Protein Aggregation after Brain Ischemia

本项目的主要目的是研究在缺血后神经元中形成的蛋白质聚集体的毒性。短暂的脑缺血可导致海马CA1区锥体神经元在再灌流约3天后延迟性死亡，而齿状回神经元基本完好。我们最近获得了强有力的生化和形态学证据，表明缺血导致神经元死亡前蛋白质聚集体的戏剧性和渐进性积累，而在损伤后存活的神经元中几乎不存在这些聚集体。细胞内囊泡膜、内质网、线粒体和树突质膜上蛋白质聚集物的聚集最为明显。通过转基因过表达或缺血预适应诱导神经元中的分子伴侣可以阻止蛋白质聚集体的形成和缺血神经元的死亡。基于这些结果，我们提出了一个新的假说，即缺血后未折叠蛋白的过度产生会导致不可逆的蛋白聚集体的形成，最终导致神经元的迟发性死亡。具体目标是：(一)。通过共聚焦显微镜、定量电子显微镜分析和电子显微镜断层扫描研究缺血后蛋白质的进一步聚集及其与细胞死亡的关系。(Ii)。研究蛋白质聚集在脑缺血后神经细胞死亡中的重要性，方法是利用已知可增加或减少缺血细胞死亡的条件，以及使用过表达已知可保护未折叠蛋白质免于聚集的分子伴侣动物。(三)。用多种生化和分子生物学方法研究脑缺血后蛋白质聚集的机制。在临床上，几乎没有直接保护神经元免受缺血的药物。短时缺血再灌注或不完全缺血可导致短暂缺血后CA1区神经元、缺血性卒中半暗带区神经元和溶栓治疗后缺血区神经元发生慢性型选择性死亡。这种延迟性继发性神经元死亡具有临床意义，因为了解其机制将为预防神经元死亡提供途径。缺血后的蛋白质聚集可能参与了缺血性神经元的死亡。

The main objective of this project is to investigate the toxicity of protein aggregates formed in postischemic neurons. A short period of cerebral ischemia causes delayed neuronal death at about 3 days of reperfusion in hippocampal CA1 pyramidal neurons while leaving dentate gyrus neurons largely intact. We have recently obtained strong biochemical and morphological evidence that ischemia causes dramatic and progressive accumulation of protein aggregates in neurons prior to their death and that these aggregates are virtually absent in the neurons that survive the insult. The most marked accumulation of protein aggregates was found on the membranes of intracellular vesicles, the endoplasmic reticulum (ER), mitochondria and the dendritic plasmalemma. Induction of molecular chaperones in neurons by transgenic overexpression or ischemic preconditioning prevented both protein aggregate formation and ischemic neuronal death. Based on these results, we propose a new hypothesis for ischemic cell death whereby overproduction of unfolded proteins after ischemia causes formation of irreversible protein aggregates that ultimately lead to delayed neuronal death. The specific Aims are: (i). To characterize further protein aggregation after ischemia and its relationship to cell death by confocal microscopy, quantitative EM analysis and EM tomography. (ii). To investigate the importance of protein aggregation in neuronal cell death after ischemia by employing conditions known to increase or decrease the amount of ischemic cell death, and by using animals overexpressing molecular chaperones known to protect unfolded protein from aggregation. (iii). To study the mechanisms of protein aggregation after ischemia using a variety of biochemical and molecular biological methods. There is virtually no drug directly protecting neurons against ischemia in the clinic. A short period of ischemia with reperfusion or incomplete ischemia causes a slow type of selective neuronal death in CA1 neurons after transient ischemia, in the penumbra area in ischemic stroke and in the ischemic region after thrombolytic treatment. This delayed secondary neuronal death has clinical significance because understanding the mechanisms will provide avenues to prevent the neuronal death. The protein aggregation after ischemia may contribute to ischemic neuronal death.