Regulation of the Unfolded Protein Response after Acute Brain Injury

这项拟议的研究调查了新的分子靶点和过程，有望最大限度地减少大脑 缺血性脑卒中（CIS）和创伤性脑损伤（TBI）后的损害。当前的治疗策略 治疗急性脑损伤的方法在很大程度上是不成功的。我们发现依赖钙离子的 磷酸酶钙调神经磷酸酶（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质反应的细胞过程 （普遍定期审议）。UPR在应激期间减弱蛋白质翻译，并使细胞有更多的时间恢复。 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活力， 细胞培养物中的缺血条件。这项R21提案的目标是开发分子干预措施， 可用于在体内特异性调节PERK自身磷酸化。最终，由此产生的数据 建议将用于描绘在CIS和TBI期间调节UPR的治疗潜力。我们 总的假设是，在缺血条件下，CN直接与PERK相互作用，其中Ca 2+作为辅因子。 这种蛋白质复合物的形成促进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，从而减少了脑损伤。 损伤我们有两个具体目标：1）开发促进，破坏或模拟CN的分子干预措施 绑定到PERK。2)描绘了体内CN与PERK结合的Ca 2+依赖性。 生物化学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物将用于测试以下的功效： 这些分子干预在体内。共聚焦显微镜将被用来成像微区的变化 Ca 2+在ER附近。氧葡萄糖脱氢酶，一种体外缺血模型，将用于确定 PERK自身磷酸化的生理影响以及我们的分子干预。如果成功， 这些肽的开发将作为治疗脑损伤的有吸引力的治疗工具。

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 is 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, which 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 in 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.