Exosomes: A Novel Contributor of Microglia-Mediated Neuroinflammation and Neurodegeneration.

项目摘要： 阿尔茨海默病（AD）是一种毁灭性的和迅速上升的神经退行性疾病， 全球4400万人。AD病理学包括淀粉样蛋白-β（Aβ）的进行性蛋白质聚集， τ的AD的一个关键病理组成部分是由脑免疫细胞介导的神经炎症 叫做小胶质细胞。虽然小胶质细胞介导的神经炎症已经成为一种致病机制， 我们对AD中发生的小胶质细胞蛋白质变化以及小胶质细胞是如何发生变化的理解仍然存在重大差距。 可以使AD病理永久化。外泌体释放已成为小胶质细胞介导的细胞凋亡的一种机制。 神经炎症和神经变性。鉴于外泌体在大分子转移中的关键作用， 在细胞之间促进细胞间通讯，小胶质细胞来源的外泌体有可能转移到 致病性货物，可以使AD永久化。此外，小胶质细胞可以采取不同的状态，因此， 源自这些小胶质细胞状态的外来体可能具有不同的组成和不同的作用。 然而，小胶质细胞来源的外泌体在神经炎症和AD病理学中的作用仍然很差 研究了为此，迫切需要确定AD中小胶质细胞蛋白水平的变化，并确定 AD病理学中小胶质细胞衍生的外泌体的分子组成和状态特异性作用。整体 我的F31计划的目标是了解小胶质细胞和小胶质细胞衍生的外泌体如何使AD永久化 病理我将检验小胶质细胞和小胶质细胞衍生的外泌体的蛋白质谱影响 AD病理学和神经炎症。在目标1中，我将确定小胶质细胞状态对外泌体的影响。 组合物和外泌体介导的体外应答。我会将小胶质细胞转化为一种自我平衡的，抗- 使用不同的细胞因子和Aβ寡聚体的组合来治疗炎症、促炎或“AD样”状态。 然后，我将测量这些小胶质细胞释放的外泌体诱导促炎性变化的能力， 静息神经胶质细胞和神经元中的细胞毒性。利用质谱分析，我还将确定蛋白质中的差异 来源于不同小胶质细胞状态的外泌体的概况。在目标2中，我将定义小胶质细胞衍生的 与体内AD病理学相关的外泌体蛋白质组。为了实现这一点，我将雇用一只老鼠 Aβ病理模型（5XFAD），一种使用突变生物素连接酶的新型体内蛋白质标记策略 （TurboID）来生物素化小胶质细胞和小胶质细胞衍生的外泌体中的蛋白质，以及使用 质谱分析法来这些研究的结果将促进我们对神经炎症的理解。 AD发病机制的研究，有助于发现AD的治疗靶点。

PROJECT ABSTRACT: Alzheimer’s disease (AD) is a devastating and rapidly rising neurodegenerative disorder, afflicting nearly 44 million people worldwide. AD pathology includes progressive protein aggregation of amyloid-beta (Aβ) and tau. A key pathological component of AD is neuroinflammation which is mediated by immune cells of the brain called microglia. Although microglia-mediated neuroinflammation has emerged as a causal disease mechanism, there are still critical gaps in our understanding of what microglial protein changes occur in AD and how microglia can perpetuate AD pathology. Exosome release has emerged as a mechanism of microglia-mediated neuroinflammation and neurodegeneration. Given the critical role of exosomes in the transfer of macromolecules between cells to facilitate intercellular communication, it is possible that microglia-derived exosomes transfer pathogenic cargo which could perpetuate AD. Furthermore, microglia can adopt different states, and therefore, exosomes derived from these microglia states are likely to have different compositions and distinct effects. However, the role of microglia-derived exosomes in neuroinflammation and AD pathology remains poorly investigated. To this end, there is a pressing need to define microglial protein level changes in AD and define the molecular composition and state-specific effects of microglia-derived exosomes in AD pathology. The overall goal of my F31 proposal is to understand how microglia and microglia-derived exosomes perpetuate AD pathology. I will test the hypothesis that the protein profiles of microglia and microglia-derived exosomes impact AD pathology and neuroinflammation. In Aim 1, I will determine the effect of microglia state on exosome composition and exosome-mediated responses in-vitro. I will polarize microglia to either a homeostatic, anti- inflammatory, pro-inflammatory, or “AD-like” state using a combination of different cytokines and Aβ oligomers. I will then measure the ability of exosomes released by these microglia to induce pro-inflammatory changes in resting glial cells and cytotoxicity in neurons. Using mass spectrometry, I will also define differences in the protein profiles of exosomes derived from different microglial states. In Aim 2, I will define changes in microglia-derived exosomal proteome that are associated with AD pathology in-vivo. To accomplish this, I will employ a mouse model of Aβ pathology (5XFAD), a novel in-vivo protein labeling strategy which uses a mutated biotin ligase (TurboID) to biotinylate proteins in microglia and microglia-derived exosomes, and quantitative proteomics using mass spectrometry. The results from these studies will advance our understanding of neuroinflammatory mechanisms of AD pathogenesis and contribute to the discovery of therapeutic targets for AD.