European Journal of Pharmacology 670 (2011) 479–486

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

Melanocortin MC4 receptor agonists counteract late inflammatory and apoptoticresponses and improve neuronal functionality after cerebral ischemia

Luca Spaccapelo a, Alessandra Bitto b, Maria Galantucci a, Alessandra Ottani a, Natasha Irrera b,Letteria Minutoli b, Domenica Altavilla b, Ettore Novellino c, Paolo Grieco c, Davide Zaffe d,Francesco Squadrito b, Daniela Giuliani a, Salvatore Guarini a,⁎a Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, Via G. Campi 287, 41125, Modena, Italyb Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, University of Messina, Messina, Italyc Department of Pharmaceutical Chemistry and Toxicology, University of Napoli “Federico II”, Napoli, Italyd Department of Biomedical Sciences, Section of Human Morphology, University of Modena and Reggio Emilia, Modena, Italy

⁎ Corresponding author. Tel.: +0039 59 5055371; faxE-mail address: salvatore.guarini@unimore.it (S. Gua

0014-2999/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.ejphar.2011.09.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 May 2011Received in revised form 29 August 2011Accepted 7 September 2011Available online 21 September 2011

Keywords:Cerebral ischemiaMelanocortin MC4 receptorInflammationApoptosisLearning and memoryNeuroprotectionSynaptic plasticity

Indirect evidence indicates that, in cerebral ischemia, melanocortins have neuroprotective effects likelymediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in ischemicstroke, we investigated the effects of a highly selectiveMC4 receptor agonist. Gerbils were subjected to tran-sient global cerebral ischemia by occluding both common carotid arteries for 10 min. In saline-treatedstroke animals, an impairment in learning and memory occurred that, at day 11 after stroke, was associatedwith hippocampus up-regulation of tumor necrosis factor-α (TNF-α), BAX, activated extracellular signal-regulated kinases (ERK1/2), c-jun N-terminal kinases (JNK1/2) and caspase-3, down-regulation of Bcl-2,and neuronal loss. Treatment for 11 days with the selective melanocortin MC4 receptor agonist RO27-3225, as well as with the well known non-selective [Nle4,D-Phe7]α-melanocyte-stimulating hormone(NDP-α-MSH) as a reference non-selective melanocortin, counteracted the inflammatory and apoptoticresponses, as indicated by the changes in TNF-α, BAX, ERK1/2, JNK1/2, caspase-3 and Bcl-2 protein expres-sion. Furthermore, melanocortin treatment reduced neuronal loss and dose-dependently improved learn-ing and memory. These positive effects were associated with overexpression of Zif268, an immediate earlygene involved in injury repair, synaptic plasticity and memory formation. Pharmacological blockade ofMC4 receptors with the selective MC4 receptor antagonist HS024 prevented all effects of RO27-3225 andNDP-α-MSH. These data give direct evidence that stimulation of MC4 receptors affords neuroprotectionand promotes functional recovery from stroke, by counteracting prolonged and/or recurrent inflammatoryand apoptotic responses, and likely by triggering brain repair pathways.

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1. Introduction

Important developments in the field of neurocritical care haveimproved the therapeutic approaches to neurologic patients, butischemic stroke remains the leading cause of adult disability and thesecond main cause of death worldwide, the only approved therapybeing thrombolysis within 3–4.5 h of symptom onset (Adams et al.,2007; Baldwin et al., 2010; Yepes et al., 2009). Remarkable progresshas been made in the last few years in understanding the molecularmechanisms of neurodegeneration but, despite intensive investiga-tions, no innovative neuroprotective drugs have had, so far, provensuccess in advanced clinical trials. This failure is due to several con-comitants causes, such as insufficient preclinical testing in multiple

stroke models, narrow window of therapeutic opportunities, blockadeof just one of the ischemia-related mechanisms leading to neuronaldamage, toxic side effects (Gladstone et al., 2002; Leker and Shohami,2002; Moskowitz et al., 2010; O'Collins et al., 2006; Rogalewski et al.,2006; Tatro, 2006; Yepes et al., 2009).

Melanocortin peptides belong to the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) group (Catania, 2008;Catania et al., 2004; Corander et al., 2009; Patel et al., 2010; Wikbergand Mutulis, 2008). These endogenous peptides and synthetic ana-logs have protective effects in animal and human hypoxic condi-tions, such as circulatory shock and myocardial ischemia (Bazzani et al.,2001; Giuliani et al., 2010; Guarini et al., 1990, 2004; Mioni et al., 2005;Noera et al., 2001; Ottani et al., 2010). Melanocortins also afford a strongneuroprotection – in a broad therapeutic treatment window – againstinjury due to experimental global or focal cerebral ischemia (Giulianiet al., 2006a,b, 2007b, 2009; Holloway et al., 2011; Huang and Tatro,2002; Ottani et al., 2009). This effect occurs through a counteraction

Fig. 1. Experimental schedule and treatments.

