM2 for therapeutic purposes. In Aim 1, we will examine the effects of the gain-of-function Trem2T96K mutation on Aβ pathology, neurodegeneration and plaque associated-neuritic dystrophy in 5XFAD mice. We will also determine whether Trem2T96K impacts cognitive function in 5XFAD mice. In Aim 2, we will investigate the impact of the gain-of-function Trem2T96K mutation on microglial cell activation and recruitment to Aβ plaques as well as immune response to lipopolysaccharide stimulation in 5XFAD mice. RNA-seq profiling of microglia will reveal the effects of Trem2T96K on microglial gene expression signature and how they compare to disease-associated microglia. In Aim 3, we will analyze whole genome sequencing data from the NIMH family sample and NIA ADSP to identify novel AD-associated TREM2 mutations that will be characterized for their effects on ligand-dependent activation of TREM2. We will then introduce novel TREM2 gain-of-function mutations into iPSC-derived human microglia-like cells using CRISPR/Cas9 system and investigate underlying molecular mechanisms. Finally, we will predict and validate novel targeted drugs that mimic TREM2 activation and/or oppose TREM2 loss-of-function using computational approaches. The major goals of this proposal are to comprehensively assess the effects of TREM2 gain-of-function mutations on AD pathogenesis and explore underlying molecular networks, which will facilitate AD therapeutics targeting TREM2.