Using Peripheral Microglial Exosomes to predict brain inflammation in the human Parkinson’s brain

在这个提议中，我们将确定我们是否可以使用已经脱落到细胞外的小胶质细胞外囊泡（EV）。 脑脊髓液（CSF）和血清中的小胶质细胞的活化状态预测帕金森氏症的大脑（PD）。 目前，该领域的一个主要障碍是没有建立和验证的方法来检测大脑 炎症反应的神经退行性疾病在生活中。小胶质细胞，CNS的常驻免疫细胞 它不断地在大脑中巡逻，寻找由大量免疫刺激剂引起的感染或炎症的迹象。 小胶质细胞的作用是清除对中枢神经系统的潜在威胁，但它们的化学特征是基于它们的 持续释放（细胞因子）。这些细胞因子激活邻近的小胶质细胞， 被认为是驱动疾病发病机制的事件级联。虽然细胞因子通常被认为是 为了作为可溶性分子起作用，最近的努力已经表明细胞因子被封装在EV中。这些EV 含有大量炎症相关的mRNA，这些mRNA编码与其他信号传导相关的精氨酸相关基因， 这些分子已知能反映母体细胞的生理状态。与小胶质细胞不同，电动汽车可以通过 血脑屏障在渗漏和发炎的条件下，这两者都是已知的生理过程， 警局为了确定我们是否可以鉴定外周中的小胶质细胞EV，我们将表征从外周中分离的EV。 疾病相关的小胶质细胞（DAM）使用RNAseq和液相色谱-质谱（LC/MS）。 这些新的数据将被用于生成外围发现工作的目标。该提案旨在解决 两个未满足的需求1）在活体中检测脑炎症的可能性和2）区分EV的能力 从外周血单核细胞释放的那些。我们假设 从血清/CSF中提取的小胶质细胞EV将反映来自DAM的EV谱。为了验证我们的假设，我们 建议1）分析疾病相关小胶质细胞（DAM）中的膜结合蛋白和EV货物，2） 分析从PD外周血分离的外周EV中的膜结合蛋白和EV货物 单核细胞，3）鉴定和验证用于抗体的独特的小胶质细胞特异性EV膜抗原 4）使用这些经验证的抗体从CSF和血清中拉下小胶质细胞特异性EV。 申请中提出的所有组织均采集自死亡前的PD患者， 并且具有病理学证实的疾病而没有合并症。我们相信这些人 aims本身具有很高的影响力和新颖性，但集体可以为我们提供预测大脑的必要工具。 炎症

In this proposal, we will determine if we can use microglial extracellular vesicles (EV) that have been shed into the cerebral spinal fluid (CSF) and serum to predict the microglial activation state in the Parkinson’s brain (PD). Currently, a major obstacle in the field is that there are no established and validated methods to detect brain inflammation in response to neurodegeneration during life. Microglia, the resident immune cell of the CNS constantly patrol the brain, looking for signs of infection or inflammation caused by a host of immune stimulants. The role of microglia is to clear potential threats to the CNS, but their chemical signatures based on their presence are continuously released (cytokines). These cytokines activate neighboring microglia initiating a cascade of events that are believed to drive disease pathogenesis. Although cytokines are generally considered to function as soluble molecules, recent efforts have shown that cytokines are encapsulated in EVs. These EVs contain a host of inflammatory-associated mRNAs that encode cytokine-associated genes among other signaling molecules that are known to reflect the physiological state of the parent cell. Unlike microglia, EVs can cross the blood-brain barrier under leaky and inflamed conditions, both of which are known physiological processes in PD. To determine if we can identify microglial EVs in the periphery we will characterize EVs isolated from disease-associated microglia (DAM) using RNAseq and Liquid chromatography-mass spectrometry (LC/MS). This novel data will be used to generate targets for discovery work in the periphery. This proposal aims to address two unmet needs 1) the possibility to detect brain inflammation in the living and 2) the ability to distinguish EVs released from microglia from those released from peripheral blood mononuclear cells. We hypothesize that microglial EVs extracted from the serum/CSF will reflect EV profiles from DAM. To test our hypothesis, we propose to 1) Analyze membrane-bound proteins and EV cargo in disease-associated microglia (DAM), 2) analyze membrane-bound proteins and EV cargo in peripheral EVs isolated from PD peripheral blood mononuclear cells, 3) identify and validate unique microglial-specific EV membrane antigens for antibody discovery work and 4) use these validated antibodies to pull down microglial-specific EVs from CSF and serum. All the tissues proposed in the application were collected from premortem PD patients who have since passed and have pathological confirmation of disease without comorbidities. We believe that each of these individual aims is high impact, and novel on their own, but collectively could provide us the tools necessary to predict brain inflammation.