The effects of APOE genotype in homeostatic microglial function in preclinical APOE mouse model

项目摘要 阿尔茨海默病（AD）是一种破坏性的神经退行性疾病。衰老与人类基因组的α 4等位基因 APOE基因是AD最强的遗传危险因子。APOE是大脑中主要的胆固醇转运蛋白 促进神经元生长，碎片清除，主要由星形胶质细胞产生。在炎症 小胶质细胞产生APOE作为免疫调节剂。慢性炎症加剧了 AD病理学进展。先前的研究表明APOE 4基因型在APOE水平降低中的作用 脑中的蛋白质，基线时炎症增加，树突棘简化。研究 本文提出的目的是了解AD发病前小胶质细胞功能的APOE 4相关改变。我们产生 在CX 3CR 1启动子下表达GFP的新型人APOE敲入小鼠，以研究APOE的作用 基因型在稳态小胶质细胞功能中的作用到目前为止，在我的论文中，我已经研究了APOE 4的干扰， 稳态小胶质细胞功能，并发现APOE 4改变了小胶质细胞的监视和对 通过下调趋化性嘌呤能受体的损伤。我还研究了 年龄，性别和APOE基因型在小胶质细胞功能中的作用，并发现年龄影响小胶质细胞对 这些改变发生在APOE 4大脑中更早。 本F99/K 00提案将包括以下研究计划中提出的2个目标。在目标1中，我将 确定小胶质细胞在稳态功能和对 淀粉样蛋白（A β）。目的1a将检查来自APOE 3和APOE 4小胶质细胞的全转录组RNA-seq。我们将 鉴定APOE 3和APOE 4小胶质细胞中差异表达的基因并研究特异性途径 参与改变体内平衡的小胶质细胞功能。目的1b将研究APOE基因型对 小胶质细胞在AD发作前识别AD的能力。我们将研究小胶质细胞对急性暴露于 APOE基因型影响小胶质细胞对APOE单体和寡聚体的亲和力。Aim 1c将研究APOE 蛋白质与噬菌体的相互作用及其对吞噬作用的影响。我们将研究APOE-A β相互作用及其 对小胶质细胞识别A β和摄取的影响。在目标2中，我将继续在博士后环境中培养技能 研究衰老过程中小胶质细胞与外周免疫系统相互作用的机制， 神经元变性为了实现这一目标，我将首先确定理想的博士后实验室，然后获得 博士后职位我的目标是在一个重视创新的机构完成我的博士后研究， 科学严谨、培训和专业发展。为了获得这个奖学金，我的赞助商和我是 密切合作，确定会议和邀请演讲者，这将使我能够扩大我的网络 并确定理想的实验室设置。F99/K 00将极大地帮助我完成我的博士学位，并获得 我正在争取的博士后奖学金。

Project Summary Alzheimer's disease (AD) is a devastating neurodegenerative condition. Aging and the 4 allele of the APOE gene are the strongest genetic risk factor for AD. APOE is the main cholesterol transporter in the brain that promotes neuronal growth, debris clearance, and it is mainly produced by astrocytes. Upon inflammatory insults APOE is produced by microglia to act as an immunomodulator. Chronic inflammation exacerbates the progression of AD pathology. Previous studies show an effect of APOE4 genotype in decreased levels of APOE protein in the brain, increased inflammation at baseline, and simplification of dendritic spines. The studies proposed here aim to understand APOE4-related alteration in microglial function prior AD onset. We generated a novel human APOE knock-in mice expressing GFP under the CX3CR1 promoter to study the effects of APOE genotype in homeostatic microglia function. So far in my thesis, I have examined the disturbances of APOE4 in homeostatic microglial function and found that APOE4 altered microglia surveillance and response to damage through the downregulation of chemotactic purinergic receptors. I have also studied the effects of aging, sex, and APOE genotype in microglial function and found that aging affects microglia response to damage, and these alterations occur earlier in APOE4 brains. This F99/K00 proposal will encompass 2 aims presented in the following research plan. In Aim 1, I will identify the molecular mechanism underlying microglial alteration in homeostatic function and response to amyloid  (A). Aim 1a will examine whole transcriptome RNA-seq from APOE3 and APOE4 microglia. We will identify genes differentially expressed in APOE3 and APOE4 microglia and investigate specific pathways involved in altering homeostatic microglial function. Aim 1b will examine the effects of APOE genotype on microglia's ability to recognize A prior AD onset. We will investigate microglia response to acute exposure to A and if APOE genotype affects microglia affinity to A monomers and oligomers. Aim 1c will study APOE protein interaction with A and its effect on phagocytosis. We will investigate APOE-A interactions and their effect on microglia recognition of A and uptake. In Aim 2, I will continue to build skills in a post-doctoral setting researching the mechanisms underlying microglia crosstalk with the peripheral immune system during aging and neuronal degeneration. To achieve this, I will first identify the ideal postdoctoral lab and then obtain the postdoctoral position. I aim to complete my postdoctoral fellowship in an institution that values innovation, scientific rigor, training, and professional development. To obtain this fellowship, my sponsor and I are working closely in identifying conferences and inviting speakers that will allow me to expand my network and identify the ideal lab setting. The F99/K00 will greatly assist me in both completing my PhD and obtaining the postdoctoral fellowship I am striving for.