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of the main ischemia-related, early developed mechanisms of damage,and is associated with long-lasting improvement in functional recoveryaccompanied by overexpression of Zif268, an immediate early generequired for synaptic plasticity and memory formation and involvedin injury repair (Giuliani et al., 2006a,b, 2007b, 2009).

Indirect evidence from our studies suggests that these neuropro-tective effects are mediated by melanocortin MC4 receptors withinthe central nervous system (CNS) (Giuliani et al., 2006a,b, 2007b,2009). To gain direct insight into the role of MC4 receptors in strokeconditions, we investigated the effects of a melanocortin agonist,highly selective at MC4 receptors, in a gerbil model of transient globalbrain ischemia, by exploring the late inflammatory and apoptoticmachineries and neuronal functionality.

2. Materials and methods

2.1. Transient global brain ischemia

Male Mongolian gerbils (Charles River Breeding Laboratories,Calco, Italy) weighing 70–80 g were used. Housing conditions and ex-perimental procedures were in strict accordance with the EuropeanCommunity regulations on the use and care of animals for scientificpurposes (CEE Council 89/609; Italian D.L. 22-1-92 No. 116) andwere approved by the Committee on Animal Health and Care of Mo-dena and Reggio Emilia University. The animals were acclimatizedto our housing conditions for at least 1 week before use, and werekept in air-conditioned colony rooms (temperature 21±1 °C, humid-ity 60%) on a natural light/dark cycle, with food in pellets and tapwater available ad libitum. Surgical procedures, stroke induction,and animal sacrifice at the end of the observation period, were per-formed under general anesthesia with chloral hydrate, 400 mg/kgintraperitoneally (i.p.) (Sigma). Ischemic stroke was induced byoccluding with atraumatic clips both common carotid arteries for10 min (Giuliani et al., 2006a,b, 2009; Liu et al., 1998). Rectaland cranial (left temporalis muscle) temperatures were monitoredwith temperature probes, from the induction of anesthesia and for11 days, and were maintained close to 37 °C by means of heatinglamps. After stroke induction, gerbils were allowed to recoverfrom anesthesia and surgery for 3 days before starting behavioralstudies. Sham ischemic animals underwent to the same surgicalprocedure except for carotid occlusion. Body weight was recordedthroughout the 11-day observation period.

2.2. Drugs and treatment schedules

Butir-His-D-Phe-Arg-Trp-Sar-NH2 trifluoracetate salt (RO27-3225,selective agonist at melanocortin MC4 receptors: Benoit et al., 2000;Giuliani et al., 2007a) and Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 trifluoracetate salt (NDP-α-MSH, whichacts at melanocortin MC1, MC3, MC4 and MC5 receptors: Catania etal., 2004; Giuliani et al., 2007a) were synthesized in our laboratoryby conventional solid phase chemistry, purified by RP-HPLC andchecked for proper molecular weight by mass-spectroscopy, as previ-ously reported (Grieco et al., 2002, 2003). Cys-Nle-Arg-His-D-Nal-Arg-Trp-Gly-Cys (HS024; cyclic MSH analog with S-S bridge betweentwo Cys) was purchased from Neosystem (Strasbourg, France). Alldrugs were dissolved in saline (1 ml/kg) and administered i.p. Controlanimals (ischemic or sham ischemic) received equal volume of salineby the same route. RO27-3225 and NDP-α-MSH were administeredevery 12 h (for 11 days) starting 3 h after the ischemic episode.RO27-3225 and NDP-α-MSH were also assessed on learning andmemory in sham gerbils. Pretreatment with HS024 (potent and high-ly selective melanocortin MC4 receptor antagonist) (Kask et al., 1998)or saline, when done, was performed i.p. 20 min before each adminis-tration of RO27-3225, NDP-α-MSH or saline. All drugs and salinewere administered during the daytime (7:00–8:00 a.m. and 7:00–

8:00 p.m.). The doses of RO27-3225 (45–180 μg/kg), NDP-α-MSH(340 μg/kg) and HS024 (130 μg/kg) were selected by a pilot studyand on the basis of previous investigations (Bitto et al., 2011; Giulianiet al., 2006a,b, 2007a,b, 2009; Kask et al., 1998; Ottani et al., 2009).Experimental schedule (chosen on the basis of our previous studies)and treatments are depicted in Fig. 1.

