Stress Proteins in Brain Cell Injury

描述(申请人提供)：中风是世界范围内主要的死亡原因之一，也是导致长期残疾的主要原因。尽管许多临床试验都是针对中风患者的，但到目前为止，只有溶栓疗法成为一种有效的治疗方法。尽管如此，新的治疗靶点已经出现。长期以来，钙调节失调一直与中风时的脑损伤有关。我们之前在这笔赠款上的工作以及其他人的工作表明，热休克蛋白70 kDa(HSP70)家族的伴侣蛋白或应激蛋白，即使在缺血后表达，也可以保护大脑。BCL2家族蛋白对细胞凋亡的调控是决定缺血结局的另一个重要因素。这些途径共同控制细胞内钙稳态以及细胞的凋亡和坏死性死亡。内质网(ER)和线粒体在称为线粒体相关ER膜(MAM)的特定位置相互作用，以调节细胞钙稳态和细胞死亡。MAM是应激/伴侣蛋白和BCL2家族相互作用的关键部位，是决定脑缺血预后的关键因素。我们最近发现，过表达内质网伴侣和HSP70家族成员GRP78可以保护线粒体功能，减少线粒体钙超载，并改善应激后脑细胞的存活。我们还发现，在星形胶质细胞中，GRP78在应激后移位到线粒体内膜，这是已知对神经元生存不可或缺的中枢神经系统胶质细胞。在本提案的目标1中，我们将通过比较野生型和线粒体靶向的GRP78来研究易位的GRP78在线粒体功能中的作用。MiRNAs转录后基因沉默的发现导致了人类疾病中新的假说的爆炸性增长。关于miRNAs在脑缺血中的研究是最近的，大多数都集中在miRNAs的轮廓变化上。我们最近报道，减少或阻断miR-181，一种富含大脑的miRNA，在损伤后的最初阶段保护大脑免受中风的影响。目的2是一个翻译目标，我将继续这些研究，以确定改变miR-181对中风患者长期行为结果的影响，测试治疗后，并确定miR-181是否对雌性动物也有效。我们最近证明miR-181可以靶向GRP78和抗凋亡的BCL2家族成员BCL2和MCL1。因此，我们将研究miR-181在MAM内质网-线粒体钙转运中的作用。使用计算的miRNA靶标预测，我们确定miR-200可能靶向GRP75、线粒体伴侣和BCL2。这项建议的目的3将重点放在通过降低miR-181水平来保护的机制上，详细研究对内质网和线粒体钙离子以及线粒体功能的影响。总体而言，这一提议检验了一个新的假设：miRNAs作为伴侣和BCL2家族成员的主要调节者，影响脑缺血后的钙稳态、线粒体-ER串扰和预后。

DESCRIPTION (provided by applicant): Stroke is one of the leading causes of death worldwide and a major cause of long-term disability. Although many clinical trials have targeted stroke patients, only thrombolysis has so far emerged as an effective treatment. Nonetheless, new therapeutic targets have emerged. Calcium dysregulation has long been implicated in brain injury in the setting of stroke. Our prior work on this grant, as well as work from others, has shown that the heat shock protein 70 kDa (HSP70) family of chaperones or stress proteins, even if expressed after ischemia, can protect the brain. The regulation of apoptosis by the BCL2 family of proteins is another important determinant of ischemic outcome. These pathways converge to control cellular calcium homeostasis and both apoptotic and necrotic cell death. The endoplasmic reticulum (ER) and mitochondria interact at specific sites called the mitochondrial associated ER membrane (MAM) to regulate cellular calcium homeostasis and cell death. MAM is a critical site of stress/chaperone protein and BCL2 family interaction, and is central to determining the outcome from cerebral ischemia. We recently found that overexpressing GRP78, an ER chaperone and HSP70 family member, preserves mitochondrial function, reduces mitochondrial Ca2+ overload, and improves brain cell survival after stress. We also found that GRP78 translocates to the mitochondrial inner membrane after stress in astrocytes, CNS glial cells that are known to be integral to neuronal survival. In Aim 1 of this proposal we will investigate the role of translocated GRP78 in mitochondrial function by comparing wild type and mitochondrially targeted GRP78. The discovery of posttranscriptional gene silencing by miRNAs has led to an explosion of new hypotheses in human disease. Studies of miRNAs in cerebral ischemia are recent, and most have focused on profiling changes in miRNAs. We recently reported that reducing or blocking miR-181, a brain-enriched miRNA, protects the brain from stroke in the initial post-injury period. Aim 2 is a translational aim and ill follow up these studies to determine the effects of altering miR-181 on long term behavioral outcome from stroke, test post-treatment, and determine whether miR-181 is also effective in female animals. We recently demonstrated that miR-181 can target both GRP78 and anti-apoptotic BCL2 family members BCL2 and MCL1. Therefore, the role of miR-181 in ER-mitochondrial calcium transfer in MAM will be studied. Using computational miRNA target prediction we identified miR-200 as potentially targeting GRP75, a mitochondrial chaperone, and BCL2. Aim 3 of this proposal will focus on the mechanism of protection by reducing miR-181 levels investigating in detail effects on ER and mitochondrial Ca2+, and mitochondrial function. Overall this proposal exams a novel hypothesis: that miRNAs act as master regulators of chaperones and BCL2 family members influencing Ca2+ homeostasis, mitochondria-ER crosstalk, and outcome after cerebral ischemia.