病理过氧化氢对超氧化物歧化酶致病结构形成的调控

A major pathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, is the oligomers or fibrils formed by copper, zinc superoxide dismutase (SOD1) and TAR DNA-binding protein 43 kDa (TDP-43) in motor neuron cells. In this study, we plan to use pathological concentration of hydrogen peroxide to trigger the oligomerization and fibrillization of wild-type human SOD1 both in vitro and in SH-SY5Y neuroblastoma cells or N2a neuroblastoma cells, and investigate the regulation of the formation of disease-causing structures of SOD1 and TDP-43 by pathological hydrogen peroxide. Oxidized SOD1 oligomers will be then used to induce the fibrillization of wild-type human TDP-43 and its pathological mutants. We attempt to analyze the structures of SOD1-TDP-43 complex, SOD1 fibrils and TDP-43 fibrils on the molecular level, and to identify the structural features and pathological posttranslational modifications of both SOD1 fibrils and TDP-43 fibrils. DyLight dyes will be used to label oxidized SOD1 oligomers that enter into neuronal cells through endocytosis. Oxidized SOD1 oligomers will be used to induce the autophagy and apoptosis of neuronal cells, and the mislocalization, pathological posttranslational modifications, and aggregation of endogenous SOD1 and TDP-43 will be detected. We will detect pathological posttranslational modifications such as sulfenic acid modification of SOD1 and TDP-43 in pathological samples from ALS patient's brain and spinal cord, in order to explain the role of pathological posttranslational modifications in the pathogenesis of ALS. We hope to demonstrate that pathological hydrogen peroxide could trigger the fibrillization of wild-type SOD1 via sulfenic acid modification of this enzyme in living neuronal cells, accompanied by cytoplasm mislocalization and fibrillization of wild-type TDP-43, thereby inducing neuronal apoptosis and causing the initiation and development of sporadic ALS. Information obtained from this study can enhance our understanding of how hydrogen peroxide regulates SOD1/TDP-43 aggregation and toxicity, and should be useful in the development of new therapeutic regents for ALS.

肌萎缩性脊髓侧索硬化症(ALS)是一种致命的神经退行性疾病，其重要病理特征之一是铜锌超氧化物歧化酶(SOD1)和TAR DNA结合蛋白43(TDP-43)在运动神经元中形成的纤维化聚集体。本项目用病理过氧化氢诱导人野生型SOD1在体外和神经细胞中形成寡聚体和纤维，并用SOD1寡聚体诱导人TDP-43生长纤维，解析SOD1-TDP-43复合物的结构，鉴定SOD1和TDP-43形成纤维的结构特征和病理翻译后修饰，研究病理过氧化氢对ALS相关重要蛋白质致病结构形成的调控；DyLight标记SOD1寡聚体通过胞吞进入神经细胞，检测神经细胞自噬和凋亡及内源SOD1和TDP-43的错误定位和病理翻译后修饰；检测ALS病人临床样本中SOD1和TDP-43的病理翻译后修饰；阐明病理过氧化氢对SOD1和TDP-43积聚和细胞毒性的调控机制及翻译后修饰在ALS发病机制中的作用，为相关药物开发提供新的理论依据。

肌萎缩侧索硬化症（ALS）是一种致命的神经退行性疾病，其重要病理特征之一就是由铜锌超氧化物歧化酶（SOD1）以及TAR DNA结合蛋白43（TDP-43）在中枢神经系统的运动神经元以及星形胶质细胞中形成纤维样聚集体。我们用基于双甲酮的次磺酸化检测抗体，检测到无论在体外还是在活的神经细胞中，病理浓度过氧化氢都可以使得野生型SOD1发生次磺酸化修饰，而且这种可逆的氧化修饰对于SOD1生长纤维和发挥病理功能至关重要，质谱检测确定其修饰位点为第111位的半胱氨酸；我们运用免疫共沉淀和酶联免疫的方法检测了ALS病人的脑脊液样本和相同年龄段的对照组病人脑脊液样本，发现散发型ALS病人脑脊液样本中的次磺酸化修饰SOD1水平显著高于对照组，因此次磺酸化修饰SOD1可能是散发型ALS早期诊断生化指标和早期ALS的生物标记物；次磺酸化修饰的SOD1寡聚体能够诱导内源SOD1在胞质中发生异常积聚，同时诱导内源TDP-43错误定位和在胞质中发生异常积聚，最终导致神经细胞发生凋亡；据此我们建立了病理浓度过氧化氢对SOD1和TDP-43积聚和细胞毒性的调控模型。我们在还原条件下制备了高度均一的全长人SOD1淀粉样纤维，发现这些纤维具有显著的细胞毒性，运用冷冻电镜结合三维重构技术，首次在原子水平上解析了全长SOD1纤维的高分辨率冷冻电镜结构（2.95 Å），发现SOD1纤维核心由其N端的3-55区域和C端的86-153区域组成，这两个区域主要通过3对盐桥紧密结合在一起；SOD1纤维由单两股原纤维以左手螺旋的方式缠绕而成；纤维核心的疏水氨基酸侧链向内折叠形成5个疏水空腔，起到了稳定淀粉样纤维结构的作用；通过比较脱辅基SOD1二聚体结构，我们提出在SOD1错误折叠过程中，脱辅基SOD1亚基的一个α-螺旋和由8个β-折叠组成的β-桶状结构转变成SOD1纤维的13个β-折叠结构。上述研究不仅揭示了病理过氧化氢以及次磺酸化修饰在散发型ALS发病机制中的作用，而且在原子水平上揭示了SOD1由生理型向病理型结构转变的机制，也为ALS相关药物开发提供了新的理论依据。

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