PP2Ac甲基化在锰暴露诱导神经细胞自噬障碍中的作用研究

The methylation of C- terminal leucine residue of PP2A catalytic subunit is important for PP2A executing its dephosphorylation function, methylated by leucine carboxyl methyltransferase-1,LCMT-1. Dephosphorylatoin of ULK1 (Unc-51 like kinase-1) is a key event to initiate autophagy. Neurodegenerative change due to excessive Mn exposure is associated to abnormal autophagy. Our previous studies showed PP2Ac methylation decreased upon manganese exposure. In view of other literatures and our own results, we speculate that autophagy impaired by manganese might due to PP2A catalytic subunit demethylation and its catalytic activity loss, ULK1 dephosphorylation following mammalian target of rapamycin complex 1(mTORC1) activation through manganese disruption ROS generation. The present study is designed to investigate the impact of high level Mn exposure on autophagy in vivo and vitro. We also discuss the pathways of PP2A-C methylation regulated by Protein Phosphatase Methyl-Esterase inhibitor (ABL-127), antioxidant (N-acetyl-L-cysteine, NAC) and transfection LCMT-1 overexpression into SK-N-SH neurons and their effects on ULK1 dephosphorylation and autophagy. This study will help to insight to manganese neurotoxicity and to provide some new strategies for prevention and treatment of neurodegenerative diseases upon elevated manganese exposure.

亮氨酸甲基转移酶(leucine carboxyl methyltransferase-1,LCMT-1 )催化PP2Ac甲基化对PP2A发挥去磷酸化功能具有重要作用，ULK1去磷酸化是自噬启动的关键事件。甲基化的PP2A通过调控mTORC1/ULK1而调控自噬。锰诱导神经细胞退行性变与抑制自噬有关。前期研究提示锰暴露诱导PP2Ac去甲基化。根据前期结果及文献推测锰通过氧化应激诱导PP2Ac去甲基化，PP2A活性下降，mTORC1磷酸化活化而对ULK1抑制增强，抑制细胞自噬，从而发生退行性改变。本研究拟通过细胞实验与动物实验相结合，研究锰对神经细胞自噬的影响，探讨补充ABL-1、抗氧化剂（NAC）及转染过表达LCMT-1质粒调控PP2A-C甲基化后UKL1去磷酸状态及神经细胞自噬的变化。本研究为深入理解锰神经毒性，改善锰诱导的神经退行性疾病，提供防治方法的新思路。

自噬是生物通过囊泡吞噬自身细胞质、异常蛋白或受损细胞器并转运至溶酶体与之结合形成自噬溶酶体，继而降解内容物的进化保守过程。该自我更新过程异常与神经退行性病变有关。mTORC1/ULK1通路激活自噬，异三聚体PP2A 与锰诱导的神经损伤相关。该酶的催化亚基C（PP2Ac）在游离羧基末端L309位点由LCMT1和PME进行可逆性地甲基化修饰，调控全酶的去磷酸化作用。本研究通过体内外模型探讨锰通过PP2Ac甲基化调控mTORC1/ULK1诱导神经细胞自噬异常。体内实验发现锰诱导的学习记忆能力损伤与细胞自噬异常有关，mTORC1/ULK1通路激活且PP2Ac去甲基化增高。进一步地，通过化学修饰（PME-1抑制剂ABL-127）及基因调控（LCMT1过表达）上调体外细胞PP2Ac甲基化修饰，结果表明提高PP2Ac甲基化水平有助于改善锰诱导的N2a细胞自噬异常，且该保护效应通过mTORC1/ULK1通路发挥作用，ULK1是mTORC1下游的效应因子，受mTORC1信号通路的负调节。