Characterization of epsilon-sarcoglycan interacting proteins in mouse brain

描述（由申请人提供）：肌阵挛是指肌肉或肌肉群突然、不自主的抽搐。肌张力障碍被定义为持续肌肉收缩的综合征，经常导致扭曲和重复运动，或异常姿势。肌张力障碍通常被认为是一种中枢神经系统疾病。遗传性肌阵挛-肌张力障碍（M-D），以前被称为遗传性原发性肌阵挛或遗传性（酒精反应性）肌阵挛性肌张力障碍，是一种常染色体显性遗传的肌张力障碍，具有不完全的肌张力。Zimprich及其同事首先在编码？的基因中发现了功能缺失突变。肌聚糖（？- SG;基因名称：人中的SGCE和小鼠中的Sgce）。SGCE在胚胎发育和成人中广泛表达。SGCE在几乎所有测试的大脑区域中表达。我们建立了DYT11肌张力障碍的Sgce敲除模型。我们发现，Sgce基因敲除小鼠表现出肌阵挛、肌张力障碍的运动缺陷、焦虑、抑郁以及多巴胺能和多巴胺能系统的变化。此外，我们还开发了一种特异于？SG.虽然基因敲除小鼠复制了大多数DYT11症状，但DYT11的功能仍然存在。SG及其突变形式在引起M-D中的作用在很大程度上是未知的。具体来说，在分子水平上，无论？SG在中央突触中形成复合体，如果是，则该复合体的性质尚不清楚。这些未知因素阻碍了充分理解M-D发病机制的努力，从而阻碍了为患者制定有效的治疗策略。进一步的详细分析？SG综合体应提供一个独特的机会，以澄清？SG和SGCE突变的病理生理作用。详细了解DYT11肌张力障碍的病理生理学也将加速药物发现过程，以开发肌阵挛-肌张力障碍和相关肌张力障碍和肌阵挛疾病的治疗。 我们研究的广泛的长期目标是使用转基因小鼠来确定：1）？SG在体内2）5-SG蛋白的丢失如何导致M-D。本申请的目的是进一步表征？- SG蛋白复合物使用Sgce敲除小鼠和我们已经制造的单克隆抗体，将使我们能够回答这些问题。我们假设？- SG与其他蛋白质相互作用并在突触处发挥作用。鉴定这些相互作用的蛋白质可以阐明？突触传递中的SG。我们进一步假设？- SG与其他蛋白质相互作用，SG导致突触处复合物的减少。复合物的减少导致回路水平的突触传递和可塑性改变，以及系统水平的肌阵挛、指示肌张力障碍的运动缺陷或两者。这项研究的基本原理是，一旦确定SGCE在导致大脑运动控制功能障碍中的作用，就可以开发纠正M-D的可能干预措施。 我们特别准备好进行拟议的研究，因为我们已经创建了一个模拟DYT11患者的Sgce突变小鼠系。此外，我们还开发了其他遗传和抗体工具，可以严格检验上述假设。我们的另一个优势是我们能够使用的多学科方法，包括分子，遗传，解剖，生物化学，神经生理和行为技术。这项工作将在一个研究环境中进行，许多NIH资助的研究人员和共享NIH资助的核心资源，重点是使用神经系统疾病的动物模型。我们计划用以下具体目标来检验我们的假设：具体目标1：为了鉴定相互作用的蛋白质，我们将从WT和Sgce KO小鼠脑中制备突触体组分。免疫沉淀的？- SG复合物将使用针对？的特异性单克隆抗体进行。SG，然后通过凝胶电泳分离这些蛋白质。仅在WT小鼠脑样品中出现的条带应为？- SG与Sgce KO小鼠脑组织样品中出现的相互作用蛋白条带应为免疫沉淀实验中非特异性沉淀的假阳性条带。将分离蛋白条带并通过质谱法进行测序。 具体目标2：分析损失？SG对相互作用蛋白的表达水平的影响，我们将从WT和Sgce KO小鼠脑制备突触体组分，并通过Western印迹用针对所鉴定蛋白的抗体分析相互作用蛋白的水平。 具体目标3：分析损失？SG对突触传递和可塑性的影响，我们将测定海马CA1区Schaffer侧支通路的输入输出关系、成对脉冲比和长时程增强。 上述特定目标的成功完成将产生一个候选突触蛋白的列表，这些蛋白与？SG和损失的影响？- SG对其表达水平和突触传递的影响。结果应该有助于我们确定？- SG在体内以及Sgce突变如何导致M-D。这些结果将显著增加我们对M-D病理生理学的理解，这最终有助于M-D患者和其他肌张力障碍和肌阵挛患者的治疗方法的发展。

DESCRIPTION (provided by applicant): Myoclonus refers to sudden, involuntary jerking of a muscle or group of muscles. Dystonia is defined as a syndrome of sustained muscle contractions, frequently causing twisting and repetitive movements, or abnormal postures. Dystonia is generally believed to be a disorder of the central nervous system. Inherited myoclonus- dystonia (M-D), previously referred to as hereditary essential myoclonus or hereditary (alcohol-responsive) myoclonic dystonia, is an autosomal dominant dystonia with incomplete penetrance. Zimprich and colleagues first identified loss-of-function mutations in a gene coding for ?-sarcoglycan (?-SG; gene name: SGCE in human and Sgce in mouse). SGCE is widely expressed in embryonic development and in adults. SGCE is expressed in almost all brain regions tested. We have established a Sgce knockout model of DYT11 dystonia. We found that the Sgce knockout mice showed myoclonus, motor deficits indicative of dystonia, anxiety, depression, and changes in the dopaminergic and serotonergic systems. Furthermore, we have developed a monoclonal antibody specific for ?-SG. While the knockout mice replicated most of the DYT11 symptoms, the function of ?-SG and the role of its mutated forms in causing M-D are largely unknown. Specifically, at molecular level, whether ?-SG forms a complex in the central synapse, if yes, the nature of this complex is not known. These unknowns hamper efforts to adequately understand the pathogenesis of M-D, thus preventing the development of effective therapeutic strategies for patients. Further detailed analysis of the ?-SG complex should provide a unique opportunity for clarifying the functional role of ?