2.3. Assessment of spatial learning and memory

We used the Morris water-maze test with minor modifications, aspreviously described (Giuliani et al., 2006a,b, 2007b, 2009). This testmeasures animal's ability to learn, remember and go to a place inspace defined only by its position relative to distal extramaze cues.The apparatus consisted of a circular white pool filled to a depth of15 cmwith water (27 °C) rendered opaque with milk powder. Gerbilswere trained to find the spatial location of a little platform of clearPerspex hidden by arranging for its top surface to be 1 cm belowthe water level; the platform occupied a fixed position at 20 cmfrom the pool wall. In each training, latency to escape onto the hiddenplatform was recorded. On the day before starting training, each ger-bil was given 60 s of adaptation to the pool. According to Morris' testschedules (Giuliani et al., 2006a,b, 2007b, 2009), animals were thensubjected to a first 5-day training sequence (to assay learning) start-ing at day 4 after ischemia induction, followed at day 3 after the endof learning assay (that is, day 11 after ischemia) by a second 1-daytraining (to assay memory) (Fig. 1). Tests were performed between10.00 a.m. and 03.00 p.m. in a sound-proof room. The pool wasdrained and cleaned each day at the end of testing.

2.4. Isolation of cytoplasmic and nuclear proteins and western blotanalysis

At day 11 after stroke, after the last behavioral test (and within90 min) the whole hippocampus was dissected from brains andimmediately frozen into liquid nitrogen and stored at −80 °C, topermit adequate preservation of the phosphorylation state. Afterhomogenization in 0.5 ml lysis buffer [25 mM Tris/HCL, pH 7.4,1.0 mM EGTA, 1.0 mM EDTA, 0.5 mM phenyl methylsulfonyl fluo-ride, aprotinin, leupeptin, pepstatin A (10 μg/ml each) and Na3VO4

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100 mM] with an ultra-turrax (Ika, Germany) homogenizer, the ho-mogenate was subjected to centrifugation at 15,000 g for 15 min: thesupernatant containing cytoplasmic proteins was collected and storedat −80 °C. The pellets after a single wash with the hypotonic buffer[10 mM Hepes pH 7.9, 1.0 mM EGTA, 1.0 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A (10 μg/mleach) and Na3VO4 100 mM] were incubated on ice for 30 min, mixedfrequently and centrifuged for 15 min at 4 °C: the supernatants werecollected as nuclear extracts and stored at −80 °C. The concentrationof total proteins was determined using the Bio-Rad protein assay kit(Bio-Rad, Richmond, CA, USA), as previously described (Giuliani et al.,2006b, 2009; Ottani et al., 2009). Cytoplasmic proteins were used toassess extracellular signal-regulated kinases (ERK1/2), c-junN-terminalkinases (JNK1/2), caspase-3, tumor necrosis factor-α (TNF-α), BAX andBcl-2, whereas nuclear proteins were used for Zif268 analysis. Forwestern blot analysis, proteins (30 μg for each sample) after dena-turation were electrophoretically separated and transferred ontonitrocellulose membranes. Staining of the blots with Ponceau'ssolution showed that total protein amount was equal in each lane.The blots were then blocked and incubated with a primary antibodyfor phosphorylated ERK1/2 and JNK1/2, caspase-3 (Bio-Vision, CA,USA), TNF-α, BAX, Bcl-2 (Chemicon International, CA, USA), and Zif268(Egr1: Abcam, UK), overnight at 4 °C. The day after, the membraneswere incubated with a specific secondary antibody peroxidase-conjugated (Pierce, UK) for 1 h at room temperature, and thenanalyzed by the enhanced chemiluminescence system (Amersham,UK). To prove equal loading, the blots were analyzed for β-actinexpression (house-keeping gene) using an anti-β-actin antibody(Cell Signaling, Charlottesville, VA, USA). Protein signal was quanti-fied by scanning densitometry using a bio-image analysis system(Bio-Profil, Celbio, Italy), and expressed as relative integrated inten-sity in comparison with those of control normal animals measuredwith the same batch (Giuliani et al., 2006b, 2009; Ottani et al.,2009, 2010). In each sample, protein analysis was performed usingfour different gels.

