Biochemistry and Cell Biology of alpha-Synucleinopathies

帕金森病、路易体痴呆和多系统萎缩是被称为突触核蛋白病的神经退行性疾病。它们的特征在于α-突触核蛋白（α-SN）作为纤维状内含物在选择性脆弱的神经元和神经胶质细胞中积累。我们对α-SN内含物形成和α-SN毒性的细胞机制了解甚少。为了更好地理解α-SN聚集的过程，我们的实验室使用了人类神经元 [BE（2）-M17 D]细胞和四环素关闭（TetOff）诱导机制产生细胞 过表达野生型人α-SN。诱导7天后，在这些细胞中检测到肌氨酰不溶性α-SN聚集体。将我们的细胞模型暴露于鱼藤酮（一种有效的线粒体复合物I抑制剂），导致在5天内形成RIPA缓冲液不溶性、较高分子量肌氨酰可溶性α-SN物质（与寡聚体一致）和肌氨酰不溶性α-SN。在Western印迹上检测到寡聚体为含有截短的α-SN的离散条带，并且它们与在Ser 129或泛素处磷酸化的α-SN的抗体没有免疫反应性。这种低聚物在没有鱼藤酮处理的细胞中几乎检测不到，可以作为α-SN聚合的种子，或者当它们达到临界浓度时对细胞有毒。在下文提出的研究中，我们计划进一步表征α-SN寡聚体，重点确定（i）暴露于与PD相关的其他神经毒素的细胞中是否产生类似的寡聚体，以及是否可以在家族性和散发性突触核蛋白病的大脑中发现，（ii）其形成的机制，以及（iii）其在α-SN组装和神经毒性中的作用。此外，我们将确定（iv）是否过表达α-SN改变神经元细胞中的基因表达谱。这些研究将有助于我们了解α-SN包涵体的形成及其对神经元的选择性神经毒性。

Parkinson disease, dementia with Lewy bodies and multiple system atrophy are neurodegenerative disorders referred to as synucleinopathies. They are characterized by accumulation of alpha-synuclein (alpha-SN) as fibrillar inclusions in selectively vulnerable neurons and gila. Our understanding of the cellular mechanisms underlying the formation of alpha-SN inclusions and the toxicity of alpha-SN is poorly understood. In order to better understand the process of alpha-SN aggregation, our laboratory has used human neuronal [BE(2)-M17D] cells and the tetracycline-off (TetOff) inducible mechanism to generate cells overexpressing wild-type human alpha-SN. After 7 days of induction, sarkosyl-insoluble alpha-SN aggregates are detected in these cells. Exposure of our cell model to rotenone, a potent mitochondrial complex I inhibitor, led to the formation of RIPA buffer-insoluble, higher molecular weight sarkosyl-soluble alpha-SN species (consistent with oligomers) and sarkosyl-insoluble alpha-SN within five days. The oligomers were detected on Western blots as discrete bands containing truncated alpha-SN, and they exhibited no immunoreactivity with antibodies to alpha-SN phosphorylated at Ser129 or ubiquitin. Such oligomers, which were barely detectable in cells without rotenone treatment, may serve as seeds for alpha-SN polymerization or may be toxic to cells when they reach a critical concentration. In the studies proposed below, we plan to characterize alpha-SN oligomers further, focusing on determining (i) whether similar oligomers are produced in cells exposed to other neurotoxins relevant to PD and can be found in brains of familial and sporadic synucleinopathies, (ii) the mechanisms involved in their formation, and (iii) their role in alpha-SN assembly and neurotoxicity. In addition, we will determine (iv) whether overexpressing alpha-SN alters the gene expression profile in neuronal cells. These studies will contribute to our understanding of the formation of alpha-SN inclusions and their selective neurotoxicity for neurons.