Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, J.B. Winsloews Vej 21, st, DK5000 Odense C, Denmark; Oxford Parkinson's Disease Centre, Department of Physiology, Anatomy and Genetics, South Parks Road, University of Oxford, OX1 3QX Oxford, UK.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK5230 Odense M, Denmark.
Neurobiol Dis. 2019 Dec;132:104581. doi: 10.1016/j.nbd.2019.104581. Epub 2019 Aug 21.
Mutations in parkin, encoded by the PARK2 gene, causes early-onset familial Parkinson's disease (PD), but dysfunctional parkin has also been implicated in sporadic PD. By combining human isogenic induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) and a novel large-scale mass spectrometry based proteomics and post-translational modification (PTM)-omics approach, we have mapped changes in protein profiles and PTMs caused by parkin deficiency in neurons. Our study identifies changes to several proteins previously shown to be dysregulated in brains of sporadic PD patients. Pathway analysis and subsequent in vitro assays reveal perturbations in migration and neurite outgrowth in the PARK2 KO neurons. We confirm the neurite defects using long-term engraftment of neurons in the striatum of immunosuppressed hemiparkinsonian adult rats. The GTP-binding protein RhoA was identified as a key upstream regulator, and RhoA activity was significantly increased in PARK2 KO neurons. By inhibiting RhoA signalling the migration and neurite outgrowth phenotypes could be rescued. Our study provides new insight into the pathogenesis of PD and demonstrates the broadly applicable potential of proteomics and PTMomics for elucidating the role of disease-causing mutations.
Parkin 基因突变，由 PARK2 基因编码，导致早发性家族性帕金森病（PD），但功能失调的 parkin 也与散发性 PD 有关。通过将具有和不具有 PARK2 敲除（KO）的人同源诱导多能干细胞（iPSC）与一种新型基于大规模质谱的蛋白质组学和翻译后修饰（PTM）组学方法相结合，我们已经绘制了由神经元中 parkin 缺乏引起的蛋白质图谱和 PTM 变化。我们的研究确定了几个先前在散发性 PD 患者大脑中失调的蛋白质的变化。通路分析和随后的体外试验揭示了 PARK2 KO 神经元中迁移和神经突生长的干扰。我们使用免疫抑制的半帕金森成年大鼠纹状体中长期移植神经元来证实神经突缺陷。GTP 结合蛋白 RhoA 被鉴定为关键的上游调节剂，并且 PARK2 KO 神经元中的 RhoA 活性显著增加。通过抑制 RhoA 信号，可挽救迁移和神经突生长表型。我们的研究为 PD 的发病机制提供了新的见解，并证明了蛋白质组学和 PTM 组学在阐明致病突变作用方面具有广泛的应用潜力。
