Androgen Receptor Overexpression Is Neuroprotective in Experimental Stroke

REVIEW

Androgen Receptor Overexpression Is Neuroprotectivein Experimental Stroke

Patricia Ayala & Masayoshi Uchida & Kozaburo Akiyoshi & Jian Cheng &

Joel Hashimoto & Taiping Jia & Oline K. Ronnekleiv & Stephanie J. Murphy &

Kristine M. Wiren & Patricia D. Hurn

Received: 25 February 2011 /Revised: 31 March 2011 /Accepted: 4 April 2011 /Published online: 15 April 2011# Springer Science+Business Media, LLC 2011

Abstract Male sex is a known risk factor in human stroke.However, the role of the cognate receptor for androgens—the androgen receptor (AR)—in stroke outcome remainsunclear. Here, we found that AR mRNA is downregulatedin the peri-infarct tissue of gonadally intact male micesubjected to middle cerebral artery occlusion (MCAO) and6 h reperfusion. We then used genetically engineered miceoverexpressing AR in brain (AR-Tg) to compare outcomesfrom MCAO in intact or castrated males and to evaluate theneuroprotective role of dihydrotestosterone (DHT) replace-ment in AR-Tg castrates. A further evaluation of ARoverexpression in ischemic paradigms was performed usingrat PC12 cells transfected with human AR and treated withoxidative and apoptotic stressors. We then studied the roleof DHT in cultures overexpressing AR. Our results show(1) ischemia alters the expression of AR by decreasing ARmRNA levels, (2) AR overexpression is protective in vivo

against MCAO in intact and castrated AR-Tg mice and invitro against oxidative and apoptotic stressors in AR-PC12cells, and (3) DHT does not enhance the protectiontriggered by AR overexpression in AR-Tg castrated micenor in AR-PC12 cells.

Keywords Cerebral ischemia . Stroke . Androgen receptor .

DHT. Transgenic mice . PC12

Introduction

Sexual dimorphism in human stroke outcome has been welldocumented. Although male sex is a known risk factor [1],the role of androgens in male cerebral ischemia has beenunder-investigated. Low testosterone levels are associatedwith poor outcome after acute ischemia in men [2], yetfindings from the few studies on the effect of androgens inrodent stroke are inconsistent. For instance, testosteroneand dihydrotestosterone (DHT) have been shown to eitherprotect against or exacerbate damage and dysfunction aftercerebral ischemia [3–5]. Such variable results may haveemerged from the use of different androgen doses oranimals in different stages of life [1, 6]. To elucidate thisapparent discrepancy, we previously performed extensivestudies on adult and aged castrated C57/BL6 male mice andrats supplemented with different doses of testosterone orDHT and subjected to cerebral ischemia. Our findingsindicate that the neuroprotection afforded by androgens, asmeasured histologically or by functional outcome, in malemice and rats is dose- and age-dependent [1, 5]. Paradox-ically, both protection against and exacerbation of ischemicdamage can be blocked by the androgen receptor (AR)antagonist flutamide, suggesting that AR is involved inboth outcomes [5, 6].

Section Disease-Related Neuroscience

P. Ayala (*) :M. Uchida :K. Akiyoshi : J. Cheng :S. J. Murphy :K. M. Wiren : P. D. HurnDepartment of Anesthesiology and Perioperative Medicine,Oregon Health & Science University,3181 SW Sam Jackson Park Rd,Portland, OR 97239-3098, USAe-mail: [email protected]

O. K. Ronnekleiv : P. D. HurnDepartment of Physiology and Pharmacology,Oregon Health & Science University,Portland, OR 97239-3098, USA

J. Hashimoto : T. Jia :K. M. WirenDepartment of Behavioral Neuroscience,Oregon Health & Science University; Research Service,Portland Veterans Affairs Medical Center P3-R&D39,Portland, OR 97239, USA

Transl. Stroke Res. (2011) 2:346–357DOI 10.1007/s12975-011-0079-z

ARs are transcription factors mainly activated byandrogens. ARs are also activated by non-androgenicfactors such as growth factors and signaling molecules [7–10]. Yet, the molecular mechanism by which AR mightcontribute to neuroprotection remains largely unknown. Itis not clear at present whether AR expression in brain isaffected by ischemia, and hence whether the neuroprotec-tive outcome is determined by AR availability as either anessential signaling mechanism for androgens, an enhancedsignaling through non-androgenic agonists, or by activationthrough ligand-independent pathways.

Thus, we set out to determine (1) whether ARs arepresent in the cortex and striatum, the regions involved inour standard rodent model of focal cerebral ischemia, and,if so, whether ischemia alters AR expression, and (2)whether AR density is a limiting factor for the effects ofandrogens on ischemic outcomes. To answer these ques-tions, we quantified AR mRNA levels in cortex andstriatum—brain regions differentially affected by stroke—and studied the effect of AR overexpression in stroke invivo using intact and castrated transgenic mouse linesexpressing both endogenous AR and targeted rat AR, andin vitro using a cell line stably transfected with human AR.

