Abstract 16: Inhibiting Ezh2 Is Neuroprotective After Stroke In Aged Mice

Abstract

Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme, a key modulator of microglia polarization. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke and microglial over-expressing microglial EZH2 exacerbates stroke outcome in vivo . Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. Microglial EZH2 over-expressing was performed using lentiviral vectors. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated with tamoxifen injection in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , P<0.01, N=5/each group). EZH2 deletion reduced brain infarct volume (29.27±2.23% vs. 6.07±0.88%, P<0.001, N=7/each group) and improved functional outcome assayed by adhesive removal test (59±13.1 seconds, N=7 in control vs. 26.28±4.1 seconds, N=12 in KO, P<0.01). Mechanistically, microglial EZH2 deletion led to a decrease in expression of M1 marker iNOS (170±14.78 vs. 76.65±11.38 cells/mm 2 , P<0.05, N=4/each group), an increase in M2 marker Arg1 (96.64±11.48 vs. 203.3±22.02 cells/mm 2 , P<0.05, N=4/each group) co-stained in microglia (Iba1). We additionally found over-expressing EZH2 in microglial cells exacerbated stroke outcome including performance in adhesive tape removal and hang-wire tests 3 days after stroke. We observed decreased Arg1 and increased iNOS in stroke mice with over-expression of EZH2 (n= 5/6, p=<0.05). Conclusions: Genetic deletion of EZH2 in microglia improved stroke outcome in aged while overexpression of EZH2 exacerbated stroke outcome. The effect of protective effect of EZH2 inhibition may be due to limiting microglial M1 polarization and enhancing M2 polarization.