Role of Protein Phosphatase-1 in Cerebral Ischemia and Cell Death

描述（由申请人提供）：由于心脏骤停导致的全脑缺血导致神经功能缺损，需要昂贵的长期医疗保健。尽管有这种重大的临床和经济影响，目前还没有具体的药物治疗。全脑缺血与广泛的坏死和凋亡性细胞死亡有关;这些过程受到几种机制的严格调控，包括蛋白磷酸化的关键作用。虽然蛋白激酶参与控制全脑缺血中的细胞死亡是公认的，但蛋白磷酸酶的作用受到的关注相对较少。蛋白磷酸酶-1是丝氨酸/苏氨酸蛋白磷酸酶亚家族的成员，其参与细胞死亡的调节。我们已经鉴定并纯化了哺乳动物脑中蛋白磷酸酶-1的几种新的多聚体形式。其中一种称为蛋白磷酸酶-1IC（PP-1IC）的活性在猪和狗的全脑缺血模型以及缺血的细胞培养模型中被体内激活。基于这些新的发现，我们假设PP-1IC是连接全脑缺血和细胞死亡的信号转导通路的一个组成部分。为了确定PP-1IC的激活机制和在全脑缺血中的作用，我们建议纯化和表征PP-1IC全酶从对照和缺血猪前脑心脏骤停复苏和再灌注后。将通过质谱法测定从对照和缺血脑中纯化的天然PP-1IC的分子组成，并将通过体外重建鉴定的组分来研究PP-1IC调节的机制。将开发PP-1和其他试剂的特异性膜渗透抑制剂，并用于确定PP-1IC在脑缺血细胞培养模型中作为细胞死亡介质的作用。本研究基于以下假设探讨PP-1IC缺血激活的机制：依赖性蛋白激酶II（CaMKII）和/或蛋白激酶C4（PKC 4）作为上游调节剂起作用。这些研究将阐明调节脑中天然PP-1全酶活性的生理和病理生理机制，并确定PP-1在全脑缺血中控制细胞死亡的作用。这种功能性蛋白质组学方法的目标是开发合理的基于机制的治疗方法，以减轻缺血性脑细胞死亡，并具有临床转化的长期目标。公共卫生相关性：由于心脏骤停引起的全脑缺血导致神经功能损害，需要昂贵的长期医疗保健。尽管有巨大的社会，医学和经济影响，目前没有具体的药物治疗。这些研究将阐明调节脑中天然蛋白磷酸酶-1全酶的生理和病理生理机制，并确定它们在全脑缺血中控制细胞死亡的作用。该方法旨在开发基于合理机制的蛋白磷酸酶-1抑制剂，以减轻缺血性细胞死亡和神经损伤。

DESCRIPTION (provided by applicant): Global cerebral ischemia due to cardiac arrest results in debilitating neurological impairment necessitating costly long-term health care. Despite this major clinical and economic impact, there is currently no specific medical therapy. Global cerebral ischemia is associated with extensive necrotic and apoptotic cell death; these processes are tightly regulated by several mechanisms, including a critical role for protein phosphorylation. While the involvement of protein kinases in the control of cell death in global cerebral ischemia is well established, the role of protein phosphatases has received relatively little attention. Protein phosphatase-1 is a member of the serine/threonine protein phosphatase subfamily that has been implicated in the regulation of cell death. We have identified and purified several novel multimeric forms of protein phosphatase-1 in mammalian brain. The activity of one of these, termed protein phosphatase-1IC (PP-1IC), is activated in vivo in pig and dog models of global cerebral ischemia as well as in cell culture models of ischemia. Based on these novel findings, we hypothesize that PP-1IC is a component of the signal transduction pathways that link global cerebral ischemia to cell death. In order to determine the mechanisms of activation and the role of PP-1IC in global cerebral ischemia, we propose to purify and characterize the PP-1IC holoenzyme from control and ischemic pig forebrain following cardiac arrest with resuscitation and reperfusion. The molecular compositions of native PP-1IC purified from control and ischemic brain will be determined by mass spectrometry, and the mechanisms of PP-1IC regulation will be studied by reconstitution of the identified components in vitro. Specific membrane permeable inhibitors of PP-1 and other reagents will be developed and used to determine the role of PP-1IC as a mediator of cell death in cell culture models of cerebral ischemia. The mechanisms of ischemic activation of PP-1IC will be investigated based on the hypothesis that the Ca2+regulated protein kinases, Ca2????dependent protein kinase II (CaMKII) and/or protein kinase C4 (PKC4) function as upstream regulators. These studies will elucidate physiological and pathophysiological mechanisms that regulate native PP-1 holoenzyme activity in brain and define the role of PP-1 in the control of cell death in global cerebral ischemia. This functional proteomics approach is targeted to the development of rational mechanism-based therapies to attenuate ischemic brain cell death, with a long-term goal of clinical translation. PUBLIC HEALTH RELEVANCE: Global cerebral ischemia due to cardiac arrest results in debilitating neurological impairment necessitating costly long-term health care. Despite an immense social, medical and economic impact, there is currently no specific pharmacological therapy. The proposed studies will elucidate physiological and pathophysiological mechanisms that regulate native protein phosphatase-1 holoenzymes in brain and determine their role in the control of cell death in global cerebral ischemia. This approach is targeted to the development of rational mechanism-based protein phosphatase-1 inhibitors to attenuate ischemic cell death and neurological injury.