Materials and Methods

All experiments were performed in accordance with theNIH guidelines for research animal care and were approvedby the Institutional Animal Care and Use Committee atOregon Health and Science University.

Preparation for In Vivo Studies

Experimental Groups

Experiments were performed on C57/BL6, hemizygous ARtransgenic B6D2F2 (AR-Tg) [11], and B6D2F2 (WT) malemice. Animals were maintained on a 12/12-h light/darkcycle and permitted ad libitum access to water and standardlab chow.

Hormone Assays

Serum levels of total and free testosterone and DHT weremeasured by radioimmunoassay (RIA; Diagnostic ProductsCorp, Los Angeles, CA, USA) as previously described [3, 5].

Castration and Hormone Pellet Implantation

Castration and hormone pellet implantation (0.5 mg DHT)were performed 1 week before middle cerebral arteryocclusion (MCAO) as previously described [5].

Physiological Measurements

In a separate cohort of animals, laser-Doppler flowmetry(LDF), mean arterial blood pressure, arterial blood gases(pH, PaO2, and PaCO2), temporalis muscle temperature, andblood glucose values were measured immediately prior toMCAO and at 45 and 90 min MCAO in intact WT (n=3)and AR-Tg (n=3) to ensure equivalency of physiologicalvariables between these two groups.

Middle Cerebral Artery Occlusion (MCAO) Modeland Infarct Volume Assessment

Mice were randomized for surgery at approximately16 weeks of age, based on a weight of 22–30 g. Transientfocal cerebral ischemia was induced using reversibleMCAO via the intraluminal filament technique, and infarctvolume assessment was carried out as previously described[5]. Mice were subjected to 90 min MCAO followed by24 h reperfusion for infarct volume assessment. For tissuedamage analysis, mice were decapitated at 24 h reperfusion.The brain was removed and sectioned into seven 2-mm-thick coronal sections. The slices were incubated in 2%2,3,5-triphenyltetrazolium chloride (TTC, Sigma, St. Louis,MO, USA) for 10 min on each side at 37°C, fixed in 10%formalin overnight, and then photographed. Images wereanalyzed with Image Analysis Software (Sigma Scan Pro,Jandel, San Rafael, CA, USA). Infarction size wasexpressed as a percentage of the contralateral structure asa correction for edema as previously described [5, 12, 13].For quantitative real-time polymerase chain reaction(qPCR), animals were treated with 2 h MCAO and 6 hreperfusion.

Tissue Preparation for Quantitative Real-Time PCR

Separate cohorts of animals were used for quantitative real-time PCR (qPCR) analysis. Male mice with or withoutMCAO and reperfusion were sedated with ketamine HCl(100 mg/mL, 120 μL) and killed by decapitation. A brainslicer (EM Corporation, Chestnut Hill, MA, USA) was usedto produce 1-mm frontal blocks, which were placed in RNAlater (Ambion, Austin, TX, USA) for tissue RNA preser-vation. Thereafter, the preoptic area, hippocampus, andcortex were dissected under the guidance of a dissectingmicroscope. For qPCR measurement of AR in differentcortex regions, brain slices that encompass the middlecerebral artery (MCA) territory were microdissected fromcortical regions (peri-infract zone, ischemic core, andtransition between peri-infract zone and ischemic core) aspreviously described [3]. To account for the effects ofanesthesia and surgical stress, qPCR measurement of ARfrom contralateral side was performed as control. For

Transl. Stroke Res. (2011) 2:346–357 347

analysis of the AR transgene expression, brain, calvaria,femur, ear, fat, heart, intestine, kidney, liver, lung, muscle,skin, spleen, tendon, and thymus from AR-Tg mice werecollected for RNA isolation from AR-Tg male mice.Tissues were snap-frozen and then stored at −80°C.

RNA Extraction and Reverse Transcription

Total RNA was extracted from the dissected tissues underRNase-free conditions using the Qiagen RNeasy kitaccording to the manufacturer's protocol and was quantifiedwith a NanoDrop ND-100 spectrophotometer (NanoDropTechnologies, Wilmington, DE, USA). cDNA was synthe-sized from 200 ng of total RNAwith 50 U murine leukemiavirus reverse transcriptase (ABI, Foster City, CA, USA),4 μL 5× Go Taq Flexi Buffer (Promega, Madison, WI,USA), 5 mM MgCl2, 0.625 mM dNTP, 100 ng randomhexamer primers (Promega), 15 U Rnasin (Promega), and10 mM dithiothreitol (DTT) in diethyl pyrocarbonate(DEPC)-treated water (Ambion) in a total volume of20 μL. Reverse transcription was conducted according tothe following protocol: 42°C for 60 min, 99°C for 5 min,and 4°C for 5 min. The cDNA was diluted 1:10 withnuclease-free water (Ambion) for a final estimated cDNAconcentration of 1 ng/μL and stored at −20°C.

