Investigating the role of an EIF2B3 variant as an Alzheimer's disease risk modifier

项目摘要 人类遗传学研究提供了一种强有力的方法来识别与阿尔茨海默氏症相关的基因和途径 疾病（AD）。载脂蛋白E同种型4（APOE4）是散发性阿尔茨海默病最强的遗传危险因素 疾病尽管APOE4对AD风险有很强的影响，但APOE4存在广泛的临床结局。 携带者：有些人可能早在30岁时就出现认知障碍，而其他携带者仍然存在 100岁以上的认知正常。APOE4携带者临床表现的大范围表明 存在调节APOE相关风险的遗传变异。为了研究这一假设，我们进行了 在发病年龄分布极端的APOE4携带者的分层全基因组关联研究， 识别改变风险的变量。我们鉴定了EIF2B3中编码氨基酸取代的单一变体， S404A作为候选风险修改器。eIF2B3是eIF2B的亚基，eIF2B是eIF2的鸟嘌呤交换因子（GEF）， 参与翻译控制和综合应激反应。我们的实验室最近发现APOE4 诱导鼠和人iPSC衍生的细胞中组成性上调的ISR和异常蛋白质翻译， 小胶质细胞，表明APOE4单独可导致细胞应激水平增加1，2。此外，委员会认为， EIF2B2是另一种eIF2B亚基，是在AD GWAS3中鉴定的AD风险基因。本提案的总体目标是 直接检验EIF2B3变体通过ISR失调调节APOE 4相关风险的假设 从而在疾病的背景下改变小胶质细胞的功能。在目标1中，我将联合收割机结合无细胞体外荧光 GEF测定和结构化学，以确定EIF2B3S404A对eIF2B结构完整性的直接影响 和酶活性。目标2将确定EIF2B3S404A对 使用人诱导多能干细胞（iPSC）衍生的小胶质细胞和APOE4小胶质细胞功能的体外研究 活细胞成像在疾病相关的压力的背景下。最后，在目标3中，我将使用小说 涉及将人小胶质细胞前体细胞直接注射到小鼠脑中的异种移植方法 评估EIF2B3S404A在体内和疾病背景下对APOE4小胶质细胞功能的影响。 总之，这里的结果将证实或反驳EIF2B3变体调节APOE 4相关的细胞凋亡的假设。 通过改变小胶质细胞功能来降低风险。在整个项目中，我将测试ISR抑制剂（ISRIB）的有效性。 药物在体外减轻EIF2B3S404A诱导的对EIF2B活性的影响以及在体外和体内的小胶质细胞中的作用。的 通过了解变异影响的方式，这里的工作有可能极大地影响公共卫生。 免疫细胞功能和改变疾病风险，并通过潜在地鉴定用于治疗AD的新型药物。 这里提出的奖学金培训计划将为我提供技术技能，科学知识，关键 从艾莉森·高特博士的指导和伊坎学院的协作环境中获得的思维技能 在西奈山的医学大，装备我与必要的技能，成功地过渡到一个职业生涯 作为一个独立的科学家。

Project Summary Human genetic studies provide a powerful approach to identify genes and pathways associated with Alzheimer's disease (AD). Apolipoprotein E isoform 4 (APOE4) is the strongest genetic risk factor for sporadic Alzheimer's disease. Despite the strong effect of APOE4 on AD risk, a wide range of clinical outcomes exist within APOE4 carriers: some can develop cognitive impairment as early as 30 years of age while other carriers remain cognitively normal beyond 100 years. The large range in clinical manifestation amongst APOE4 carriers suggests that there are genetic variants that modulate APOE-associated risk. To investigate this hypothesis, we conducted stratified genome-wide association studies of APOE4 carriers at the extremes of age at onset distribution to identify variants that modify risk. We identified a single variant in EIF2B3 encoding the amino acid substitution S404A as a candidate risk modifier. eIF2B3 is a subunit of eIF2B, a guanine exchange factor (GEF) for eIF2 that is involved in translational control and the integrated stress response. Our lab has recently shown that APOE4 induces a constitutively upregulated ISR and aberrant protein translation in murine and human iPSC-derived microglia, demonstrating that APOE4 alone can lead to increased levels of cellular stress1,2. Furthermore, EIF2B2, another eIF2B subunit, is an AD risk gene identified in AD GWAS3. The overall goal of this proposal is to directly test the hypothesis that EIF2B3 variants modulate APOE4-associated risk by dysregulating the ISR and thus alter microglial cell function in the context of disease. In Aim 1, I will combine cell-free in vitro fluorescent GEF assays and structural chemistry to determine the effects of EIF2B3S404A directly on eIF2B structural integrity and enzymatic activity, in the absence of cellular stress. Aim 2 will determine the impact of EIF2B3S404A on APOE4 microglia cell function in vitro using human induced pluripotent stem cell (iPSCs)- derived microglia and live-cell imaging in the context of disease-related stressors. Lastly, in Aim 3, I will employ novel xenotransplantation methods involving direct injection of human microglia precursor cells into the mouse brain to evaluate the effect of EIF2B3S404A on APOE4 microglia cell function in vivo and in the context of disease. Together, the results here will confirm or refute the hypothesis that EIF2B3 variants modulate APOE4-associated risk by altering microglial cell function. Throughout the project, I will test the efficacy of an ISR inhibitor (ISRIB) drug at alleviating EIF2B3S404A -induced effects on EIF2B activity in vitro and in microglia in vitro and in vivo. The work here has the potential to greatly impact public health through understanding the way in which variants affect immune cell function and modify disease risk and by potentially identifying a novel drug for the treatment of AD. The fellowship training plan proposed here will provide me with the technical skills, scientific knowledge, critical thinking skills gained from Dr. Alison Goate's mentorship and the collaborative environment at the Icahn School of Medicine at Mount Sinai at large, equipping me with the necessary skills for a successful transition to a career as an independent scientist.