Deacetylation of mitochondrial proteins protect neurons from ischemic injury

描述（由申请人提供）：氧化应激发生在活性氧（ROS）如超氧化物和过氧亚硝酸盐过量产生时，是缺血性卒中中DNA损伤的主要因素。因此，调控ROS的产生是缺血性卒中治疗的关键靶点。翻译后赖氨酸乙酰化近年来成为基因表达和酶活性的重要调节因子。赖氨酸残基被一组乙酰转移酶乙酰化，这些乙酰转移酶通过它们在细胞区室中的定位获得特异性。乙酰基的去除（或去乙酰化）是由去乙酰化酶催化的，包括sirtuins。Sirtuins是一个依赖于nad的去乙酰化酶家族，与代谢、细胞存活机制和寿命改变有关。在已知的sirtuins（或Sirts）中，有三个位于线粒体内（Sirt3， -4和-5）。特别是Sirt3调节线粒体蛋白的乙酰化水平，我们将研究Sirt3去乙酰化酶活性如何减少氧化损伤。我们最近报道Sirt3降低超氧阴离子水平，防止线粒体去极化，减少暴露于NMDA诱导的神经元死亡。此外，新的初步数据表明Sirt3在体外缺血模型中具有保护作用。我们假设Sirt3依赖性保护通过调节抗氧化防御的酶的去乙酰化起作用，并且增加Sirt3活性通过增强这些抗氧化系统来促进神经元存活。目前，唯一批准的治疗急性中风的方法是溶栓，不幸的是，这增加了脑出血和进一步脑损伤的风险。因此，鉴定和表征Sirt3的保护作用，对鉴定小分子调节剂和药物具有重要意义

DESCRIPTION (provided by applicant): Oxidative stress occurs with excessive generation of reactive oxygen species (ROS) like superoxide and peroxynitrite, and is a principal factor in the damage to DNA that occurs in ischemic stroke. Therefore, regulation of ROS production is a key target for ischemic stroke therapy. Post-translational lysine acetylation has recently emerged as an important regulator of gene expression and enzyme activity. Lysine residues are acetylated by a group of acetyltransferases which gain specificity through their localization in cellular compartments. Removal of the acetyl group (or deacetylation) is catalyzed by deacetylases, including the sirtuins. Sirtuins are a family of NAD-dependent deacetylases that have been implicated in metabolism, cell survival mechanisms, and alteration of life span. Of the known sirtuins (or Sirts), three are localized within the mitochondria (Sirt3, -4, and -5). In particular Sirt3 regulates acetylation level of mitochondrial proteins, and we will study how Sirt3 deacetylase activity reduces oxidative injury. We recently reported that Sirt3 reduces superoxide anion levels, prevents mitochondrial depolarization and reduces neuronal death induced by exposure to NMDA. Further, new preliminary data implicates Sirt3 in protection in an in vitro ischemia model. We hypothesize that Sirt3-dependent protection works through deacetylation of enzymes that regulate antioxidant defenses, and that increasing Sirt3 activity promotes neuronal survival by enhancing these antioxidant systems. Currently, the only approved treatment for acute stroke is thrombolysis, which unfortunately increases the risk of brain hemorrhage and further brain injury. Therefore, it would be of tremendous benefit to identify and characterize the protective effect of Sirt3 in order to identify small molecule modulators and drug design for treatment intervention. We will use cultured mouse cortical neurons exposed to oxygen and glucose deprivation (or OGD) to simulate ischemic stroke (to identify mechanisms), and a mouse in vivo stroke model (for translational studies). We will characterize a novel mechanism to increase Sirt3 protein and activity through activation of AMPK. In animals, we will compare the difference in ROS production between normal mice and mice deficient of Sirt3 protein (Sirt3 knockout, or Sirt3-ko), and if these Sirt3-ko mice are more vulnerable to ischemic injury. In cell culture, we will characterize the role of Sirt3 by comparing the effect of OGD in normal, Sirt3-deficient, and Sirt3 overexpressing cells. In Sirt3-deficient cells, we will test the effect of re-introducing normal Sirt3, inactive Sirt3, or non-mitochondrial Sirt3. The goals of thi project are to determine 1) if Sirt3 reduces brain injury and behavioral deficits after MCAo, 2) if Sirt3 regulates reactive oxygen species (ROS) levels with OGD injury in cultured mouse cortical neurons, 3) how Sirt3 enhances antioxidant defenses in cultured mouse cortical neurons, and 4) how Sirt3 reduces ischemic injury in a mouse stroke model.