SENSING HYPOXIA IN THE CNS USING HERPES VECTORS

使用疱疹病毒载体感知中枢神经系统缺氧

基本信息

批准号：
6477039
负责人：
MARC W HALTERMAN
金额：
$ 1.33万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-12-01 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/6477039
关键词：
apoptosis biological signal transduction cerebral ischemia /hypoxia embryo /fetus hypoxia gel mobility shift assay gene expression genetic transcription herpes simplex virus 1 tissue /cell culture transcription factor transfection /expression vector

项目摘要

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.

大脑缺血性损伤会导致严重的发病率围产期以及生命后期几十年的死亡率。对完整动物和体外的研究已经证实中枢神经系统缺血期间发生的极端生理扰动触发依赖于新基因的延迟形式的神经元死亡转录和缺氧引发的基因反应先于激活延迟性细胞凋亡。一种方法是确定新的治疗策略，以保护对抗这种形式的神经元细胞死亡是通过检查指导中枢神经系统缺氧反应基因表达的机制。系统发育上保守的缺氧反应表现在哺乳动物系统通过转录激活和后转录 mRNA 稳定。在外围，转录已知事件是通过缺氧诱导介导的转录因子 HIF-1alpha，与同源顺式元件结合一组受限基因的启动子区域，从而模拟基因转录率。有关使用的信息各种细胞内的这种缺氧反应机制中枢神经系统（神经元、星形细胞和小胶质细胞）的区室及其然而，与凋亡性神经元损失的关系尚不明确。广义上来说，我们假设，在缺血后中枢神经系统缺氧调节的早期基因表达在细胞区室中表现出异质性在 CNS 中，并且该响应触发一个序列，该序列要么直接或间接引起延迟性神经元死亡。我们计划利用缺氧反应性 HSV 病毒载体来绘制区域和隔室内缺氧信号的时间演变缺血性小鼠中枢神经系统。后续研究将利用 HIF 神经元用于表征 HIF 异构体诱导的异构体特异性抗体，细胞定位并通过 EMSA 确认 DNA 结合反应性 supershift 测定均在缺氧条件下进行。我们假设这些实验将表征异质缺氧反应和将定义缺血诱导的中枢神经系统转录中的离散因素激活。我们的长期目标是确定早期应对措施缺血性大脑，随后识别缺氧状态下的调节节点信号级联可以选择性地调节缺氧基因的激活。这些发现将突出新的治疗策略对抗缺氧引起的迟发性神经元死亡。