Real-Time PCR Quantification Assays

Quantitative real-time PCR analysis was performed on theABI 7500 Fast System using Taqman universal PCR mastermix according to the manufacturer's specifications. Taqmangene expression assays containing primers and probes formouse AR (assay ID: Mm00442688_m1), Bax (assay ID:Mm00432050_m1), and β-actin (assay ID: 4352341E)were prepared by ABI. The target gene-specific probeswere labeled using the reporter dye FAM, and the β-actininternal control probe was labeled with reporter dye VIC.Multiplex PCR was performed with AR or Bax and β-actinprimers and probes using the two-step PCR protocol: 95°Cfor 10 min (initial denaturing), followed by 40 cycles ofamplification at 94°C for 15 s (denaturing), and 60°C for1 min (annealing). All samples were run in triplicate.

Primer Efficiency

Standard curves for AR, Bax, and β-actin were preparedwith serial dilutions of POA cDNA in triplicate todetermine the percentage efficiency [E=10(−1/m)−1;m=slope] of each amplification (Pfaffl et al. [14]). Thelinearity correlation coefficient, R2, was 0.985 for AR, 0.98for Bax, and 0.997 for β-actin; and the efficiencies were94% for AR, 100% for Bax, and 90.3% for β-actin.Therefore, the amplification efficiencies for the target and

reference genes in both assays were within the range(90–100%) needed for using the comparative CT methodfor quantification purposes [14, 15].

Real-Time PCR Analysis

Relative quantification analysis was performed using thecomparative CT method [14, 15]. Data were expressed asan n-fold change in gene expression normalized to areference gene and relative to a calibrator sample (cortexor cerebellum). The reference gene β-actin was used tonormalize the target genes for the amount of total RNA inthe reverse transcription. To determine the CT (cyclethreshold) for each transcript, the threshold was set at thelowest point of the exponential curve where the slope of thecurve was the steepest and above the baseline of the first 15cycles. The data are reported as relative mRNA expression.For analysis, the mean±SEM of the n-fold difference wasdetermined for ipsilateral and contralateral sides, and eacharea was analyzed statistically using a two-tailed Student's ttest (p<0.05 was considered significant, *p<0.05, **p<0.01).

Immunohistochemistry

Brain slices were placed in freshly prepared 3% H2O2 inmethanol for 10 min to inhibit endogenous peroxidaseactivity. A high-temperature antigen unmasking techniquewas performed by immersing slides in boiling 0.01 Mcitrate buffer (pH 6.0) for 15 min, then slices weresubjected to immunohistochemical staining as previouslydescribed [11]. Non-specific binding was blocked withnormal goat serum, then brain slices were incubated withpolyclonal rabbit AR antibody (PA1-111A), purchased fromAffinity Bioreagents Inc. (Golden, CO, USA). The PA1-111A AR antibody maps to the N terminus of the receptorand does not recognize other members of the steroidreceptor family. Controls for nonspecific binding wereincubated with rabbit nonimmune IgG. Brain slices wereincubated with secondary biotinilated anti-rabbit antibodyand ABC reagent (Vector Laboratories, Inc., Burlingame,CA, USA), then processed for horseradish peroxidase/3,3′-diaminobenzidine tetramethyl chlorie (DAB) using theABC elite system (Vector Laboratories) according to themanufacturer's instructions. Slides were counterstained withhematoxylin followed by ethanol dehydration, and thencleared in xylene and mounted in Permount (VectorLaboratories, Inc.).

PC12 Culture

PC12, a pheochromocytoma cell line, was purchased fromAmerican Type Culture Collection (ATCC, Manassas, VA,

348 Transl. Stroke Res. (2011) 2:346–357

USA). Wild-type cells (WT-PC12) were stably co-transfectedwith the expression plasmid pCMV-AR containing full-lengthhuman AR and pRSNneo (AR-PC12) [16] or control vectors(pCol2.3 gal and pRSNneo) using FuGENE HD Transfec-tion reagent (Roche Applied Science, Indianapolis, IN,USA). The pCMV-AR vector was kindly provided to us byDr. Marco Marcelli.

AR-PC12 and wild-type and vector-PC12 controls weregrown in Corning cell culture flasks with CellBIND surface(Fisher Scientific, Tustin, CA, USA) containing RPMI1640, 20 mM HEPES (Invitrogen, Carlsbad, CA, USA),1 mL/L penicillin-streptomycin, 15% horse serum/5% fetalbovine serum, and 100 μg/mL G418 (Invitrogen) except forWT-PC12. Cultures were maintained at 37°C in a humid-ified incubator with room air/5% CO2.

For testing the effect of serum deprivation on cellgrowth, cultures were plated in serum-supplemented mediaat a density of 1×105 cells/well in 24-well plates. After24 h, cells were rinsed with serum-free media. Cultureswere then grown in serum-supplemented or serum-freemedia for 24 or 48 h, and the number of live cells wascounted. For testing the effect of DHT on cultures, cellswere plated at a density of 1×105 cells/well in 24-wellplates. After 24 h, cultures were switched to 2% horseserum medium for 24 h and finally to serum-free medium[17]. Cultures were pretreated with 10 nM DHT or ethanolas vehicle control for 1 or 2 h before testing. Finally, cellswere exposed to H202, thapsigargin (Th), or serumwithdrawal for 18 h. Cell viability was measured byMTT staining as described below. DHT was solubilized in100% ethanol, and Th was solubilized in dimethylsulfoxide (DMSO). Drugs were diluted in serum-freemedium to a final concentration of vehicles of <0.1%.Vehicle controls for DHT and Th consisted of ethanol orDMSO, respectively.

