Synaptic Mechnisms in Drosophila Neurodegeneration Model

描述(由申请人提供)：推动这项建议的假设是 突触前功能障碍是导致细胞死亡的常见原因 在多种遗传性神经退行性疾病中。这一假设是基于 观察到：1)突触功能在神经元存活中起中介作用 发育，2)强烈损害突触前功能的突变导致 大量进行性神经元变性，3)大量突触前蛋白 直接牵涉到神经退行性疾病和神经元 功能障碍/突触丧失已知先于一段相当长的时期 细胞死亡在这些疾病中的表现。然而，到目前为止，还没有 突触功能障碍介导神经元死亡的直接证据已建立 在神经退行性疾病状态下。 这项提议目标是分析两种基因的突触维持 神经退行性疾病模型：帕金森病果蝇模型 帕金森病(PD)是一种典型的“蛋白质储存”病，尼曼-皮克C型(NP-C)是一种 典型的“脂肪堆积症”。果蝇之所以被选中，是因为它的吸引力 神经退行性变的一种新的分子遗传学模型 历史是突触研究的最重要的遗传模型。Pd和NP-C是 被选为代表大量相关的神经退行性变 精神错乱。最近通过建立果蝇帕金森病模型 人突触前蛋白α-突触核蛋白的转基因过表达 显示出准确概括了人类帕金森病的诊断特征。一个 通过突变建立果蝇NP-C模型 已知的人类NP-C病因--内源性NPC I基因功能丧失 疾病。 具体地说，这项提议是对突触进行年龄递进的研究 果蝇PD和NP-C模型中突触维持的相关机制 与神经退行性变的发生、发展和流行有关。第一个目标 是通过产生荧光标记来改进果蝇模型 水平可逆调节的α-突触核蛋白和NPCI蛋白 通过依赖温度的泛化策略。第二，确认 这些模型中神经退行性变的大体特征与行为分析和 神经系统/神经结构的检查。第三，也是大多数 重要的是，要分析这些脑区突触的发育、功能和维持 模特们。化验将包括电生理测量 神经传递，蛋白质的定量荧光光学成像和 突触前终末的脂质动力学及超微结构研究 突触前结构。总之，这些研究将使我们能够得出结论 确定PD和NP-C的突触维持是否受到损害， 是导致神经细胞死亡和 这些疾病状态下的神经退化。

Description (Provided by applicant): The hypothesis driving this proposal is that presynaptic dysfunction is a common causative factor leading to cell death in multiple inherited neurodegenerative diseases. This hypothesis is based on the observations that 1) synaptic function mediates neuronal survival during development, 2) mutations which strongly impair presynaptic function result in massive, progressive neuronal degeneration, 3) a number of presynaptic proteins have been directly implicated in neurodegenerative diseases and 4) neuronal dysfunction/synapse loss is known to precede by a substantial period the manifestation of cell death in these diseases. To date, however, there is no established direct evidence of synaptic dysfunction mediating neuronal death during neurodegenerative disease states. The goal of this proposal is to assay synaptic maintenance in two genetic models of neurodegenerative diseases: Drosophila models of Parkinson's Disease (PD), a classic "protein storage" disease, and Niemann-Pick Type C (NP-C), a classic "lipid storage" disease. Drosophila was selected for its attractive properties as a new molecular genetic model of neurodegeneration, and its long history as the foremost genetic model for synaptic studies. PD and NP-C were selected as representative of a large number of related neurodegenerative disorders. The Drosophila PD model has been recently established through transgenic over-expression of human alpha-synuclein (a presynaptic protein) and shown to accurately recapitulate the diagnostic features of human PD. A Drosophila model of NP-C is being established through mutation (loss-of-function) of the endogenous NPC I gene, the known cause of human NP-C disease. Specifically, this proposal is to conduct age-progressive studies of synaptic mechanisms in Drosophila PD and NP-C models to correlate synaptic maintenance with the onset, progression and prevalence of neurodegeneration. The first aim is to improve Drosophila models by generating fluorescently tagged alpha-synuclein and NPCI proteins whose levels can be reversibly regulated through a temperature-dependent ubiquination strategy. Secondly, to confirm gross features of neurodegeneration in these models with behavioral assays and examination of nervous system/neuronal architecture. Third, and most importantly, to assay synaptic development, function and maintenance in these models. Assays will include electrophysiological measurements of neurotransmission, quantitative fluorescent optical imaging of protein and lipid dynamics in the presynaptic terminal and ultrastructural studies of presynaptic architecture. Together, these studies will allow a conclusive determination of whether synaptic maintenance is compromised in PD and NP-C, and is the causative factor that leads to neuronal cell death and neurodegeneration in these disease states.