Deacetylation of mitochondrial proteins protect neurons from ischemic injury

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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.