Regulation of the Unfolded Protein Response after Acute Brain Injury

描述（由申请人提供）：拟议的研究旨在研究新的分子靶点和过程，以期最大限度地减少脑缺血（CIS）和创伤性脑损伤（TBI）后的脑损伤。目前对抗急性脑损伤的治疗策略在很大程度上是不成功的。我们发现Ca2+依赖性磷酸酶钙调磷酸酶（CN）可以与内质网（ER）中的应激传感器PERK结合，增加其自身磷酸化并增强称为未折叠蛋白反应（UPR）的细胞过程。UPR在压力下减弱蛋白质的翻译，给细胞更多的时间来恢复。值得注意的是，我们的初步数据表明，CN的这种新的保护作用增加了细胞培养中缺血条件下的细胞活力。这项R21提案的目标是开发可用于体内特异性调节PERK自磷酸化的分子干预措施。最终，从该提案中产生的数据将用于描述在CIS和TBI期间调节UPR的治疗潜力。我们的总体假设是，在缺血条件下，CN直接与PERK相互作用，Ca2+作为辅助因子。该蛋白复合物的形成促进PERK二聚化/寡聚化和自磷酸化。这反过来又增强了对蛋白质翻译和细胞活力的抑制，从而减少了损伤后的脑损伤。我们有两个具体目标：1)开发促进、破坏或模拟CN与PERK结合的分子干预措施。2)在体内描述CN结合对PERK的Ca2+依赖性。生化分析和生物物理技术将用于绘制CN和PERK的结合相互作用，并产生肽片段。星形胶质细胞的原代培养将用于测试这些分子干预在体内的功效。共聚焦显微镜将用于成像Ca2+微域的变化在ER附近。氧葡萄糖剥夺，一个体外缺血模型，将被用来确定PERK自磷酸化的生理影响以及我们的分子干预。如果成功，这些肽的开发将成为治疗脑损伤的有吸引力的治疗工具。

DESCRIPTION (provided by applicant): The proposed research investigates novel molecular targets and processes that promise to minimize brain damage after cerebral ischemic stroke (CIS) and traumatic brain injury (TBI). Current therapeutic strategies to combat acute brain injuries have been largely unsuccessful. We discovered that the Ca2+ dependent phosphatase calcineurin (CN) can bind to PERK, a stress sensor in the endoplasmic reticulum (ER), increases its auto-phosphorylation and enhance a cellular process called the Unfolded Protein Response (UPR). The UPR attenuates protein translation during stress and gives the cell more time to recover. Significantly, our preliminary data suggest that this new protective role for CN increases cell viability after ischemic conditions in cell culture. The goal of this R21 proposal i to develop molecular interventions that can be used to specifically regulate PERK auto-phosphorylation in vivo. Ultimately, data generated from this proposal will be used to delineate the therapeutic potential of regulating the UPR during CIS and TBI. Our overall hypothesis is that, under ischemic conditions, CN directly interacts with PERK with Ca2+ as a co-factor. The formation of this protein complex promotes PERK dimerization/oligomerization and auto-phosphorylation. This, in turn, enhances inhibition of protein translation and cell viability, whic reduces brain damage after injury. We have two Specific Aims: 1) Develop molecular interventions that promote, disrupt or mimic CN binding to PERK. 2) Delineate the Ca2+ dependence of CN binding to PERK in vivo. Biochemical assays and biophysical techniques will be used to map the binding interaction of CN and PERK and to generate the peptide fragments. Primary cultures of astrocytes will be used to test the efficacy of these molecular interventions i vivo. Confocal microscopy will be used to image changes in microdomains of Ca2+ near the ER. Oxygen Glucose Deprivation, an in vitro model of ischemia, will be used to determine the physiological impact of PERK auto-phosphorylation as well as our molecular interventions. If successful, the development of these peptides will serve as attractive therapeutic tools for the treatment of brain injuries.