DA神经元α-syn蛋白积聚诱发的ER应激与HtrA2/Nurr1分子调控紊乱的的机制研究

Aberrant α-synuclein aggregation in Dapamnergic Neuron (DAergic neuron) is a pathological marker of Parkinson's disease.Much molecular including Nurr1 and HtrA2 may participate in the process of Dopaminergic cell appotosis through endoplasmic reticulum stress (ER) in DAergic cells. We found: ①6-OHDA resulted in HtrA2 dysfunction, mediated α-synuclein aggregation related ER stress in DAergic neuron. The α-synuclein aggregation blocked Wnt signal pathway and resulted in degeneration of DAergic neuron. ② Nurr1 plays key role in DAergic cell's development, survival and its functional maintain and Nurr1 defection results in DAergic dysfunction of Neurons impacted by environment toxin. However, the molecular mechanism and their interaction relationship among the genes such as HtrA2,α-synuclein and Nurr1 in Parkinson disease is still far from clear. In this project, using PD cell and animal models such as HtrA2，Nurr1+/- iPS cell and HtrA2-/-mice as well as Nurr1+/- mice, we plan to analyze the association of Wnt/GSK3β pathway with α-synuclein aggregation mediated by ER stress from HtrA2 dysfunction and Nurr1 deficiency, and detect the molecule interaction between α-synuclein, HtrA2 andNurr1. We hope to deeply underst the process of Dopaminergic cell appotosis and the mechanism of Wnt/GSK3β pathway mediated by ER stress from HtrA2 and Nurr1 in DAergic neuron and assess their neural protection by these target molecules.

α-synuclein在DA神经元内积聚是帕金森病的病理学标志，多种分子参与DA神经元的内质网（ER）应激。我们发现：①Nurr1是DA神经元抗凋亡的关键基因，其缺损增加神经元对毒素的敏感性。②6-OHDA损伤DA神经元的HtrA2功能，使α-syn蛋白积聚，诱发ER应激，阻止Wnt信号的细胞内传导，GSK3β活性升高，导致DA神经元变性。但我们对α-syn、HtrA2、Nurr1及Wnt/GSK3β等分子在细胞凋亡过程中的作用及其相互之间的调控关系并不完全清楚。本项目以人HtrA2、Nurr1缺陷iPS细胞和HtrA2-/-小鼠、Nurr1+/-杂合鼠等为基础，通过毒素干预和基因沉默等方法，分析不同条件下，模型细胞内α-syn积聚/细胞变性介导的ER应激的分子途径，检测上述信号分子的相互调控关系，从而揭示重要分子参与DA神经元凋亡的ER应激的分子机制及关键靶点的神经保护作用。

我们在筛查α-突触核蛋白（a-synuclein）磷酸化相关基因变异的基础上，构建了PD离体/在体模型，应用分子、蛋白、免疫组化等技术，分析了DA神经元内α-突触核蛋白积聚介导细胞变性的规律，以及HtrA2、α-syn、Nurr1等分子之间的相互作用关系，从而揭示了HtrA2、Nurr1、Wnt等重要分子参与DA神经元变性过程中ER应激反应的分子病理学机制：①首次证明双向特异性酪氨酸磷酸化调节性激酶1A(DYRK1A) rs8126696-T可能是中国男性罹患PD危险因素，首次发现IGF1基因rs972936-C增加PD的罹患风险；②在6-OHDA损伤DA神经元变性过程中，证实铁沉积促进a-synuclein聚集及神经元之间扩散、传递，溶酶体内a-synuclein降解障碍及自噬流受阻；③在DA神经元ER应激反应过程中，相关标志蛋白Bip/Grp78和CHOP表达上调，HtrA2、Nurr1、Wnt等分子功能下调，证实HtrA2具潜在的神经修复功能；④证明α-syn聚集将阻止细胞内Wnt信号通路传导，介导关键信号分子GSK3β磷酸化，而Wnt重组蛋白则对抗凋亡，恢复细胞功能，这种保护作用不仅激活Wnt通路，还可影响Bax、Bcl-2水平，同时抑制ER应激相关蛋白Bip和CHOP表达。总之，我们的研究结果揭示了DA神经元变性过程中ER应激的分子信号通路及其神经保护机制，明确了HtrA2功能异常对细胞内蛋白积聚/降解的影响，以及HtrA2和α-syn、Nurr1等重要分子之间的相互调控关系，证明α-syn等蛋白积聚介导的ER应激可能取决于DA神经元内HtrA2、Nurr1等基因的表达水平及功能，Wnt通路信号分子在级联反应的重要作用，以及HtrA2、Wnt/GSK3β/β-catenin及Nurr1为靶点的神经保护作用。