Targeting the integrated stress response to protect oligodendrocyte lineage cells - Resubmission 01

 描述(由申请人提供)：先进的医疗干预措施大大提高了严重早产儿的存活率。不幸的是，弥漫性白质损伤(DWMI)是这些儿童常见的并发症，导致慢性神经功能障碍。目前还没有有效的治疗方法来预防或治疗弥漫性心肌梗塞。对少突胶质细胞前体细胞(OPC)的选择性损伤和由此导致的髓鞘生成障碍是弥漫性心肌梗塞的核心。提高少突胶质细胞系细胞的存活率和功能的策略可能会对临床有益。DWMI是由缺氧、缺血和感染引起的氧化应激、谷氨酸毒性和炎症，这些似乎对OPC具有选择性毒性。整合应激反应(ISR)是一种保守的应激诱导信号通路，可被多种细胞毒性损伤激活并对其提供保护。ISR信号导致真核细胞翻译起始因子2α亚基(EIF2a)的磷酸化，从而抑制整体蛋白质合成和细胞保护基因的选择性表达。这一应用的重点是使用体外和体内模型系统来研究ISR保护OPC免受早产相关压力的能力。我们的假设是，少突胶质细胞ISR活性的增强可以为重度早产儿提供治疗优势，从而改善DWMI。在拟议的研究中，我们将确定DWMI相关的细胞毒性损伤是否激活少突胶质细胞系细胞中的ISR，以及ISR的操作是否改变了这些细胞对细胞毒性损伤的反应。在目标1中，我们将检测OPC在体外对模拟缺氧-缺血的缺氧-葡萄糖剥夺(OGD)或体外间歇性低氧的反应，这两种反应都与DWMI的诱导有关。这些研究将包括从具有基因扰动的小鼠突变中分离出的OPC，这些突变可以减弱或增强细胞的ISR反应。将对OPC的存活和成熟程度进行量化。在目标2中，我们将使用两种DWMI小鼠模型：间歇性低氧模型和炎症模型，这两种模型都会导致中枢神经系统髓鞘形成的延迟。在这些模型中，将使用ISR突变体检测ISR在少突胶质细胞反应中所起的作用：将检测OPC和少突胶质细胞数量，以及髓鞘形成和行为参数。在目标3中，我们将在前两个目标描述的体外和体内模型中确定已被证明可增强/延长ISR反应的药物吧，是否会对OPC提供保护。这些主要药物研究的证据将检验ISR的药理增强将对弥漫性心肌梗死有治疗益处的假设。总之，这些研究将为我们提供相当大的洞察力，了解ISR在寡突胶质细胞对细胞毒性侮辱的反应中所起的作用，这些细胞毒性侮辱被认为是诱导DWMI的关键。重要的是，这项工作有可能为这种毁灭性的神经疾病提供一种新的治疗方法的基础。

 DESCRIPTION (provided by applicant): Advanced medical interventions have resulted in greatly increased survival of severe preterm infants. Unfortunately, diffuse white matter injury (DWMI) is a frequent complication in these children resulting in chronic neurological disability. There are no effective therapies for the prevention or treatment of DWMI. Selective damage to oligodendrocyte progenitor cells (OPCs) and the resultant impaired myelinogenesis are central to DWMI. Strategies that would increase the survival and function of oligodendrocyte lineage cells would likely be of clinical benefit. DWMI is initiated by hypoxia, ischemia, and infection tht lead to oxidative stress, glutamate toxicity, and inflammation, which appear to be selectively toxic to OPCs. The integrated stress response (ISR) is a conserved stress-induced signaling pathway that is activated by, and provides protection to, a variety of cytotoxic insults. ISR signaling leads to the phosphorylation of the a subunit of eukaryotic translation initiation factor2 (eIF2a), resulting in inhibition of global protein synthesis and the selective expression of cytoprotective genes. The focus of this application is to investigate the ISR's ability to protect OPCs from stresses associated with premature birth using in vitro and in vivo model systems. Our hypothesis is that the enhancement of ISR activity in oligodendroglial lineage cells could provide therapeutic benefit to severe preterm infants, resulting in the amelioration of DWMI. In the proposed studies we will determine whether DWMI-associated cytotoxic insults activate the ISR in oligodendroglial lineage cells and whether the manipulation of the ISR modifies the response of these cells to the cytotoxic insults. In aim 1 we will examine the response of OPCs in vitro to either oxygen-glucose deprivation (OGD) that models hypoxia- ischemia or in vitro intermittent hypoxia, both of which are associated with DWMI induction. These studies will include OPCs isolated from mouse mutants with genetic perturbations that either diminish or enhance the cell's ISR response. Survival and maturation of the OPCs will be quantitated. In aim 2 we will use two mouse models of DWMI: an intermittent hypoxia model and an inflammation model, both of which result in delayed CNS myelination of the CNS. The role that the ISR plays in the oligodendroglial cells response in these models will be examined using the ISR mutants: OPC and oligodendrocyte numbers will be examined, as well as myelination and behavioral parameters. In aim 3 we will determine if the drug guanabenz, which has been show to enhance/prolong the ISR response, will provide protection to OPCs in the in vitro and in vivo models described in the first two aims. These proof of principle drug studies will test the hypothesis that the pharmacological enhancement of the ISR will have therapeutic benefit for DWMI. In total, these studies will provide us with considerable insight into the role that the ISR plays in the response of oligodendroglial cells to the cytotoxic insults that are believed to be critical to the induction of DWMI. Importantly, the work has the potential to provide the foundation for a novel therapeutic approach for this devastating neurological disorder.