Ang-(1-7)通过改善多巴胺能神经元的自噬障碍减轻帕金森病α-syn病理的机制研究

Angiotensin-(1-7)(Ang-(1-7)) has been newly identified as an important component of renin-angiotensin system. Several line of evidence suggests that Ang-(1-7) has protective effect on a rat model of Parkinson's disease, but its role in the pathology of α-synuclein(α-syn) in PD remains unclear. Our preliminary study showed that the level of Ang-(1-7) and its Mas receptor in the substantia nigra of hSYNA53T transgenic mice decreased with the pathological progress of α-syn. Besides, we also found that Ang-(1-7) could regulate the autophagic activity of dopaminergic(DA) neurons in hSYNA53T transgenic mice. Based on these findings, we hypothesized that Ang-(1-7) could alleviate the α-syn pathology by ameliorating the dysfunctional autophagy in DA neurons. In this project, we will firstly investigate the effect of Ang-(1-7) on ethology, α-syn pathology and dysfunctional autophagy in DA neurons by injection of Ang-(1-7) to the substantia nigra of hSYNA53T transgenic mice. In addition, we intend to observe the influence of Ang-(1-7) on autophagic flux and clearance of α-syn by culture of primary DA neurons from hSYNA53T transgenic mice. The completion of this project will not only clarify the role of Ang-(1-7) in the pathology of α-syn in PD and the underlying mechanisms, but also provide a new target for the therapies of this devastating disease.

血管紧张素-(1-7)（Ang-(1-7)）是新发现的肾素-血管紧张素系统的重要活性肽，研究表明其对帕金森病（PD）大鼠模型具有神经保护作用，但在PD的α-突触核蛋白（α-syn）病理中的作用尚不明确。预实验发现：hSYNA53T转基因小鼠黑质Ang-(1-7)及Mas受体含量伴随α-syn的病理进程逐步降低；且Ang-(1-7)可调控此模型小鼠黑质多巴胺（DA）能神经元的自噬水平。我们推测：Ang-(1-7)可通过改善DA能神经元的自噬障碍减轻PD的α-syn病理。本项目拟通过黑质区注射Ang-(1-7)，在体探究其对hSYNA53T转基因小鼠行为学特征、α-syn病理及DA能神经元自噬障碍的影响；而后培养黑质原代神经元，离体研究Ang-(1-7)对DA能神经元自噬流的调控及对α-syn清除的影响，从而阐明Ang-(1-7)对PD的α-syn病理的作用和机制，为PD的治疗提供新思路。

近来研究已证实，中枢肾素-血管紧张素系统与帕金森病（PD）有着密不可分的关系，而其旁路Angiotensin-(1-7) /Mas受体轴与PD的关联也受到日益重视，但具体机制目前尚不明确。PD的特征性病理改变之一是黑质中α-突触核蛋白（α-synuclein）异常聚集所致的路易小体的形成，而α-synuclein的主要降解方式为自噬，因此自噬障碍导致的α-synuclein清除受阻是PD的重要病理机制。我们课题组的前期研究证实，Angiotensin-(1-7)可能参与调控多巴胺能神经元的自噬水平。因此，本研究拟通过制作PD动物和细胞模型，从在体和离体层面探究Angiotensin-(1-7)在PD发病机制中的作用。首先对雄性Sprague-Dawley大鼠腹腔注射鱼藤酮四周来制作PD动物模型。同时，对大鼠右侧黑质连续注射Angiotensin-(1-7)）四周，通过动物行为学实验、免疫组化、蛋白免疫印迹法，观察Angiotensin-(1-7) 对PD大鼠的帕金森病症状及黑质中α-synuclein聚集的影响。接着，从Sprague-Dawley乳鼠黑质中提取原代多巴胺能神经元，经鱼藤酮、Angiotensin-(1-7)和（或）其阻断剂A-779处理24小时后，通过蛋白免疫印迹、caspase-3活性测定、免疫荧光法，观察Angiotensin-(1-7) 对PD细胞模型中神经元的凋亡及α-synuclein聚集的影响。最后，利用鱼藤酮、Angiotensin-(1-7)和（或）自噬抑制剂3-甲基腺嘌呤处理原代多巴胺能神经元24小时，通过蛋白免疫印迹、免疫荧光法，观察Angiotensin-(1-7)对PD细胞模型的自噬障碍及对α-synuclein清除的影响。结果发现：Angiotensin-(1-7)可缓解PD大鼠的帕金森病症状，同时减轻其黑质中α-synuclein的聚集；Angiotensin-(1-7)可通过与Mas受体结合，减轻PD细胞模型中神经元的凋亡及α-synuclein的聚集；Angiotensin-(1-7)可通过改善PD细胞模型的自噬障碍进而增加对α-synuclein的清除。这提示Angiotensin-(1-7)对PD潜在的治疗价值，为PD及其他共核蛋白病的临床干预和药物研发提供了新的靶点和理论依据。