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一个主要健康问题。缺血性细胞死亡的一个显著特征是易损细胞蛋白质合成受到不可逆转的抑制。内质网(ER)功能受损被认为是缺血导致翻译停止的机制。我们的假设是，脑缺血后内质网功能受损和随后在脆弱神经元中发现的长期蛋白质合成抑制在短暂性脑缺血引发的病理过程中发挥重要作用，最终导致神经细胞死亡。到目前为止，这一假设仅基于相关证据：仍缺乏对该假设的证明。我们的具体目的是验证这样一种假设，即通过促进蛋白质合成的恢复和内质网功能的恢复，可以减少缺血细胞死亡的程度。为此，我们将利用短暂性缺血后脆弱神经元中发现的代谢模式，在这种模式下，蛋白质合成受到严重抑制，应激基因的转录被激活。我们将用转基因神经细胞进行实验，这些基因结构展示了三种不同的成分，将确保恢复功能所需的蛋白质在缺血后特异性地合成。这些构建体将包含编码一种蛋白质的基因，该蛋白质被认为有助于蛋白质合成的恢复和ER功能的恢复，一个序列将在与蛋白质合成抑制相关的条件下激活相应mRNA的翻译，以及一个启动子，该启动子具有一个共同序列，用于结合已知的在缺血后激活的转录因子。编码潜在保护性蛋白的基因将包括针对ER、Gadd34和GRP78的Bcl-2，我们将使用具有热休克因子结合序列的启动子。稳定转染的细胞随后将暂时暴露在缺氧/葡萄糖剥夺中，这是一种严重的应激形式，会导致代谢紊乱，类似于短暂性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抵御代谢应激条件。这个拟议的项目将使我们能够建立所需的条件性基因表达平台，以在未来的动物实验中确认缺血诱导的内质网功能受损和随后关闭的翻译在导致神经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脑和其他器官中与整体蛋白质合成抑制和转录因子激活相关的各种病理状态下神经细胞死亡的机制。 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，中风影响着美国70多万公民，导致每年超过16万人死亡，每年直接和间接成本为550亿美元。这项拟议的项目旨在阐明缺血/中风导致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，以改善美国的公共健康，从而降低与中风治疗相关的成本。

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.