Department of Physiology, Guilin Medical University, Guilin, Guangxi, China.
Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guilin Medical University, Guilin, Guangxi, China.
CNS Neurosci Ther. 2023 Dec;29(12):3952-3966. doi: 10.1111/cns.14319. Epub 2023 Jun 23.
Chromosome 9 open reading frame 72 (C9orf72) is one of the most dazzling molecules in neurodegenerative diseases, albeit that its role in Parkinson's disease (PD) remains unknown. This article aimed to explore the potential mechanism of C9orf72 involved in the pathogenesis of PD.
The expression and phosphorylation levels of C9orf72 were examined by Western blotting, RT-PCR, and immunoprecipitation using PD models. Multiple bioinformatics software was used to predict the potential phosphorylation sites of C9orf72 by Cdk5, followed by verification of whether Cdk5-inhibitor ROSCOVITINE could reverse the degradation of C9orf72 in PD. By constructing the sh-C9orf72-knockdown adenovirus and overexpressing the FLAG-C9orf72 plasmid, the effects of C9orf72 knockdown and overexpression, respectively, were determined. A short peptide termed Myr-C9orf72 was used to verify whether interfering with Cdk5 phosphorylation at the Ser9 site of the C9orf72 protein could alleviate autophagy disorder, neuronal death, and movement disorder in PD models.
The expression level of the C9orf72 protein was significantly reduced, albeit the mRNA expression was not changed in the PD models. Moreover, the phosphorylation level was enhanced, and its reduction was mainly degraded by the ubiquitin-proteasome pathway. The key nervous system kinase Cdk5 directly phosphorylated the S9 site of the C9orf72 protein, which promoted the degradation of the C9orf72 protein. The knockdown of C9orf72 aggravated autophagy dysfunction and increased neuronal loss and motor dysfunction in substantia nigra neurons of PD mice. The overexpression of C9orf72 alleviated autophagy dysfunction in PD neurons. Specifically, interference with Cdk5 phosphorylation at the S9 site of C9orf72 alleviated autophagy dysfunction, neuronal death, and motor dysfunction mediated by C9orf72 protein degradation during PD.
Cumulatively, our findings illustrate the importance of the role of C9orf72 in the regulation of neuronal death during PD progression via the Cdk5-dependent degradation.
染色体 9 开放阅读框 72（C9orf72）是神经退行性疾病中最耀眼的分子之一，尽管其在帕金森病（PD）中的作用尚不清楚。本文旨在探讨 C9orf72 参与 PD 发病机制的潜在机制。
通过 Western blot、RT-PCR 和免疫沉淀法检测 PD 模型中 C9orf72 的表达和磷酸化水平。使用多种生物信息学软件预测 Cdk5 对 C9orf72 的潜在磷酸化位点，然后验证 Cdk5 抑制剂 ROSCOVITINE 是否能逆转 PD 中 C9orf72 的降解。通过构建 sh-C9orf72 敲低腺病毒和过表达 FLAG-C9orf72 质粒，分别确定 C9orf72 敲低和过表达的影响。使用短肽 Myr-C9orf72 验证干扰 C9orf72 蛋白 Ser9 位点的 Cdk5 磷酸化是否能减轻 PD 模型中的自噬紊乱、神经元死亡和运动障碍。
C9orf72 蛋白的表达水平显著降低，尽管 PD 模型中的 mRNA 表达没有改变。此外，磷酸化水平增强，其减少主要通过泛素-蛋白酶体途径降解。关键的神经系统激酶 Cdk5 直接磷酸化 C9orf72 蛋白的 S9 位点，促进 C9orf72 蛋白的降解。C9orf72 的敲低加重了自噬功能障碍，并增加了 PD 小鼠黑质神经元的神经元丢失和运动功能障碍。C9orf72 的过表达减轻了 PD 神经元中的自噬功能障碍。具体而言，干扰 Cdk5 对 C9orf72 S9 位点的磷酸化减轻了 C9orf72 蛋白降解介导的自噬功能障碍、神经元死亡和运动功能障碍。
总之，我们的研究结果表明，C9orf72 通过 Cdk5 依赖性降解在调节 PD 进展过程中的神经元死亡中起重要作用。
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