Fishman-Jacob Tali, Reznichenko Lydia, Youdim Moussa B H, Mandel Silvia A
Eve Topf and National Parkinson Foundation Centers for Neurodegenerative Diseases and the Department of Molecular Pharmacology, Faculty of Medicine, Technion, 31096 Haifa, Israel.
J Biol Chem. 2009 Nov 20;284(47):32835-45. doi: 10.1074/jbc.M109.034223. Epub 2009 Sep 11.
The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.
本研究的目的是基于沉默SKP1A基因开发一种散发性帕金森病（PD）的新模型。SKP1A基因是泛素 - 蛋白酶体/E3连接酶复合物Skp1、Cullin 1、F - 盒蛋白的一个组成部分，研究发现其在散发性PD患者的黑质中高度降低。最初，用编码SKP1A基因小鼠转录本的短发夹RNA慢病毒或乱序载体感染源自胚胎小鼠黑质的细胞系（SN4741细胞）。SKP1A基因沉默导致对帕金森病诱导神经毒素1 - 甲基 - 4 - 苯基吡啶离子和血清饥饿诱导的神经元损伤的易感性增加，同时多巴胺能标志物多巴胺转运体和囊泡单胺转运体 - 2的表达下降。SKP1A缺陷细胞在诱导分化时细胞周期完成延迟，且无法停滞在G(0)/G(1)期。相反，细胞进入S期，形成具有聚集体特征的圆形聚集体，包括对γ - 微管蛋白、α - 突触核蛋白、泛素、酪氨酸羟化酶、Hsc - 70（70 kDa热休克同源蛋白）和蛋白酶体亚基的免疫反应性，并最终导致致死表型。相反，在蛋白酶体抑制损伤下，野生型SKP1A的稳定过表达使未处理的SN4741细胞的存活指数翻倍，这表明SKP1除了其E3连接酶活性外，在多巴胺能神经元功能中具有新的结构作用。这些结果首次将SKP1与多巴胺神经元功能和存活联系起来，表明其在散发性PD中起重要作用。总之，这个新模型在很大程度上再现了散发性PD在细胞水平上描述的分子改变，提示Skp1是散发性PD神经退行性变的潜在调节因子。
