The Role of Toxic Ion Channels in ALS Pathogenesis

描述(申请人提供)：蛋白质错误折叠在许多神经退行性疾病如阿尔茨海默氏症、肌萎缩侧索硬化症(ALS)、亨廷顿病、帕金森病和传染性海绵状脑病(如克雅氏病)中起重要作用。在肌萎缩侧索硬化症中，铜锌超氧化物歧化酶(SOD1)的错误折叠结构状态赋予了这种正常有益的蛋白功能，这对疾病的病理生理是必不可少的。然而，尽管多年的紧张研究，这种后天功能的确切性质和机制(S)仍然是未知的。大多数研究表明，类淀粉样蛋白SOD1聚集体的沉积直接影响神经功能。根据我们的初步研究，我们的总体假设是，这些错误折叠的SOD1蛋白形成“有毒离子通道”，进而产生离子失衡(如钙超载)，导致神经毒性。目的：验证突变的、可能在一定条件下，野生型SOD1蛋白共定位于亚细胞脂质膜域，特别是线粒体膜外膜的细胞质表面的假设。目的：验证突变型SOD1超微结构在“毒性通道”中发生构象变化的假说。目的：验证纳米尺度的分子间力和低聚态控制突变型或野生型SOD1分子在脂膜中插入和稳定的假说。目的4：验证突变型SOD1和可能的野生型SOD1在一定条件下使脂膜对某些离子具有通透性的假设。我们的研究设计和方法是：(目标1)使用TIRF、FRET和免疫荧光显微镜技术共同定位“有毒通道”到细胞膜，并产生针对“有毒通道”的合成抗体。(目标2)使用高分辨率、流体原子力显微镜(FAFM)和(目标3)fAFM力测量来确定亚基结构、构象变化和稳定“有毒通道”的分子间力。和(目标4)使用单通道电生理学记录来测量和调节流过“有毒通道”的离子流量，以了解通道的属性和特征。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人来说，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是由侵入宿主神经元细胞膜并引起疾病病理生理的“有毒离子通道”引起的。由于它是新的，“有毒通道”模型还没有得到广泛的接受，也没有得到大量的研究资金。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)，它支持“有毒通道”模型。如果我们的模型是正确的，拟议的工作将把我们带到正确的轨道上，找到新的有效治疗ND和其他涉及“有毒通道”的蛋白质错误折叠疾病的方法。有了成功的资金，我们相信继续我们的工作将导致新的基于抗体的诊断方法的发展，以及有效的ALS新的治疗方法。 与公共卫生相关：今天，数千万人患有神经退行性疾病(ND)的衰弱和致命症状，这些疾病可能涉及“有毒渠道”，如阿尔茨海默氏症、肌萎缩侧索硬化症(ALS)、亨廷顿氏症、帕金森氏症，以及可传播的海绵状脑病，如克雅氏病。对于这些人中的绝大多数人来说，目前还没有有效的治疗方法。这项拟议的工作将使我们走上正确的道路，找到新的有效治疗新城疫和其他涉及“有毒通道”的蛋白质错误折叠疾病的方法。

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."