β-arrestin2通过D1R信号通路参与帕金森病异动症发生的机制研究

Levodopa (L-dopa) is the gold standard for symptomatic treatment of Parkinson’s disease (PD), but long-term therapy is associated with the emergence of L-dopa-induced dyskinesia (LID). The management of LIDs is an ongoing challenge in PD treatment. Since the neurobiological mechanisms of LIDs remain unclear, clinical pharmacologically intervention is restricted. Our previous studies indicate that LID is associated with sensitized D1R transmission located on the medium spiny neurons (MSNs) of the direct striatonigral pathway. Sensitized D1R transmission may also results in augmented synthesis of DARPP32/PKA/ERK. Meanwhile, previous studies demonstrated that β-arrestin2 desensitized G protein signaling at D1R and initiates receptor endocytosis or internalization. In this study, we aim to investigate β-arrestin2 in the treatment of L-dopa–induced dyskinesia via D1R signal pathway. To begin with, detect the distribution and level of β-arrestin2, and immunofluorescence used to detect the co-expression of β-arrestin2 and D1R. Then, we will explore the influence of viral rescue or overexpression of β-arrestin2 in terms of the behavior alteration and its key biochemical markers. In addition, genetic deletion of β-arrestin2 whether or not can markedly enhancing the dyskinesia-like effects of chronic L-dopa treatment is also need to be proved? Finally, we want to test the results of modulate β-arrestin2 in the treatment of L-dopa–induced dyskinesia can be reversed by D1R agonists or antagonists. This study will further explain the mechanism of LIDs and provide theory evidences for β-arrestin2 modulator therapy for Parkinson’s disease with dyskinesia.

左旋多巴是治疗帕金森病（PD）最有效的方法，但长期左旋多巴治疗后出现的异动症（LID）严重影响患者的生活质量。在前期我们研究发现D1R超敏在LID的发生机制过程中扮演着重要的角色，且与DARPP32/PKA/ERK信号通路活性改变有关。β-arrestin2可以将D1R脱敏产生抗异动症的作用，然而对β-arrestin2 参与 LID的机制研究很少。本课题拟探讨β-arrestin2通过影响D1R信号通路活性参与LID的发生机制，包括动物行为学、受体表达、信号转导的不同层面。通过观察β-arrestin2在LID发生过程中的表达变化，了解其与 D1R 共同表达情况。分别过表达和基因沉默β-arrestin2观察大鼠行为学及D1R表达和信号通路的改变。最后探究β-arrestin2 影响 LID 的症状能否被 D1R 所逆转，及其下游信号通路的变化，为开发治疗LID的药物提供理论基础。

左旋多巴（L-DOPA）是治疗帕金森病（PD）的有效药物，但是90%的患者在长期使用L-DOPA后会出现异动症(Levodopa -induced dyskinesia，LID)等运动并发症，严重影响了药物疗效和患者的生活质量。本文通过研究β-arrestin2作为GPCR信号通路中关键的负性调节蛋白在LID发生发展过程中的具体作用。我们发现在21天的行为学AIM评分中，Sham组与PD组评分比较无统计学意义（P>0.05）,LID组与Sham组、PD组相比其AIM评分明显升高，差异有统计学意义(P<0.05)；且LID组在注射L-DOPA+苄丝肼后，避开对侧旋转高峰期，其前肢功能相较于PD组明显改善，差异有统计学意义(P<0.05)。AAV-EGFP组的AIM评分与LID组接近（P>0.05）; 过表达β-arrestin2+/+组的AIM评分较LID组与AAV-EGFP组明显下降(P<0.05)；LID组、β-arrestin2+/+、AAV-EGFP组在注射L-DOPA+苄丝肼后，避开症状高峰期，其前肢功能较PD组明显改善(P<0.05)。此外，免疫荧光染色检测TH在PD组与LID组的表达明显减少，进一步证明帕金森病模型制备成功。免疫荧光双重标记染色检测背侧纹状体中β-arrestin2与D1R或D2R共同表达情况显示β-arrestin2与D1R和D2R均存在共同表达情况，这种表达在D1R阳性神经元中比在D2R阳性神经元中增加近2.5倍，提示β-arrestin2更多可能通过直接通路发挥作用。Western blot及免疫组织化学染色检测β-arrestin2蛋白在各组大鼠纹状体区表达情况发现，与Sham组、PD组相比，β-arrestin2蛋白在LID组中的表达明显下调，而Sham组、PD组中β-arrestin2蛋白的表达无明显差异。与Sham组、PD组相比，β-arrestin2蛋白在LID组与AAV-EGFP组中的表达下调，此外，磷酸化DARPP32与磷酸化EPK1/2在LID组与AAV-EGFP组中的表达升高，在β-arrestin2+/+组中表达较LID组与AAV-EGFP组下调；Sham组、PD组中β-arrestin2蛋白的表达无明显差异。同时发现β-arrestin2+/+对于LID行为学的改善，可被D1R激动剂所逆转（SKF38393），