Transient Receptor Potential M2 Channel Research Articles

Paeonol and glycyrrhizic acid in combination ameliorate the recurrent nitroglycerin-induced migraine-like phenotype in rats by regulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway

Ethnopharmacological relevancePaeonol (PAE) and glycyrrhizic acid (GLY) are predominate components of 14 blood-entering ones of Piantongtang No. 1, which is a traditional Chinese medicine prescription for chronic migraine with minimal side effects. Both paeonol and glycyrrhizic acid exhibit analgesic, neuroprotective and anti-inflammatory properties individually. Our previous research has highlighted their combined effect (PAE + GLY) in ameliorating migraine symptoms. However, there are not yet any studies exploring the mechanism of action of PAE + GLY in the treatment of migraine. Aim of the studyThis research aimed to determine the mechanism of PAE + GLY in ameliorating the recurrent nitroglycerin-induced migraine-like phenotype in rats. Materials and methodsUsing a nitroglycerin-induced migraine model via subcutaneous injection in the neck, we evaluated the effect of PAE + GLY on migraine-like symptoms. Behavioural tests and biomarkers analysis were employed, alongside transcriptome sequencing (RNA-seq). Mechanistic insights were further verified utilising reverse transcription quantitative PCR (RT-qPCR), Western blot (WB), ELISA and immunofluorescence (IF) techniques. ResultsFollowing treatment with PAE + GLY, hyperalgesia threshold and 5-hydroxytryptamine (5-HT) levels increased, and migraine-like head scratching, histamine and calcitonin gene-related peptide (CGRP) levels were reduced. RNA-Seq experiments revealed that PAE + GLY upregulated the expression of Glutamate decarboxylase 2 (GAD2) and γ-aminobutyric acid type B receptor subunit 2 (GABBR2) genes. This upregulation activated the GABAergic synapse pathway, effectively inhibiting migraine attacks. Further validation demonstrated an increase in γ-aminobutyric acid (GABA) content in cerebrospinal fluid post PAE + GLY treatment, coupled with increased expression of dural GAD2, GABBR2 and transient receptor potential channel M8 (TRPM8). Consequently, this inhibited the expression of dural cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) and transient receptor potential channel type 1 (TRPV1), subsequently downregulating p-ERK1/2, p-AKT1, IL-1β and TNF-α. ConclusionsOur findings underscore that PAE + GLY ameliorates inflammatory hyperalgesia migraine by upregulating inhibitory neurotransmitters and modulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway.

Transient receptor potential M2 channel in the hypothalamic preoptic area and its impact on thermoregulation during menopause

BackgroundDecreased estrogen levels can cause abnormal thermosensitivity of the preoptic area (POA) in the hypothalamus during menopause, which may cause hot flashes. Thermosensitive transient receptors (ThermoTRPs) affect the thermosensitivity of neurons. It is worth exploring whether ThermoTRPs change under low estrogen state and participate in the abnormal thermoregulation of POA. MethodsAdult female Sprague–Dawley rats were randomly divided into sham operation (SHAM), ovariectomy (OVX) and estrogen treatment after ovariectomy (OVX+E) groups. Under 10 ℃, 18 ℃, 25 ℃, 37 ℃ and 45 ℃ incubations, their skin temperature was monitored and the expression of TRPA1, TRPM8, TRPM2, and TRPV1 in POA were investigated. ResultsThe skin temperature of ovariectomized rats changed faster and more dramatically under different incubation temperatures. The results at mRNA level show that only the expression of TRPM2 decreased in POA of OVX group compared with the other two groups at 25 ℃, TRPA1 expression in POA of the three groups increased at 10 ℃, TRPM8 increased at 10 ℃ and 18 ℃, TRPV1 increased at 10 ℃ and 45 ℃, while the expression of TRPM2 decreased at 10 ℃ and 18 ℃ and increased at 37 ℃ and 45 ℃. In all these cases, the magnitudes of the changes were less in the OVX group relative to the other two groups. The further immunohistochemical and Western blot results of TRPM2 and the activated TRPM2 positive cells labeled by c-Fos were consistent with the results of mRNA level. ConclusionsThe expression and thermosensitivity of TRPM2 in POA changed greatly under different incubation temperatures, but the changes in ovariectomized rats were less. This may be the key factor triggering thermoregulation dysfunction under low estrogen and may cause hot flashes.

