Perineuronal nets as a barrier for a-synuclein spreading

项目概述：帕金森病（PD）是一种进行性神经退行性疾病，其特征在于神经元中存在路易体，由聚集的错误折叠的α-突触核蛋白（α-syn）和其他蛋白质组成，导致细胞死亡。α-syn可以在神经元之间转移，导致路易体扩散。α-syn错误折叠和沉积也有助于其它突触核蛋白病。神经元周网（Perineuronalnets，PNNs）是一种存在于神经元表面的细胞外基质（extracellularmatrix，ECM）。PNN调节突触形成，控制神经可塑性，作为一个保护层，防止有毒蛋白质和氧化应激，作为一个离子库和螯合分子的神经元。PNN由透明质酸（一种糖胺聚糖）、硫酸软骨素蛋白聚糖、HAPLN（连接蛋白）和腱生蛋白-R（一种糖蛋白）组成。目前的数据显示，野生型α-syn在大鼠神经元培养物中与硫酸软骨素结合。此外，在人脑中的表征表明，PNN神经元免于路易病理，而非PNN神经元则不然。将使用荧光探针标记、原子力显微镜和/或冷冻电子显微镜分析从组织样品中分离的α-syn。(2)确定硫酸软骨素、PNN和（1）中鉴定的不同a-syn种类之间的相互作用。这将通过表达和纯化蛋白质、寡聚体/原纤维制备以及使用生物物理技术来验证这些分子相互作用来确定。(3)确定α-syn、硫酸软骨素、PNN之间的相互作用对α-syn内化和接种的影响，以及对神经元形态和生理的影响。这将通过神经元培养、免疫细胞化学、共聚焦显微镜和定量以及电生理学来确定。这项研究将提高我们对a-syn与PNNs相互作用的基本分子机制以及PNNs如何保护神经元免受a-syn相关病理的理解。

Project Summary:Parkinson's disease (PD) is a progressive neurodegenerative disorder which is characterised by the presence of Lewy bodies, made up of aggregated misfolded a-synuclein (a-syn) amongst other proteins, in neurones causing cell death. a-syn can be transferred between neurones leading to Lewy body spread. a-syn misfolding and deposition also contributes to other synucleinopathies. Perineuronal nets (PNNs) are a type of extracellular matrix (ECM) found on the surface of neurones. PNNs modulate synapse formation, control neuroplasticity, act as a protective layer against toxic proteins and oxidative stress, serve as an ion reservoir and sequester molecules on neurones. PNNs are made up of hyaluronan (a glycosaminoglycan), chondroitin sulphate proteoglycans, HAPLNs (link proteins) and Tenascin-R (a glycoprotein). Current data shows that wildtype a-syn binds to chondroitin sulphates in rat neuronal cultures. Additionally, characterisation in human brains shows that PNN neurones are spared from Lewy pathology, whilst non-PNN neurones are not.The aims of this project are to (1), characterise the different a-syn species and their structures, retrieved from human PD samples. a-syn isolated from tissue samples will be analysed using fluorescent probe labelling, atomic force microscope and/or cryo-electron microscopy. (2), Determine the interactions between chondroitin sulphates, PNNs and different a-syn species identified in (1). This will be determined through expressing and purifying proteins, oligomer/ fibril preparation and using biophysical techniques to characterise these molecular interactions. (3), Determine the impact of the interactions between a-syn, chondroitin sulphates, PNNs on: a-syn internalisation and seeding, in addition to the impact on neuronal morphology and physiology. This will be determined using neuronal culture, immunocytochemistry, confocal microscopy and quantification, and electrophysiology.This research will improve our understanding of the fundamental molecular mechanisms by which a-syn interacts with PNNs and how PNNs may offer protection to neurones against a-syn related pathologies.