SENSING HYPOXIA IN THE CNS USING HERPES VECTORS

脑缺血损伤导致大量的发病率在 围产期以及生命后期几十年的死亡率。 在完整动物身上以及在体外的研究已经证实 中枢神经系统缺血时的极端生理扰动 触发依赖于新基因的迟发性神经元死亡 转录和缺氧引发的基因反应先于 激活延迟性凋亡细胞死亡。一种方法是 确定新的治疗策略，以保护 对这种形式的神经细胞死亡的治疗是通过检查 中枢神经系统低氧反应基因表达的调控机制。 在系统发育上保守的低氧反应在 哺乳动物系统通过转录激活和后... 转录信使核糖核酸稳定。在外围，转录 已知的是，这些事件是通过缺氧诱导的 转录因子，HIF-1α，与同源顺式元件结合 一组受限基因的启动子区域，从而模拟 基因转录速率。关于利用的信息 这种缺氧反应机制在不同的细胞内 中枢神经系统(神经元、星形胶质细胞和小胶质细胞)及其 然而，缺乏与神经细胞凋亡性丢失的联系。大体上， 我们假设在缺血后早期中枢神经系统低氧调节 基因表达在细胞内表现出异质性。 并且该响应触发了一个序列，该序列 直接或间接引起迟发性神经元死亡。我们计划 利用低氧响应性HSV病毒载体绘制区域和 脑室内低氧信号的时间演变 缺血小鼠中枢神经系统。后续研究将利用HIF神经元 亚型特异性抗体用于表征HIF亚型诱导， 通过EMSA进行细胞定位和确认DNA结合活性 超移位分析都是在低氧条件下进行的。我们假设 这些实验将表征异质性低氧反应和 将确定缺血诱导的中枢神经系统转录中的离散因子 激活。我们的长期目标是在 脑缺血并随后确定低氧中的调节节点 可以选择性地调节低氧基因激活的信号级联。 这些发现将突出指导的新的治疗策略 抗缺氧所致迟发性神经元死亡。

Ischemic damage to the brain results in substantial morbidity during the perinatal period as well as mortality in the later decades of life. Studies in the intact animal as well as in vitro have established that the extreme physiologic perturbations which occur during CNS ischemia trigger a delayed form of neuronal death which is dependent on new gene transcription and that hypoxia triggered gene responses precede activation of delayed apoptotic cell death. One approach to the identification of novel therapeutic strategies which would protect against this form of neuronal cell death is through examination of the mechanisms which direct hypoxia responsive gene expression in the CNS. The phylogenetically conserved hypoxia response is manifest in the mammalian systems through transcriptional activation and post- transcriptional mRNA stabilization. In the periphery, transcriptional events are known to be mediated through the hypoxia inducible transcription factor, HIF-1alpha, which binds cognate cis elements in the promoter region of a restricted set of genes thereby simulating the rate of gene transcription. Information regarding the utilization of this hypoxia responsive mechanism within the various cellular compartments of the CNS (neuronal, astrocytic and microglial) and their relationship to apoptotic neuronal loss is lacking, however. Broadly, we hypothesize that early in the post-ischemic CNS hypoxic-regulated gene expression exhibits heterogeneity within the cellular compartments in the CNS and that this response triggers a sequence which either directly or indirectly elicits delayed neuronal death. We plan to exploit hypoxia responsive HSV viral vectors to map the regional and temporal evolution of hypoxic signaling within the compartments of the ischemic murine CNS. Subsequent studies will utilize HIF neuronal isoform specific antibodies to characterize HIF isoform induction, cellular localization and confirm DNA binding reactivity through EMSA supershift assays all under hypoxic conditions. We hypothesize that these experiments will characterize heterogeneous hypoxic response and will define discrete factors in ischemia induced CNS transcriptional activation. Our long-term goals are to identify early responses in the ischemic brain and subsequently identify regulatory nodes in the hypoxic signal cascade which can selectively modulate hypoxia gene activation. Such findings will highlight novel therapeutic strategies directed against hypoxia induced delayed neuronal death.