Abstract TP138: Succinate Dehydrogenase and Complex I Downregulation Contribute to the Therapeutic Effect of Hypothermia in Oxygen-Glucose Deprivation

Abstract

Introduction: Reperfusion injury is a significant mechanism of cellular damage in the wake of ischemic stroke. This damage is mediated by succinate, an intermediate in the citric acid cycle. Rapidly accumulated by tissues during the ischemic phase, succinate is re-oxidized by succinate dehydrogenase (SDH) upon reperfusion: this, through reverse transport of electrons at mitochondrial Complex I, is responsible for the profuse production of reactive oxygen species (ROS). Therapeutic hypothermia (TH) is a well-established neuroprotective strategy for ischemic stroke that is known, moreover, to act in part through post-ischemic suppression of ROS. In this study, we sought to elaborate the mechanism of this suppression by investigating the effects of TH on SDH and Complex I expression, and thus to assess the ability of TH to protect mitochondria against post-ischemic oxidative damage. Methods: Human neural SH-SY5Y cells were divided into 9 groups: 1) sham, 2) 2 h oxygen-glucose deprivation (OGD) without treatment, and 3-9) OGD with one of the following 7 treatments: TH (34°Cfor 4 h), pre-treatment with succinate (SDH substrate, 5 mM), with malonate (SDH inhibitor, 5 mM), with rotenone (Complex I inhibitor, 1 μM) or with a combination of succinate plus either inhibitor (malonate or rotenone) or TH. After 4 or 22 h reoxygenation, OGD cell survival was determined by cell viability and levels of ROS and LDH generation. SDH and Complex I protein expression were assessed by Western blot. Results: TH, malonate, and rotenone monotherapy significantly decreased succinate concentration, ROS production, LDH release, and increased cell viability. SDH and Complex I expression were decreased by TH. Pre-treatment with succinate yielded increased ROS production and LDH release as well as decreased cell viability. These effects were reversed following treatment with malonate, rotenone, or TH, however, suggesting that the SDH and Complex I pathway is involved in TH-regulated succinate transformation. Conclusion: TH exerts its neuroprotective effect in part by mitigating the ROS-generating oxidation of succinate during reperfusion. Our study suggests a new mechanism underlying this mitochondrial protection: downregulation of the SDH and Complex I molecular pathway.