LRRK2-p38-Drosha通过调节miRNA生成稳态参与帕金森病发病的机制研究

Parkinson’s disease is the second most common neurodegenerative disease and characterized pathologically by the progressive loss of dopaminergic (DA) neurons in the substantial nigra pars compactor (SNc). Both genetic and environmental factors play a role in the etiology of the disease. One of the most common genetic causes identified in both familial and sporadic forms of PD is the mutation of leucine-rich repeat kinase 2 (LRRK2) gene. However, how mutated LRRK2 protein leads to PD pathogenesis is poorly understood and remains an intense focus of investigation in PD field. We recently showed that the nuclear RNase IIIDrosha, the key miRNA biogenic regulator, is a converging point targeted by multiple stress signals. Stress induces a p38 MAPK-dependent phosphorylation and inhibition of Drosha, resulting in a severe loss of miRNA homeostasis and a significant increase in cellular death under stress. We have now obtained exciting preliminary findings demonstrating LRRK2 engages this p38 MAPK-Drosha pathway. We propose that LRRK2 mutant may aberrantly activate p38 MAPK, inhibit Drosha function, and lead to a global miRNA dys-homeostasis, which may sensitize to the SNc DA neurons to stress and underlie PD pathogenesis. We will use a combination of molecular, cellular, and animal model approaches to first, establish whether LRRK2 mutant aberrantly activates p38 MAPK in the SNc DA neurons; second, test whether LRRK2 mutant engages p38 MAPK to dysregulate Drosha and miRNA biogenesis; third, exam whether LRRK2-induced global miRNA dys-homeostasis triggers the death of the SNc DA neurons under stress and plays a critical role in PD pathogenesis. Successful completion of this work will reveal a novel mechanism by which PD associated LRRK2 mutation interferes with neuronal homeostasis and viability and establish the interplay between LRRK2 and Drosha/miRNA as the long-sought-after link underlying PD pathogenesis.

帕金森病（PD）主要病理特点是中脑黑质致密部多巴胺能（DA）神经元死亡。遗传因素与PD发病密切相关。LRRK2突变是常染色体显性遗传性PD最常见的原因,但LRRK2突变如何诱发DA神经元死亡目前并不清楚。我们最新研究显示miRNA生成过程中的核心RNA酶Drosha是多重应激信号通路的靶点。应激激酶p38磷酸化Drosha，导致其功能下降而诱发miRNAs稳态失衡促进细胞死亡。在预实验中我们发现LRRK2突变增强p38活性并促使Drosha水平下降。因此，我们提出LRRK2通过激活p38导致Drosha功能及稳定性受损，触发miRNAs稳态失衡而导致DA神经元死亡。本研究将采用细胞、PD动物模型证实LRRK2-p38-Drosha调节通路的存在，验证LRRK2-miRNAs稳态失衡在DA神经元死亡中的作用。该研究将从Drosha-miRNAs全新的角度阐述LRRK2突变的致病机理。

帕金森病（Parkinson’s disease, PD）以中脑黑质区多巴胺能（DA）神经元死亡为主要病理表现，发病机制仍不完全清楚。近年研究表明遗传因素与PD发病密切相关，其中，LRRK2基因突变是常染色体显性遗传帕金森病最常见的原因,因此，深入研究LRRK2调控DA神经元存活于死亡的分子机制，有助于进一步阐明PD的发病机制。在该项目的开展过程中，我们发现PD致病突变体LRRK2 G2019S通过诱导激酶活性磷酸化miRNA生成关键酶Drosha负调控其功能，提出miRNA稳态失衡是LRRK2的致病机理之一。多种应激因素包括氧化应激等也是帕金森发病的重要原因，我们发现在氧化应激诱导PD动物模型中，Drosha能够被应激激酶p38MAPK磷酸化，进而被钙蛋白酶切割降解。增强Drosha表达可保护小鼠PD模型中DA神经元存活，减轻鼠的运动障碍。这些发现表明Drosha在DA神经元的存活中起着至关重要的作用，提示遗传、环境应激等导致的DA神经元Drosha功能障碍可能是PD发病的重要机理。分子伴侣介导的自噬（CMA）是一种选择性降解系统，既往研究表明其对于神经元稳态的维持至关重要，CMA自噬活性的降低与衰老、神经退行性疾病的发病密切相关，但目前仍不完全清楚CMA自噬是如何发挥保护作用的。我们发现在氧化应激过程中，CMA能够激活并降解机体中主要的抗氧化应激转录因子Nrf2的关键上游负调节因子Keap1的水平，从而稳定并上调Nrf2水平及功能，进而促进了DA神经元细胞在氧化应激下的存活。这些发现阐明了氧化应激下CMA发挥神经元保护作用的具体机制，建立了CMA -Nrf2抗氧化应激调节通路，帮助我们进一步深入认识了CMA自噬与神经退行性疾病之间的关系。线粒体功能障碍是PD发病的另一个重要机制，我们发现去乙酰化酶Sirt3通过调控线粒体转录因子A（TFAM）乙酰化水平促进线粒体DNA复制和转录，进而维持线粒体功能稳定，氧化应激条件下增强Sirt3功能可有效保护DA神经元存活。该研究工作揭示了Sirt3-TFAM去乙酰化通路是DA神经元实现线粒体质控的重要方式，证实了Sirt3是干预DA神经元死亡的潜在靶点。