Takeru Shimizu1, Robert M Dietz2, Ivelisse Cruz-Torres3, Frank Strnad1, Ana K Garske1, Myriam Moreno1, Venugopal R Venna4, Nidia Quillinan1, Paco S Herson5. 1. Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA. 2. Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA. 3. Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA. 4. Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA. 5. Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA. Electronic address: paco.herson@ucdenver.edu.
Abstract
INTRODUCTION: TRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor. METHODS: Fluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2-3months) and aged (18-20months) mice were subjected to 60min middle cerebral artery occlusion (MCAO) and injected with tat-M2NX, control scrambled peptide (tat-SCR) or clotrimazole (CTZ) either 20min prior or 3h after reperfusion. Infarct size was assessed using TTC staining. RESULTS: TRPM2 inhibition by tat-M2NX was observed by decreased Ca(2+) influx following H2O2 exposure human TRPM2 expressing cells. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR. No effect of tat-M2NX on infarct size was observed in female mice. Importantly, male TRPM2(-/-) mice were not further protected by tat-M2NX, demonstrating selectivity of tat-M2NX. Administration of tat-M2NX 3h after reperfusion provided significant protection to males when analyzed at 24h or 4days after MCAO. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice. CONCLUSIONS: These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a clinically relevant therapeutic window.
INTRODUCTION:TRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor. METHODS: Fluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2-3months) and aged (18-20months) mice were subjected to 60min middle cerebral artery occlusion (MCAO) and injected with tat-M2NX, control scrambled peptide (tat-SCR) or clotrimazole (CTZ) either 20min prior or 3h after reperfusion. Infarct size was assessed using TTC staining. RESULTS:TRPM2 inhibition by tat-M2NX was observed by decreased Ca(2+) influx following H2O2 exposure humanTRPM2 expressing cells. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR. No effect of tat-M2NX on infarct size was observed in female mice. Importantly, male TRPM2(-/-) mice were not further protected by tat-M2NX, demonstrating selectivity of tat-M2NX. Administration of tat-M2NX3h after reperfusion provided significant protection to males when analyzed at 24h or 4days after MCAO. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice. CONCLUSIONS: These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a clinically relevant therapeutic window.
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