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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2013 Jun 26;33(10):1549–1555. doi: 10.1038/jcbfm.2013.105

Androgen and PARP-1 regulation of TRPM2 channels after ischemic injury

Takeru Shimizu 1, Tara A Macey 2, Nidia Quillinan 1, Jelena Klawitter 1, Anne-Laure L Perraud 3, Richard J Traystman 1,4, Paco S Herson 1,4,*
PMCID: PMC3790922  PMID: 23801245

Abstract

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.

Keywords: ischemia, neuroprotection, PARP-1, stroke, TRPM2

Introduction

The calcium-permeable TRPM2 ion channel is expressed in neurons in the cortex, hippocampus, striatum, and brainstem, among other regions1, 2, 3, 4, 5, 6 and is activated after oxidative stress, induced by exogenous hydrogen peroxide.2, 7, 8, 9 Transient receptor potential M2 (TRPM2) channels were initially identified and extensively characterized by our group,7, 8, 10, 11 and demonstrated to contribute to oxidative stress-induced cell death. Indeed, the most well-characterized role for TRPM2 is as an executioner of cell death after oxidative stress, leading to excessive Ca2+ influx and consequent cell death (For review, see1, 2, 9, 12, 13). This observation provided a strong rationale to hypothesize that TRPM2 channels have a significant role in a variety of neurodegenerative diseases whose etiologies involve oxidative stress, including ischemic stroke. Indeed, we recently demonstrated that genetic knockdown (shRNA) or TRPM2 inhibition with clotrimazole (CTZ) reduced neuronal damage after middle cerebral artery occlusion (MCAO) or in vitro ischemia in male brain while having no effect in the female brain.14, 15 The aim of the current study is to determine the mechanism of TRPM2 regulation in the male brain.

Early work using experimental stroke models demonstrated robust gender differences in tissue outcomes, largely focused on the protective role of female sex steroids.16, 17, 18 Emerging data suggest distinct cell-death pathways are triggered in male and female brains. In males, cell death after cerebral ischemia is mediated predominantly by excessive reactive oxygen species production and subsequent overactivation of poly (ADP-ribose) polymerase-1 (PARP-1), ultimately leading to mitochondrial dysfunction, release of apoptosis-inducing factor and cell death. In contrast, in females, cell death involves caspase-dependent apoptosis.19, 20, 21 The male-specific overactivation of PARP-1 in ischemic cell death is particularly relevant because PARP-1-generated adenosine-5′-diphosphoribose (ADPr) directly activates TRPM2 channels after exposure to exogenous oxidants.22, 23 Transient receptor potential M2 channels are the only known ion channels to be directly activated by intracellular ADPr, therefore we hypothesize that TRPM2 is a downstream mediator of PARP-1-induced cell death. Several studies report that PARP inhibitors, and deletion of the PARP-1 gene, provides protection in a sexually dimorphic manner, protecting male, but not female, brain.19, 20, 24, 25, 26, 27 The mechanism of this sex-specific response after PARP inhibition remains unknown. In the current study, we sought to extend the therapeutic window of the protective effect of TRPM2 channel inhibition on ischemic stroke, and directly tested the hypothesis that PARP-1 activation is involved in activation of TRPM2 channels using castrated male mice and PARP-1 knockout mice.

Materials and Methods

Experimental Animals

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado, Denver and conformed to the National Institutes of Health guidelines for the care and use of animals in research. TRPM2 KO mice28 were bred in-house and used at 9 to 10 weeks of age (20 to 30 g). Wild-type C57Bl/6 mice (males and females, 9 to 10 weeks of age, weighing 20 to 30 g) were purchased from Charles River Laboratories (Wilmington, MA, USA). Male PARP knockout mice (129S-Parp1tm1Zqw/J) and corresponding WT mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were permitted ad libitum access to water and standard lab chow. All experiments were performed in a masked randomized manner, with a separate investigator generating experimental code using computer-generated random number generator to randomize.

Castration and Dihydrotestosterone Replacement

Castration and dihydrotestosterone (DHT) treatment were performed 7 days before MCAO surgery. Gonadectomy was performed under isoflurane anesthesia, as previously described.29 Dihydrotestosterone was administered by subcatenous implant of a continuous release pellet (5 mg).