Cell Viability Assays

Cell viability was determined spectrophotometrically usingthe 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazoliumbromide (MTT) assay as described [18]. Cells were lysedwith DMSO, and MTT reduction was determined fluoro-metrically at 540 nm.

Statistics

All data are expressed as mean±SEM. Infarct volumes andhormone assay data were analyzed using one-way analysisof variance (ANOVA) with post hoc Student–Newman–Keuls multiple range test or Dunn's method for multiplegroups as indicated and unpaired t test when comparing twogroups. Physiological data were analyzed by two-wayANOVAwith a post hoc Student–Newman–Keuls to correct

for multiple comparisons. p<0.05 was considered statisti-cally significant. All statistical analyses were performedusing SigmaStat Statistical Software, Version 3.0 (SPSS,Inc., Chicago, IL, USA).

Results

Androgen Receptor mRNA Levels Decreaseafter Cerebral Ischemia

Relative AR expression in naïve C57/BL6 male mousebrain was first evaluated. AR mRNA levels were measuredin cortex, hippocampus, and the preoptic area of thehypothalamus (POA) by qPCR. AR mRNA was moreabundant in mouse PAO than in cortex and hippocampus(4.59±0.59, 2.54±0.45, and 0.99±0.10, respectively;Fig. 1). Little is known about the effect of ischemia onthe expression of AR, particularly in cortex and striatum,two regions greatly affected by MCAO, our model ofexperimental stroke; therefore, we studied whether ARexpression is affected by MCAO. Gonadally intact C57/BL6 male mice were treated with focal cerebral ischemiafor 2 h. Cortex and striatum tissues were microdissectedafter 6 h reperfusion from different regions in the ipsilateral(IL) and contralateral (CL) sides (Fig. 2a), and AR mRNAlevels were measured by qPCR (Fig. 2b). AR mRNA waswidely distributed in the cortex across regions in the CLside, with 1.7-fold higher in region b compared to regionsa, c, and d. Even though AR mRNA was also present instriatum, levels were only a 25% fraction of cortex region a(Fig. 2b). As expected, ischemia resulted in a decrease inAR mRNA in the IL side when compared to the CL side.AR expression was decreased by approximately 30% in thetransition zone in cortex (region b; 1.55±0.05 in CL vs.

POA Cortex Hippocampus0.0

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Fig. 1 AR mRNA levels in male mouse brain. The distribution of ARmRNA in naïve C57/BL6 male mouse brain was determined in cortex,hippocampus, and preoptic hypothalamus (POA) by qPCR. Data areexpressed in fold change in gene expression normalized to β-actin as areference gene. Values are means±SEM; n=4, *p<0.05


1.07±0.11 in IL). AR mRNA in the ischemic core (regionsc and d) was highly affected (region c, 1.10±0.22 in CL vs.0.74±0.10 in IL; region d, 0.94±0.7 in CL vs. 0.47±0.02 inIL). AR mRNA was also decreased in striatum (region St)by 70% after MCAO (0.30±0.02 in CL vs. 0.10±0.04 inIL; Fig. 2b). Finally, AR mRNA levels in the peri-infarctzone (region a) in cortex (IL side) were unaffected whencompared to the same region in the CL side (Fig. 2b). Thereduction in AR mRNA levels after MCAO correlated withan increased expression of Bax mRNA, a protein involvedin apoptosis (region b, 0.43±0.01 in CL vs. 0.73±0.04 inIL; region c, 0.36±0.02 in CL vs. 0.60±0.04 in IL; regiond, 0.33±0.01 in CL vs. 0.52±0.02 in IL; striatum, 0.11±0.01 in CL vs. 0.2±0.01 in IL; Fig. 2c), indicating that the

reduction of AR mRNA is not explained by a generalmRNA downregulation post-ischemia.

Androgen Receptor Transgenic Mouse OverexpressesAndrogen Receptor Transgene in Brain Tissue

To determine whether increasing AR density enhancesandrogen sensitivity and allows androgens to provide morerobust protection in mice, we used an AR transgenic (AR-Tg)mouse model with overexpression targeted to mesenchymalprogenitors and cells of the osteoblast lineage as previouslypublished [11]. AR-Tg mice express both endogenous ARand rat AR transgene, and serum testosterone and estradiollevels are not significantly different between transgenic andWT littermates [11]. AR-Tg mice were previously charac-terized for the expression of the AR transgene in a variety oftissues [11]. Here, we measured AR transgene expression byqPCR analysis in brain from AR-Tg mice and found thatafter calvaria, AR transgene is up to 100-fold more abundantin brain than in other tissues tested such as femur, ear, fat,heart, intestine, kidney, and liver (Table 1). We nextevaluated AR protein expression in vivo by immunocyto-chemical analysis as previously described [11]. We detectedimmuno complexes after DAB staining, AR is brown, andafter counterstained with hematoxylin, the nucleus is purple.Figure 3 shows AR immunoreactivity in the majority of cellsin AR-Tg—but not WT—brain. Because of elevated ARexpression in the brain and thus increased sensitivity tocirculating androgens, AR-Tg mice, therefore, provide anideal model to study AR neuroprotection.

