Project 1

肌萎缩侧索硬化症的发病机制与SOD1的聚集和纤颤有关 硬化症(ALS)，这是该计划项目更新的一个主要新目标，开发更好的生物检测方法来 研究这些多聚体SOD1在与疾病更相关的系统中的毒性 人类。在项目2中，我们进一步开发和使用了人体细胞培养系统，该系统可以对重要细胞进行近距离建模 运动神经元变性的生物学方面？我们最近开发的人类胚胎干细胞来源 运动神经元(HESC-MN)系统。单元格与其他模型系统相比具有明显的优势，因为它们代表 肌萎缩侧索硬化症中退化的主要细胞类型，它们完全是人类。这些细胞表达识别神经元 标记物表现出成熟运动神经元的典型电生理功能，并可与 其他神经元和非神经元细胞。转染这些细胞表达ALS-SOD1蛋白引起 对细胞存活和形态的有害影响。对这个项目来说很重要的是，我们最近展示了 外源添加的ALS-SOD1蛋白多聚体被很好地吸收。我们将利用这些细胞来研究 SOD1蛋白多聚体和聚集体形成不同阶段的毒性及其与细胞毒性的关系 运动神经元变性的进展。该研究计划概述了高度协作、循序渐进的 运动神经元自发和诱导突变及WT SOD1聚集体形成的评价方法， 随后研究了SOD1聚集体对神经退行性机制的影响， 最后，通过使用SODl聚集的药物抑制剂来研究SODl减少 聚集可以防止运动神经元死亡。

Aggregation and fibrillation of SODl have been implicated in disease mechanisms of Amyotrophic Lateral Sclerosis (ALS), and it is a major new goal of this Program Project renewal to develop better biological assays to study the toxicity of these multimeric forms of SODl in systems that will be more relevant to the disease in humans. In Project 2 we further develop and use a human cell culture system that closely models important cell biological aspects of motor neuron degeneration¿our recently developed human embryonic stem cell-derived motor neuron (HESC-MN) system. The cells have distinct advantages over other model systems as they represent the major cell type that degenerates in ALS and they are fully human. The cells express identifying neuronal markers, exhibit electrophysiological function typical for mature motor neurons, and can be co-cultured with other neuronal and non-neuronal cells. Transfection of these cells to express ALS-SODl proteins causes deleterious effeds on cell survival and morphology. Importantly for this project, we have recently shown that exogenously added ALS-SODl protein multimers are taken up quite well. We will utilize these cells to study the toxicity of SODl protein multimers and aggregates at different stages of their formation and relate it to the progression of motor neuron degeneration. This research plan outlines a highly collaborative, step-by-step approach to evaluate spontaneous and induced mutant and WT SODl aggregate formation in motor neurons, followed by an investigation of the consequences of SODl aggregates on neurodegenerative mechanisms and, finally, by using pharmacological inhibitors of SODl aggregation to investigate whether reduced SODl aggregation can prevent motor neuron death.