Deacetylation of mitochondrial proteins protect neurons from ischemic injury

描述(由申请人提供)：氧化应激伴随着超氧化物和过氧亚硝酸盐等活性氧物种(ROS)的过度产生而发生，是缺血性中风中发生的DNA损伤的主要因素。因此，调节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是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基转移酶乙酰化，这些乙酰基转移酶通过定位于细胞室而获得特异性。去乙酰基(或去乙酰化)是由包括sirtuins在内的去乙酰酶催化的。Sirtuins是一类依赖NAD的脱乙酰酶家族，参与新陈代谢、细胞生存机制和寿命的改变。在已知的sirtuins(或Sirts)中，有三个定位于线粒体(SIRT3、-4和-5)。特别是SIRT3调节线粒体蛋白质的乙酰化水平，我们将研究SIRT3脱乙酰酶活性如何减少氧化损伤。我们最近报道，SIRT3可降低超氧阴离子水平，防止线粒体去极化，并减少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依赖的保护作用是通过调节抗氧化防御的酶的脱乙酰化来发挥作用的，并且增加SIRT3的活性通过增强这些抗氧化系统来促进神经元的存活。目前，唯一被批准的急性中风治疗方法是溶栓治疗，不幸的是，这会增加脑出血和进一步脑损伤的风险。因此，鉴定和表征SIRT3的保护作用对于鉴定小分子调节剂和药物将大有裨益。 治疗干预设计。我们将使用培养的小鼠皮质神经元暴露在缺氧和葡萄糖剥夺(或OGD)中来模拟缺血性中风(以确定机制)，并使用小鼠体内中风模型(用于翻译研究)。我们将描述一种通过激活AMPK来增加SIRT3蛋白和活性的新机制。在动物中，我们将比较正常小鼠和SIRT3蛋白缺陷小鼠(SIRT3基因敲除，或SIRT3-KO)小鼠之间的ROS产生的差异，以及这些SIRT3-KO小鼠是否更容易受到缺血损伤。在细胞培养中，我们将通过比较OGD在正常、SIRT3缺陷和SIRT3过表达细胞中的作用来表征SIRT3的作用。在缺乏SIRT3的细胞中，我们将测试 重新引入正常SIRT3、失活SIRT3或非线粒体SIRT3的效果。这个项目的目标是确定1)SIRT3是否减少了MCAO后的脑损伤和行为缺陷，2)如果 3)SIRT3如何增强培养的小鼠皮质神经元的抗氧化防御能力，以及4)SIRT3如何减少小鼠中风模型中的缺血性损伤。

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.