Biochemistry and Cell Biology of alpha-Synucleinopathies

帕金森病、路易体痴呆和多系统萎缩是一种神经退行性疾病，称为突触核病。它们的特点是α-突触核蛋白(α-SN)在选择性脆弱的神经元和GILA中以纤维包涵体的形式积聚。我们对α-SN包裹体形成的细胞机制和α-SN的毒性了解甚少。为了更好地了解α-SN聚集的过程，我们实验室使用了人神经元 [Be(2)-M17D]细胞及其四环素关闭(TetOff)诱导机制 过表达野生型人α-SN。诱导7天后，在这些细胞中检测到不溶于肌糖的α-SN聚集体。我们的细胞模型暴露于鱼藤酮，一种有效的线粒体复合体I抑制剂，导致在五天内形成不溶于RIPA缓冲液的、较高分子量的肌糖可溶的α-SN物种(与低聚体一致)和肌糖不溶的α-SN。这些寡聚体在Western blotts上被检测为含有截短的α-SN的离散条带，它们与Ser129或泛素磷酸化的α-SN抗体没有免疫反应性。在没有鱼藤酮处理的情况下，这些低聚物在细胞中几乎检测不到，它们可能作为α-SN聚合的种子，或者当它们达到临界浓度时可能对细胞有毒。在下面提出的研究中，我们计划进一步确定α-SN寡聚体的特征，重点是确定(I)在暴露于其他与帕金森病相关的神经毒素的细胞中是否产生类似的寡聚体，并在家族性和散发性突触核病症的脑中发现类似的寡聚体，(Ii)它们形成的机制，以及(Iii)它们在α-SN组装和神经毒性中的作用。此外，我们将确定(Iv)过度表达α-SN是否会改变神经细胞中的基因表达谱。这些研究将有助于我们理解α-SN包裹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.