The Role of Toxic Ion Channels in ALS Pathogenesis

描述（由申请人提供）：已知蛋白质错误折叠在许多神经变性疾病中起中心作用，所述神经变性疾病例如阿尔茨海默氏病、肌萎缩侧索硬化（ALS）、亨廷顿氏病、帕金森氏病和传染性海绵状脑病例如克雅氏病。在ALS中，铜-锌超氧化物歧化酶（SOD 1）的错误折叠结构状态赋予这种通常有益的蛋白质一种对疾病病理生理学至关重要的功能。然而，尽管经过多年的深入研究，这种获得性功能的确切性质和机制仍然难以捉摸。大多数研究认为，淀粉样SOD 1蛋白聚集体的沉积直接影响神经功能。基于我们的初步研究，我们的总体假设是，这些错误折叠的SOD 1蛋白形成“有毒离子通道”，这反过来又会造成离子失衡（如钙超载），导致神经毒性。目标1：为了检验突变体的假设，也许在某些条件下，野生型SOD 1蛋白共定位于亚细胞脂质膜结构域，特别是线粒体外膜的细胞质面。目的2：验证突变型和野生型SOD 1的超微结构在“毒性通道”中发生构象变化的假设。“目标3：为了验证纳米级分子间力和寡聚状态控制突变体和可能的野生型SOD 1分子在脂质膜中的插入和稳定的假设。目标4：为了检验突变型SOD 1和野生型SOD 1在某些条件下使脂质膜可被某些离子渗透的假设。我们的研究设计和方法是：（目的1）利用TIRF、FRET和免疫荧光显微镜技术将“毒性通道”共定位于细胞膜，并合成抗“毒性通道”的抗体。（目标2）使用高分辨率，流体原子力显微镜（fAFM）和（目标3）fAFM力测量，以确定亚基结构，构象变化和分子间的力量，稳定“有毒通道。”目标4：使用单通道电生理学记录来测量和调节通过“毒性通道”的离子流，以了解通道特性和特征。今天，数以千万计的人患有神经退行性疾病（ND）的衰弱和致命症状。对于这些人中的绝大多数，目前还没有有效的治疗方法。我们提出了一个新的模型，描述了某些形式的ND，由于“有毒离子通道”侵入宿主神经元细胞膜，并引起疾病的病理生理。由于是新的，“有毒通道”模型还没有得到广泛的接受或大量的研究资金。然而，在本申请中提出的是新的和令人信服的实验证据（关于ALS），其支持“毒性通道”模型。如果我们的模型是正确的，那么这项工作将使我们走上正确的道路，为ND和其他涉及“有毒通道”的蛋白质错误折叠疾病找到新的有效治疗方法。“随着资金的成功，我们相信我们工作的继续将导致新的基于抗体的诊断方法的开发，以及针对ALS的有效的新治疗方法的开发。 公共卫生相关性：今天，数以千万计的个体遭受潜在地涉及“毒性通道”的神经变性疾病（ND）的衰弱和致命症状，所述神经变性疾病例如阿尔茨海默氏病、肌萎缩侧索硬化症（ALS）、亨廷顿氏病、帕金森氏病和传染性海绵状脑病例如克雅氏病。对于这些人中的绝大多数，目前还没有有效的治疗方法。这项工作将使我们走上正确的道路，为ND和其他涉及“有毒通道”的蛋白质错误折叠疾病找到新的有效治疗方法。"

DESCRIPTION (provided by applicant): Protein misfolding is known to play a central role in numerous neurodegenerative diseases such as Alzheimer's, amyotrophic lateral sclerosis (ALS), Huntington's, Parkinson's and transmissible spongiform encephalopathies such as Creutztfeld-Jakob's disease. In ALS, the misfolded structural state of copper-zinc super-oxide dismutase (SOD1) imparts a function to this normally beneficial protein that is essential to disease pathophysiology. However, the exact nature and mechanism(s) of this acquired function have remained elusive despite years of intense research. Most research has proposed that the deposition of aggregates of amyloid-like SOD1 protein directly affects neurological function. Based on our preliminary studies, our overall hypothesis is that these misfolded SOD1 proteins form "toxic ion channels," which in turn create ionic imbalances (such as calcium overload) that result in neurotoxicity. Aim1: To test the hypothesis that a mutant and perhaps, under certain conditions, wild-type SOD1 protein co-localize to subcellular lipid membrane domains especially the cytoplasmic face of the outer mitochondrial membrane. Aim2: To test the hypothesis that the ultrastructure of mutant and perhaps wild-type SOD1 undergoes conformational changes in "toxic channels." Aim 3: To test the hypothesis that nano-scale intermolecular forces and oligomeric states govern the insertion and stabilization of mutant and perhaps wild-type SOD1 molecules in lipid membrane. Aim 4: To test the hypothesis that mutant SOD1 and perhaps wild-type SOD1, under certain conditions, render the lipid membrane permeable by certain ions. Our research design and methods are to: (Aim 1) Use TIRF, FRET and immuno-fluorescence microscopy techniques to co-localize the "toxic channels" to cellular membranes and also generate a synthetic antibody against "toxic channels." (Aim 2) Use high resolution, fluid atomic force microscopy (fAFM) and (Aim 3) fAFM force measurements to determine the subunit structure, conformational changes and the intermolecular forces that stabilize "toxic channels." And (Aim 4) uses single-channel electrophysiology recordings to measure and modulate ion flow through the "toxic channels" in order to understand channel properties and characteristics. Today tens of millions of individuals suffer the debilitating and fatal symptoms of neurodegenerative diseases (ND). For the vast majority of these individuals, no effective treatment exists currently. We have proposed a new model describing certain forms of ND as resulting from "toxic ion channels" which invade host neuronal cell membranes and give rise to disease pathophysiology. Since it is new, the "toxic channel" model has not yet gained wide acceptance or significant research funding. However, presented in this application is new and compelling experimental evidence (regarding ALS) which supports the "toxic channel" model. If our model is correct, the proposed work will be putting us on the right track to finding new effective treatments for ND and other protein misfolding diseases involving "toxic channels." With successful funding we are convinced that continuation of our work will lead to the development of new antibody-based diagnostics, as well as effective new therapeutic treatments for ALS. PUBLIC HEALTH RELEVANCE: Today tens of millions of individuals suffer the debilitating and fatal symptoms of neurodegenerative diseases (ND) potentially involving "toxic channels," such as Alzheimer's, amyotrophic lateral sclerosis (ALS), Huntington's, Parkinson's, and transmissible spongiform encephalopathies such as Creutzfeldt-Jakob diseases. For the vast majority of these individuals, no effective treatment exists currently. The proposed work will be putting us on the right track to finding new effective treatments for ND and other protein misfolding diseases involving "toxic channels."