Parkin and Its Regulation of Neuronal Apoptosis

描述（由申请人提供）：帕金森氏病是第二常见的神经退行性疾病，帕金森氏病的常染色体隐性突变。为了测试parkin作为多亚基组成部分的工作模型，scf样泛素连接酶复合物保护多巴胺神经元免于凋亡，该复合物的其他组成部分，包括self -10和cullin-1，将在小鼠原代神经元培养中受到RNA干扰而下调。这些成分的下调将被评估为多巴胺神经元凋亡的增强，并与下调parkin本身的影响进行比较。为了验证帕金森相关复合物的候选底物cyclin E是凋亡级联反应的关键介质这一假设，我们将评估在parkin、self -10或cullin-1下调的情况下，cyclin E相关活性的药理学抑制对多巴胺神经元的拯救作用。最后，在相同的原代培养系统中，慢病毒介导的parkin过表达将被评估为多巴胺神经元免受神经毒素的保护，并与突变形式的parkin过表达进行比较，包括临床定义的突变和泛素同源性和环结构域中缺失的形式。

DESCRIPTION (provided by applicant): Mutations in parkin underlie an autosomal recessive form of Parkinson's disease, the second most common neurodegenerative disease. To test a working model of parkin as a component of a multi-subunit, SCF-like ubiquitin ligase complex that protects dopamine neurons from apoptosis, other components of the complex, including sel-10 and cullin-1, will be dowregulated by RNA interference in murine primary neuronal cultures. Downregulation of these components will be evaluated for potentiation of dopamine neuron apoptosis and compared to the effects of downregulating parkin itself. To test the hypothesis that a candidate substrate of the parkin-associated complex, cyclin E, is a key mediator of the apoptotic cascade(s) against which wildtype parkin normally protects neurons, pharmacological inhibition of cyclin E-associated activity will be evaluated for rescue of dopamine neurons in the context of parkin, sel-10, or cullin-1 downregulation. Finally, lentivirus-mediated overexpression of parkin in the same primary culture system will be assessed for protection of dopamine neurons from neurotoxins as compared to overexpression of mutant forms of parkin, including clinically defined mutations and forms deleted in the ubiquitin homology and RING domains.