The Role of Toxic Ion Channels in ALS Pathogenesis

项目摘要/摘要 众所周知，蛋白质错误折叠在许多神经退行性疾病中起着核心作用，例如 阿尔茨海默氏症、肌萎缩侧索硬化症、亨廷顿病、帕金森病和遗传性海绵状 脑病，如克雅氏病。在肌萎缩侧索硬化症中，铜锌错折叠的结构状态 超氧化物歧化酶(SOD1)赋予这种正常情况下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，这种后天功能的确切性质和机制(S)仍然难以捉摸 尽管进行了多年的紧张研究。大多数研究都提出，淀粉样蛋白聚集体的沉积- 与SOD1蛋白一样，SOD1蛋白直接影响神经功能。根据我们的初步研究，我们的整体 假说是这些错误折叠的SOD1蛋白形成了“有毒离子通道”，进而产生离子通道 导致神经毒性的失衡(如钙超载)。 目的：验证一个突变体，也许在某些条件下，野生型SOD1蛋白共同作用的假说。 定位于亚细胞脂质膜域，特别是外线粒体的细胞质表面 薄膜。目的：验证突变型和野生型SOD1超微结构的假说。 在“有毒通道”中经历构象变化。目的3：检验纳米尺度的假说 分子间作用力和寡聚态支配着突变的插入和稳定，也许是野生的- 脂膜中有SOD1型分子。目标4：检验突变的SOD1，也许还有野生型- SOD1型，在一定条件下，使脂膜对某些离子具有通透性。 我们的研究设计和方法是：(Aim1)使用TIRF、FRET和免疫荧光显微镜 共同定位“有毒通道”到细胞膜并产生合成抗体的技术 反对“有毒渠道”。(AIM2)使用高分辨率流体原子力显微镜(FAFM)和(AIM 3)FAFM 作用力测量以确定亚基结构、构象变化和分子间作用力 这稳定了“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“有毒通道”的离子流动，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的衰弱和致命症状 (ND)。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。我们已经提出了一个 描述某些形式的ND是由侵入宿主神经元的“有毒离子通道”引起的新模型 细胞膜和引起疾病的病理生理学。因为它是新的，所以“有毒通道”模型并没有 但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和 令人信服的实验证据(关于肌萎缩侧索硬化症)，支持“有毒通道”模型。如果我们的模型是 正确，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 其他与“有毒通道”有关的蛋白质错误折叠疾病。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的发展，以及有效的 肌萎缩侧索硬化症的新疗法。

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.