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Fig. 2 Cerebral ischemia results in decreased AR mRNA. a Schematicrepresentation of microdissected cortex and striatum tissue. b–cGonadally intact C57/BL6 male mice were treated with focal cerebralischemia for 2 h followed by 6 h reperfusion. mRNA expression levelswere determined by qPCR and are expressed in fold change normalizedto contralateral side. b AR mRNA and (c) Bax mRNA. IL ipsilateral,CL contralateral. Values are means±SEM; n=4, *p<0.05 fromcontralateral side. IL regions a (peri-infract zone), b (transition zone),and c and d (ischemic core) were microdissected from cortex; St,microdissected tissue from striatum

Table 1 AR transgene expression in AR-Tg mice

Tissue Expressiona SEM

Brain 1.00 0.072

Calvaria 11.72 1.780

Femur 0.38 0.134

Ear 0.02 0.004

Fat 0.01 0.003

Heart 0.13 0.011

Intestine 0.07 0.015

Kidney 0.18 0.010

Liver 0.07 0.002

Lung 0.78 0.096

Muscle 0.46 0.067

Skin 0.11 0.026

Spleen 0.11 0.011

Tendon 0.13 0.006

Thymus 0.26 0.049

a Expression normalized to brain


Androgen Receptor Overexpression is Neuroprotective

Gonadally intact male mice from AR-Tg line 104 and WTmale mice were subjected to 90 min MCAO. No differ-ences in intra-ischemic physiologic parameters and LDFreduction were observed among experimental groups(Table 2). The plasma testosterone levels from intactanimals in our study were similar to endogenous levels

observed in intact naïve male mice [5]. Infarct volumes wereassessed at 24 h reperfusion (Fig. 4a). AR-Tg 104 malessustained smaller infarct volumes relative to WT males incortex (21.5±1.9% vs. 40.6±4.7%), striatum (92.3±3.1% vs.110.9±4.1%), and hemisphere (29.6±2.1% vs. 46.3±2.8%;Fig. 4a). There were no differences in physiological variablesduring the ischemic insult between the groups; mean arterialblood pressure, arterial blood gases, and glucose remained

Fig. 3 AR transgenic mice over-express AR in brain. Representa-tive immunohistochemistry ofAR staining in prefrontal cortexof AR transgenic (AR-Tg) andwild-type (WT) male mice.Objective, ×20. Arrows showexamples of AR staining

Table 2 LDF and temporalis muscle temperature in 90 min MCAO+24 h reperfusion

Group LDF (%) Temporalis muscle temperature (°C)

MCAO, min Reperfusion, 5 min Pre-MCAO MCAO, min Reperfusion, 5 min

Intact

WT-Tg 16±1 101±4 37.9±0.1 36.9±0.1 36.7±0.1

AR-Tg 104 17±1 98±4 37.0 37.0 37.0

AR-Tg 106

Castrated

WT-Tg CAST 14±3 78±12 36.9±0.3 37.3±0.1 36.9±0.2

WT-Tg CAST+DHT 14±4 82±22 36.8±0.1 37.1±0.2 36.8±0.2

AR-Tg CAST 16±1 101±4 37.0±0.1 36.7±0.1 36.7±0.1

AR-Tg CAST+DHT 17±1 98±5 36.8±0.1 36.8±0.1 36.8±0.1

Values are mean±SEM

Intact gonadally intact male mice, Wt-Tg wild-type male mice, AR-Tg 104 androgen receptor transgenic mice line 104, AR-Tg 106 androgenreceptor transgenic mice line 106, CAST untreated castrated male mice, CAST+DHT castrated male mice+dihydrotestosterone, LDF, laser-Doppler flowmetry, MCAO middle cerebral artery occlusion


within physiologic limits (pH 7.30±0.01 in WT vs. 7.30±0.03 on AR-Tg 104; PaCO2 30±4 mmHg in WT vs. 34±1 mmHg in AR-Tg 104; PaO2 155±7 mmHg in WT vs.151±6 mmHg in AR-Tg 104; HCO3 15.8±0.4 U in WT vs.12.7±1.2 U in AR-Tg 104; glucose 140±24 mg/dL in WTvs. 162±25 mg/dL in AR-Tg 104; and mean arterial bloodpressure of 72±2 mmHg in WT vs. 71±2 mmHg in AR-Tg104 mice).