TRPM3-Induced Gene Transcription Is under Epigenetic Control.

Transient receptor potential M3 (TRPM3) cation channels regulate numerous biological functions, including gene transcription. Stimulation of TRPM3 channels with pregnenolone sulfate activates stimulus-responsive transcription factors, which bind to short cognate sequences in the promoters of their target genes. In addition, coregulator proteins are involved that convert the chromatin into a configuration that is permissive for gene transcription. In this study, we determined whether TRPM3-induced gene transcription requires coactivators that change the acetylation pattern of histones. We used compound A485, a specific inhibitor of the histone acetyltransferases CBP and p300. In addition, the role of bromodomain proteins that bind to acetylated lysine residues of histones was analyzed. We used JQ1, an inhibitor of bromodomain and extra terminal domain (BET) family proteins. The results show that both compounds attenuated the activation of AP-1 and CREB-regulated gene transcription following stimulation of TRPM3 channels. Inhibition of CBP/p300 and BET proteins additionally reduced the transcriptional activation potential of the transcription factors c-Fos and Elk-1. Transcriptional upregulation of the interleukin-8 gene was attenuated by A485 and JQ1, indicating that proinflammatory cytokine expression is controlled by CBP/p300 and bromodomain proteins. We conclude that TRPM3-induced signaling involves transcriptional coactivators and acetyl-lysine-bound bromodomain proteins for activating gene transcription.

Abstract WP248: Extended Therapeutic Window Of A Novel Peptide Inhibitor Of Trpm2 Channels On Memory Following Focal Cerebral Ischemia

Adult stroke is the a leading cause of mortality and morbidity, with significant sequelae including memory deficits. Despite intense research, no pharmacological interventions are currently available to improve cognitive outcome following ischemic stroke (MCAO). Studies suggest that the non-selective transient receptor potential M2 (TRPM2) channels may contribute to brain injury, specifically in males. This study used electrophysiology and neurobehavior to investigate whether TRPM2 could restore cognitive impairments 30 days after MCAO. To assess the functional role of TRPM2 channels, our novel TRPM2 channel antagonist tat-M2NX was administered intravenously 29 days following MCAO, with contextual fear conditioning performed to measure memory function in mice on day 31. Sham operated mice exhibited intact spatial memory, as indicated by high an increase in freezing behavior (65.44% ± 4.167, N=6) whereas) whereas MCAO mice treated with tat-Scr treated reduced freezing behavior (36.7%, ± 24.4 N=7). . Administration of tatM2NX resulted in improved memory function (72.26% ± 10.98 n=9) compared to tatSCR-treated mice. To assess the effect of MCAO on hippocampal long-term potentiation (LTP), a well-accepted model of learning and memory, extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 31 days after MCAO. Under control conditions, a physiological theta burst stimulation (40 pulses, 100Hz) resulted in LTP that increased the slope of fEPSP to 70.8%, n=5 of baseline MCAO injured mice had impaired LTP (121.1%, n=5). However, mice treated with tat-M2NX on day 30 and recorded on day 31 preserved LTP (185.4, n=5), consistent with improved memory function above. This study suggests that TRPM2 channels contribute to the functional memory impairment seen after MAO at delayed timepoints. Therefore, targeting TRPM2 channel activity may be a potential therapeutic approach to improve long-term functional outcome following MCAO.

Functional Restoration following Global Cerebral Ischemia in Juvenile Mice following Inhibition of Transient Receptor Potential M2 (TRPM2) Ion Channels.