Middle Cerebral Artery Occlusion Model

Transient focal cerebral ischemia (60 minutes for wild-type animals and 90 minutes for knockout animals) was induced using reversible MCAO through the intraluminal filament techniques as previously described.30 Briefly, mice were anesthetized with isoflurane (induction 3.0% and maintenance 1.5% to 2.0%, delivered through a face mask in oxygen-enriched air). Head and body temperature was monitored and maintained at 36.5±1.0°C throughout the MCAO surgery with an electrical heating pad and heating lamp. A laser Doppler probe (Moor Instruments, Oxford, England, UK) was placed over the ipsilateral cortex to assure adequate occlusion. Probe placement was established in a similar location for all mice by making a small incision (probe hole) in the middle of a line drawn between the outer canthus (lateral corner of the eye) and ear canal. All mice had a similar level of occlusion throughout, reduced to less than 20% of baseline (Table 1). Clotrimazole (30 mg/kg) or vehicle was administered via subcutaneous injection (50 μL/10 g body weight) 120 minutes after occlusion (60 minutes after reperfusion).

Table 1. Physiologic data measured during MCAO surgery.

  LDF (% of baseline)
 Temperature (°C)
  Just before reperfusion Reperfusion 5 minutes Rectal Tympanic
Male WT (n=7) 11±2 86±10 36.6±0.1 36.8±0.2
Male TRPM2 KO (n=5) 13±2 70±8 36.5±0.1 36.8±0.1
Male TRPM2 KO+CTZ (n=7) 9±1 91±7 36.6±0.2 37.1±0.2
Male TRPM2 KO+Veh (n=7) 12±2 74±7 36.5±0.0 36.8±0.1
Female WT (n=7) 7±1 93±9 36.7±0.1 36.6±0.1
Female TRPM2 KO (n=7) 8±1 88±12 36.4±0.1 36.5±0.2
Intact male+Veh (n=8) 17±2 87±10 36.2±0.2 35.8±0.3
Intact male+CTZ (n=8) 22±4 94±6 36.2±0.2 36.1±0.3
CAST+Veh (n=9) 12±2 98±8 35.9±0.3 36.1±0.3
CAST+CTZ (n=11) 13±1 84±6 35.8±0.2 36.0±0.2
CAST+DHT+Veh (n=12) 12±1 93±3 35.5±0.2 35.5±0.2
CAST+DHT+CTZ (n=11) 14±2 95±4 35.5±0.3 35.7±0.3
PARP-1 KO+Veh (n=7)* 11±1 90±7 36.7±0.2 36.6±0.3
PARP-1 KO+CTZ (n=6)* 9±2 98±9 36.5±0.2 36.3±0.4
PARP activity male (n=8)* 8±1 83±5 36.7±0.2 36.5±0.3
PARP activity female (n=8)* 9±2 73±4 36.8±0.1 36.5±0.3
PARP activity male CAST (n=8)* 11±2 75±9 36.5±0.2 36.8±0.2
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Abbreviations: ANOVA, analysis of variance; CAST, untreated castrated male mice; CTZ, clotrimazole; DHT, dihydrotestosterone; KO, knockout; LDF, laser-Doppler flowmetry; MCAO, middle cerebral artery occlusion; PARP KO, poly(ADPribose) polymerase-1 knockout mice; TRPM2 KO, transient receptor potential M2 knockout mice; Veh, vehicle (corn oil); WT, wild type.

No differences among groups were observed at baseline or during MCAO surgery. All measurements are within physiologic range and are reported as mean±s.e.m. No statistical difference was observed between groups using one-way ANOVA.

Infarct Volume Analysis

At 24 hours after reperfusion, mice were anesthetized with isoflurane 5.0% and the animals were decapitated for brain removal. If subarachnoid hemorrhage was observed, the mouse was excluded from the study. A total of 163 mice were used in the study, with 13 being excluded because of subarachnoid hemorrahge and 3 for premature death, with no differences among groups. Brains were sliced into five 2-mm-thick coronal sections and were placed in a 1.2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, St Louis, MO, USA) for 30 minutes at 37°C and fixed in 10% formalin for 24 hours. Both sides of each stained coronal slice were photographed using a digital camera (Leica Microsystems, Buffalo Grove, IL, USA), and infarction was measured with ImageJ (NIH, Bethesda, MD, USA) and integrated across all five slices. To account for the effect of edema, the infarct volume of ipsilateral hemisphere was estimated indirectly and expressed as a percentage of the contralateral hemisphere.

