TRPM2在全脑缺血/再灌注损伤引发海马CA1区神经元迟发性死亡中的作用

As an important member of TRP channel family, transient receptor potential melastatin 2 (TRPM2) is activated by oxidative stress such as ADPR, H2O2 and some other oxidative metabolites, and permeat calicum and zinc. TRPM2 is highly expressed in central nervous system, which implies an important role of TRPM2 in neuronal disease such as AD, ischemia and so on. Our previous studies have shown that TRPM2 knocking-out could significantly decrease the number of hippocampal delayed neuronal death caused by global ischemia or reperfusion injury. The H2O2 induced hippocampal neuronal intracellular zinc release that was mediated by TRPM2 channel has also been observed. It has been reported that calcium overloading and zinc accumulation are key factors in delayed neuronal death in hippocampal CA1 area caused by global ischemia or reperfusion injury, but its exact mechanism is still unclear. TRPM2 knocking-out mice and kinds of techniques such as fluorescence imaging, electronicphysiology, pharmacology, biological chemistry and behavior testing will be employed in this project, hoping to elaborate the role of TRPM2 in global ischemia or reperfusion caused hippocampal delayed neuronal death, which will find a new potential clinical therapeutic target for brain ischemia in future.

Transient receptor potential melastatin 2（TRPM2）属于TRP家族，主要受氧化应激产物ADPR和H2O2等激活，并通透钙离子和锌离子。TRPM2在中枢神经系统中高表达，提示其在神经系统中具有重要功能。申请人前期研究显示TRPM2敲除小鼠能显著减少全脑缺血/再灌注损伤引发的海马神经元迟发性死亡。研究还发现TRPM2介导了H2O2诱导海马神经元胞内锌离子释放现象。已知钙超载和锌离子累积在全脑缺血导致神经元迟发性死亡中具有关键作用，但具体机制并不清楚。本项目拟用TRPM2基因敲除小鼠，组合荧光成像、电生理，药理学，生化和行为学等实验手段，从细胞、脑片和整体动物水平研究TRPM2与全脑缺血/再灌注损伤引发锌离子累积和钙超载的关系，阐明TRPM2在全脑缺血/再灌注损伤导致海马神经元迟发性死亡中的作用及其机制，为今后临床治疗脑缺血疾病提供潜在的有效治疗靶点。

Transient receptor potential melastatin 2（TRPM2）属于TRP家族，主要受氧化应激产物ADPR和H2O2等激活，并通透钙离子和锌离子。TRPM2在中枢神经系统中高表达，提示其在神经系统中具有重要功能。本项目通过使用TRPM2基因敲除小鼠，并组合荧光成像、电生理，药理学，生化和行为学等实验手段，从细胞、脑片和整体动物水平研究TRPM2与全脑缺血/再灌注损伤中引发锌离子累积和钙超载的关系。体内、体外缺血再灌注建模结果显示，TRPM2基因敲除可有效减少[Zn2+]c、活性氧生成，CA1神经元死亡以及缺血后的功能认知障碍。延时成像结果显示，TRPM2缺陷对缺血阶段诱导的[Zn2+]c增加没有影响，但是可以显著减少再灌注阶段胞质Zn2+累积以及活性氧的诱导的[Zn2+]c增加。这些结果首次证明了TRPM2通道在再灌注过程中[Zn2+]c激活和CA1神经元迟发性死亡中有关键作用。接下来，我们为了证明TRPM2在缺血/再灌注损伤中的作用是否与线粒体自噬相关，检测了两条经典的线粒体自噬通路（PINK1/Parkin和Bnip3/Nix），但结果显示这两条通路并未参与到TRPM2在海马CA1神经元迟发死亡过程中。除此之外，我们进一步在Camk II Cre/TRPM2 FloxP小鼠（选择性在前脑兴奋性神经元敲除TRPM2小鼠）进行全脑缺血/再灌注损伤，结果显示该小鼠能够显著减轻海马CA1区锥体神经元死亡，进一步明确TRPM2在这一过程中具有重要作用。本项目阐明了TRPM2通道是在缺血再灌注阶段引起了胞内锌离子增高,进而介导神经元迟发性死亡的机制，为今后临床治疗脑缺血疾病提供新的治疗靶点。