Mechanisms of MAP2 degradation after in vitro ischemia.

描述(改编自申请人的摘要)：本报告的目的 应用是阐明MAP2降解的早期细胞触发因素 在大鼠海马片上进行体外缺血。Map2是一个枢纽 细胞骨架蛋白在缺血后迅速降解，可能起关键作用 在神经元不可逆的形态损伤中的作用，最终导致 神经退行性变。MAP2退化的最早时间点将是 确定的和神经元内发生这种情况的空间位置将 有洞察力。NNDA和内源性谷氨酸受体的作用及其机制 钙的作用将被视为MAP2崩溃的可能触发因素。 将使用共聚焦激光扫描来识别有害钙的来源 显微镜和钙通道阻滞剂。钙调神经磷酸酶和钙蛋白酶的作用 因为负责MAP2分解的酶将通过 使用免疫印迹技术和钙蛋白酶抑制剂测量MAP2水平。 MAP2上磷酸化改变的准确位置将被确定。 利用MALDI-TOF质谱仪及磷酸酶钙调神经磷酸酶的作用 在这种情况下，去磷酸化将使用这种特定的抑制剂进行检测 酵素。这个项目的结果将提供重要的新信息 关于这一关键细胞骨架蛋白的损伤机制，并将进一步 目的是了解细胞骨架的损伤是如何参与细胞死亡的 中风后。

DESCRIPTION (Adapted from applicant's abstract): The objective of this application is to elucidate the early cellular triggers of MAP2 degradation following in vitro ischemia in the rat hippocampal slice. MAP2 is a pivotal cytoskeletal protein that is degraded rapidly after ischemia and may play a key role in the irreversible morphological damage in neurons, leading ultimately to neurodegeneration. The earliest timepoint of MAP2 degradation will be determined and the spatiai location within the neurons where this occurs will be discerned. The role of NNDA and inetabotropic glutamate receptors and the role of calcium will be examined as possible triggers of the MAP2 breakdown. The source of damaging calcium will be identified using confocal laser scanning microscopy and calcium pathway blockers. The roles of calcineurin and calpain as the enzymes responsible for the MAP2 breakdown will be elucidated by measuring MAP2 levels using immunoblotting techniques and calpain inhibitors. The precise location of altered phosphorylation on MAP2 will be identified using MALDI-TOF mass spectrometry and the role of the phosphatase calcineurin in that dephosphorylation will be assayed using specific inhibitors of this enzyme. The results from this project will provide important new information about the mechanism of damage to this key cytoskeletal protein and will further the goal to understand how damage to the cytoskeleton is involved in cell death after stroke.