Shutdown of Translation and Ischemia/Stroke-Induced Cell Death

描述（由申请人提供）：缺血/卒中是全球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脆弱细胞中蛋白质合成的不可逆抑制。内质网（ER）功能受损已被确定为缺血诱导的翻译关闭的机制。我们的假设是，缺血后ER功能受损以及随后在脆弱神经元中发现的蛋白质合成的长期抑制在短暂性脑缺血引发并最终导致神经元细胞死亡的病理过程中发挥着重要作用。到目前为止，这一假设仅基于相关证据：仍然缺乏对该假设的证明。我们的具体目的是检验这一假设，即缺血性细胞死亡的程度可以通过促进蛋白质合成的恢复和ER功能的恢复来降低。为此，我们将利用短暂缺血后脆弱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。我们将进行实验与神经元细胞转染基因结构表现出三个不同的组件，将保证恢复功能所需的蛋白质是专门合成后缺血。所述构建体将包含编码被认为促进蛋白质合成的恢复和ER功能的恢复的蛋白质的基因、在与抑制蛋白质合成相关的条件下激活相应mRNA翻译的序列、以及具有用于结合已知在缺血后被激活的转录因子的共有序列的启动子。编码潜在保护性蛋白的基因包括靶向ER的Bcl-2、GADD 34和GRP 78，我们将使用具有热休克因子结合序列的启动子。然后将稳定转染的细胞短暂暴露于氧/葡萄糖剥夺，这是一种严重的应激形式，导致代谢紊乱，模拟由短暂缺血诱导的代谢紊乱。然后，我们将研究蛋白质合成的恢复和ER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条件基因表达平台，以在未来的动物实验中确认缺血诱导的ER功能受损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，条件基因表达的方法将使我们能够阐明神经元细胞死亡的机制，在各种病理状态的大脑和其他器官与全球蛋白质合成的抑制和转录因子的激活。 公共卫生相关性：中风是发病率和死亡率的主要原因，根据美国心脏协会的数据，中风影响美国超过70万公民，每年导致超过16万人死亡，每年直接和间接费用为550亿美元。该项目旨在阐明缺血/中风引起的神经细胞死亡的机制。因此，该项目将有助于建立新的治疗干预途径，以改善美国的公共卫生，从而降低与中风治疗相关的费用。

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.