Poly (ADP-ribose) Polymerase Activity Assay and ADPribose Measurements

At 24 hours reperfusion, PARP activity in the peri-infarct (penumbral) zones was evaluated by quantifying polyADP-ribose (PAR) using ELISA kit (HT PARP in vivo Pharmocodynamic Assay II, TREVIGEN, Gaithersburg, MD, USA) according to the manufacturer's instructions. For each animal, tissue was collected from cortical ipsilateral peri-infarct zones and the corresponding sites of the contralateral sides. One slice for each brain was stained using TTC to identify the infarcted region and used as a guide for accurate selection of peri-infarct tissue. These tissues were snap frozen and kept at −80°C until use. Chemiluminescent readings were measured using a microplate reader (Synergy 2, BioTek, Winooski, VT, USA). Data were normalized to mean luminescence from non-ischemic cortex within each group.

ADPribose was measured using tandem mass spectrometry (LC-MS/MS) as previously described.31, 32 Briefly, peri-infarct and contralateral cortical tissues were obtained 24 hours after reperFfusion and homogenized in ice cold 12% perchloric acid. Samples were redissolved in water and an Agilent series 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) coupled to an ABSciex API4000 triple stage quadrupole mass spectrometer (ABSciex, Foster City, CA, USA) equipped with an electrospray ionization source was employed for quantitation of ADPribose according to the previously published method for quantitation of nucleotide mono, di, and triphosphates.31 Sample extracts were loaded onto the C18 cartridge desalting column and washed with 1 mL/minute of 97% 4 mmol/L dibutylammonium formate (pH 6.5) buffer and 3% methanol for 0.7 minutes. Thereafter, the switching valve was activated and the analytes were back-flushed with 100% methanol from the desalting column onto the analytical column (Synergi Polar 4 u column (250 × 3 mm, 4 μm particle size, Phenomenex, Torrance, CA, USA)) connected to the API4000. The LC mobile phase consisted of 2 mmol/L dibutylammonium formate buffer (pH 6.5) in water (mobile A) and methanol (mobile B). The following LC gradient was run at 0.6 mL/minute: 0 to 1 minute: 95% mobile A; 1 to 3 minutes: 95% to 70% mobile A; 3 to 10 minutes: 70% to 45% mobile A; 10 to 14 minutes: 45% to 10% mobile A; 14 to 14.8 minutes: 10% to 95% mobile A; 14.8 to 18 minutes: held at 95% mobile A. MS data were acquired in negative electrospray ionization mode with an ion voltage of −4.2 kV. After mass transitions were used: ADPR (quantitation): 558.2→345.9; ADPR (verification); 558.2→210.7; internal standard 6-aminohexyl-ADP: 525.0→233.0. Nebulizer gas was at 30, and so was the heater gas (both nitrogen). The collision gas and curtain gas were at 10 and 20, respectively (both nitrogen). The source temperature was kept at 400°C.Data were acquired and processed for calibration and quantification of all samples using the Analyst software 1.4.2 and data are presented as the ratio of peri-infarct/contralateral cortical values.

Quantitative Real-Time Polymerase Chain Reaction

Ipsilateral and contralateral cortices were collected, snap frozen, and stored at −80°C. Total RNA was isolated using the RNAqueous-4PCR kit (Ambion, Austin, TX, USA) per the manufacturer's instructions. Briefly, approximately 10 mg of tissue was lysed in lysis buffer and total RNA was isolated and eluted from a column with 50 μL RNase-free elution buffer, and further treated with Turbo DNase (Ambion, Austin, TX). First strand cDNA was reverse transcribed from 500 ng total RNA with High Capacity cDNA archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time polymerase chain reaction (PCR) reactions were run in triplicate using Taqman universal PCR mastermix (Applied Biosystems) on an ABI 7300 Real-time PCR system. FAM labeled probe/primer sets used to detect 18S, TRPM2 and PARP-1 were synthesized by Applied Biosytems. The housekeeping gene 18S was assayed for each sample using 5 ng of cDNA and 50 ng of cDNA for TRPM2 and PARP-1. Expression levels were calculated using the ΔΔCT method relative to 18S and normalized to non-ischemic cortex.

