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一种持续肌肉收缩的综合征，经常引起扭曲和重复运动，或异常姿势。肌张力障碍通常被认为是一种中枢神经系统紊乱。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，是一种不完全外显的常染色体显性肌张力障碍。Zimprich和他的同事们首先在编码？-肌聚糖（?-SG，基因名称：人SGCE，小鼠SGCE）。SGCE在胚胎发育和成人中广泛表达。SGCE几乎在所有测试的大脑区域表达。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萎缩、运动障碍、焦虑、抑郁以及多巴胺能和血清素能系统的变化。此外，我们还开发了一种针对？-SG的单克隆抗体。虽然基因敲除小鼠复制了DYT11的大部分症状，但？-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，是否？-SG在中枢突触形成复合体，如果是，这种复合体的性质尚不清楚。这些未知因素阻碍了充分了解M-D发病机制的努力，从而阻碍了对患者有效治疗策略的发展。进一步详细分析？-SG复合体应该提供一个独特的机会来阐明？-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机制，也将加速药物发现进程，开发肌阵挛性肌张力障碍及相关肌张力障碍和肌阵挛疾病的治疗方法。我们研究的广泛的、长期的目标是使用转基因小鼠来确定：1)？2) 5-SG蛋白的丢失如何导致M-D。此应用程序的目的是进一步描述？-SG蛋白复合物使用Sgce敲除小鼠和我们已经制作的单克隆抗体这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中起作用。这些相互作用蛋白的鉴定可能会阐明？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，损失？-SG导致突触复合体的减少。复合体的减少在回路水平上导致突触传递和可塑性的改变，在系统水平上导致肌阵挛，运动障碍，或两者兼而有之。这项研究的基本原理是，一旦确定了SGCE在导致大脑运动控制功能障碍中的作用，就可以开发出纠正M-D的可能干预措施。我们特别准备进行拟议的研究，因为我们已经创造了一个模仿DYT11患者的Sgce突变小鼠系列。此外，我们已经开发了其他基因和抗体工具，可以严格测试上述假设。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，生化，神经生理学和行为技术。这项工作将在一个研究环境中进行，有许多美国国立卫生研究院资助的研究人员，并共享美国国立卫生研究院资助的核心资源，这些资源集中在使用神经疾病的动物模型上。我们计划通过以下具体目的来验证我们的假设：具体目的1：为了鉴定相互作用的蛋白质，我们将从WT和Sgce KO小鼠大脑中制备突触体组分。？-SG复合物将使用针对？-SG，然后用凝胶电泳分离这些蛋白质。只出现在WT小鼠脑样本中的条带应该是？-SG及其相互作用蛋白和Sgce KO小鼠脑样品中出现的条带应该是免疫沉淀实验中非特异性沉淀的假阳性条带。蛋白质条带将被分离并通过质谱测序。具体目标2：分析？-SG对相互作用蛋白表达水平的影响，我们将制备WT和Sgce KO小鼠大脑的突触体组分，并通过Western blot对鉴定的蛋白进行抗体分析相互作用蛋白的水平。具体目标3：分析？-SG对突触传递和可塑性的影响，我们将确定海马CA1 Schaffer侧通路的输入输出关系、配对脉冲比和长期增强。上述特异性目标的成功完成将产生一系列候选突触蛋白，这些候选突触蛋白与？-SG和损失的影响？-SG对其表达水平和突触传递的影响。结果应该可以帮助我们确定？-SG在体内以及Sgce突变如何导致M-D。该结果将显著增加我们对M-D的病理生理学的理解，最终有助于M-D患者以及其他肌张力障碍和肌阵挛患者的治疗方法的开发。

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.