Shutdown of Translation and Ischemia/Stroke-Induced Cell Death

描述（由申请人提供）：缺血/中风是世界范围内的主要健康问题。缺血性细胞死亡的一个突出特征是易损细胞中蛋白质合成的不可逆抑制。内质网（ER）功能的损害已被确定为缺血导致翻译关闭的机制。我们的假设是，在易损神经元中发现的缺血后内质网功能损伤和随后的蛋白质合成长期抑制在短暂性脑缺血引发的病理过程中起重要作用，最终导致神经元细胞死亡。迄今为止，这一假设仅基于相关的证据：对这一假设的证明仍然缺乏。我们的具体目的是验证一个假设，即通过促进蛋白质合成的恢复和内质网功能的恢复可以减少缺血性细胞死亡的程度。为此，我们将利用在短暂缺血后易损神经元中发现的代谢模式，其中蛋白质合成受到严重抑制，应激基因的转录被激活。我们将对神经细胞进行实验，这些神经细胞转染了具有三种不同成分的遗传结构，这些成分将保证在缺血后特异性合成恢复功能所需的蛋白质。这些构建体将包含一种蛋白质的基因编码，这种蛋白质被认为可以促进蛋白质合成的恢复和内质网功能的恢复，一种序列可以在蛋白质合成受到抑制的情况下激活相应mRNA的翻译，以及一种启动子，该启动子具有已知在缺血后激活的转录因子结合的一致序列。编码潜在保护蛋白的基因将包括靶向内质网的Bcl-2、GADD34和GRP78，我们将使用具有热休克因子结合序列的启动子。然后，稳定转染的细胞将短暂地暴露于缺氧/葡萄糖剥夺中，这是一种严重的应激形式，会引起代谢紊乱，类似于短暂缺血引起的代谢紊乱。然后，我们将研究蛋白质合成的恢复和内质网功能的恢复是否确实有助于细胞承受代谢应激条件。该项目将使我们能够建立所需的条件基因表达平台，在未来的动物实验中，证实缺血诱导的内质网功能损伤和随后的翻译关闭在导致神经元细胞死亡的病理过程中的作用。此外，条件基因表达方法将使我们能够阐明大脑和其他器官的各种病理状态下神经元细胞死亡的机制，这些死亡与抑制全局蛋白合成和转录因子的激活有关。

DESCRIPTION (provided by applicant): Ischemia/stroke presents a major health problem worldwide. A prominent feature of ischemic cell death is an irreversible suppression of protein synthesis in vulnerable cells. Impairment of endoplasmic reticulum (ER) function has been identified as the mechanism underlying the shutdown of translation induced by ischemia. Our hypothesis has been that the post-ischemic impairment of ER function and subsequent long-lasting suppression of protein synthesis found in vulnerable neurons play an important role in the pathological process triggered by transient cerebral ischemia and culminating in neuronal cell death. To date this hypothesis has been based only on correlative evidence: a proof of the hypothesis is still lacking. Our Specific Aim is to test the hypothesis that the extent of ischemic cell death can be reduced by facilitating a recovery of protein syn- thesis and restoration of ER function. To this end, we will take advantage of the metabolic pattern found in vulnerable neurons after transient ischemia where protein synthesis is severely suppressed and transcription of stress genes is activated. We will perform experiments with neuronal cells transfected with genetic constructs exhibiting three distinct components that will guarantee that the protein required to restore function is synthesized specifically after ischemia. The constructs will contain the gene coding for a protein that is believed to facilitate recovery of protein synthesis and restoration of ER function, a sequence that will activate translation of the respective mRNA under conditions associated with suppression of protein synthesis, and a promoter with a consensus sequence for the binding of transcription factors known to be activated after ischemia. The genes coding for a potentially protective protein will include Bcl-2 targeted to the ER, GADD34, and GRP78, and we will use promoters with binding sequences for heat shock factors. Stably transfected cells will then be transiently exposed to oxygen/glucose deprivation, a severe form of stress causing metabolic disturbances that mimic those induced by transient ischemia. We will then investigate whether recovery of protein synthesis and restoration of ER function do indeed help cells to withstand the metabolic stress conditions. The proposed project will allow us to establish the conditional gene expression platform needed to confirm, in future animal experiments, the role of ischemia-induced impairment of ER function and subsequent shutdown of translation in the pathological process resulting in neuronal cell death. Furthermore, the conditional gene expression approach will enable us to elucidate mechanisms of neuronal cell death in various pathological states of the brain and other organs associated with suppression of global protein synthesis and activation of transcription factors. PUBLIC HEALTH RELEVANCE: Stroke is a major cause of morbidity and mortality, which according to the American Heart Association affects more than 700,000 citizens in the United States, resulting in more than 160,000 deaths per year and $55 billion direct and indirect annual costs. The proposed project is designed to elucidate the mechanisms underlying neuronal cell death caused by ischemia/stroke. The project will thus help to establish new avenues of therapeutic intervention to improving public health in the United States and therefore reducing the costs associated with stroke treatments.