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

探索微泡穿越血脑屏障的运输作为一种新型的α-突触核蛋白清除机制和帕金森病生物标志物的来源

• 批准号: 9751979
• 负责人: Min Shi
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751979
• 关键词: Age; Animal Model; Animals; Astrocytes; Biological Markers; Blood; Blood - brain barrier anatomy; Brain; Cell membrane; Cerebrospinal Fluid; Data; Diagnostic; Disease; Disease Progression; Endocytic Vesicle; Etiology; Foundations; Future; Goals; Human; Immunoassay; In Vitro; Injections; Investigation; Label; Lewy Bodies; Measures; Membrane; Modeling; Molecular; Multiple System Atrophy; Mus; Neural Cell Adhesion Molecule L1; Neuraxis; Neuroglia; Neurons; Oligodendroglia; Parkinson Disease; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Peripheral; Plasma; Play; Process; Proteins; Rest; Role; Route; Sampling; Severity of illness; Source; Specificity; System; Techniques; Technology; Testing; Transport Vesicles; Transportation; Vesicle; Vesicle Transport Pathway; Work; alpha synuclein; base; brain cell; cell type; cellular pathology; cohort; disorder control; exosome; experimental study; extracellular; extracellular vesicles; human disease; human subject; improved; in vivo; microvesicles; nanoparticle; neuropathology; new therapeutic target; novel; novel marker; novel therapeutics; overexpression; peripheral blood; prion-like; synucleinopathy

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.

摘要 虽然α-突触核蛋白（α-syn）主要是一种胞浆蛋白，但细胞外α-syn被认为在细胞内起重要作用。 在PD病理学中的作用，有助于关键过程，如Lewy的进行性朊病毒样扩散 整个大脑的身体病理学，以及神经元和神经胶质中细胞病理学的起始。在神经元 系统中，发现α-syn通过非经典途径输出，并在细胞外分泌 囊泡（EV）。除了对了解α-syn在PD发病机制中的作用的意义外， 将α-syn定位于EV提供了一种利用外周α-syn作为PD生物标志物的独特策略， 特异性靶向源自大脑的EV中的α-syn。事实上，我们最近已经证明，α-syn 从大脑进入血液，并且这种中枢神经系统（CNS）衍生的α-syn的一部分， 来自血浆的，包含在外来体（内吞来源的小EV）内。此外，这种外围可访问 外泌体α-syn与脑脊液（CSF）总α-syn在区分对照与PD方面的表现相似 人类实验对象有趣的是，虽然CSF α-syn在PD中减少，但CNS衍生的外泌体α-syn在PD中减少。 增加，表明EV中α-syn从脑中的清除可能在PD中上调。但只有 一小部分脑室内注射的α-syn与血液外泌体相关; 其余的人住在哪个房间里还不知道。例如，目前还不清楚脱落的微泡是否 (MVs直接从质膜出芽的囊泡）参与α-syn分泌，尤其是从 CNS衍生的血浆EV作为PD生物标志物的前景，也不清楚什么类型的细胞可以 参与产生穿过血脑屏障的含α-syns的囊泡。最后，α-syn分泌的作用， EV在PD发病机制中的作用仍有待确定。在这里，我们建议调查是否α-syn运输 通过EV从大脑进入血液可能提供了一种清除有毒α-syn物质的机制， 以及在PD患者中改变的含EV的α-syn的潜在细胞来源，其可能适合于使用 作为诊断或进展生物标志物。我们将首先检查总数和经过修正的 来源于神经元、星形胶质细胞和少突胶质细胞的血浆EV中的（磷酸化和寡聚）α-syn。 该蛋白质将使用灵敏的定量免疫测定法以及纳米颗粒分析法来表征。 跟踪分析（NTA），以确定不同类型EV中α-syn形式的分布。两种类型的 分析将用于比较PD患者、健康对照和多系统分析患者的样本。 萎缩，一种相关但不同的突触核蛋白病，以少突胶质细胞包涵体为特征。然后我们将开发一个 适用于研究含EV的α-syn可穿过BBB和 进入血浆，并开始研究含α-顺式异构体的EV跨膜转运的潜在机制。 BBB.这些实验将是有价值的，为未来的研究，旨在开发生物标志物的PD 进展，以及寻求针对这种潜在清除机制的新的治疗方法。