BRAIN ISCHEMIA--MU-CALPAIN ACTIVITY & EIF-4E DEGRADATION

出现大量神经元超微结构和生化异常 在脑缺血和再灌流期间；然而，病因 这些现象之间的相互关系还没有得到很好的理解。Dr.Neumar 最近发现(1)大脑eIF-4E水平在 (2)钙激活的Mu可使eIF-4E在体外迅速降解。 钙调蛋白。缺血时eIF-4E的蛋白降解可能是由于钙离子的作用。 诱导了Mu-calain的激活。EIF-4E与mRNA的结合是一种速率限制 Mu-calain在eIF-4E翻译起始和降解中的作用 可以将已知的缺血时钙内流现象联系起来 以及随后抑制的蛋白质合成。 提出的假说是：(1)体内缺血和钙超载 体外引起神经元内Mu-calain激活和eIF-4E降解，(2) 缺血诱导的Mu-calain激活和eIF-4E丢失最多 在选择性脆弱的神经元中突出，以及(3)用 钙蛋白酶抑制剂可降低MU-钙蛋白酶自身蛋白水解酶活性和 EIF-4E的降解。Neumar博士将利用这些假说进行研究 (1)大鼠全脑缺血再灌流模型的建立 大鼠停搏复苏及(II)肌内钙超载 神经分化的NB-104细胞。针对eIF-4E和EIF-4E的抗体 激活的Mu-calain将在Western blots中用于表征(I)在 钙离子诱导的培养神经元在大鼠脑缺血再灌注损伤中的作用 再灌流诱导MU-calain激活和eIF-4E降解。灯 电子显微镜和免疫组织化学将提供局部 Mu-calain激活和eIF-4E的超微结构定位 神经元降解与超微结构同步评价 受伤。一种经细胞处理的Mu-calain抑制剂的作用将是 在上述所有实验中进行评估。

Numerous neuronal ultrastructural and biochemical abnormalities develop during brain ischemia and reperfusion; however, the causal interrelationships of these phenomena are not well understood. Dr.Neumar has recently found that (1) brain eIF-4E levels decline sharply during ischemia and (2) eIF-4E is rapidly degraded in vitro by Ca2+-activated mu- calpain. Proteolysis of eIF-4E during ischemia could be due to calcium- induced mu-calpain activation. mRNA binding by eIF-4E is a rate limiting step in translation initiation, and degradation of eIF-4E by mu-calpain could connect the recognized phenomena of calcium influx during ischemia and subsequent depressed protein synthesis. The hypotheses proposed are that (1) ischemia in vivo and Ca2+ overloading in vitro cause mu-calpain activation and eIF-4E degradation in neurons, (2) ischemia-induced activation of mu-calpain and loss of eIF-4E are most prominent in selectively vulnerable neurons, and (3) pretreatment with a calpain inhibitor reduces both mu-calpain autoproteolytic activation and degradation of eIF-4E. Dr. Neumar will study these hypotheses utilizing ((i) an in vivo model of complete brain ischemia and reperfusion by cardiac arrest and resuscitation in rats and (ii) inophore-induced Ca2+ overload in neuronally-differentiated NB-104 cells. Antibodies specific to eIF-4E and activated mu-calpain will be used in Western blots to characterize (i) in cultured neurons Ca2+-induced and (ii) in rat brains ischemia and reperfusion-induced mu-calpain activation and eIF-4E degradation. Light and electron microscopy with immunohistochemistry will provide regional and ultrastructural localization of mu-calpain activation and eIF-4E degradation with simultaneous ultrastructural evaluation for neuronal injury. The effect of a cell-permeant mu-calpain inhibitor will be evaluated in all of the above experiments.