SIRT2 Inhibition Confers Neuroprotection by Downregulation of FOXO3a and MAPK Signaling Pathways in Ischemic Stroke
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Sirtuin 2 (SIRT2) is a family member of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases which appears to have detrimental roles in an array of neurological disorders such as Parkinson’s disease (PD) and Huntington’s disease (HD). In light of the recently emerging roles of sirtuins in normal physiology and pathological conditions such as ischemic stroke, we investigated the role of SIRT2 in ischemic stroke-induced neuronal cell death. Primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) under in vitro ischemic conditions, and subsequently tested for the efficacy of SIRT2 inhibitors AK1 and AGK2 in attenuating apoptotic cell death caused by OGD. We have also evaluated the effect of SIRT2 inhibition in C57BL/6 mice subjected to 1 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion, which is a model for ischemic reperfusion injury in vivo. Significant reductions in apoptotic cell death were noted in neurons treated with AK1 or AGK2, as evidenced by reduced cleaved caspase-3 and other apoptotic markers such as Bim and Bad. In addition, downregulation of phosphorylated-AKT and FOXO3a proteins of the AKT/FOXO3a pathway, as well as a marked reduction of JNK activity and its downstream target c-Jun, were also observed. When tested in animals subjected to MCAO, the neuroprotective effects of AGK2 in vivo were evidenced by a substantial reduction in ipsilateral infarct area and a significant improvement in neurological outcomes. A similar reduction in the levels of pro-apoptotic proteins in the infarct tissue, as well as downregulation of AKT/FOXO3a and JNK pathway, were also noted. In summary, the current study demonstrated the neuroprotective effects of SIRT2 inhibition in ischemic stroke, and identified the downregulation of AKT/FOXO3a and MAPK pathways as intermediary mechanisms which may contribute to the reduction in apoptotic cell death by SIRT2 inhibition.
KeywordsIschemic stroke SIRT2 AK1 AGK2 Apoptosis MAPK FOXO3a
The authors would like to thank Dr. Kang Sungwook for his assistance with cell culture work, Ms. Poh Luting for her assistance in immunoblotting, and Ms. Lee Shu Ying for her guidance on confocal microscopy. The authors would also like to thank Dr. David Fann for his critical review of the manuscript.
DTS and TVA conceived and designed the experiments. DTS, LJW, BSH, and TVA performed the experiments. DTS and TVA were involved in drafting and editing the manuscript, and interpreted primary data. All authors read and approved the final manuscripts.
Compliance with Ethical Standards
All studies were performed under research protocol approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore (NUS) in accordance with the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines. Every effort was made to reduce animal suffering.
Conflict of Interest
The authors declare that they have no conflict of interest.
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