Hippocampal cell death and cognitive dysfunction are common following global cerebral ischemia across all ages, including children. Most research has focused on preventing neuronal death. Restoration of neuronal function after cell death is an alternative approach (neurorestoration). We previously identified transient receptor potential M2 (TRPM2) ion channels as a potential target for acute neuroprotection and delayed neurorestoration in an adult CA/CPR mouse model. Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) in juvenile (p20-25) mice was used to investigate the role of ion TRPM2 channels in neuroprotection and ischemia-induced synaptic dysfunction in the developing brain. Our novel TRPM2 inhibitor, tatM2NX, did not confer protection against CA1 pyramidal cell death but attenuated synaptic plasticity (long-term plasticity (LTP)) deficits in both sexes. Further, in vivo administration of tatM2NX two weeks after CA/CPR reduced LTP impairments and restored memory function. These data provide evidence that pharmacological synaptic restoration of the surviving hippocampal network can occur independent of neuroprotection via inhibition of TRPM2 channels, providing a novel strategy to improve cognitive recovery in children following cerebral ischemia. Importantly, these data underscore the importance of age-appropriate models in disease research.

Research progress on transient receptor potential melastatin 2 channel in nervous system diseases.

Transient receptor potential M2 (TRPM2) ion channel is a non-selective cationic channel that can permeate calcium ions, and plays an important role in neuroinflammation, ischemic reperfusion brain injury, neurodegenerative disease, neuropathic pain, epilepsy and other neurological diseases. In ischemic reperfusion brain injury, TRPM2 mediates neuronal death by modulating the different subunits of glutamate N-methyl-D-aspartic acid receptor in response to calcium/zinc signal. In Alzheimer's disease, TRPM2 is activated by reactive oxygen species generated by β-amyloid peptide to form a malignant positive feedback loop that induces neuronal death and is involved in the pathological process of glial cells by promoting inflammatory response and oxidative stress. In epilepsy, the TRPM2-knockout alleviates epilepsy induced neuronal degeneration by inhibiting autophagy and apoptosis related proteins. The roles of TRPM2 channel in the pathogenesis of various central nervous system diseases and its potential drug development and clinical application prospects are summarized in this review.

The decrease in hippocampal transient receptor potential M2 (TRPM2) channel and muscarinic acetylcholine receptor 1 (CHRM1) is associated with memory loss in a surgical menopause rat model

IntroductionThe aim of the study was to investigate the association of transient receptor potential M2 (TRPM2) channel and muscarinic acetylcholine receptor 1 (CHRM1) activity with the memorial functions that are deteriorated in surgical menopause.Material and methodsA total of 14 female rats were randomly divided into 2 groups: group (G)1: sham group; group (G)2: surgical menopause group, the group in which bilateral ovariectomy was performed. Fourteen days after the surgical procedure, learning and memorial tests were performed in G1 and G2 for a totally 13 days. The time required for the rats to find the cheese in the labyrinth was recorded and statistical evaluation of it was performed between groups. On the 14th day of the memory test, the rats were decapitated and the brain tissues were fixed in 10% formalin. Hippocampal TRPM2 and CHRM1 gene expression was evaluated with RNA isolation, complementary DNA (cDNA) synthesis and quantitative real-time PCR (qRT-PCR) analysis. TRPM2 and CHRM1 immunoreactivity was evaluated in hippocampal tissue with the immunohistochemical method. Histo-score was calculated regarding the diffuseness of and severity of the staining; and statistical analyses were performed.ResultsIn the ovariectomized group, the mean time required for the rats to find the cheese was statistically significantly elongated (39.29 ±4.0 s vs. 29.86 ±2.6 s). When the hippocampal TRPM2 and CHRM1 gene expression and immunoreactivity were compared with the sham group, there was a statistically significant decrease in the surgical menopause group (p < 0.05).ConclusionsIn surgical menopause, in deterioration of memorial functions, hippocampal TRPM2 channel and CHRM1 activity plays an important role.

