天冬酰胺内肽酶剪切α-突触核蛋白介导帕金森病发病的机制研究

Recently it has been reported that the truncation of α-synuclein plays important roles in the pathogenesis of Parkinson’s disease (PD). However, the exact mechanisms underlying α-synuclein truncation remain elusive. We found that asparagine endopeptidase (AEP) is upregulated in the substantia nigra of PD patients, where it cleaves α-synuclein and generates the a.a.1-103 fragment of α-synuclein. However, the mechanisms mediating α-synuclein cleavage by AEP in the substantia nigra remain unclear. Moreover, there is no direct evidence demonstrating the cleavage of α-synuclein by AEP plays important roles in PD. In the present project, we will use the in vitro PD models and animal models such as α-synuclein transgenic mice and AEP gene knockout mice, as well as viral vector-mediated overexpression of α-synuclein in the rat substantia nigra. We will apply behavioral tests and classic cell biological and molecular biological methods to investigate the molecular mechanisms of AEP activation and α-synuclein cleavage during aging, to demonstrate the effect of AEP-mediated α-synuclein cleavage on its aggregation and neurotoxicity, and to illustrate the critical role of α-synuclein cleavage in the pathogenesis of PD. The results of the current project will verify the role of AEP in the degeneration of dopaminergic neurons in PD, and provide novel target for the diagnosis and treatment of PD.

新近研究发现α-突触核蛋白的剪切在帕金森病（PD）发病中起到关键作用，但其机制不明。申请人前期研究发现PD患者黑质组织中天冬酰胺内肽酶（AEP）活性升高，并剪切α-突触核蛋白，形成其1-103片段。目前尚不明确多巴胺能神经元内AEP特异性活化，并剪切α-突触核蛋白的机制，也不清楚AEP对α-突触核蛋白的剪切是否直接参与PD的发病。本课题拟采用体外PD模型和α-突触核蛋白转基因小鼠、AEP基因敲除小鼠，以及大鼠黑质立体定位病毒注射等模型，联合运用动物行为学以及经典的细胞、分子生物学方法，重点探讨黑质多巴胺能神经元老化过程中AEP特异性活化并剪切α-突触核蛋白的机制，明确AEP剪切对α-突触核蛋白聚集和神经毒性的影响，以阐明AEP介导的α-突触核蛋白剪切在PD发病中的核心作用。本课题的研究结果将为AEP介导PD多巴胺能神经元损伤提供实验依据，为深入研究PD的早期诊治策略提供新的分子靶标。

帕金森病（Parkinson’s disease, PD）的病因和发病机制不明，其特征性的病理改变为黑质致密部多巴胺能神经元变性缺失，残存的神经元内路易小体形成。α-突触核蛋白的异常聚集在路易小体的形成和神经元损伤中起到关键作用，但其异常聚集的分子机制尚不明确。在本课题中，我们重点研究了天冬酰胺内肽酶（asparagine endopeptidase, AEP）剪切α-突触核蛋白介导PD发病的分子机制。我们发现AEP特异性剪切α-突触核蛋白的N103位点，形成α-syn(1-103)片段。质谱分析和免疫组织化学染色发现PD患者脑内存在大量AEP剪切形成的α-syn(1-103)片段，而且小鼠脑内AEP的活性和α-syn(1-103)片段的表达水平均随年龄的增长而逐渐增高。体外研究发现α-syn(1-103)片段更易聚集形成不溶性的聚集体，导致神经损伤。小鼠脑内过表达α-syn(1-103)片段能够诱导黑质内α-突触核蛋白聚集、多巴胺能神经元损伤和动物行为学障碍，而敲除小鼠AEP的表达能够部分阻断α-突触核蛋白诱导的小鼠PD样病理学、形态学和行为学变化。为了进一步证实AEP对α-突触核蛋白的剪切在PD发病中的作用，我们在小鼠脑内过表达不能被AEP剪切的α-突触和蛋白突变体（N103A），发现N103A突变体能够阻断α-syn(1-103)片段的生成，并减缓α-突触核蛋白在脑内的聚集，减轻小鼠的PD样行为学改变。本研究阐明了AEP通过剪切α-突触核蛋白介导PD发病的分子机制，提示AEP可以作为PD治疗的新靶点。