BRAIN TORSIN-A AND CHILDHOOD ONSET DYSTONIA

脑扭转 A 型和儿童期发作的肌张力障碍

• 批准号: 6478533
• 负责人: PULLANIPALLY SHASHIDHARAN
• 金额: $ 28.18万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478533
• 关键词: PC12 cells; adenosine triphosphate; age difference; behavior test; binding proteins; brain; chimeric proteins; disease /disorder model; dystonia; genetically modified animals; green fluorescent proteins; human tissue; immunocytochemistry; immunoprecipitation; laboratory mouse; molecular pathology; monoclonal antibody; point mutation; protein localization; protein protein interaction; yeast two hybrid system

Childhood onset dystonia is an autosomal dominant movement disorder that has been linked to a three base pair (GAG) deletion in the DYT1 gene localized to chromosome 9. The DYT1 gene encodes a novel ATP binding protein of 332 amino acid residues called torsinA (Ozelius et al, 1997). We have recently demonstrated that torsinA indeed binds to ATP and is expressed in brain and other peripheral tissues (Shashidharan et al, 2000a). In the brain, torsinA expression is restricted to neurons as shown by in situ hybridization (Augood et al, 1999) and immunohistochemical techniques (Shashidharan et al, 2000a; Walker et al., 2001a). Neither the cellular function of torsinA nor its role in dystonia is known. In order to better understand the cellular function of torsinA and its role in childhood-onset dystonia, we have: a) developed transgenic mice expressing human mutant (deltaE)-torsinA, b) generated PC12 clonal cell lines expressing normal and (deltaE)-torsinA, c) developed and characterized polyclonal and monoclonal antibodies to human torsinA d) cloned and characterized normal and deltaE-torsinA cDNAs, e) generated fusion proteins with GST and GFP, f) obtained postmortem brain tissue from a patient with childhood-onset dystonia, g) established a program to obtain postmortem brains by rapid autopsy from patients with dystonia. Equipped with these we have begun investigations into the cellular function of torsinA and its role in childhood onset dystonia. Our long-term objective is to better understand the pathogenesis and cellular dysfunction in DYT1 dystonia and develop rational methods for therapeutic intervention. In this grant proposal we will carry out the following specific aims: AIM 1: To characterize DYT1 transgenic mice expressing human torsinA by biochemical, molecular and immunohistochemical techniques and behavioral studies. Our preliminary studies revealed that 35 percent of transgenic mice expressing deltaE-torsinA develop abnormal hyperkinetic motor function. The symptoms develop between 3-10 weeks of life in mice. Interestingly the age of onset and penetrance level exhibited by transgenic mice is comparable to that of the human disorder. The age of onset in humans is between ages 5-12 and the penetrance level is 30-40 percent. AIM 2: To investigate the biochemical properties of torsinA. Our preliminary studies utilizing co-immunoprecipitation techniques revealed that torsinA may interact with other proteins. Whether these interacting proteins are necessary for the biological function of torsinA remains to be further elucidated. AIM 3: To isolate and identify protein partners of torsinA. TorsinA belongs to AAA+ family, a class of proteins associated with complex assembly, operation and disassembly of cellular proteins. The carboxy terminal region of torsinA, that undergoes a point mutation in DYT1 dystonia, is thought to be involved in either formation of an oligomeric ring structure or in an interaction with other proteins. Our initial studies described in Specific aim 2 will provide us with insights into the presence of interacting proteins with torsinA, which will be further investigated by yeast two-hybrid system.

儿童期发病的肌张力障碍是一种常染色体显性运动障碍，与位于 9 号染色体的 DYT1 基因中的三碱基对 (GAG) 缺失有关。DYT1 基因编码一种新型 ATP 结合蛋白，由 332 个氨基酸残基组成，称为 torsinA（Ozelius 等人）等人，1997）。 我们最近证明torsinA确实与ATP结合并在大脑和其他外周组织中表达（Shashidharan等人，2000a）。在大脑中，torsinA 表达仅限于神经元，如原位杂交（Augood 等人，1999）和免疫组织化学技术（Shashidharan 等人，2000a；Walker 等人，2001a）所示。 TorsinA 的细胞功能及其在肌张力障碍中的作用尚不清楚。 为了更好地了解 torsinA 的细胞功能及其在儿童期肌张力障碍中的作用，我们：a) 开发了表达人类突变体 (deltaE)-torsinA 的转基因小鼠，b) 生成了表达正常和 (deltaE)-torsinA 的 PC12 克隆细胞系torsinA，c) 开发并表征了人torsinA 的多克隆和单克隆抗体 d) 克隆并表征了正常和 deltaE-torsinA cDNA，e) 生成了融合蛋白GST 和 GFP，f）从患有儿童期肌张力障碍的患者身上获得死后脑组织，g）建立了一个通过快速尸检从肌张力障碍患者身上获得死后大脑的计划。 有了这些，我们已经开始研究 torsinA 的细胞功能及其在儿童期肌张力障碍中的作用。 我们的长期目标是更好地了解 DYT1 肌张力障碍的发病机制和细胞功能障碍，并开发合理的治疗干预方法。 在这项拨款提案中，我们将实现以下具体目标： 目标 1：通过生化、分子和免疫组织化学技术以及行为研究来表征表达人 torsinA 的 DYT1 转基因小鼠。 我们的初步研究表明，35% 表达 deltaE-torsinA 的转基因小鼠出现异常的多动运动功能。 这些症状在小鼠出生后 3-10 周内出现。 有趣的是，转基因小鼠表现出的发病年龄和外显率水平与人类疾病相当。 人类发病年龄为 5-12 岁，外显率为 30-40%。 目的2：研究torsinA的生化特性。 我们利用免疫共沉淀技术进行的初步研究表明，torsinA 可能与其他蛋白质相互作用。 这些相互作用的蛋白对于torsinA的生物学功能是否是必需的仍有待进一步阐明。 目标 3：分离并鉴定 torsinA 的蛋白伴侣。 TorsinA 属于 AAA+ 家族，一类与细胞蛋白质的复杂组装、操作和分解相关的蛋白质。 TorsinA 的羧基末端区域在 DYT1 肌张力障碍中发生点突变，被认为参与寡聚环结构的形成或与其他蛋白质的相互作用。 具体目标 2 中描述的初步研究将使我们深入了解与 torsinA 相互作用的蛋白质的存在，这将通过酵母双杂交系统进行进一步研究。