Protein Aggregation after Brain Ischemia

本项目的主要目的是研究缺血后神经元中形成的蛋白质聚集体的毒性。短暂的脑缺血在再灌注约3天时引起海马CA1锥体神经元的迟发性神经元死亡，而留下齿状回神经元基本上完好无损。我们最近获得了强有力的生物化学和形态学证据表明，缺血导致神经元死亡前蛋白质聚集体的急剧和进行性积累，并且这些聚集体在存活的神经元中几乎不存在。最显着的蛋白质聚集体的积累被发现在细胞内的囊泡，内质网（ER），线粒体和树突质膜的膜。通过转基因过表达或缺血预处理诱导神经元中的分子伴侣可防止蛋白聚集体形成和缺血性神经元死亡。基于这些结果，我们提出了一个新的假设缺血性细胞死亡，即缺血后过度生产的未折叠蛋白导致形成不可逆的蛋白质聚集体，最终导致迟发性神经元死亡。具体目标是：（一）。通过共聚焦显微镜、定量EM分析和EM断层扫描进一步表征缺血后蛋白质聚集及其与细胞死亡的关系。（ii）。通过采用已知可增加或减少缺血性细胞死亡量的条件，以及通过使用过表达已知可保护未折叠蛋白免于聚集的分子伴侣的动物，研究蛋白聚集在缺血后神经元细胞死亡中的重要性。（iii）。应用多种生物化学和分子生物学方法研究缺血后蛋白质聚集的机制。临床上几乎没有直接保护神经元免受缺血的药物。短暂的缺血再灌注或不完全缺血导致CA1神经元在短暂缺血后、缺血性卒中的半影区和溶栓治疗后的缺血区域中缓慢类型的选择性神经元死亡。这种迟发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。缺血后蛋白质聚集可能参与缺血性神经元死亡。

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.