纹状体NK1受体通过受体-受体作用机制减少多巴胺D1受体内化参与帕金森病异动症

Levodopa and associated drugs-induced dyskinesia has limited their use in the treatment of Parkinson’s disease (PD). Pulsatile dopamine stimulation and hypersensitive D1 receptor induced changes of signal conduction play a pivotal role in the emergence of levodopa-induced dyskinesia (LID). Under the support of National Natural Foundation of China, we found that nigral substance P is involved in the occurrence of LID by enhancing dopamine release through a positive feedback, in which presynaptic mechanism was involved. But whether postsynaptic substance P receptor (NK1 receptor) is involved in LID remains unknown. Recently, we found that NK1 and D1 receptor in postsynaptic membrane of striatum formed complex by coimmunoprecipitation. Co-labeling test showed that co-localization of both in the striatum. Additionally, NK1 and D1 receptor in the postsynaptic membrane of striatum are simultaneously increased in mouse models of LID, whereras intrastriatal injection of NK1 antagonist reduces the occurrence of LID. So we hypothesized that NK1 receptor was involved in the occurrence of LID by reducing internalization of D1 receptor via receptor-receptor mechanism, namely postsynaptic mechanism. In order to test this hypothesis, the present proposal will investigate the mechanism of postsynaptic NK1 receptor in the occurrence of LID by in vitro and in vivo studies (using coimmunoprecipitation, double staining, electrophysiology and other technologies) , which will provide a new target in the treatment of LID.

左旋多巴类药物治疗帕金森病（PD）导致的异动症（LID）是其治疗的难点，波动性多巴胺刺激和多巴胺D1受体敏感性异常升高及其介导的信号转导通路改变在LID的发生中处于核心地位。我们在上一国家自然科学基金资助下发现黑质P物质通过与多巴胺分泌间形成正反馈参与LID的发生，这主要涉及突触前机制，而突触后P物质受体（NK1受体）是否参与LID的发生尚不清楚。近期我们利用免疫共沉淀发现纹状体突触后膜NK1受体与D1受体形成复合物，免疫双标提示二者共表达，LID小鼠模型纹状体突触后膜上NK1及D1受体的表达同时升高，纹状体内微注射NK1受体拮抗剂减少了LID的发生。因此我们提出如下假说：NK1受体通过受体-受体机制减少D1受体的内化参与LID的发生，为验证此假说，本课题拟采用免疫共沉淀、免疫双标、神经电生理等技术从体内外两方面深入探讨突触后膜NK1受体在LID发生中的具体机制，为治疗LID提供新的靶点。

帕金森病（PD）是一种典型的神经变性疾病，重复左旋多巴治疗后导致的异动症（LID）是其治疗的难点，其发生机制与纹状体的可塑性改变有关。在本自然科学基金的资助下我们发现PC12细胞中NK1受体（NK1R）与TH共表达，给予PC12细胞NK1受体拮抗剂后，PC12细胞中络氨酸羟化酶（TH）、钙离子/钙调素依赖性蛋白激酶Ⅱ（CaMKⅡ）的磷酸化水平降低，NK1R拮抗剂减少PC12细胞中DA的释放量，敲减PC12细胞中的NK1R后，我们发现PC12细胞中磷酸化的CaMKⅡ、TH水平均显著降低，并且钙离子的内流减少。同时，神经电生理结果则显示，P物质可以诱发钙内流，而NK1R拮抗剂可阻断P物质诱发的钙内流。在黑质纹状体脑片中我们发现突触后膜中NK1受体与D1受体形成复合物，免疫双标提示二者共表达，而给予不同剂量的NK1受体拮抗剂后，纹状体中多巴胺D1受体介导的信号转导道路中相关分子标记物如细胞外激酶（ERK1/2）以及环磷腺苷效应元件结合蛋白（CREB）等即早基因的表达下调，同时我们给予PD小鼠重复左旋多巴处理后，发现PD小鼠呈现出经典的异动症症状，而且，LID小鼠模型中纹状体突触后膜上NK1及D1受体的表达同时升高，而给予NK1受体拮抗剂可以减少PD小鼠的AIM评分，与此同时，NK1受体拮抗剂并未影响左旋多巴的抗PD作用，这表明NK1受体拮抗剂是一种理想的抗异动症药物。最后，我们发现NK1受体拮抗剂减少PD小鼠纹状体中相关信号转导分子如磷酸化DARPP-32、ERK1/2、GluR1以及FosB的表达。以上结果表明纹状体NK1受体参与了异动症的发生，而拮抗NK1受体有助于减少异动症的发生。