Mechanisms of synaptic dysfunction in Parkinson's and other synuclein-linked dise

摘要 该项目的长期目标是确定引起细胞和分子机制， 帕金森病（PD）和其他相关神经系统疾病患者的突触缺陷。 这些疾病的病理标志包括突触处的突触核蛋白水平异常， 整个神经元。突触核蛋白参与突触囊泡的形成， 运输，囊泡运输途径的确切步骤，这些步骤被改变的 突触核蛋白仍不清楚。因此，目前还不可能制定有针对性的战略， PD中的突触功能。这里提出的实验通过识别 精确的突触囊泡运输缺陷导致突触处突触核蛋白过量， 从而导致这些缺陷，以及扭转这些缺陷的新策略。这些实验利用了 两个模型突触，非常适合研究突触囊泡贩运，使用急性 和基因干扰结合高定量生化分析来测量 突触核蛋白相互作用，设计试剂干扰这些相互作用，并详细超微结构 分析提供了最好的机会，以确定细胞和分子机制，导致 突触核蛋白诱导的突触囊泡运输缺陷。在最初的研究中， 导致突触囊泡丢失，脑池增加，网格蛋白包被轮廓改变，符合 抑制网格蛋白介导的突触囊泡再循环。展望未来，拟议的实验是 旨在确定过量的野生型突触核蛋白和PD相关的细胞机制， 突变（例如A30P、E46K、A53T）引起囊泡运输缺陷（目的1和2）。实验将 还研究了突触核蛋白如何与特定的突触结合配偶体（例如PI（4，5）P2和 未包被的ATP酶）导致突触囊泡运输缺陷， 破坏这些相互作用将被评估为逆转突触缺陷的可能手段（Aim 2和3）。所提出的实验是创新的，因为他们是第一个使用组合， 定量生化结合试验、急性扰动、受控刺激条件，以及 详细的超微结构分析，以确定精确的突触囊泡运输缺陷引起的 过量的突触核蛋白或其突变，这非常适合于总体目标。了实验 意义重大，因为它们代表了理解产生机制的第一步 突触缺陷，它们提供了可能的靶向分子策略，用于改善突触缺陷， 功能因此，这些研究对减缓或停止神经变性有直接的意义， 认知缺陷和PD和其他突触核蛋白病中的痴呆。

ABSTRACT The long term goal of this project is to identify the cellular and molecular mechanisms that give rise to the synaptic defects in patients with Parkinson's disease (PD) and other related neurological disorders. The pathological hallmark of these diseases includes abnormal levels of ¿-synuclein at synapses and throughout the neuron. While it is generally agreed that synucleins participate in synaptic vesicle trafficking, the exact steps of the vesicle trafficking pathway that are perturbed by altered levels of synuclein remain unclear. Thus, at present, it is not possible to design targeted strategies for improving synaptic function in PD. Experiments proposed here take the first steps toward this by identifying the precise synaptic vesicle trafficking defects caused excess synuclein at synapses, the mechanisms giving rise to these defects, and new strategies for reversing them. The experiments take advantage of two model synapses that are ideally suited for studies of synaptic vesicle trafficking, using both acute and genetic perturbations. The combination of highly quantitative biochemical assays to measure synuclein interactions, design of reagents to perturb these interactions, and detailed ultrastructural analyses provides the best opportunity to identify the cellular and molecular mechanisms leading to synuclein-induced synaptic vesicle trafficking defects. In initial studies, excess wild type synuclein causes a loss of synaptic vesicles, increased cisternae, and altered clathrin-coated profiles, consistent with inhibiting clathrin-mediated synaptic vesicle recycling. Going forward, proposed experiments are aimed at identifying the cellular mechanisms by which excess wild type ¿-synuclein and PD-related mutations (e.g. A30P, E46K, A53T) cause vesicle trafficking defects (Aims 1 and 2). Experiments will also investigate how synuclein interactions with specific synaptic binding partners (e.g. PI(4,5)P2 and the uncoating ATPase) contribute to the synaptic vesicle trafficking defects, and targeted strategies for disrupting these interactions will be assessed as a possible means for reversing synaptic defects (Aim 2 and 3). The proposed experiments are innovative because they are the first to use a combination of quantitative biochemical binding assays, acute perturbations, controlled stimulation conditions, and detailed ultrastructural analyses to identify the precise synaptic vesicle trafficking defects caused by excess synuclein or its mutations, which is ideally suited for the overall goal. The experiments are significant because they represent the first steps toward understanding the mechanisms giving rise to the synaptic defects, and they provide possible targeted, molecular strategies for improving synaptic function. Thus, these studies have direct implications for slowing or halting the neurodegeneration, cognitive deficits, and dementia in PD and other synucleinopathies.