During the process of generating transgenic mice withrecombinant DNA, the transferred sequences are integratedrandomly on the chromosome, and variable expression ofthe targeted genes may occur due to the site of integration.Therefore, a second AR-Tg mice line derived fromindependent founders, AR-Tg 106 [11] again with similartestosterone levels to WT, was also evaluated. Gonadallyintact AR-Tg 106 mice were exposed to focal cerebralischemia, and infarct volumes were assessed as describedabove. AR-Tg 106 mice had smaller infracts than WT incortex (15.2±1.5% vs. 39.9±7.7%), striatum (78.2±5% vs.110.4±7.2%), and total hemisphere (17±1.4% vs. 34.7±1.4%; Fig. 4b). No differences in intra-ischemic physiolog-ic parameters and LDF reduction were observed (Table 2).AR-Tg 106 mice were used for the following experimentsand referred to as AR-Tg.

DHT Is Not More Efficacious in AR-Tg Mice

Given that AR overexpression improved outcome ingonadally intact mice producing endogenous AR ligands,we speculated that exogenous androgen would be evenmore effective in the AR-enhanced brain. We first evaluatedprotection against ischemia-reperfusion injury in castratedAR-Tg and WT male mice. Surprisingly, AR-Tg castrateswere protected relative to WT controls in cortex andhemisphere (Fig. 5), suggesting that androgen availabilityis not required for the potential benefit of AR over-expression. Indeed, castrates implanted with 0.5 mg DHT1 week prior to MCAO, which results in low, physiolog-ically relevant plasma steroid levels [1, 5], did notdemonstrate further improved ischemic outcomes relativeto castrates with AR overexpression alone. Plasma DHTlevels were lower in castrated (0.19±0.09 pg/mL; n=12)versus in DHT-replaced mice (0.97±0.32 pg/mL; n=18)post-reperfusion (Dzien and Hurn, personal communica-tion), and testosterone levels were under the detection limitsin castrated animal in both experimental groups (Dzien andHurn, personal communication). No differences on intra-ischemic physiologic parameters and LDF reduction wereobserved in any of the experimental groups (Table 2).

Androgen Receptor Expression Protects PC12 Cellsfrom Serum Withdrawal

PC12 cells ordinarily lack ARs and are sensitive to serumwithdrawal [19]. We transfected this cell line with a humanAR expression construct (AR-PC12) and confirmed AR

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Fig. 4 AR overexpression is neuroprotective in AR transgenic mice.Animals were treated with focal cerebral ischemia and infarct volumes(percent contralateral structure) from two mouse lines derived fromindependent founders were assessed. a Intact AR transgenic mice(AR-Tg line 104, n=10) and wild-type mice (WT, n=10). b Intact ARtransgenic mice (AR-Tg line 106, n=10) and wild-type mice (WT, n=10). Values are means±SEM; *p<0.05

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Fig. 5 DHT does not protect AR transgenic mice from MCAO.Animals were treated with focal cerebral ischemia and infarct volumes(percent contralateral structure) in untreated castrated mice (AR-TgCAST, n=10; WT CAST, n=10) and in castrates implanted with0.5 mg DHT (AR-Tg CAST+DHT, n=10; WT CAST+DHT, n=10)were assessed. Values are means±SEM; *p<0.05


expression, and lack of expression in wild-type or vector-transfected controls (data not shown). Transfected cellsgrew robustly in serum-supplemented media, and cellnumber increased by 200% in AR-PC12 as compared towild-type controls (Fig. 6). Cell death in response to serumwithdrawal was strongly reduced in AR-PC12 cultures.

DHT Does Not Elicit Protection from Injury in AR-PC12 Cells

AR-PC12 cell viability was measured in cultures treatedwith H2O2. Viability was increased from 20% to 200% inAR-PC12 cells compared to wild-type or vector PC12cultures (Fig. 7a, b). Next, cultures were incubated with theapoptotic agent thapsigargin (Th), which triggers intracel-lular calcium elevation [20]. Cell viability was 45% higherin AR-PC12 cells than the control cultures (Fig. 7c),indicating that AR expression protected AR-PC12 cellsfrom calcium-induced apoptosis. Consistent with our invivo findings, DHT did not provide enhanced protectionagainst H2O2 and Th (Fig. 6a–c), or serum withdrawal(Fig. 8) in AR-PC12 cells.

Discussion

The present study demonstrates three important findings.First, AR mRNA is decreased in peri-infarct tissue afterMCAO, suggesting limited AR density and androgen-ARsignaling during ischemia-reperfusion injury. Second, ARoverexpressing mice are protected from experimentalstroke, presumably because gonadal androgens capitalizeon enhanced AR availability and initiate protectivereceptor-mediated mechanisms. This finding indicates thatAR availability can shape ischemic outcomes in the male.

