Deacetylation of mitochondrial proteins protect neurons from ischemic injury

线粒体蛋白的去乙酰化可保护神经元免受缺血性损伤

基本信息

批准号：
8837070
负责人：
Conrad Alano
金额：
$ 33.25万
依托单位：
NORTHERN CALIFORNIA INSTITUTE/RES/EDU
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8837070
关键词：
Acetylation Acetyltransferase Address Animal Model Animals Antioxidants Behavioral Boxing Brain Injuries Brain hemorrhage Caloric Restriction Cause of Death Cell Culture Techniques Cell Death Cell Nucleus Cell Survival Cells Cessation of life Citric Acid Cycle Cytosol DNA DNA Damage Data Deacetylase Deacetylation Defense Mechanisms Drug Design Enzymes Excision Exposure to Family Gene Expression Generations Glucose Glutathione Goals In Vitro Infarction Injury Intervention Ischemia Ischemic Stroke Isocitrate Dehydrogenase Knock-out Longevity Lysine Mediating Metabolism Mitochondria Mitochondrial Proteins Modeling Mus N-Methylaspartate Nervous System Trauma Neurons Oxidants Oxidative Stress Oxygen Peroxonitrite Production Protein Acetylation Proteins Reactive Oxygen Species Regulation Regulator Genes Reperfusion Injury Reporting Risk Rodent Model Role Sirtuins Specificity Stroke Superoxide Dismutase Superoxides System Testing United States Work acute stroke cell injury deprivation design disability enzyme activity gene therapy human SOD2 protein in vivo neuronal survival novel novel strategies overexpression prevent protective effect small molecule stroke therapy thrombolysis translational study

项目摘要

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.

描述（由申请人提供）：氧化应激发生在过度产生活性氧（ROS）等超氧化物和过氧亚硝酸盐，并且是缺血性中风中DNA损害的主要因素。因此，ROS产生的调节是缺血性中风治疗的关键目标。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剂。赖氨酸残基被一组乙酰基转移酶通过乙酰基乙酰化，通过其定位在细胞室中获得特异性。去除乙酰基（或脱乙酰化）是由包括sirtuins在内的脱乙酰基酶催化的。 Sirtuins是NAD依赖性脱乙酰基酶的家族，与代谢，细胞存活机制和寿命改变有关。在已知的Sirtuins（或Sirts）中，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脱乙酰基酶活性如何减少氧化损伤。我们最近报道，SIRT3降低了超氧化阴离子水平，可防止线粒体去极化并减少暴露于NMDA引起的神经元死亡。此外，新的初步数据暗示SIRT3在体外缺血模型中的保护中。我们假设SIRT3依赖性保护通过调节抗氧化剂防御的酶的脱乙酰化来起作用，并且增加SIRT3活性通过增强这些抗氧化剂系统来促进神经元存活。目前，唯一的急性中风治疗方法是溶栓，不幸的是，这增加了脑出血和进一步的脑损伤的风险。因此，识别和表征SIRT3的保护作用以识别小分子调节剂和药物将是巨大的好处治疗干预的设计。我们将使用暴露于氧气和葡萄糖剥夺（或OGD）的培养的小鼠皮质神经元来模拟缺血性中风（以识别机制），并在体内卒中模型（用于翻译研究）。我们将通过激活AMPK来表征一种新的机制来增加SIRT3蛋白和活性。在动物中，我们将比较缺乏SIRT3蛋白（SIRT3敲除或SIRT3-KO）的正常小鼠和小鼠之间ROS产生的差异，如果这些SIRT3-KO小鼠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，SIRT3缺陷型和SIRT3过表达细胞中的作用来表征SIRT3的作用。在SIRT3缺陷的细胞中，我们将测试重新引入正常的SIRT3，无活性SIRT3或非线粒体SIRT3的效果。该项目的目标是确定1）SIRT3是否减少了MCAO之后的脑损伤和行为缺陷，2）如果 SIRT3在培养的小鼠皮质神经元中用OGD损伤调节活性氧水平（ROS）水平，3）SIRT3如何增强培养的小鼠皮质神经元中的抗氧化剂防御，4）SIRT3如何减少小鼠中风模型中缺血性损伤。