SIRT2 Inhibition Confers Neuroprotection by Downregulation of FOXO3a and MAPK Signaling Pathways in Ischemic Stroke

  • David T. She
  • Lap Jack Wong
  • Sang-Ha Baik
  • Thiruma V. Arumugam


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.


Ischemic 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.

Author Contributions

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.

Funding Information

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. 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. CrossRefGoogle Scholar
  2. 2.
    Doyle KP, Simon RP, Stenzel-Poore MP (2008) Mechanisms of ischemic brain damage. Neuropharmacology 55:310–318. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bansal S, Sangha KS, Khatri P (2013) Drug treatment of acute ischemic stroke. Am J Cardiovasc Drugs 13:57–69. CrossRefPubMedGoogle Scholar
  4. 4.
    Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431–1568CrossRefPubMedGoogle Scholar
  5. 5.
    Moskowitz MA, Lo EH, Iadecola C (2010) The science of stroke: mechanisms in search of treatments. Neuron 67:181–198. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11:700–714. CrossRefPubMedGoogle Scholar
  7. 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. CrossRefPubMedGoogle Scholar
  8. 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. CrossRefPubMedGoogle Scholar
  9. 9.
    Aune SE, Herr DJ, Kutz CJ, Menick DR (2015) Histone deacetylases exert class-specific roles in conditioning the brain and heart against acute ischemic injury. Front Neurol 6:145. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Schweizer S, Meisel A, Märschenz S (2013) Epigenetic mechanisms in cerebral ischemia. J Cereb Blood Flow Metab 33:1335–1346. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    She DT, Jo D-G, Arumugam TV (2017) Emerging roles of sirtuins in ischemic stroke. Transl Stroke Res 8:405–423. CrossRefGoogle Scholar
  12. 12.
    Haigis MC, Sinclair DA (2010) Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol 5:253–295. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Landry J, Sutton A, Tafrov ST, Heller RC, Stebbins J, Pillus L, Sternglanz R (2000) The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases. Proc Natl Acad Sci U S A 97(11):5807–5811. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Oellerich MF, Potente M (2012) FOXOs and sirtuins in vascular growth, maintenance, and aging. Circ Res 110:1238–1251. CrossRefPubMedGoogle Scholar
  15. 15.
    Bordone L, Guarente L (2005) Calorie restriction, SIRT1 and metabolism: understanding longevity. Nat Rev Mol Cell Biol 6:298–305. CrossRefPubMedGoogle Scholar
  16. 16.
    North BJ, Marshall BL, Borra MT, Denu JM, Verdin E (2003) The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol Cell 11:437–444CrossRefPubMedGoogle Scholar
  17. 17.
    Perrod S, Cockell MM, Laroche T, Renauld H, Ducrest AL, Bonnard C, Gasser SM (2001) A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast. EMBO J 20:197–209. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Vaquero A, Scher MB, Lee DH, Sutton A, Cheng HL, Alt FW, Serrano L, Sternglanz R et al (2006) SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis. Genes Dev 20:1256–1261. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Outeiro TF, Kontopoulos E, Altmann SM, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM et al (2007) Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson’s disease. Science 317:516–519. CrossRefPubMedGoogle Scholar
  20. 20.
    Chopra V, Quinti L, Kim J, Vollor L, Narayanan KL, Edgerly C, Cipicchio PM, Lauver MA et al (2012) The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington’s disease mouse models. Cell Rep 2:1492–1497. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    de Oliveira RM, Sarkander J, Kazantsev AG, Outeiro TF (2012) SIRT2 as a therapeutic target for age-related disorders. Front Pharmacol 3:82. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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. CrossRefPubMedGoogle Scholar
  23. 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. CrossRefPubMedGoogle Scholar
  24. 24.
    You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, Erlacher M, Villunger A, Mak TW (2006) FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. J Exp Med 203:1657–1663. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Haoues M, Refai A, Mallavialle A, Barbouche MR, Laabidi N, Deckert M, Essafi M (2014) Forkhead box O3 (FOXO3) transcription factor mediates apoptosis in BCG-infected macrophages. Cell Microbiol 16:1378–1390. CrossRefPubMedGoogle Scholar
  26. 26.
    Wada T, Penninger JM (2004) Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23:2838–2849. CrossRefPubMedGoogle Scholar
  27. 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. CrossRefPubMedGoogle Scholar
  28. 28.
    Fann DY-W, Lee SY, Manzanero S et al (2013) Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke. Cell Death Dis 4:e790. CrossRefPubMedGoogle Scholar
  29. 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. CrossRefPubMedGoogle Scholar
  30. 30.
    Zhao H, Sapolsky RM, Steinberg GK (2006) Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke. Mol Neurobiol 34:249–270. CrossRefPubMedGoogle Scholar
