Exploring microvesicular transport across the blood-brain barrier as a novel a-synuclein clearance mechanism and source of Parkinson's disease biomarkers

摘要 虽然α-突触核蛋白(α-syn)主要是一种胞内蛋白，但细胞外α-syn被认为起着重要的作用 在帕金森病的病理过程中发挥重要作用，如路易氏病的进行性、普利子样的扩散 整个大脑的躯体病理，以及神经元和神经胶质细胞病理的启动。在神经元中 系统中，α-syn被发现通过非经典途径输出并在细胞外分泌。 水泡(EVS)。除了它对理解α-SYN在帕金森病发病机制中的作用的意义外， α-SYN到EVS的本地化为利用外周α-SYN作为PD生物标志物提供了一种独特的策略，通过 特别针对源自大脑的电动汽车中的α-SYN。事实上，我们最近已经证明了α-syn 从大脑到血液，这种源自中枢神经系统的α-SYN的一小部分被恢复 来自血浆的，包含在外体(小的内源性EV)中。此外，该外围设备可访问 外体α-SYN与全脑脊液α-SYN在区分PD和对照组方面的表现相似 人类受试者。有趣的是，在帕金森病患者脑脊液α-SYN减少的同时，中枢神经系统来源的外体α-SYN减少 增加，提示α-SYN在EVS中从脑中的清除可能在PD中上调。然而，只有 侧脑室注射的一小部分α-SYN与血液外切体有关； 其余人所在的车厢尚不清楚。例如，尚不清楚微泡是否会脱落。 (MV，直接从质膜发芽的小泡)参与α-SYN的分泌，特别是从 中枢神经系统来源的血浆EVS作为PD生物标志物的前景，也不清楚哪些类型的细胞可能 参与生成跨越血脑屏障的含α-SYN的囊泡。最后，α-SYN的分泌通过 EVS在帕金森病发病机制中的作用尚不清楚。在这里，我们建议调查α-SYN是否传输了 通过EVS从大脑到血液可能提供了一种清除有毒α-SYN物种的机制，如 以及作为PD患者EV-α-SYN改变的潜在细胞来源，可能适合使用 作为诊断或进展的生物标志物。我们将首先检查全部和翻译后修改 (磷酸化和寡聚体)来自神经元、星形胶质细胞和少突胶质细胞的血浆EV中的α-SYN。 这种蛋白质将使用灵敏的、定量的免疫分析方法以及纳米颗粒进行表征。 跟踪分析，以确定α-SYN形式在不同类型电动汽车中的分布。这两种类型的 分析将用于比较帕金森病患者、健康对照组和多系统患者的样本 萎缩，一种相关但不同的以少突胶质细胞包涵体为特征的联核病。然后，我们将开发一种 适合研究含EV的α-SYN跨越血脑屏障机制的动物模型 进入血浆，并开始研究含有α-SYN的EV跨 BBB。这些实验将对未来旨在开发帕金森病生物标记物的研究具有价值 进展，以及寻找针对这一潜在清除机制的新的治疗方法。

Abstract Although α-synuclein (α-syn) is largely a cytosolic protein, extracellular α-syn is believed to play an important role in the pathology of PD, contributing to key processes such as the progressive, prion-like spread of Lewy body pathology throughout the brain, and initiation of cellular pathology in neurons and glia. In neuronal systems, α-syn was found to be exported through a non-classical pathway and secreted in extracellular vesicles (EVs). In addition to its implications for understanding the role of α-syn in PD pathogenesis, the localization of α-syn to EVs presents a unique strategy for utilizing peripheral α-syn as a PD biomarker, by specifically targeting α-syn in EVs originating in the brain. Indeed, we have recently demonstrated that α-syn crosses from brain to blood, and that a fraction of this central nervous system (CNS)-derived α-syn, recovered from plasma, is contained within exosomes (small EVs of endocytic origin). Further, this peripherally accessible exosomal α-syn performed similarly to cerebrospinal fluid (CSF) total α-syn in differentiating control from PD human subjects. Intriguingly, while CSF α-syn is decreased in PD, CNS-derived exosomal α-syn was increased, suggesting that clearance of α-syn from the brain in EVs may be up-regulated in PD. However, only a small portion of intracerebroventricularly-injected α-syn was associated with blood exosomes; the compartment in which the rest resided is not known. For example, it is unclear whether shedding microvesicles (MVs, vesicles that bud directly from the plasma membrane) are involved in α-syn secretion, especially from the perspective of CNS-derived plasma EVs as PD biomarkers, nor is it clear what types of cells may participate in generating α-syn-containing vesicles that cross the BBB. Finally, the role of α-syn secretion via EVs in PD pathogenesis remains to be defined. Here, we propose to investigate whether α-syn transported from the brain to the blood via EVs potentially provides a mechanism for clearance of toxic α-syn species, as well as a potential cellular source of EV-contained α-syn altered in PD patients, which may be suitable for use as diagnostic or progression biomarkers. We will first examine the total and post-translationally modified (phosphorylated and oligomeric) α-syn in plasma EVs derived from neurons, astrocytes, and oligodendrocytes. This protein will be characterized both using sensitive, quantitative immunoassays, as well as Nanoparticle Tracking Analysis (NTA), to determine the distribution of α-syn forms in different types of EVs. Both types of analysis will be used to compare samples from PD patients, healthy controls, and patients with multiple system atrophy, a related but distinct synucleinopathy featuring oligodendrocyte inclusions. We will then develop an animal model suitable for studying the mechanisms by which EV-contained α-syn may cross the BBB and enter the plasma, and begin to study the potential mechanisms of α-syn-containing EV transport across the BBB. These experiments will be valuable for future studies aimed at developing biomarkers for PD progression, as well as for seeking novel therapeutic treatments aimed at this potential clearance mechanism.