Deacetylation of mitochondrial proteins protect neurons from ischemic injury

描述（由申请人提供）：氧化应激伴随活性氧（ROS）（如超氧化物和过氧亚硝酸盐）的过度生成而发生，是缺血性卒中中发生的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最近已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基转移酶乙酰化，乙酰基转移酶通过其在细胞区室中的定位获得特异性。乙酰基的去除（或脱乙酰化）由脱乙酰酶催化，包括沉默调节蛋白。Sirtuins是NAD依赖性脱乙酰基酶家族，其与代谢、细胞存活机制和寿命的改变有关。在已知的sirtuins（或Sirtuins）中，有三种位于线粒体内（Sirt 3、Sirt 4和Sirt 5）。特别是Sirt 3调节线粒体蛋白的乙酰化水平，我们将研究Sirt 3脱乙酰酶活性如何减少氧化损伤。我们最近报道，Sirt 3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Sirt 3在体外缺血模型中具有保护作用。我们假设Sirt 3依赖性保护作用通过调节抗氧化防御的酶的脱乙酰化起作用，并且增加Sirt 3活性通过增强这些抗氧化系统来促进神经元存活。目前，唯一批准的急性中风治疗方法是溶栓，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Sirt 3的保护作用以鉴定小分子调节剂和药物组合物将是非常有益的。 设计治疗干预措施。我们将使用培养的小鼠皮质神经元暴露于氧和葡萄糖剥夺（或OGD），以模拟缺血性中风（以确定机制），和小鼠体内中风模型（用于翻译研究）。我们将描述一种通过激活AMPK来增加Sirt 3蛋白和活性的新机制。在动物中，我们将比较正常小鼠和Sirt 3蛋白缺陷小鼠（Sirt 3敲除或Sirt 3-ko）之间ROS产生的差异，以及这些Sirt 3-ko小鼠是否更容易受到缺血性损伤。在细胞培养中，我们将通过比较OGD在正常、Sirt 3缺陷和Sirt 3过表达细胞中的作用来表征Sirt 3的作用。在Sirt 3缺陷细胞中，我们将测试 重新引入正常Sirt 3、失活Sirt 3或非线粒体Sirt 3的效果。该项目的目标是确定1）Sirt 3是否减少MCAo后的脑损伤和行为缺陷，2）如果 Sirt 3在培养的小鼠皮层神经元中调节OGD损伤的活性氧（ROS）水平，3）Sirt 3如何增强培养的小鼠皮层神经元中的抗氧化防御，以及4）Sirt 3如何减少小鼠中风模型中的缺血性损伤。 公共卫生相关性：缺血性中风是美国第三大死亡原因和残疾的主要原因。目前，唯一批准的急性中风治疗方法是溶栓，不幸的是，这会增加脑出血和进一步脑损伤的风险。这将是巨大的好处，缺血性中风的细胞死亡机制的特点。我们建议研究Sirt 3活性如何减少中枢神经系统的氧化损伤，以开发预防和治疗缺血性损伤的新方法。

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. PUBLIC HEALTH RELEVANCE: Ischemic stroke is the third leading cause of death and a leading cause of disability in the United States. Currently, the only approved treatment for acute stroke is thrombolysis, which unfortunately increases the risk of brain hemorrhage and further brain injury. It would be of tremendous benefit to characterize cell death mechanisms in ischemic stroke. We propose to study how Sirt3 activity reduces oxidative injury in the CNS in order to develop novel approaches to prevent and treat ischemic injury.