Imaging and functional regulation of microenvironment in subcellular organelle

过量谷氨酸作为一种兴奋毒性诱导剂，在急性和慢性神经元细胞死亡（包括缺血、创伤、亨廷顿病、阿尔茨海默病和肌萎缩性侧索硬化症）中起重要作用。尽管经过多年的研究，谷氨酸诱导的兴奋性毒性神经元细胞死亡的确切机制尚未得到很好的表征。钙调磷酸酶（CaN）和钙蛋白酶（Calcineurin, Ca^<2+>/钙调蛋白（CaM）依赖性蛋白磷酸酶和Ca^<2+>依赖性半胱氨酸蛋白酶）分别被认为是参与神经元细胞死亡的两个最重要的Ca^<2+>依赖性效应因子。尽管can介导的神经元细胞死亡机制仍有争议，但钙调磷酸酶引发退行性神经元线粒体功能障碍的一种机制已得到强烈支持。另一方面，钙调磷酸酶通过切割细胞骨架蛋白调节生理信号转导。膜蛋白和酶，也被认为在神经细胞死亡的一种形式中起着重要作用，涉及多种疾病的发病机制，如脑损伤、阿尔茨海默病和局灶性或全局性脑缺血。calpain的一些底物，如细胞周期蛋白依赖性激酶5（Cdk5）的特异性激活剂p35，已被认为是诱导神经元细胞死亡的关键分子。在本研究中，我们发现在谷氨酸神经兴奋毒性过程中，钙调磷酸酶A（calcalpain）在体内和体外直接被钙蛋白酶裂解，导致该酶转化为活性形式。质谱分析鉴定出了三个裂解位点，其中两个为本构活性位点。裂解的CnA过表达可诱导半胱天冬酶活性和神经元细胞死亡。钙蛋白酶抑制剂不仅能阻断钙调磷酸酶的裂解，而且在体外和体内对神经元细胞死亡具有保护作用。这些结果表明，钙调神经磷酸酶是钙蛋白酶的一个重要靶点，钙蛋白酶介导的钙调神经磷酸酶的激活会引发兴奋性神经变性。这进一步说明了磷酸酶和蛋白酶在病理生理上的直接串扰。少

Excessive glutamate as an excitotoxic inducer plays an important role in acute and chronic neuronal cell death including ischemia, trauma, Huntington's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Despite years of investigation, the precise mechanism involved in glutamate-induced excitotoxic neuronal cell death has not been well characterized.Calcineurin(CaN) and calpain, a Ca^<2+>/calmodulin (CaM)-dependent protein phosphatase and a Ca^<2+>-dependent cysteine protease, respevtively, are thought to be two of the most important Ca^<2+>-dependent effectors involved in neuronal cell death. Although the mechanism of CaN-mediated neuronal cell death remains controversial, one proposed mechanism that calcineurin triggers dysfunction of the mitochondria in degenerative neurons has been strongly supportedOn the other hand, calpain regulates physiological signal transduction by cleaving cytoskeletal proteins, membrane proteins and enzymes and is also thought to play a critic … More al role in a form of neuronal cell death involved in the pathogenesis of several diseases such as brain injury, Alzheimer's disease and focal or global cerebral ischemia. Some substrates of calpain such as p35, a specific activator of cyclin-dependent kinase 5(Cdk5) have been suggested as key molecules in the induction of neuronal cell death.In this research, we showed that during glutamate neuroexcitotoxicity, calcineurin A(CnA) is directly cleaved by calpain in vitro and in vivo, resulting in the enzyme being converted to an active form. Mass spectrometry identified the three cleavage sites in CnA and two of them were constitutively active forms. Overexpression of the cleaved CnA induced caspase activity and neuronal cell death. Calpain inhibitors not only blocked the cleavage of calcineurin, but also protected against neuronal cell death in vitro and in vivo. These results indicate that calcineurin is a crucial target for calpain and the calpain-mediated activation of calcineurin triggers excitotoxic neurodegeneration. This further shows the direct cross talk between phosphatase and protease in pathopysiology. Less