Department of Physiology and Medical Physics, Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
J Neurosci. 2012 Feb 1;32(5):1847-58. doi: 10.1523/JNEUROSCI.2345-11.2012.
Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.
谷氨酸受体过度摄取 Ca(2+)导致的兴奋毒性导致中风、创伤和癫痫发作后的神经元损伤。细胞内 Ca(2+)水平的升高激活了一组具有木瓜蛋白酶样活性的钙依赖性蛋白酶，即钙蛋白酶。在这里，我们研究了钙蛋白酶激活在 NMDA 诱导的兴奋性毒性损伤中的作用，这种损伤发生在胚胎期（E16-E18）的鼠皮质神经元中，这些神经元：
经历了兴奋性坏死，其特征是 Ca(2+)稳态的即刻失调、线粒体膜电位（Δψ(m)）的持续去极化以及对 bax 基因缺失不敏感；
经历了兴奋性细胞凋亡，其特征是 NMDA 诱导的细胞内 Ca(2+)增加的恢复、对 bax 基因缺失的敏感性以及延迟的 Δψ(m)去极化和 Ca(2+)失调；
对兴奋性毒性损伤具有耐受性。
有趣的是，用钙蛋白酶抑制剂 calpeptin 处理、过表达内源性钙蛋白酶抑制剂 calpastatin 或基因沉默钙蛋白酶可保护神经元免受兴奋性细胞凋亡，但对兴奋性细胞坏死没有影响。calpeptin 在 bax 缺失神经元中没有发挥保护作用，但对 bid 缺失神经元的保护作用与野生型细胞相似。为了确定钙蛋白酶在兴奋性细胞凋亡过程中何时被激活，我们使用钙蛋白酶敏感的Förster 共振能量转移探针通过延时荧光显微镜监测钙蛋白酶的激活动力学。我们观察到延迟的钙蛋白酶激活，它发生在线粒体参与之后，直接发生在神经元死亡之前。相比之下，我们在兴奋性细胞坏死或对兴奋性毒性损伤具有耐受性的神经元中没有检测到明显的钙蛋白酶活性。
在器官型海马切片培养物中进行的氧/葡萄糖剥夺诱导损伤证实，钙蛋白酶仅在 bax 依赖性细胞凋亡中被特异性激活，并且在这种情况下作为下游细胞死亡执行者发挥作用。