  31. 31.
    Sun J, Nan G (2016) The mitogen-activated protein kinase (MAPK) signaling pathway as a discovery target in stroke. J Mol Neurosci 59:90–98. CrossRefPubMedGoogle Scholar
  32. 32.
    Ramakrishnan G, Davaakhuu G, Kaplun L, Chung WC, Rana A, Atfi A, Miele L, Tzivion G (2014) Sirt2 deacetylase is a novel AKT binding partner critical for AKT activation by insulin. J Biol Chem 289:6054–6066. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC et al (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868CrossRefPubMedGoogle Scholar
  34. 34.
    Wang J, Koh H-W, Zhou L, Bae UJ, Lee HS, Bang IH, Ka SO, Oh SH et al (2017) Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1. Hepatology 65:225–236. CrossRefPubMedGoogle Scholar
  35. 35.
    Wang Y, Mu Y, Zhou X, Ji H, Gao X, Cai WW, Guan Q, Xu T (2017) SIRT2-mediated FOXO3a deacetylation drives its nuclear translocation triggering FasL-induced cell apoptosis during renal ischemia reperfusion. Apoptosis 22:519–530. CrossRefPubMedGoogle Scholar
  36. 36.
    Xie XQ, Zhang P, Tian B, Chen XQ (2016) Downregulation of NAD-dependent deacetylase SIRT2 protects mouse brain against ischemic stroke. Mol Neurobiol 54:7251–7261. CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang L, Qu Y, Tang J, Chen D, Fu X, Mao M, Mu D (2010) PI3K/Akt signaling pathway is required for neuroprotection of thalidomide on hypoxic-ischemic cortical neurons in vitro. Brain Res 1357:157–165. CrossRefPubMedGoogle Scholar
  38. 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:, 188
  39. 39.
    Li D, Li X, Wu J, Li J, Zhang L, Xiong T, Tang J, Qu Y et al (2015) Involvement of the JNK/FOXO3a/Bim pathway in neuronal apoptosis after hypoxic-ischemic brain damage in neonatal rats. PLoS One 10:e0132998. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Yin J, Han P, Tang Z, Liu Q, Shi J (2015) Sirtuin 3 mediates neuroprotection of ketones against ischemic stroke. J Cereb Blood Flow Metab 35:1783–1789. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Li J, Flick F, Verheugd P, Carloni P, Lüscher B, Rossetti G (2015) Insight into the mechanism of intramolecular inhibition of the catalytic activity of sirtuin 2 (SIRT2). PLoS One 10:e0139095. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Voelter-Mahlknecht S, Ho AD, Mahlknecht U (2005) FISH-mapping and genomic organization of the NAD-dependent histone deacetylase gene, Sirtuin 2 (Sirt2). Int J Oncol 27:1187–1196PubMedGoogle Scholar
  43. 43.
    North BJ, Verdin E (2007) Interphase nucleo-cytoplasmic shuttling and localization of SIRT2 during mitosis. PLoS One 2:e784. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Flick F, Lüscher B (2012) Regulation of sirtuin function by posttranslational modifications. Front Pharmacol 3:29. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Fischer A, Mühlhäuser WWD, Warscheid B, Radziwill G (2017) Membrane localization of acetylated CNK1 mediates a positive feedback on RAF/ERK signaling. Sci Adv 3:e1700475. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Modur V, Nagarajan R, Evers BM, Milbrandt J (2002) FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression. Implications for PTEN mutation in prostate cancer. J Biol Chem 277:47928–47937. CrossRefPubMedGoogle Scholar
  47. 47.
    Boccitto M, Kalb RG (2011) Regulation of Foxo-dependent transcription by post-translational modifications. Curr Drug Targets 12:1303–1310. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Hafeez A, Elmadhoun O, Peng C et al (2014) Reduced apoptosis by ethanol and its association with PKC-δ and Akt signaling in ischemic stroke. Aging Dis 5:366–372. PubMedPubMedCentralGoogle Scholar
  49. 49.
    Bhuiyan MIH, Jung SY, Kim HJ, Lee YS, Jin C (2011) Major role of the PI3K/Akt pathway in ischemic tolerance induced by sublethal oxygen-glucose deprivation in cortical neurons in vitro. Arch Pharm Res 34:1023–1034. CrossRefPubMedGoogle Scholar
  50. 50.
    Franke TF, Kaplan DR, Cantley LC (1997) PI3K: downstream AKTion blocks apoptosis. Cell 88:435–437CrossRefPubMedGoogle Scholar
  51. 51.
    Liu Q, Qiu J, Liang M, Golinski J, van Leyen K, Jung JE, You Z, Lo EH et al (2014) Akt and mTOR mediate programmed necrosis in neurons. Cell Death Dis 5:e1084. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 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. CrossRefPubMedGoogle Scholar
  53. 53.
    Dhanasekaran DN, Reddy EP (2008) JNK signaling in apoptosis. Oncogene 27:6245–6251. CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Rothgiesser KM, Erener S, Waibel S, Luscher B, Hottiger MO (2010) SIRT2 regulates NF-κB dependent gene expression through deacetylation of p65 Lys310. J Cell Sci 123:4251–4258. CrossRefPubMedGoogle Scholar
  55. 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. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Physiology, Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore
  2. 2.Neurobiology/Ageing Programme, Life Sciences InstituteNational University of SingaporeSingaporeSingapore
  3. 3.Biomedical Institute for Global Health Research and Technology (BIGHEART)National University of SingaporeSingaporeSingapore
  4. 4.School of PharmacySungkyunkwan UniversitySuwonRepublic of Korea

Personalised recommendations