The Role of Toxic Ion Channels in ALS Pathogenesis

项目总结/摘要 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， 阿尔茨海默氏症、肌萎缩性侧索硬化症（ALS）、亨廷顿氏症、帕金森氏症和传染性海绵状 克雅氏病等脑病。在ALS中，铜锌的错误折叠结构状态 超氧化物歧化酶（SOD 1）赋予这种通常有益的蛋白质一种对疾病至关重要的功能 病理生理学然而，这种获得性功能的确切性质和机制仍然难以捉摸 尽管经过多年的深入研究大多数研究认为，淀粉样蛋白聚集体的沉积- SOD 1蛋白直接影响神经功能。根据我们的初步研究， 一种假说是，这些错误折叠的SOD 1蛋白形成“有毒离子通道”，其反过来产生离子通道。 失衡（如钙超载）导致神经毒性。 目的1：为了验证突变体和野生型SOD 1蛋白在一定条件下共同作用的假设， 定位于亚细胞脂质膜结构域，特别是线粒体外膜的胞质面 膜的目的2：验证突变型和野生型SOD 1的超微结构 在“有毒通道”中经历构象变化。目的3：检验纳米尺度 分子间力和寡聚状态控制着突变体和野生型的插入和稳定， 型SOD 1分子。目的4：为了验证突变SOD 1和野生SOD 1的假设， 在某些条件下，SOD 1型使脂质膜可被某些离子渗透。 我们的研究设计和方法是：（目的1）利用TIRF、FRET和免疫荧光显微技术 将“毒性通道”共定位于细胞膜并产生合成抗体的技术 “有毒渠道”。（目标2）使用高分辨率的流体原子力显微镜（fAFM）和（目标3）fAFM 力测量以确定亚基结构、构象变化和分子间力 稳定“有毒通道”。和（目标4）使用单通道电生理记录来测量和 调节通过“毒性通道”的离子流，以了解通道的性质和特征。 今天，数以千万计的人遭受神经退行性疾病的衰弱和致命的症状 （ND）。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 新模型描述某些形式的ND是由侵入宿主神经元的“有毒离子通道”引起的 细胞膜并引起疾病病理生理学。既然是新的，“有毒通道”模式就没有 但却获得了广泛的接受或大量的研究资金。然而，本提案中提出的是新的， 令人信服的实验证据（关于ALS），支持“有毒通道”模型。如果我们的模型是 正确的，拟议的工作将使我们走上正确的轨道，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。通过成功的融资，我们相信 我们的工作的继续将导致新的抗体为基础的诊断，以及有效的发展。 ALS的新治疗方法

Project Summary/Abstract 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: (Aim1) 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". (Aim2) 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) use 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 proposal 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.