Primary Cell Culture

Experiments were performed on sex-stratified mouse cortical neuronal cultures, as described previously.14, 15 Briefly, male and female embryonic day 17 embryos, identified by laparotomy to inspect gonads, obtained from WT and TRPM2 KO mice were rapidly removed from timed pregnant mice under isoflurane anesthesia. The isolated cortices were digested with papain (20 mg/mL; Worthington Biochemical, Lakewood, NJ, USA) and the digested pieces were triturated and filtered through cell sorting nylon mesh (70 mm; BD Biosciences, Bedford, MA, USA). Cells were plated at a concentration of 2.8 × 105 cells per well (24-well plate) coated with poly-D-lysine. In vitro ischemia, oxygen glucose deprivation was produced by transferring cells after 10 to 11 days in vitro to glucose-free medium and placing in an anaerobic chamber for 2 hours (Coy Laboratory Products, Grass Lake, MI, USA). Re-oxygenation was initiated by transferring neuronal cultures to the aerobic incubator and cell viability was determined 24 hours later using the MTT assay, as described previously.14, 15

Statistical Analysis

All data are presented as mean±s.e.m. Each n represents an individual animal. All experiments were performed in a randomized masked manner. Statistical significance was determined using students t-test (unpaired, 2-tailed, if P<0.05) for two groups and one-way analysis of variance with Newman–Keuls post hoc analysis for multiple groups. Two-way analysis of variance with Newman–Keuls post hoc analysis was used to determine the influence of TRPM2 inhibitors and sex on neuronal viability. Statistical significance was set at P<0.05.

Results

Effect of Transient Receptor Potential M2 Knockout on Infarct Volumes and Cell Death in Male and Females

Male and female wild type (WT) and TRPM2 KO mice were subjected to 60-minute MCAO and infarct volume of total hemisphere was analyzed. Male TRPM2 KO mice has smaller infarct volumes compared with matched WT mice, 49.1±3.4% (n=7) in WT vs. 27.3±4.3% (n=7) in TRPM2 KO (Figure 1A; P<0.05). In contrast, TRPM2 KO had no effect on infarct volumes in female mice, 42.3±2.5% (n=7) in WT vs. 45.2±4.3% (n=7) in TRPM2 KO (Figure 1A).

Figure 1.

Figure 1

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Transient receptor potential M2 (TRPM2) knockout (KO) reduces infarct volume, specifically in males. (A) Quantification of infarct volume in male and female wild type (WT) and TRPM2 KO mice and male TRPM KO mice administered CTZ. Data presented as % of infarct relative to contralateral hemisphere. (B) Quantification of neuronal survival after oxygen glucose deprivation in male and female cultures obtained from WT and TRPM2 KO mice. Data presented as cell survival. Data presented as scatter plot showing each individual data point and horizontal bars represent mean±s.e.m. Infarct volume and neuronal viability compared using one-way analysis of variance followed by Neuman–Keuls post hoc analysis (*P<0.05).

To confirm the hypothesis that TRPM2 is involved in sex-specific neuronal cell death, cortical neurons from male and female WT and TRPM2 KO embryos were cultured separately, as described previously.14, 15 Male neurons cultured from TRPM2 KO mice had significantly improved survival after in vitro ischemia (oxygen glucose deprivation) compared with parallel neuronal cultures obtained from WT mice (Figure 1B). In contrast, no difference in neuronal survival after oxygen glucose deprivation was observed in female neurons obtained from WT and TRPM2 KO mice (Figure 1B).