Reversal of Global Ischemia-Induced Cognitive Dysfunction by Delayed Inhibition of TRPM2 Ion Channels

Hippocampal injury and cognitive impairments are common after cardiac arrest and stroke and do not have an effective intervention despite much effort. Therefore, we developed a new approach aimed at reversing synaptic dysfunction by targeting TRPM2 channels. Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) in mice was used to investigate cognitive deficits and the role of the calcium-permeable ion channel transient receptor potential-M2 (TRPM2) in ischemia-induced synaptic dysfunction. Our data indicates that absence (TRPM2-/-) or acute inhibition of TRPM2 channels with tatM2NX reduced hippocampal cell death in males only, but prevented synaptic plasticity deficits in both sexes. Remarkably, administration of tatM2NX weeks after injury reversed hippocampal plasticity and memory deficits. Finally, TRPM2-dependent activation of calcineurin-GSK3β pathway contributes to synaptic plasticity impairments. These data suggest persistent TRPM2 activity following ischemia contributes to impairments of the surviving hippocampal network and that inhibition of TRPM2 channels at chronic time points may represent a novel strategy to improve functional recovery following cerebral ischemia that is independent of neuroprotection.

Stimulation of TRPM3 channels increases the transcriptional activation potential of Elk-1 involving cytosolic Ca2+, extracellular signal-regulated protein kinase, and calcineurin

Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration.

Stimulation of transient receptor potential M3 (TRPM3) channels increases interleukin-8 gene promoter activity involving AP-1 and extracellular signal-regulated protein kinase

Stimulation of Ca2+ permeable TRPM3 (transient receptor potential melastatin-3) channels with the steroid ligand pregnenolone sulfate activates stimulus-responsive transcription factors, including the transcription factor AP-1 (activator protein-1). As part of a search for AP-1-regulated target genes we analyzed the gene encoding interleukin-8 (IL-8) in HEK293 cells expressing TRPM3 channels. Here, we show that stimulation of TRPM3 channels activated transcription of an IL-8 promoter-controlled reporter gene that was embedded into the chromatin of the cells. Mutational analysis of the IL-8 promoter revealed that the AP-1 binding site of the IL-8 promoter was essential to connect TRPM3 stimulation with the transcription of the IL-8 gene. Genetic experiments revealed that the basic region leucine zipper proteins c-Jun and ATF2 and the ternary complex factor Elk-1 are essential to couple TRPM3 channel stimulation with the IL-8 gene. Moreover, we identified extracellular signal-regulated protein kinase (ERK1/2) as signal transducer connecting TRPM3 stimulation with enhanced transcription of the IL-8 gene. Furthermore, we show that stimulation of TRPC6 (transient receptor potential canonical-6) channels with its ligand hyperforin also increased IL-8 promoter activity, involving the AP-1 binding site within the IL-8 gene, suggesting that activation of IL-8 gene transcription may be a common theme following TRP channel stimulation.

Extracellular Signal-Regulated Protein Kinase, c-Jun N-Terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1

Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca2+ ions into the cells and a rise in the intracellular Ca2+ concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca2+ /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. J. Cell. Biochem. 118: 2409-2419, 2017. © 2017 Wiley Periodicals, Inc.

The coding of cutaneous temperature in the spinal cord.

The spinal cord is the initial stage that integrates temperature information from peripheral inputs. Here we used molecular genetics and in vivo calcium imaging to investigate the coding of cutaneous temperature in the spinal cord in mice. We found that heating or cooling the skin evoked robust calcium responses in spinal neurons, and their activation threshold temperatures distributed smoothly over the entire range of stimulation temperatures. Once activated, heat-responding neurons encoded the absolute skin temperature without adaptation and received major inputs from transient receptor potential (TRP) channel V1 (TRPV1)-positive dorsal root ganglion (DRG) neurons. By contrast, cold-responding neurons rapidly adapted to ambient temperature and selectively encoded temperature changes. These neurons received TRP channel M8 (TRPM8)-positive DRG inputs as well as novel TRPV1(+) DRG inputs that were selectively activated by intense cooling. Our results provide a comprehensive examination of the temperature representation in the spinal cord and reveal fundamental differences in the coding of heat and cold.

Meningeal transient receptor potential channel M8 activation causes cutaneous facial and hindpaw allodynia in a preclinical rodent model of headache.