-SG and the pathophysiological roles of SGCE mutations. Detailed understanding of pathophysiology of DYT11 dystonia would also accelerate the drug discovery process to develop treatment for myoclonus-dystonia and related dystonia and myoclonus disorders. The broad, long-term objective of our research is to use transgenic mice to determine: 1) the functional role of ?-SG in vivo 2) how the loss of 5-SG protein leads to M-D. The objective of this application is to further characterize ?-SG protein complex using the Sgce knockout mice and monoclonal antibody that we have already made that will enable us to answer these questions. We hypothesize that ?-SG interacts with other proteins and functions at the synapse. Identification of these interacting proteins may elucidate the function of ?-SG in the synaptic transmission. We further hypothesize that ?-SG interacts with other proteins and loss of ?-SG leads to the reduction of the complex at the synapse. The reduction of the complex leads to altered synaptic transmission and plasticity at circuit level, and myoclonus, motor deficits indicative of dystonia, or both, at system level. The rationale for the proposed research is that once the roles of SGCE in causing dysfunction of movement control in the brain are determined, possible interventions to correct M-D can be developed. We are particularly well prepared to undertake the proposed research because we have created a line of Sgce mutant mice that mimic the DYT11 patients. Furthermore, we have developed other genetic and antibody tools that can critically test the above hypothesis. Our other strength is the multidisciplinary approaches we are able to use, which include molecular, genetic, anatomical, biochemical, neurophysiologic, and behavioral techniques. The work will be conducted in a research environment with many NIH-funded investigators and shared NIH-funded core resources that are focused on using animal models of neurological disorders. We plan to test our hypothesis with the following Specific Aims: Specific Aim 1: To identify the interacting proteins, we will prepare the synaptosomal fractions from WT and Sgce KO mouse brains. Immunoprecipitation of the ?-SG complex will be performed using specific monoclonal antibody against ?-SG followed by separation of these proteins by gel electrophoresis. The bands only appeared in the WT mouse brain samples should be ?-SG and the interacting proteins and the bands appears in the Sgce KO mouse brain samples should be false positive bands that non-specifically precipitated in the immunoprecipitation experiment. Protein bands will be isolated and sequenced by Mass Spectrometry. Specific Aim 2: To analyze the effect of loss of ?-SG on the expression levels of the interacting proteins, we will prepare synaptosomal fractions from WT and Sgce KO mouse brains and analyze the levels of the interacting proteins by Western blot with antibodies against the identified proteins. Specific Aim 3: To analyze the effect of loss of ?-SG on the synaptic transmission and plasticity, we will determine input output relationship, paired pulse ratio, and long-term potentiation of hippocampal CA1 Schaffer collateral pathway. The successful completion of the above Specific Aims will produce a list of candidate synaptic proteins that interact with ?-SG and the effects of loss of ?-SG on their expression levels and synaptic transmission. The results should help us to determine the function of ?-SG in vivo and how the mutation of Sgce causes M-D. The results should significantly increase our understanding of the pathophysiology of M-D, which can ultimately aid the development of therapeutic treatments for M-D patients and other dystonia and myoclonus patients.