SIRT2 Inhibition Confers Neuroprotection by Downregulation of FOXO3a and MAPK Signaling Pathways in Ischemic Stroke
- 313 Downloads
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.
This work was supported by the Singapore National Medical Research Council Research Grant (NMRC/CBRG/0102/2016 and NMRC/CBRG/0036/2017) and the Singapore Ministry of Education Academic Research Fund Tier 1 Grant (R-185-000-285-112) to TVA and the Swee-Liew Wadsworth Concept Grant (Research) to DTS. DTS is a recipient of the National University of Singapore Research Scholarship.
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.
- 1.Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T et al (2012) Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet (London, England) 380:2095–2128. https://doi.org/10.1016/S0140-6736(12)61728-0 CrossRefGoogle Scholar
- 7.Ni Y, Gu W-W, Liu Z-H, Zhu YM, Rong JG, Kent TA, Li M, Qiao SG et al (2018) RIP1K contributes to neuronal and astrocytic cell death in ischemic stroke via activating autophagic-lysosomal pathway. Neuroscience 371:60–74. https://doi.org/10.1016/j.neuroscience.2017.10.038 CrossRefPubMedGoogle Scholar
- 8.Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L et al (2018) Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 25:486–541. https://doi.org/10.1038/s41418-017-0012-4 CrossRefPubMedGoogle Scholar
- 12.Haigis MC, Sinclair DA (2010) Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol 5:253–295. https://doi.org/10.1146/annurev.pathol.4.110807.092250 CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Deng R, Tang J, Xie B-F, Feng GK, Huang YH, Liu ZC, Zhu XF (2010) SYUNZ-16, a newly synthesized alkannin derivative, induces tumor cells apoptosis and suppresses tumor growth through inhibition of PKB/AKT kinase activity and blockade of AKT/FOXO signal pathway. Int J Cancer 127:220–229. https://doi.org/10.1002/ijc.25032 CrossRefPubMedGoogle Scholar
- 23.Sunters A, Fernández de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA, Coffer PJ, Medema RH et al (2003) FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 278:49795–49805. https://doi.org/10.1074/jbc.M309523200 CrossRefPubMedGoogle Scholar
- 27.Fann DY-W, Lim Y-A, Cheng Y-L, Lok KZ, Chunduri P, Baik SH, Drummond GR, Dheen ST et al (2017) Evidence that NF-κB and MAPK signaling promotes NLRP inflammasome activation in neurons following ischemic stroke. Mol Neurobiol 55:1082–1096. https://doi.org/10.1007/s12035-017-0394-9 CrossRefPubMedGoogle Scholar
- 29.Kramer M, Dang J, Baertling F, Denecke B, Clarner T, Kirsch C, Beyer C, Kipp M (2010) TTC staining of damaged brain areas after MCA occlusion in the rat does not constrict quantitative gene and protein analyses. J Neurosci Methods 187:84–89. https://doi.org/10.1016/j.jneumeth.2009.12.020 CrossRefPubMedGoogle Scholar
- 38.Dong Y, Liu HD, Zhao R, Yang CZ, Chen XQ, Wang XH, Lau LT, Chen J, Yu ACH (2009) Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3gamma in astrocyte. J Neurochem 109 Suppl:182–8. doi: https://doi.org/10.1111/j.1471-4159.2009.05974.x, 188
- 52.Li Y, Matsumori H, Nakayama Y, Osaki M, Kojima H, Kurimasa A, Ito H, Mori S et al (2011) SIRT2 down-regulation in HeLa can induce p53 accumulation via p38 MAPK activation-dependent p300 decrease, eventually leading to apoptosis. Genes Cells 16:34–45. https://doi.org/10.1111/j.1365-2443.2010.01460.x CrossRefPubMedGoogle Scholar
- 55.Spires-Jones TL, Fox LM, Rozkalne A, Pitstick R, Carlson GA, Kazantsev AG (2012) Inhibition of sirtuin 2 with sulfobenzoic acid derivative AK1 is non-toxic and potentially neuroprotective in a mouse model of frontotemporal dementia. Front Pharmacol 3:42. https://doi.org/10.3389/fphar.2012.00042 CrossRefPubMedPubMedCentralGoogle Scholar