Synaptic Mechnisms in Drosophila Neurodegeneration Model

果蝇神经退行性变模型中的突触机制

• 批准号: 6344133
• 负责人: Kendal Broadie
• 金额: $ 37.48万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344133
• 关键词: Drosophilidae; Niemann Pick disease; Parkinson's disease; alpha synuclein; cell death; electron microscopy; electrophysiology; gene mutation; genetic models; genetically modified animals; green fluorescent proteins; immunocytochemistry; model design /development; neural degeneration; neural transmission; neurons; pathologic process; radionuclide double label; synapses; temperature sensitive mutant; tissue /cell culture

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.

描述（由申请人提供）：驱动该提案的假设是 突触前功能障碍是导致细胞死亡的常见原因 多种遗传性神经退行性疾病这个假设是基于 观察结果表明：1）突触功能介导神经元的存活， 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 直接与神经退行性疾病有关， 已知功能障碍/突触丧失先于一个相当长的时期， 这些疾病中的细胞死亡表现。然而，迄今为止， 建立了突触功能障碍介导神经元死亡的直接证据 在神经退行性疾病状态下。 这项提议的目的是分析两个遗传性神经元中突触的维持。 神经退行性疾病的模型：帕金森病的果蝇模型 (PD)，一种典型的“蛋白质储存”疾病，和尼曼-匹克C型（NP-C），一种 典型的“脂质沉积”病果蝇被选中是因为它的吸引力 作为一种新的神经退行性疾病的分子遗传模型， 作为突触研究的最重要的遗传模型。PD和NP-C是 选择作为大量相关神经退行性疾病的代表 紊乱果蝇PD模型最近通过以下方法建立 人α-突触核蛋白（突触前蛋白）的转基因过表达， 显示准确地概括了人类PD的诊断特征。一 果蝇NP-C基因突变模型的建立 内源性NPC I基因（人类NP-C的已知原因）的（功能丧失） 疾病 具体地说，这项建议是进行突触的年龄进展研究， 果蝇PD和NP-C模型中与突触维持相关的机制 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的 其水平可可逆调节的α-突触核蛋白和NPCI蛋白 通过一种依赖于温度的泛素化策略。第二，确认 在这些模型中用行为测定法观察神经变性的大体特征， 检查神经系统/神经元结构。第三，也是最 重要是，为了测定这些细胞中突触发育、功能和维持， 模型试验将包括电生理学测量， 神经传递，蛋白质的定量荧光光学成像， 突触前终末脂质动力学和超微结构研究 突触前结构总之，这些研究将使一个结论性的 确定PD和NP-C中突触维持是否受损， 并且是导致神经元细胞死亡的致病因子， 神经退行性疾病。