过氧化应激对分子伴侣介导自噬的调节机制及其在多巴胺神经元死亡中的作用

Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by a loss of dopaminergic neurons in the substantia nigra (SN).The key question to PD is why and how DA neurons dies in the disease. Our own studies of an important neuronal survival factor MEF2D, have shown that CMA regulates the constitutive levels of MEF2D in neurons. High levels of wt or mt α-synuclein which cause Parkinsonism can dysregulate the degradation process of MEF2D by Chaperone-mediated autopahgy(CMA). These observations strongly support the exciting hypothesis that dys-regulation of CMA plays a critical role in the pathogenic process of PD. However, convincing evidence establishing a clear dysregulation of CMA pathway by stress in PD is lacking. Chaperone-mediated autophagy is a form of autophagy. The levels of LAMP2A at lysosomal membrane directly relate to activity of CMA. Hsp90, as a critical chaperone in CMA, is involved in regulation of LAMP2A stabilization and its organization to multimeric complex at lysosome membrane. Exposure to neurotoxins and oxidative stress are considered to be one of the important factors either triggering and/or facilitating the PD pathogenic process. Oxidative stress induces the activity of p38 MAPK. Our preliminary data showed that Hsp90 is phosphorylated by p38 in vitro. In a dopaminergic neuronal cell line, oxidative stress increased the phosphorylation level of Hsp90. In 6-OHDA PD animal model, p38 inhibitor can decrease LAMP2A level in lysosomes of SNc. In this application, we propose to combine molecular and cellular methods and animal models to determine 1) in Aim 1, whether phosphorylation of Hsp90 by p38 MAPK modulates CMA activity in DA neurons;2)in Aim 2,whether the regualtion of CMA activity plays an important role in DA neuronal death and degradaion of alpha-synuclein. This study will identify the molecular link between oxidative stress and CMA and reveal how dysregulation of CMA may undermine DA neuronal viability.This novel mechanism may be relevant to the pathogenesis of PD and provide basis for developing therapeutic strategies.

帕金森病（PD）是一种发病机制不清、以多巴胺（DA）神经元死亡为主要特点的疾病。过氧化应激是其主要致病因素。我们的研究显示分子伴侣介导自噬（CMA）与PD发病密切相关，PD致病蛋白α-synulein大量表达时，抑制神经存活因子MEF2D通过CMA降解，诱发DA神经元死亡。但PD发病过程中CMA功能的调节机制和作用仍未阐明。自噬溶酶体膜LAMP2A的稳定性决定CMA功能，Hsp90在稳定LAMP2A和保持其活性中起着关键性的作用。预实验表明p38磷酸化Hsp90；过氧化应激可通过激活p38调节溶酶体膜LAMP2A水平。本课题在此基础上，以p38 磷酸化Hsp90和对其功能调节为切入点，在细胞和动物模型中研究过氧化应激对LAMP2A和CMA功能的调节机制，以及该调节作用对PD关键蛋白代谢和DA神经元存活死亡的直接影响。阐明CMA功能变化在PD发病中的作用，进一步丰富PD的分子发病机制。

应激条件下神经元稳态失衡是神经元慢性变性、死亡的重要原因。以往研究表明，作为选择性降解系统，伴侣介导的自噬（CMA）对于应激条件下细胞稳态的维持是至关重要的，CMA自噬功能的降低与神经退行性疾病的发病密切相关。然而，对于应激条件下CMA自噬发挥保护作用的具体途径以及CMA自噬是如何受到调节的却了解甚少。阐明这两个关键环节将有助于深入认识CMA自噬与神经退行性疾病之间的关系。在该项目的开展过程中，我们发现在氧化应激条件下，内质网应激通过p38MAPK激酶激活CMA自噬。多种PD致病因素可以激活该信号通路，抑制该通路将加重多巴胺能神经元的死亡。该研究首次证实了激酶-CMA自噬调控方式，并将氧化应激、CMA自噬与神经元稳态三者联系起来。CMA自噬通过选择性的降解多种底物发挥细胞保护作用。PARK7是一种常染色体隐性家族性PD基因，在维护线粒体功能方面扮演重要角色。我们发现PARK7可通过CMA介导的溶酶体依赖性途径降解。在PD动物模型中，调节CMA自噬-PARK7通路可以抑制神经毒素引发的线粒体形态和功能损害。该研究从线粒体功能的角度阐明了CMA自噬发挥细胞保护作用的新的机制，为研究CMA自噬的在应激条件下对神经元的保护作用开辟了新的方向。MicroRNA是细胞小分子非编码RNA，几乎参与所有重要的细胞生物学过程，但应激如何调节miRNA生成则不清楚。Ⅲ型RNA酶Drosha是调控miRNA合成过程的关键分子。在本项目的开展过程中，我们发现氧化应激通过激活p38MAPK抑制Drosha的功能，导致与神经元存活相关的miRNA表达失调，过表达Drosha 可减轻病理应激诱发的神经元死亡。该研究工作揭示了应激条件下p38 MARK--Drosha--microRNA全新的调节通路，以Drosha 为核心建立细胞稳态应激调节新模式，从全新的角度探索了AD、PD 的发病机制。