Extended therapeutic window of a novel peptide inhibitor of TRPM2 channels following focal cerebral ischemia.
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.



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.



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.



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.
Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Takeru
T
Shimizu
Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Robert M
RM
Dietz
Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Ivelisse
I
Cruz-Torres
Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Frank
F
Strnad
Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Ana K
AK
Garske
Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Myriam
M
Moreno
Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA.
Venugopal R
VR
Venna
Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO, USA; Neuronal Injury Program, University of Colorado School of Medicine, Aurora, CO, USA.
Nidia
N
Quillinan
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.
Paco S
PS
Herson
D000367:Age Factors; D000818:Animals; D020520:Brain Infarction / Q000188:drug therapy / Q000209:etiology; D002545:Brain Ischemia / Q000150:complications / Q000188:drug therapy*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005260:Female; D057809:HEK293 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D018696:Neuroprotective Agents / Q000627:therapeutic use*; D010455:Peptides / Q000627:therapeutic use*; D012737:Sex Factors; D050053:TRPM Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018696:Neuroprotective Agents; D010455:Peptides; D050053:TRPM Cation Channels; C544443:TRPM2 protein, mouse
Aging; Cerebral ischemia; Experimental stroke; Sex; TRPM2 channel