CELL BIOLOGY OF ALPHA-SYNUCLEIN IN PARKINSON'S DISEASE

帕金森病中α-突触核蛋白的细胞生物学

• 批准号: 6393174
• 负责人: MICHAEL GEBHARD SCHLOSSMACHER
• 金额: $ 11.77万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6393174
• 关键词: 6 hydroxydopamine; Lewy body; Parkinson's disease; alpha synuclein; brain cell; disease /disorder model; dopamine; family genetics; gene mutation; genetically modified animals; laboratory mouse; mesencephalon; neural degeneration; neuropathology; protein isoforms; tissue /cell culture; transfection

DESCRIPTION (Adapted from the applicant's abstract): PD is an age-related, progressive movement disorder characterized by resting tremor, hypokinesia, rigidity and clinical response to dopaminergic therapy. Neuropathologically, PD is defined by the relatively selective loss of dopaminergic neurons in the midbrain and the presence of intraneuronal inclusions known as Lewy bodies. There are several known etiologies of the Parkinsonism syndrome which include infectious, toxic, and other neurodegenerative disorders and "idiopathic" PD. Focusing on knowledge derived from a rare form of familial PD, the investigator will examine how two recently identified mutations in the alpha-synuclein (alphaS) gene lead to this neurologic condition. The central hypothesis of this research training application is that heterozygous mutations of alphaS initiate the selective degeneration of predominantly dopaminergic neurons in the midbrain of affected patients. These genotypes represent the first clue to the molecular pathogenesis of neuronal dysfunction and cell death in PD. Using biochemical and cell and molecular biological techniques, the investigator plans to study the normal and abnormal function of alphaS in cell culture and examine its effects in vivo as well. The investigator will pursue 4 specific aims, proposing: (1) to characterize the constitutive expression of alphaS in mammalian cells in comparison with human and rodent brain; to that end, the investigator will first express alphaS protein in bacteria and generate high-affinity antibodies to synthetic and bacterially expressed proteins; (2) to systematically explore suitable neural cell models of alphaS overexpression in both human and rodent dopaminergic cell cultures of mesencephalic and non-mesencephalic origin and in rodent oligodendrocytes and will investigate the effects of wild type and mutant human alphaS gene expression in primary neurons and midbrain parenchyma of transgenic mice; (3) to localize alphaS in transfected and primary cells morphologically in order to help determine its function and to search immunocytochemically and biochemically for evidence of insoluble or oligomerized isoforms; and (4) to analyze the phenotypic consequences of mutant alphaS on the metabolism of dopaminergic cell markers (e.g., TH, VMAT-2) and to subject such cells to known mesencephalic neuronal toxins (e.g., MPP+, 6-OH-dopamine). Through these 4 specific aims, the investigator seeks to create dynamic and manipulatable cellular models for alphaS mutation-related dopaminergic neuronal dysfunction. Such cell models should provide important pathogenetic information and should ultimately be useful in screening potential therapeutic compounds.

描述（改编自申请人摘要）：PD是一种与年龄相关的进行性运动障碍，其特征为静息性震颤、运动障碍、僵硬和对多巴胺能治疗的临床反应。神经病理学上，PD被定义为中脑多巴胺能神经元的相对选择性丧失和被称为路易体的神经元内包涵体的存在。帕金森综合征有几种已知的病因，包括感染性、毒性和其他神经退行性疾病和“特发性”帕金森病。聚焦于一种罕见的家族性帕金森病的知识，研究者将研究最近发现的α -突触核蛋白（α has）基因的两种突变是如何导致这种神经系统疾病的。本研究训练应用的中心假设是α α α的杂合突变启动了受影响患者中脑主要多巴胺能神经元的选择性变性。这些基因型代表了PD中神经元功能障碍和细胞死亡的分子发病机制的第一条线索。利用生物化学和细胞分子生物学技术，研究α has在细胞培养中的正常和异常功能，以及在体内的作用。研究者将追求4个具体目标，提出：(1)将α has在哺乳动物细胞中的组成表达与人类和啮齿动物的大脑进行比较；为此，研究者将首先在细菌中表达alpha has蛋白，并生成针对合成蛋白和细菌表达蛋白的高亲和力抗体；(2)系统探索人类和啮齿动物中脑和非中脑来源的多巴胺能细胞培养物以及啮齿动物少突胶质细胞中α - has过表达的合适神经细胞模型，并研究野生型和突变型人类α - has基因在转基因小鼠原代神经元和中脑组织中的表达影响；(3)在转染细胞和原代细胞中进行α - has的形态学定位，以帮助确定其功能，并在免疫细胞化学和生物化学上寻找不溶性或寡聚的亚型的证据；(4)分析突变型α - has对多巴胺能细胞标记物（如TH、VMAT-2）代谢的表型影响，并将这些细胞置于已知的中脑神经元毒素（如MPP+、6-羟基多巴胺）下。通过这4个特定的目标，研究者试图创建动态和可操作的细胞模型，用于α has突变相关的多巴胺能神经元功能障碍。这种细胞模型应该提供重要的发病信息，并最终用于筛选潜在的治疗化合物。