Effect of Post-Middle Cerebral Artery Occlusion Administrations of Transient Receptor Potential M2 Channel Inhibitor Clotrimazole on Infarct Volumes in Male Mice

Male mice were subjected to 60-minute MCAO and infarct volume of total hemisphere was analyzed. The TRPM2 inhibitor CTZ (30 mg/kg) was injected 120 minutes after occlusion and infarct volume analyzed 24 hours after reperfusion. All mice had similar reduction in relative blood flow measured by laser Doppler flowmetry (Table 1). Male WT mice treated with CTZ had smaller total infarct volumes compared with vehicle-treated male mice, 46.2±3.1% (n=8) in vehicle vs. 32.5±3.6% (n=8) in CTZ (Figure 2; P<0.05). To confirm specificity of CTZ neuroprotection, CTZ was administered to male TRPM2 KO mice 120 minutes after occlusion. CTZ did not reduce infarct volumes in male TRPM2 KO mice compared with vehicle-treated TRPM2 KO mice, 27.3±4.3% (n=7) in vehicle TRPM2 KO and 25.1±3.9% (n=7) in CTZ TRPM2 KO (Figure 1A, P<0.05).

Figure 2.

Figure 2

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Clotrimazole (CTZ) reduces infarct volume after middle cerebral artery occlusion. Representative 2,3,5-triphenyltetrazolium chloride stained brain sections (2 mm) with vehicle (A) and CTZ (B) treated male animals (n=8 per group). (C) Quantification of infarct volume. Data presented as % of infarct relative to contralateral hemisphere. Data presented as scatter plot showing each individual data point and horizontal bars represent mean±s.e.m. Infarct volume compared using two-tailed t-test (*P<0.05).

Effect of Androgen/Androgen Receptor Signaling on Clotrimazole Protection in Male Mice

In order to assess the role of androgen receptor (AR) signaling, mice were castrated 1 week before MCAO to remove endogenous androgens. In contrast to intact male mice, administration of CTZ (30 mg/kg) had no effect on infarct volume in castrated male mice, 44.6±3.1% (n=9) in CAST vehicle mice vs. 39.5±3.3% (n=9) in CAST CTZ-treated mice (Figure 3). Androgen replacement using implanted DHT pellets rescued CTZ protection in castrated mice. Mice implanted with DHT (5 mg) had comparable infarcts compared with CAST mice (44.6±3.1% (n=9) in CAST mice vs. 40.4±2.3 (n=12) in CAST+DHT mice). Dihydrotestosterone-replaced mice treated with CTZ had significantly reduced infarcts compared with vehicle-treated mice, 40.4±2.3 (n=12) in DHT vehicle mice vs. 31.5±2.8% (n=9) in DTH CTZ-treated mice.

Figure 3.

Figure 3

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Androgen signaling is required for clotrimazole (CTZ) protection. Infarct volumes were assessed in castrated (CAST)- and vehicle-treated mice (CAST vehicle, n=9), CAST- and CTZ-treated mice (CAST CTZ, n=11), CAST- and dihydrotesosterone (DHT)-replaced and vehicle-treated mice (CAST DHT, n=12), and CAST- and DHT-replaced and CTZ-treated mice (CAST DHT CTZ, n=9). Data presented as % of infarct relative to contralateral hemisphere. Data presented as scatter plot showing each individual data point and horizontal bars represent mean±s.e.m. Infarct volume compared using one-way analysis of variance followed by Neuman–Keuls post hoc analysis (*P<0.05).

Effect of Poly (ADP-ribose) Polymerase-1 -1 Gene Deletion on Clotrimazole Protection in Male Mice

Gonadally intact male PARP-1 KO mice were subjected to 90-minute MCAO and total infarct (hemisphere) was analyzed. Ischemic duration was increased to 90 minutes to obtain large infarcts, avoiding a floor effect and thus allowing the determination of the role of inhibition of TRPM2 channels in the absence of PARP-1. Administration of CTZ did not further decrease total infarct volumes compared with vehicle treated animals, 23.0±2.3% (n=6) vs. 26.3±1.8% (n=7; Figure 4). These data suggest that PARP-1 is upstream of TRPM2 channel activation.

Figure 4.

