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，弥漫性白色物质损伤（DWMI）是一个常见的并发症，在这些儿童导致慢性神经功能障碍。没有有效的治疗方法来预防或治疗DWMI。选择性损伤少突胶质细胞祖细胞（OPC）和由此产生的受损髓鞘是核心DWMI。增加少突胶质细胞系细胞的存活和功能的策略可能具有临床益处。DWMI由缺氧、缺血和感染引发，导致氧化应激、谷氨酸毒性和炎症，这些似乎对OPCs具有选择性毒性。整合应激反应（ISR）是一种保守的应激诱导的信号通路，其被多种细胞毒性损伤激活并提供保护。ISR信号转导导致真核生物翻译起始因子2（eIF 2a）α亚基的磷酸化，从而抑制整体蛋白质合成和细胞保护基因的选择性表达。本申请的重点是使用体外和体内模型系统研究ISR保护OPC免受与早产相关的应激的能力。我们的假设是，在少突胶质细胞系细胞ISR活性的增强，可以提供治疗的好处，严重早产儿，在DWMI的改善。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。在目的1中，我们将检查OPCs在体外对模拟缺氧-缺血的氧-葡萄糖剥夺（OGD）或体外间歇性缺氧的反应，这两者都与DWMI诱导相关。这些研究将包括从具有减少或增强细胞ISR反应的遗传扰动的小鼠突变体中分离的OPC。将对OPC的存活和成熟进行定量。在目标2中，我们将使用两种DWMI小鼠模型：间歇性缺氧模型和炎症模型，这两种模型均导致CNS的CNS髓鞘形成延迟。将使用ISR突变体检查ISR在这些模型中在少突胶质细胞反应中发挥的作用：将检查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，我们将确定已显示增强/延长ISR反应的药物胍那苄是否将在前两个目标中描述的体外和体内模型中为OPC提供保护。这些原理药物研究的证据将检验ISR的药理学增强将对DWMI产生治疗获益的假设。总的来说，这些研究将为我们提供相当大的洞察的作用，ISR在响应少突胶质细胞的细胞毒性损伤，被认为是至关重要的诱导DWMI。重要的是，这项工作有可能为这种毁灭性的神经系统疾病的新治疗方法提供基础。

 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.