Wang Yi, Liu Jia, Chen Min, Du Tingting, Duan Chunli, Gao Ge, Yang Hui
Department of Neurobiology, Capital Medical University, Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, No. 10, XiToutiao outside the YouAnmen, Fengtai District, Beijing 100069, China; Department of Clinical Laboratory, China Rehabilitation Research Center, School of Rehabilitation Medicine, Capital Medical University, No. 10, North Road, Fengtai District, Beijing 100068, China.
Department of Neurobiology, Capital Medical University, Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, No. 10, XiToutiao outside the YouAnmen, Fengtai District, Beijing 100069, China.
Int J Biochem Cell Biol. 2016 Jun;75:34-44. doi: 10.1016/j.biocel.2016.03.007. Epub 2016 Mar 21.
Rotenone has been shown to induce many parkinsonian features and has been widely used in chemical models of Parkinson's disease (PD). Its use is closely associated with α-synuclein (α-syn) phosphorylation both in vivo and in vitro. However, the mechanisms whereby rotenone regulates α-syn phosphorylation remain unknown. Protein phosphatase 2A (PP2A) has been shown to play an important role in α-syn dephosphorylation. We therefore investigated if rotenone caused α-syn phosphorylation by down-regulation of PP2A activity in mice. Rotenone increased the phosphorylation of α-syn at Ser129, consistent with the inhibition of PP2A activity by increased phosphorylation of tyrosine 307 at the catalytic subunit of PP2A (pTyr307 PP2Ac). We further explored the interactions among rotenone, PP2A, and α-syn in SK-N-SH cells and primary rat cortical neurons. Rotenone inhibited PP2A activity via phosphorylation of PP2Ac at Tyr307. The reduction in PP2A activity and rotenone cytotoxicity were reversed by treatment with the PP2A agonist, C2 ceramide, and the Src kinase inhibitor, SKI606. Immunoprecipitation experiments showed that rotenone induced an increase in calmodulin-Src complex in SK-N-SH cells, thus activating Src kinase, which in turn phosphorylated PP2A at Tyr307 and inhibited its activity. C2 ceramide and SKI606 significantly reversed the rotenone-induced phosphorylation and aggregation of α-syn by increasing PP2A activity. These results demonstrate that rotenone-reduced PP2A activity via Src kinase is involved in the phosphorylation of α-syn. These findings clarify the novel mechanisms whereby rotenone can induce PD.
鱼藤酮已被证明可诱发许多帕金森病特征，并已广泛应用于帕金森病（PD）的化学模型中。其使用在体内和体外均与α-突触核蛋白（α-syn）磷酸化密切相关。然而，鱼藤酮调节α-syn磷酸化的机制仍不清楚。蛋白磷酸酶2A（PP2A）已被证明在α-syn去磷酸化中起重要作用。因此，我们研究了鱼藤酮是否通过下调小鼠PP2A活性导致α-syn磷酸化。鱼藤酮增加了Ser129位点α-syn的磷酸化，这与PP2A催化亚基酪氨酸307（pTyr307 PP2Ac）磷酸化增加导致的PP2A活性抑制一致。我们进一步探讨了鱼藤酮、PP2A和α-syn在SK-N-SH细胞和原代大鼠皮层神经元中的相互作用。鱼藤酮通过Tyr307位点磷酸化PP2Ac抑制PP2A活性。PP2A激动剂C2神经酰胺和Src激酶抑制剂SKI606处理可逆转PP2A活性降低和鱼藤酮细胞毒性。免疫沉淀实验表明，鱼藤酮诱导SK-N-SH细胞中钙调蛋白-Src复合物增加，从而激活Src激酶，进而使PP2A在Tyr307位点磷酸化并抑制其活性。C2神经酰胺和SKI606通过增加PP2A活性显著逆转了鱼藤酮诱导的α-syn磷酸化和聚集。这些结果表明，鱼藤酮通过Src激酶降低PP2A活性参与了α-syn磷酸化。这些发现阐明了鱼藤酮可诱发PD的新机制。