Figure 4

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Clotrimazole (CTZ) does not decrease infarct volumes in PARP-1 knockout mice. Clotrimazole was administered via subcutaneous injection 60 minutes after reperfusion in vehicle-treated mice (n=7) and CTZ-treated mice (n=6). Data presented as % of infarct relative to contralateral structure. Data presented as scatter plot showing each individual data point and horizontal bars represent mean±s.e.m. No statistical difference between vehicle and control using two-tailed t-test.

To confirm the involvement of PARP activity after ischemic insult, we measured the PAR level in tissues from peri-infarct regions and the corresponding zones of the contralateral sides from male and female animals 24 hours after reperfusion (Figure 5A). Poly (ADP-ribose) polymerase activity in peri-infarct regions had 1.7-fold increase compared with contralateral cortex in male animals (n=8) whereas there was no change in female animals, 1.01-fold change compared with contralateral cortex (n=8; Figure 5B). Because sexual dimorphism was observed in the PARP activity, we examined the effect of testosterone removal on the PARP activity. Unlike gonadally intact male animals, there was no relative increase in PARP activity in the ipsilateral cortex in castrated animals, 0.86-fold change compared with contralateral cortex (n=8; Figure 5B). We used tandem mass spectrometry (LC-MS/MS) to directly measure the effect of ischemia on levels of ADPribose, the TRPM2 agonist. ADPribose levels increase 5.3-fold in the peri-infarct region intact male animals (n=4), with no increase in ADPribose levels in the peri-infarct region of female animals (n=5) or castrated male animals (n=5; Figure 5C).

Figure 5.

Figure 5

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Poly (ADP-ribose) polymerase (PARP) activity increased in ischemic regions in male brains. (A) Representative slice stained with 2,3,5-triphenyltetrazolium chloride after tissue collection. Squares indicate peri-infarct regions (yellow) and the corresponding site of the contralateral side (black) that were collected for PARP activity assay. (B) Relative expression of PARP activity in peri-infarct regions in male brains showed 1.47-fold increase compared with contralateral side, whereas there was no difference in female brains (n=8 in male and female) or castrated (CAST) males. (C) Relative ADP-ribose levels in peri-infarct regions of male brains showed 5.3-fold increase compared with contralateral side, whereas no difference in female brains or CAST males was observed. Values are mean±s.e.m. *P<0.05.

Quantitative real-time PCR was used to assess differences in PARP-1 expression between males and females and whether ischemia causes an increase in PARP-1 expression that may account for the observed increase in PARP activity. Expression of PARP-1 messengerRNA relative to the housekeeping gene 18S was similar in non-ischemic males and females (1.26±0.17 and 1.3±0.39 respectively; n=4). Poly (ADP-ribose) polymerase-1 expression was unchanged by ischemia in males and females as assessed by the ratio of PARP-1 messenger RNA in ipsilateral: contralateral hemispheres (0.93±0.13 and 0.94±0.19 respectively; n=4).

Discussion

This study provides mechanistic insight into the sex-specific role of TRPM2 in neuronal injury after experimental stroke. We show that the removal of androgens prevents neuroprotection by the TRPM2 inhibitor CTZ. We also observed a lack of neuroprotection by CTZ in PARP-1 knockout, suggesting that PARP-1 activity is upstream of TRPM2 activation. Further, we show that PARP-1 activity is selectively increased in males after ischemia and this ischemia-induced activity requires androgens. This is a novel example of an important neuronal death mechanism that is engaged in a sexually dimorphic manner.

