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

许多神经元超微结构和生化异常发展 在脑缺血和再灌注期间;然而， 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-4 E水平在 缺血和（2）eIF-4 E在体外被Ca 2+激活的mu- 钙蛋白酶 缺血期间eIF-4 E的蛋白水解可能是由于钙- 诱导的μ-钙蛋白酶激活。 mRNA与eIF-4 E的结合是一个限速因子， 翻译起始中的步骤和μ-钙蛋白酶对eIF-4 E的降解 可以将缺血期间钙内流的公认现象 随后抑制蛋白质合成。 所提出的假说是：（1）体内缺血和钙超载 体外引起神经元中μ-钙蛋白酶激活和eIF-4 E降解，（2） 缺血诱导的μ-钙蛋白酶激活和eIF-4 E丢失是最常见的 突出的选择性脆弱的神经元，和（3）预处理与 钙蛋白酶抑制剂降低μ-钙蛋白酶自身蛋白水解激活和 eIF-4 E的降解。 Neumar博士将研究这些假设， ((i)完全脑缺血和心脏再灌注的体内模型 在大鼠中的停搏和复苏和（ii）在大鼠中的无机载体诱导的Ca 2+超载 神经元分化的NB-104细胞 eIF-4 E特异性抗体和 活化的μ-钙蛋白酶将用于蛋白质印迹以表征（i）在 培养的神经元Ca ~（2+）诱导;（ii）大鼠脑缺血， 再灌注诱导的μ-钙蛋白酶活化和eIF-4 E降解。 光 电子显微镜和免疫组织化学将提供 μ-钙蛋白酶激活和eIF-4 E的超微结构定位 神经元降解同时进行超微结构评价 损伤 细胞渗透性μ-钙蛋白酶抑制剂的作用将是 在所有上述实验中进行了评估。

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.