SK通道与Orai1途径的反馈调控关系对帕金森病多巴胺能神经元钙离子稳态的影响机制

In the early stage of pathological changes of Parkinson’s disease, the imbalance of calcium homeostasis in dopaminergic (DA) neurons is considered to be one of the key factors triggering apoptosis. Previous studies have shown that the Store Operated Ca2+ Entry (SOCE) pathway is directly related to DA neurons with endoplasmic reticulum stress, calcium overload and other important cellular regulatory processes. We previously have found that SK channel currents were enhanced after treatment of 6-OHDA in rat DA neurons; knocking down the expression of STIM1 and Orai1, which are important molecules in SOCE pathway, could decrease the oxidative stress; preliminary results indicated that the expression of Orai1 and SK channels coexisted in DA neurons. Together with reported results, we hypothesize that the SOCE pathway, represented by STIM1-Orai1 in this study, may have feedback regulatory relationship with SK channels, which fine tune the cellular calcium homeostasis, affect neuronal stress responses, modulate the firing pattern and influence the cell death and survival. This study manage to study the role of SOCE pathway in endoplasmic reticulum stress and the dynamic changes of cellular calcium concentration, to explore the potential feedback relationship between SK channels and STIM1-Orai1 pathway in regulating cellular calcium homeostasis, and to discover the effect and underlying mechanism of neuronal protection to DA neurons in PD pathogenesis. This study will provide novel theoretical and experimental basis for recognizing the role of SK channels and STIM1-Orai1 pathway in pathogenesis in Parkinson’s diseases.

在帕金森病病理过程中，多巴胺（DA）能神经元的钙离子稳态失衡被认为是引起细胞死亡的重要触发因素。既往研究表明，钙库操纵的钙通道（SOCE）途径与DA能神经元内质网应激、钙超载等重要过程直接相关。我们前期发现，在6-OHDA处理的大鼠DA能神经元中，SK通道电流有增强；而下调SOCE途径Stim1-Orai1的表达后，DA能神经元氧化应激水平降低；免疫染色提示在DA能神经元中，Orai1与SK通道共存表达。结合以往研究，我们推测以STIM1-Orai1为代表的SOCE途径可能与SK通道之间存在反馈调节，从而参与调控细胞Ca2+稳态和细胞应激反应，最终对细胞存活死亡产生影响。本课题拟从SK通道与STIM1-Orai1途径在病理状态下与Ca2+稳态反馈调控联系入手，探索其对DA能神经元的保护作用。这将为认识SK通道与STIM1-Orai1途径在帕金森病理过程中的作用和机制提供新的理论和实验依据。

在帕金森病病理过程中，多巴胺（DA）能神经元的钙离子稳态失衡被认为是引起细胞死亡的重要触发因素。既往研究表明，钙库操纵的钙通道（SOCE）途径与DA能神经元内质网应激、钙超载等重要过程直接相关。我们前期发现，在6-OHDA处理的大鼠DA能神经元中，SK通道电流有增强。基于上述分析及前期预实验结果，本课题借助我们在帕金森病模型大鼠SNc DA能神经元电生理领域积累的研究基础，在SNc脑片、培养的DA 能细胞和PC12细胞层面上，通过PD造模药物6-OHDA的处理，采用全细胞膜片钳技术、脑片培养技术、钙离子成像技术、免疫组化/激光共聚焦技术等，探讨了6-OHDA造模条件下SNc DA能神经元中以 STIM1-Orai1为代表的SOCE途径与SK通道之间的反馈调控关系。我们的结果表明：下调SOCE途径Stim1-Orai1的表达后，DA能神经元氧化应激水平降低；免疫染色提示在DA能神经元中，Orai1与SK通道共存表达。在6-OHDA处理情况下，钙库中大量内源性的Ca2+释放进入细胞质中，而钙库Ca2+储存的耗竭激活了STIM1-Orai1通路为代表的SOCE途径；该途径参与到SNc DA能神经元的内质网应激过程中，改变了正常 Ca2+内环境稳态的调节，促进了 Ca2+大量内流进入胞浆，最终引发细胞死亡。而激活的SK通道则与Orai1途径之间存在反馈调控关系，通过调控Orai1表达、阻断其与SK通道之间的反馈途径，进而减弱内源性钙库释放的 Ca2+离子流，最终维护了神经元钙离子稳态，从而发挥神经元的保护作用。本课题从SK通道与STIM1-Orai1途径在病理状态下与Ca2+稳态反馈调控联系入手，验证了其对DA能神经元的保护作用。这为深入理解SK通道与STIM1-Orai1途径在帕金森病理过程中的作用和机制提供了新的理论和实验依据。