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.

儿童发作肌张力障碍是一种常染色体显性运动障碍，它与位于染色体9染色体的Dyt1基因中的三个碱基对（GAG）缺失有关。Dyt1基因编码332个称为Torsina的332氨基酸残基的ATP结合蛋白（Zelelius et and，1997年）。 我们最近证明，龙田确实与ATP结合，并在大脑和其他外周组织中表达（Shashidharan等，2000a）。在大脑中，Torsina表达仅限于神经元，如原位杂交所示（Augood等，1999）和免疫组织化学技术（Shashidharan等，2000a; Walker等，2001a）。 Torsina的细胞功能和其在肌张力障碍中的作用都不是已知的。 为了更好地理解Torsina的细胞功能及其在儿童期肌肌张力障碍中的作用，我们有：a）发达的转基因小鼠表达人突变体（Deltae）-torsina，b）产生的PC12克隆细胞系表达正常和（deltae），（deltoe） - torsina，c） Deltae-torsina cDNA，e）与GST和GFP产生的融合蛋白，f）从患有儿童发作的肌张力障碍患者的患者中获得了死后脑组织，G）建立了一项程序，通过从患有肌张力肌的患者的快速尸检获得了一项程序，以获取后大脑。 配备了这些，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理和细胞功能障碍，并开发用于治疗干预的合理方法。 在这项赠款建议中，我们将执行以下特定目的：目标1：通过生化，分子和免疫组织化学技术和行为研究来表征表达人躯干的Dyt1转基因小鼠。 我们的初步研究表明，表达三角洲龙骨的35％的转基因小鼠会发展出异常的超动运动功能。 症状在小鼠生命的3-10周之间发展。 有趣的是，转基因小鼠表现出的发病和外渗水平的年龄与人类疾病的年龄相当。 人类的发病年龄在5-12岁之间，外渗水平为30-40％。 目的2：研究Torsina的生化特性。 我们利用共免疫沉淀技术的初步研究表明，Torsina可能与其他蛋白质相互作用。 这些相互作用的蛋白是否对于田氏的生物学功能是必需的，尚待进一步阐明。 目标3：隔离和鉴定Torsina的蛋白质伴侣。 Torsina属于AAA+家族，AAA+家族是一类与复杂组装，操作和拆卸细胞蛋白有关的蛋白质。 Torsina的羧基末端区域（在Dyt1 dystonia中经历了点突变）被认为参与了寡聚环结构的形成或与其他蛋白质的相互作用。 我们在特定目标2中描述的最初研究将为我们提供有关与Torsina相互作用蛋白质存在的见解，这将通过酵母两杂交系统进一步研究。