Migraine headache is a neurological disorder affecting millions worldwide. However, little is known about the mechanisms contributing to migraine. Recent genome-wide association studies have found single nucleotide polymorphisms in the gene encoding transient receptor potential channel M8. Transient receptor potential channel M8 is generally known as a cold receptor but it has been implicated in pain signaling and may play a role in migraine pain. In order to investigate whether transient receptor potential channel M8 may contribute to the pain of migraine, the transient receptor potential channel M8 activator icilin was applied to the dura mater using a rat behavioral model of headache. Cutaneous allodynia was measured for 5 hours using Von Frey filaments. Dural application of icilin produced cutaneous facial and hind paw allodynia that was attenuated by systemic pretreatment with the transient receptor potential channel M8-selective antagonist AMG1161 (10 mg/kg p.o.). Further, the anti-migraine agent sumatriptan (0.6 mg/kg s.c.) or the non-selective NOS inhibitor L-NAME (20 mg/kg i.p.) also attenuated allodynia when given as a pretreatment. These data indicate that transient receptor potential channel M8 activation in the meninges produces behaviors in rats that are consistent with migraine and that are sensitive to pharmacological mechanisms known to have efficacy for migraine in humans. The findings suggest that activation of meningeal transient receptor potential channel M8 may contribute to the pain of migraine.

Sex Steroids Do Not Modulate TRPM2-Mediated Injury in Females following Middle Cerebral Artery Occlusion(1,2,3).

Calcium-permeable transient receptor potential M2 (TRPM2) ion channel activation contributes to cerebral ischemic injury specifically in males. In male mice, circulating androgens are required for TRPM2 inhibition with clotrimazole (CTZ) to provide protection following experimental stroke. Sufficient levels of circulating androgens are necessary to support ischemia-induced activation of poly ADP ribose polymerase (PARP) and consequent activation of TRPM2 channels. In this study, we tested whether differences in sex steroids contribute to the lack of CTZ neuroprotection in females. Middle cerebral artery occlusion (MCAO) was performed using adult female mice that were hormonally intact, ovariectomized (OVX) or dihydrotestosterone (DHT) treated. CTZ or vehicle was administered at the time of reperfusion, animals were euthanized 24 h later and brains and serum were collected. Infarct analysis revealed no effect of CTZ in intact females or females lacking endogenous sex steroids (OVX). Interestingly, treatment of female mice with the potent androgen receptor agonist DHT had no effect on ischemic injury and did not permit CTZ neuroprotection. Similarly, DHT-treated females did not exhibit increased levels of ADPribose, the TRPM2 ligand generated by PARP, following ischemia. No differences in TRPM2 or androgen receptor expression were observed between males and females. These data suggest that the lack of TRPM2 activation in females following experimental stroke is not due to the presence of estrogen or the absence of androgens. In conclusion, our data demonstrate that while circulating androgens are necessary for PARP-mediated TRPM2 injury in males, they are not sufficient to produce TRPM2 activation in females.

Androgen and PARP-1 Regulation of TRPM2 Channels after Ischemic Injury

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel was recently demonstrated to have a sexually dimorphic contribution to ischemic brain injury, with inhibition or knockdown of the channel protecting male brain preferentially. We tested the hypothesis that androgen signaling is required for this male-specific cell-death pathway. Additionally, we tested the hypothesis that differential activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is responsible for male-specific TRPM2 channel activation and neuronal injury. We observed that administration of the TRPM2 inhibitor clotrimazole (CTZ) 2 hours after onset of ischemia reduced infarct volume in male mice and that protection from ischemic damage by CTZ was abolished by removal of testicular androgens (castration; CAST) and rescued by androgen replacement. Male PARP-1 knockout mice had reduced ischemic damage compared with WT mice and inhibition of TRPM2 with CTZ failed to reduce infarct size. Lastly, we observed that ischemia increased PARP activity in the peri-infarct region of male mice to a greater extent than female mice and the difference was abolished in CAST male mice. Data presented in the current study indicate that TRPM2-mediated neuronal death in the male brain requires intact androgen signaling and PARP-1 activity.