We recently demonstrated that genetic or pharmacological inhibition of TRPM2 reduces ischemic injury in a sexually dimorphic manner, protecting males while having no effect in females.14 We confirm that important observation in this study using TRPM2 KO mice and neurons. Similarly, there is extensive evidence that inhibition of PARP-1 activity reduces ischemic injury in the male mouse brain specifically.20, 26, 27, 33 Therefore, we hypothesized that TRPM2 activation after cerebral ischemia requires PARP-1 activity. Indeed, TRPM2-mediated cell death after oxidative stress has been demonstrated to depend on the functional activity of PARP-1 and consequent production of ADPribose.22, 23 Consistent with this in vitro observation, we demonstrate for the first time that neuroprotection after TRPM2 inhibition is lost in PARP-1 knockout mice. Further, our data show that ischemia-induced activity of PARP is greater in the male brain compared with the female brain and that increased PARP activity is lost after removal of endogenous androgens (CAST). This is particularly interesting as the PARP activity assay performed in our study detects ADP-ribose, the ligand which activates TRPM2. This observation coupled with the loss of neuroprotection after TRPM2 inhibition in castrated, WT and PARP-1 knockout mice leads us to the conclusion that PARP-1 signaling is necessary for TRPM2 engagement in male ischemic brain injury. While this data are consistent with reduced PARP-1 expression in the CAST male as previously reported,30 our data from intact males and females suggest that PARP expression alone cannot account for differences in PARP activity. We observe no difference in PARP-1 expression between males and females, yet we observe a clear difference in PARP activity and ADPribose levels in response to experimental stroke. Thus, androgen signaling may be required for both expression and function of PARP-1. Similarly, we have reported no difference in expression of TRPM2 between males and females, yet there is a selective activation of the channel in response to ischemia in males only.14, 15 This provides further evidence that cell-death mechanisms engaged in males are different than those in females and provides a link to the role of androgens in this pathway. The precise mechanism that links AR signaling to PARP activity and TRPM2 activation remains to be elucidated.

Previous studies examining the effect of CAST on infarct volume after experimental stroke have given mixed results with some reports of androgen removal being protective30, 34 or having no effect.29 While our data do not directly address the effect of androgen removal and replacement, our study did make the novel observation that CAST fundamentally alters postischemic signaling, abolishing the protective effect of TRPM2 inhibition after experimental stroke. Additionally, we observed that androgen replacement with the potent AR agonist DHT reverses the effect of CAST on TRPM2 signaling. The AR is a classic nuclear steroid receptor that is expressed throughout the brain, particularly in neurons that are sensitive to ischemic injury in our model such as cortex, striatum, and hippocampus.35, 36, 37 The AR acts transcriptionally to alter expression of genes containing an androgen response element, although the subset of genes responsible for modifying ischemic sensitivity remains unclear. In addition to genomic effects, recent reports have implicated rapid signaling as a mechanism of neuroprotection, including rapid activation of MAPK/ERK and CREB (for review, see38). Our data do not clarify the mechanism by which AR signaling alters TRPM2 activity; however, it appears clear that PARP-1 activity is regulated by androgen status. Our measurements of PARP-1 expression (mRNA) lead us to hypothesize that the effects of AR signaling we observe on TRPM2 activity are related to rapid AR signaling. Indeed, it was recently demonstrated that PARP-1 activity is stimulated by ERK phosphorylation.39 Therefore, it is possible that androgens facilitate PARP-1 activity via increased activity of ERK (Figure 6). Regardless, our data implicates AR signaling in regulating PARP-1 activity in the postischemic brain and this signaling cascade is necessary for TRPM2 engagement in ischemic injury in the male brain.

Figure 6.

Figure 6

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Model of regulation of transient receptor potential M2 (TRPM2) channel activity after cerebral ischemia in the male brain. AR, androgen receptor; CTZ, clotrimazole; DHT, dihydrotestosterone; NUDIX, ADP-ribose-binding region of C-terminus of TRPM2 channel; PARG, poly(ADP-ribose)glycohydrolase; PARP, poly(ADP-ribose) polymerase.

In conclusion, inhibition of TRPM2 channels 120 minutes after occlusion is effective to reduce infarct volume. This extends the therapeutic window from our previous studies where CTZ was administered at the time of occlusion.14 Further, the lack of protective effect of CTZ in male TRPM2 KO mice confirms the utility of this compound for dissecting the mechanism of TRPM2 channel regulation in the male brain after experimental stroke. Our data provide strong evidence that PARP-1 is required for TRPM2 activation after experimental stroke and that PARP-1 activity and thus TRPM2-mediated cell death requires the presence of androgens. Finally, our study provides a possible new therapeutic approach to the treatment of stroke in men.

The authors declare no conflict of interest.

Footnotes

Project funded by NIH grant NS058792, Walter S and Lucienne Driskill Foundation grant, AHA-Philips Resucitation Fellowhship 12POST11930031.

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