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

Abstract

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.

Targeting Oxidative Stress and Inflammation to Prevent Ischemia-Reperfusion Injury

“…They are involved in various cellular physiological processes, such as energy metabolism, oxidative stress, and apoptosis. Their ability to resist oxidative stress has been widely studied in cerebral ischemia-reperfusion (Hou et al, 2010;Rothgiesser et al, 2010;Morris et al, 2011;She et al, 2018;Wang et al, 2018;Wu D. et al, 2018;Chang et al, 2019;Duan et al, 2019;Fusco et al, 2019;Rao et al, 2019;Xian et al, 2019;Zhao B. et al, 2019). SIRT1 activates the FOXO family, and FOXO3a has the ability to resist anti-oxidative stress and enhances ROS scavenging activity (Hou et al, 2010).…”

“…Inflammatory cytokines can enhance the activity of SIRT2 in cells, enhance its deacetylation of NF-κB, reduce the transcriptional activity of NF-κB, and reduce the expression of inflammatory mediators, such as interleukin-6 (IL-6), matrix metalloproteinase 9 (MMP9), and cyclooxygenase-2 (COX-2), to exert antioxidant and anti-inflammatory effects (Rothgiesser et al, 2010). Further studies have also shown that SIRT2specific activity inhibitor can aggravate neurological impairment after CIRI, and identified the downregulation of AKT/FOXO3a and p38/MAPK pathways as intermediary mechanisms that may contribute to the reduction in apoptotic cell death caused by SIRT2 inhibition (She et al, 2018;Wu D. et al, 2018). SIRT3 enhances SOD2 activity by deacetylating its lysine residues, reducing ROS levels in cells or tissues, increasing the organism's ability to resist oxidative stress, and inhibiting apoptosis (Wang et al, 2018).…”

Notoginseng Leaf Triterpenes Ameliorates OGD/R-Induced Neuronal Injury via SIRT1/2/3-Foxo3a-MnSOD/PGC-1α Signaling Pathways Mediated by the NAMPT-NAD Pathway

“…SIRT1, SIRT2, and SIRT3 have the highest deacetylase activities and can exert great neuroprotective effects in cerebral ischemia [ 60 – 62 ]. With an NAD+-dependent deacetylase, SIRT1, SIRT2, and SIRT3 specifically promote the transcription of a set of genes related to cell survival [ 63 – 65 ], mitochondrial function [ 66 ], energy metabolism [ 26 , 66 , 67 ], oxidizing reaction [ 65 , 66 , 68 ], and inflammation [ 69 , 70 ]. SIRT1/2/3 deficiency or knockdown attenuated the neuroprotection of NAD+ [ 52 , 62 ].…”

“…SIRT1 and SIRT2 are key regulators of cellular antioxidative and antiapoptotic responses [ 26 , 71 , 72 ], and similar to SIRT1/2, NRF2 plays a crucial role in promoting mitochondrial biogenesis and regulating mitochondrial function with a relatively independent manner [ 43 , 58 , 73 ]. Under cellular stresses, SIRT1/2 can deacetylate FOXO1 and FOXO3A [ 68 , 72 , 74 ], and the transcriptional activity mediated by NRF2 is improved, which in turn, induces the increase of antioxidant genes expression, decrease of ROS production, and upregulation of mitochondrial superoxide dismutase (MnSOD) expression [ 58 , 68 ]. In this study, OGD/R induction led to the SIRT1/2 downregulation, decrease of FOXO3a nuclear localization and phosphorylated levels (Figures 5(e) – 5(g) ), increase of the ROS production, downregulation of antioxidant proteins and factors (SOD, CAT, MnSOD), and mitochondria injury (Figures 5(e) – 5(g) ).…”

“…Sirt3 is the primary mitochondria-targeted deacetylase, predominantly expressed in highly metabolic tissues, and binds to and deacetylate several metabolic and respiratory enzymes that regulate mROS generation and mitochondrial functions [ 60 , 62 , 69 ]. Mitochondrial Sirt3 induces forkhead box O3 (FoxO3a) translocation to the nucleus and augments FoxO3a-dependent antioxidant defense mechanisms [ 68 , 72 , 76 ] through upregulation of PGC-1 α [ 62 , 65 , 75 ] and SOD2 [ 66 , 75 ], which is similar to the regulatory role of SIRT1. PGC-1 α and SOD2 stimulate mitochondrial biogenesis and electron transport activity [ 14 , 62 , 65 ].…”