Regulation of Ion Channels by Pyridine Nucleotides

Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion-transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide-binding β-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP(+) to Kvβ removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K(+) transporters. These nucleotides also have been shown to modify the activity of the plasma membrane K(ATP) channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit-the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP(+) metabolite, NAADP(+), regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias.

Effects of corticotropin-releasing factor on intermediolateral cell column neurons of newborn rats

Corticotropin-releasing factor (CRF) is a neuropeptide that mediates neuroendocrine, autonomic, and behavioral processes associated with the stress response. CRF-containing fibers and receptors are found in various regions of the central nervous system including the spinal cord. Here, we report excitatory effects of CRF on sympathetic preganglionic neurons in the intermediolateral cell column (IML) of in vitro spinal cord preparations from newborn rats. We also examine the receptor subtypes that are involved in the CRF effects. Application of CRF significantly depolarized the IML neurons and increased the frequency of excitatory postsynaptic potentials (EPSPs) in the IML neurons. These effects were blocked by the CRF receptor 1 antagonist, antalarmin. Menthol, a transient receptor potential channel M8 agonist, depressed EPSPs enhanced by CRF. Our findings suggested that CRF depolarized the IML neurons via direct postsynaptic action and CRF-affected interneurons located in the spinal cord send EPSPs to IML neurons. These excitatory effects of CRF may be caused through CRF1 receptors but not CRF2 receptors.

TRPM2 channel activation following in vitro ischemia contributes to male hippocampal cell death

Hippocampal CA1 neurons are particularly sensitive to ischemic damage, such as experienced following cardiac arrest and cardiopulmonary resuscitation. In recent years transient receptor potential M2 (TRPM2) channels have been identified as mediators of ischemic damage. We previously demonstrated that neuroprotective strategies targeting TRPM2 channels preferentially protect male cortical neurons from ischemic injury both in vitro and in vivo. It is important to determine the role of TRPM2 in ischemic injury of hippocampal neurons as this population of neurons are particularly sensitive to ischemic injury and are therapeutic targets. Here we report significantly decreased neuronal cell death following in vitro ischemia preferentially in male hippocampal neurons using TRPM2 inhibitors or knockdown of TRPM2 expression. Electrophysiological characterization of sex-stratified cultures shows similar levels of functional TRPM2 channel expression in male and female hippocampal neurons under basal conditions. In contrast, recordings made during reperfusion following in vitro ischemia revealed that TRPM2 channels are activated only in male neurons, resulting in rapid and complete depolarization. These findings provide strong evidence for TRPM2 as a target for protection against cerebral ischemia in male brain and helps define a molecular cell death pathway that is differentially engaged in male and female neurons.

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death

The transient receptor potential M2 channel (TRPM2) is the Ca2+-permeable cation channel controlled by cellular redox status via β-NAD+ and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H2O2)- and ADPR-induced Ca2+ responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H2O2-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death.

Sex Differences in Neuroprotection Provided by Inhibition of TRPM2 Channels following Experimental Stroke

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel is activated following oxidative stress and has been implicated in ischemic damage; however, little experimental evidence exists linking TRPM2 channel activation to damage following cerebral ischemia. We directly assessed the involvement of TRPM2 channels in ischemic brain injury using pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of TRPM2 expression. Each of the four TRPM2 inhibitors tested provided significant protection to male neurons following in vitro ischemia (oxygen-glucose deprivation, OGD), while having no effect in female neurons. Similarly, TRPM2 knockdown by TRPM2 shRNA resulted in significantly reduced neuronal cell death following OGD only in male neurons. The TRPM2 inhibitor clotrimazole reduced infarct volume in male mice, while having no effect on female infarct volume. Finally, intrastriatal injection of lentivirus expressing shRNA against TRPM2 resulted in significantly smaller striatal infarcts only in male mice following middle cerebral artery occlusion, having no significant effect in female mice. Data presented in the current study demonstrate that TRPM2 inhibition and knockdown preferentially protects male neurons and brain against ischemia in vitro and in vivo, indicating that TRPM2 inhibitors may provide a new therapeutic approach to the treatment of stroke in men.