Using human iPSC derived microglia and chimeric models to examine the role of PLCG2 in Alzheimers disease

摘要 阿尔茨海默病(AD)是老年性痴呆症的主要原因，全世界有500多万人受到影响 单单是美国。不幸的是，目前的治疗主要是姑息疗法和几种前景看好的药物。 候选人在晚期临床试验中失败了。因此，迫切需要提高我们的认识。 阿尔茨海默病的发生和发展的机制。作为人的主要先天免疫细胞 脑、小胶质细胞参与AD的发病机制已有几十年的历史。但确切地说是如何 小胶质细胞对阿尔茨海默病的发病起作用，这种作用随病程变化的程度尚不清楚。近期 遗传学研究发现了几个在小胶质细胞中高表达的阿尔茨海默病风险基因。通过研究 在诱导多能干细胞(IPSC)来源的人小胶质细胞中的这些基因，我们和其他人的目标是推进 我们对这些AD风险基因的正常和疾病相关功能的理解。最近有一次 已发现的突变发生在富含小胶质细胞的基因PLCG2(磷脂酶C伽马2)中，并编码 一种保护性等位基因，与发展为阿尔茨海默病的几率降低有关。这种突变特别有趣 PLCG2编码一种跨膜信号转导酶，在钙信号转导中起关键作用。钙 信号转而又集中于小胶质细胞功能的几乎每一个方面，包括吞噬、定向迁移、 炎性小体激活和免疫基因转录的两个主要调节因子的诱导；NFAT和 NFkB。因此，我们建议使用CRISPR基因编辑来产生表达AD的等基因IPSCs PLCG2的保护性突变。然后我们将把这些细胞分化成小胶质细胞，并检测 PLCG2对小胶质细胞基因表达、钙信号和功能的影响。通过结合CRISPR 基因编辑、小胶质细胞分化和AD的新嵌合模型我们的目标是克服几个关键的 小胶质细胞研究的障碍和PLCG2突变对人小胶质细胞基因的影响 在体外和体内的表达和功能。 好了！

ABSTRACT Alzheimer disease (AD) is the leading cause of age-related dementia, affecting over 5 million people in the United States alone. Unfortunately, current therapies are largely palliative and several promising drug candidates have failed in late-stage clinical trials. Hence, there is an urgent need to improve our understanding of the mechanisms that drive the development and progression of AD. As the primary innate immune cell of the brain, microglia have been implicated in the pathogenesis of AD for several decades. But precisely how microglia contribute to AD and the degree to which this changes with disease duration remains unclear. Recent genetic studies have uncovered several AD risk genes that are highly expressed in microglia. By studying these genes in induced pluripotent stem cell (iPSC)-derived human microglia, we and others aim to advance our understanding of both the normal and disease-associated functions of these AD risk genes. One recently discovered mutation occurs in the microglia-enriched gene PLCG2 (Phospholipase C Gamma 2) and codes for a protective allele associated with a reduced chance of developing AD. This mutation is particularly interesting as PLCG2 encodes a transmembrane signaling enzyme that plays a critical role in calcium signaling. Calcium signaling is in turn central to nearly every facet of microglial function including phagocytosis, directed migration, inflammasome activation, and induction of two master regulators of immune gene transcription; NFAT and NfkB. We therefore propose to use CRISPR gene editing to produce isogenic iPSCs that express the AD protective mutation in PLCG2. We will then differentiate these cells into microglia and examine the impact of PLCG2 manipulations on microglial gene expression, calcium signaling and function. By combining CRISPR gene editing, microglial differentiation, and a novel chimeric model of AD we aim to overcome several critical barriers to microglial research and determine the impact of PLCG2 mutations on human microglial gene expression and function both in vitro and in vivo. !