However, protection by AR overexpression surprisinglypersists in males lacking gonadal androgens and isunchanged by DHT replacement, suggesting that AR-mediated mechanisms can use non-androgens as agonists.Alternatively, AR overexpression during development mayalter responsivity to ischemic damage. Third, transfectionof human AR into PC12 cells that ordinarily lack ARreceptors also confers protection against several insults.Consistent with the animal data, AR-mediated protection inthese cells does not require androgens. Together, these dataemphasize that AR signaling can be pivotal to improvedearly tissue and cell outcomes after experimental injury butis initiated by partners other than the nonaromatizableandrogen, DHT.

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Fig. 8 DHT does not enhance AR protection against serumwithdrawal. Cultures were grown for 18 h in the absence of serumwith (+) or without (−) DHT. Cell viability was expressed as percentof control cells. AR-PC12 (AR), wild-type (WT), and vector (V) PC12cultures. Values are means±SEM; n=4, *p<0.05 from WT and Vcontrols without DHT. #p<0.5 from WT and V controls with DHT

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100 µM H2O2 200 µM H2O2 100 nM Th

a cb

Fig. 7 Protection against H2O2 and Th is AR dependent. Cultureswere incubated with 100 μM H2O2 (a), 200 μM H2O2 (b), or 100 nMTh (c) for 18 h in serum-free media with (+) or without (−) DHT. Cellviability was expressed as percent of control cells. AR-PC12 (AR),wild-type (WT), and vector (V) PC12 cultures. Values are means±SEM; n=4, *p<0.05 from WT and V controls without DHT. #p<0.5from WT and V controls with DHT

24 h 48 h0

5

10

15

20

25

30

35

PC12 + Serum

PC12 - Serum

AR-PC12 + Serum

AR-PC12 - Serum

* *

**

**

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er o

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6

Fig. 6 AR expression in PC12 is protective against serum withdraw-al. Wild-type and AR-PC12 cultures were grown in the presence orabsence of serum for 24 or 48 h, and the number of live cells wascounted. Values are means±SEM; n=3, *p<0.05


Here, we used qPCR to study the relative AR expressionin three regions of male mouse brain and found that ARmRNA is more abundant in the preoptic area of thehypothalamus than in cortex and hippocampus. In agree-ment with our findings, AR has been previously foundhighly expressed in the hypothalamus in the adult rat brainby different methods, among others, steroid autoradiogra-phy [21, 22] and in situ hybridization [23]. AR mRNA andprotein were also found in the developing and adult ratbrain [24–26]. However, AR gene expression has beenreported more abundant in hippocampus than cortex in therat as well [27]. More recently, it has been shown that thelargest number of AR-expressing cells is in the cerebralcortex [28]. These discrepancies can be explained bydifferences in the analytical methods used. Nevertheless, acomprehensive mapping of AR in the mouse brain may berequired.

Altered AR levels as well as other steroid receptors havealso been reported after cerebral injury. Immunohistochemicalstaining of rat brain treated with MCAO showed ARupregulation in the ischemic parietal and caudate putamenareas after 24 h reperfusion [29], and the mineralocorticoidreceptor (MR) was upregulated at early time points afterischemia in human hippocampus following brief cerebralischemia [30]. Furthermore, MR mRNAwas increased in theneonatal rat exposed to hyperthermia with or without addedanoxia. Interestingly, the same report did not find variationon glucocorticoid mRNA expression in the same animals[31], suggesting that different steroid receptors may havedifferent responses after brain injury.

Estrogen receptors (ER) are also differentially modulatedby MCAO. ERα mRNA induction in the cerebral cortexappears within the first 4 h and peaks later (16 h) in theischemic injury. ERβ mRNA expression is initially in-creased; however, injury induces a significant decline inERβ in the later stages of ischemia [32].

Our MCAO model was 90 min occlusion followed by24 h reperfusion. We expected the mRNA levels to be lowby 24 h due to tissue degradation and therefore decided tomeasure mRNA levels performing a longer occlusion timefollowed by 6 h reperfusion, as previously reported [3].Here, we also quantitated AR expression levels afterMCAO and are first to report that AR mRNA levels aremodulated by ischemia in cortex and striatum after 6 hreperfusion in male mouse brain. Interestingly, this obser-vation resembles the ERβ decline cited above. We showthat the reduction in AR mRNA expression is region-specific affecting the core ischemic zone more than theischemic penumbra probably due to blood perfusion fromcollateral arteries to the penumbra area. In the present study,we did not elucidate the mechanisms of AR mRNAregulation, since we only measured AR mRNA levels atone time point after reperfusion and did not aim to study

the transcriptional or post-transcriptional regulatory path-ways of AR mRNA in ischemic mice [33].

