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

ABSTRACT: 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.

SUBMITTER: Shimizu T

PROVIDER: S-EPMC5240152 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Shimizu Takeru T Dietz Robert M RM Cruz-Torres Ivelisse I Strnad Frank F Garske Ana K AK Moreno Myriam M Venna Venugopal R VR Quillinan Nidia N Herson Paco S PS

Experimental neurology 20160615 Pt A

<h4>Introduction</h4>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.<h4>Methods</h4>Fluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2-3m ...[more]

PMID: 27317297

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Project description:Background and purposeReperfusion after transient cerebral ischemia causes severe damage to mitochondria; however, little is known regarding the continuous change in mitochondrial biogenesis during reperfusion. Mitochondrial biogenesis causes an increase in the individual mitochondrial mass of neurons and maintains their aerobic set-point in the face of declining function. The aim of this study was to examine mitochondrial biogenesis in the cortex during reperfusion following focal cerebral ischemia.MethodsMale Wistar rats were subjected to transient focal cerebral ischemia. The relative amount of cortical mitochondrial DNA was analyzed using quantitative real-time PCR at 0 h, 24 h, 72 h, and 7 d after reperfusion. Three critical transcriptional regulators of mitochondrial biogenesis were measured by semi-quantitative reverse-transcription PCR. The protein expression of cytochrome C oxidase subunits I and IV was detected by Western blotting.ResultsEvidence of increased mitochondrial biogenesis was observed after reperfusion. The cortical mitochondrial DNA content increased after 24 h, peaked after 72 h, and maintained a high level for 7 d. The cortical expression of three critical genes for the transcriptional regulation of mitochondrial biogenesis, namely, peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcription factor A, also increased at 24 h and 72 h. The expression of peroxisome proliferator-activated receptor coactivator-1α returned to the baseline level at 7 d, but two other factors maintained higher levels compared with the controls. Moreover, the expression of cytochrome C oxidase subunits I and IV was increased in the cortex.ConclusionsThese results indicate that reperfusion increased mitochondrial biogenesis following focal cerebral ischemia, and this tendency was exacerbated as the reperfusion time was extended. Reperfusion-induced mitochondrial biogenesis was mediated through up-regulation of critical transcriptional regulators of mitochondrial biogenesis.

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

Publications

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

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