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退行性疾病中起核心作用，如阿尔茨海默氏症，肌萎缩性侧索硬化症（ALS），亨廷顿氏症，帕金森病和传染性海基状脑病，如克雅氏病。在ALS中，铜锌超氧化物歧化酶（SOD1）的错误折叠结构状态赋予这种通常有益的蛋白质一种功能，这对疾病病理生理至关重要。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。大多数研究提出淀粉样SOD1蛋白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蛋白形成了“有毒离子通道”，进而造成离子失衡（如钙超载），从而导致神经毒性。目的1：验证突变型SOD1蛋白，或者在某些条件下，野生型SOD1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SOD1的超微结构在“毒性通道”中发生构象变化的假设。目的3：验证纳米级分子间力和寡聚态控制突变型和野生型SOD1分子在脂质膜中的插入和稳定的假设。目的4：验证突变型SOD1和野生型SOD1在一定条件下使脂质膜被某些离子渗透的假设。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将“有毒通道”共定位到细胞膜上，并生成针对“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。并且（Aim 4）使用单通道电生理记录来测量和调制通过“有毒通道”的离子流动，以了解通道的特性和特征。今天，数以千万计的人遭受神经退行性疾病（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。我们提出了一种新的模型，描述了某些形式的ND是由“有毒离子通道”侵入宿主神经元细胞膜并引起疾病病理生理的结果。由于这是一种新模式，“有毒渠道”模型尚未获得广泛接受或大量研究资金。然而，在本应用中提出了新的和令人信服的实验证据（关于ALS），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“毒性通道”的蛋白质错误折叠疾病寻找新的有效治疗方法。有了成功的资助，我们相信继续我们的工作将导致开发新的基于抗体的诊断方法，以及有效的ALS新治疗方法。

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."