Two AR-transgenic lines (lines 104 and 106) derivedfrom independent founders were generated with a copynumber of 5–7 (for both lines), the AR transgene relative tothe endogenous AR gene [11]. The authors were targetingAR transgene expression to mesenchymal progenitors andcells of the osteoblast lineage, where it resulted in elevatedAR expression. Notably, a survey of transgene expressionin a variety of tissues identified brain with the secondhighest level of expression, expressing up to 100-fold moreAR transgene than in any other tested tissue within the AR-Tg mice. Type I collagen promoter activity has beenpreviously described in brain tissue [34–36]. Consistentwith overexpression, we observed higher AR protein levelsin AR-Tg mice than WT mice. Our findings fromgenetically engineered mouse strains expressing bothendogenous and exogenous AR indicated that AR over-expression protects cortex, striatum, and hemispherefollowing MCAO and 24 h reperfusion in intact male micewith similar peripheral levels of testosterone. Furthermore,we showed that the depletion of androgens by castrationalso results in smaller infarct volumes in castrated AR-Tgcompared with castrated WT mice after 24 h reperfusion,suggesting that AR plays a role in neuroprotection. It isimportant to note that our animal model used controlledconditions and lacked confounding physiological variabledifferences between animals during MCAO. Earlier studiesin rats and in mice suggest that reduction of androgens viastress or castration improves histological damage aftercerebral ischemia in animals expressing endogenous AR[1, 3–5, 37, 38]. However, damaging effects of androgensin vivo have also been found [1, 3–5, 37, 38]. Finally,beneficial effects of androgens following peripheral nervedamage or brain trauma have also been reported in animals[39–41]. Our group recently reported that the effect ofandrogens on outcome after ischemia in mice and rats isdose-dependent and is blocked by the AR antagonistflutamide, suggesting that AR mediates this response toischemia [1, 5]. Differences of effect of androgens on strokecould be related to the differences in the density andactivity of ARs in response to the ligand and to theischemic stimulus.

To further analyze the neuroprotective role of AR, wealso used PC12 cells, an artificial system of in vitro cellculture. PC12, a male rat pheocytoma cell line, offers theadvantage that in its undifferentiated state, it does notexpress AR [42], but it is easily transfected with the ARgene cloned in a mammalian expression vector. Consistentwith our observations in the AR-Tg in vivo model, ARexpression triggers cell growth and protects against oxida-tive stress, apoptosis, and serum deprivation in AR-PC12cultures, although it is not clear whether this protection


resulted from an accelerated growth of AR-PC12 cells dueto AR or upregulation of antiapoptotic genes mediated byAR, or both.

Our most striking and surprising results show that DHTimplanted in castrated mice does not enhance protection inour in vivo model, the AR-Tg mice, nor AR-PC12 culturedcells. First, DHT plasma levels maintained within the lowphysiological range throughout an episode of focal cerebralischemia did not enhance neuroprotection in castratedtransgenic male mice overexpressing AR. This observationis in agreement with an earlier report indicating thatandrogens have no effect on injury in rat [43]. In contrast,our previous studies in C57/BL6 male mice after focalcerebral ischemia show that low doses of androgens areneuroprotective, a protection mediated by AR [1, 5, 6].Second, we also showed that physiological concentrationsof DHT did not enhance the protection provided by ARexpression against H2O2, Th, and serum withdrawal in AR-PC12 cultures. Taken together, these data suggest that ARactivation is mediated by different agonists in our experi-mental systems.

AR is a member of the steroid and nuclear receptorsuperfamily that also consists of the estrogen, glucocorti-coid, mineralocorticoid, progesterone, vitamin D, thyroid,and retinoic acid receptors [44]. Upon binding the andro-gens testosterone and DHT, AR undergoes a conformation-al change that affects AR interactions with proteins andDNA as well as nuclear shuttling. In the nucleus, ARmediates changes in gene expression to influence cellproliferation, differentiation, apoptosis, and metabolism.The ligand-binding domain not only interacts with testos-terone and DHT but also with a number of steroidal andnon-steroidal agonists and antagonists [45]. After castra-tion, the AR signaling system remains activated despite lowserum levels of androgens in human prostate cancerpatients [46]. A large body of evidence indicates that ARis activated by other means, e.g., alternative sources ofandrogens such as the adrenal androgens dehydroepian-drosterone (DHEA) and androstendione [47]. DHEA is awell-known neuroprotective neurosteroid [48–51], but itsneuroprotective effects are not completely understood.Although the metabolic profile of DHEA in the brain hasnot been fully elucidated, DHEA may be metabolized to theactive metabolites 7α-OH DHEA and 7β-OH DHEA [52,53]. In addition, AR can be activated by non-steroidmechanisms such as AR amplification with concomitantoverexpression of AR [10, 54] and the activation of kinasepathways [55]. Moreover, ligand-independent activationtriggers increased signaling by a number of other growthfactor receptors that can enhance AR signaling, which inturn induces downstream activation of pathways critical forgrowth and survival, including the AKT, MAPK, and STATpathways [7, 8, 56].

Conclusion

AR overexpression is neuroprotective in vivo in ARtransgenic mice and in vitro in an AR-transfected cell line,and physiological concentrations of DHT do not enhancethe protection conferred by AR overexpression. Furtherstudies are needed to elucidate the signaling mechanisms ofAR following ischemic brain injury.

Acknowledgements We thank Ms. Kathy Gage for her outstandingeditorial assistance. This research was funded by National Institute ofHealth grants NS 33668, NR 003521, and NS 49210.

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Androgen Receptor Overexpression Is Neuroprotective in Experimental Stroke