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）是与年龄相关的痴呆症的主要原因，影响世界上超过500万人。 只有美国。不幸的是，目前的治疗主要是姑息性的，几种有前途的药物 候选人在后期临床试验中失败了。因此，迫切需要提高我们的认识， 驱动AD发展和进展的机制。作为免疫系统的主要先天免疫细胞， 几十年来，小胶质细胞与AD的发病机制有关。但究竟是如何 小胶质细胞导致AD，并且这种变化随疾病持续时间的程度仍不清楚。最近 遗传学研究已经发现了在小胶质细胞中高度表达的几种AD风险基因。通过研究 这些基因在诱导多能干细胞（iPSC）衍生的人类小胶质细胞，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。一个近期 发现的突变发生在小胶质细胞富集基因PLCG 2（磷脂酶C γ 2）中， 一种与AD发病几率降低相关的保护性等位基因。这种突变特别有趣 因为PLCG 2编码在钙信号传导中起关键作用的跨膜信号传导酶。钙 信号传导又是小胶质细胞功能的几乎每一个方面的中心，包括吞噬作用，定向迁移， 炎性小体激活，并诱导免疫基因转录的两个主要调节因子; NFAT和 NfkB。因此，我们建议使用CRISPR基因编辑来产生表达AD的同基因iPSC。 PLCG 2的保护性突变。然后，我们将这些细胞分化为小胶质细胞，并检查 PLCG 2对小胶质细胞基因表达、钙信号传导和功能的操纵。通过结合CRISPR 基因编辑、小胶质细胞分化和一种新型的AD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，并确定PLCG 2突变对人类小胶质细胞基因的影响 体外和体内的表达和功能。 !

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. !