2.5. Histology

At day 11 after stroke, and within 90 min after the last behavioraltest, transcardial perfusion with 4% ice-cold phosphate-buffered para-formaldehyde was performed in the remaining gerbils, then brainswere removed and processed for histology, as previously described(Giuliani et al., 2006a,b, 2007b, 2009). Hippocampus coronal sections(7 μm-thick) were stained with hematoxylin–eosin and Nissl method.Morphological analysis was performed using an Axiophot photomicro-scope (Carl Zeiss, Jena, Germany), and evaluation of viable neurons inthe CA1 subfield using an image system (Soft Imaging System GmbH,Münster, Germany), on 5 different slides of serial sections for eachhippocampal sample. The density of neurons was estimated in a100 micron-thick band overlapping the pyramidal cell layer.

2.6. Statistical analysis

Data are shown as mean±S.E.M. All data were detected blind tothe treatment. Behavioral data were analyzed by means of one-wayrepeated measures ANOVA followed by the Tukey's multiple compar-ison test. All other data were assessed by one-way ANOVA followedby the Tukey's test.

3. Results

3.1. RO27-3225 and NDP-α-MSH improve learning and memory

Impairment in learning and memory is a typical consequence ofstroke. We investigated, therefore, the ability of gerbils subjected toa 10-min period of global brain ischemia to learn, remember and go

to the platform of the Morris apparatus. In saline-treated control ger-bils such a period of ischemia caused a significant impairment, com-pared with sham ischemic animals, in place finding both during thefirst training session (assay of learning) and during the second ses-sion (assay of memory) (Fig. 2A and B). On the other hand, in gerbilsassigned to i.p. treatment (starting 3 h after the ischemic insult) withRO27-3225 for 11 days there was a dose-dependent improvement inlearning (assessed at days 4–8 after stroke: first session) and memory(assessed at day 11 after stroke: second session) performance, thatwas statistically significant with the doses of 90 and 180 μg/kg.NDP-α-MSH, used as a reference melanocortin at a dose (340 μg/kg)equimolar (162 nmol/kg) to the highest dose of RO27-3225, as wellsignificantly improved learning and memory, compared with ische-mic control animals (Fig. 2A and B). Melanocortin-treated gerbils, infact, during the acquisition session required progressively less timeto escape to the platform, and they promptly located the platform3 days after the end of the acquisition time. In both sessions of theMorris test, the beneficial effect of RO27-3225 (selective agonist atmelanocortin MC4 receptors) and NDP-α-MSH (which acts at mela-nocortin MC1, MC3, MC4 and MC5 receptors) on learning and memorywas blunted by i.p. pretreatment with the selective MC4 receptor an-tagonist HS024 (Fig. 2A and B). Of note, HS024 significantly worsenedmemory in stroke animals (Fig. 2B).

At the doses used here, either RO27-3225 or NDP-α-MSH did notsignificantly affect learning and memory in normal control (sham)gerbils (not shown, because not clinically relevant). Similarly, treat-ment with RO27-3225 and NDP-α-MSH did not significantly affectbody weight, which resulted equivalent in all experimental groupsduring the 11-day observation period (not shown).

3.2. RO27-3225 and NDP-α-MSH modulate the inflammatory response

The hippocampus is one of the first brain targets of a global ischemicinsult. The robust inflammatory response evoked by the ischemic epi-sode begins within a few hours and evolves overmany days (Moskowitzet al., 2010). Among others, the mitogen-activated protein kinase(MAPK)members JNK1/2 and ERK1/2 are activated after brain ischemia,and their activation has been implicated in the transcription of severalgenes involved in inflammatory processes, including genes encodingfor proinflammatory cytokines (Cuny, 2009; Giuliani et al., 2006b;Leker and Shohami, 2002; Subramaniam and Unsicker, 2010; Sugino etal., 2000).

In our present study, immunoblot analysis showed high levels ofactive JNK1/2 and ERK1/2 in the whole hippocampus of saline-treatedstroke gerbils, as detected after the second session of behavioral study(day 11 after stroke) (Fig. 3A and B). In gerbils treated with RO27-3225 (at the dose maximally effective on learning and memory) orNDP-α-MSH for 11 days, starting 3 h after stroke, there was a signifi-cant reduction in JNK1/2 and ERK1/2 activities, and this protectiveeffect was prevented by gerbil pretreatment with the MC4 receptorantagonist HS024 (Fig. 3A and B).

Consistently, TNF-α hippocampal levels, assessed at the same timepoint, resulted increased in saline-treated stroke gerbils, and treat-ment with either RO27-3225 or NDP-α-MSH significantly bluntedsuch increase (Fig. 3C). Also this effect of both melanocortins was pre-vented by pretreatment with the MC4 receptor antagonist HS024(Fig. 3C).

3.3. RO27-3225 and NDP-α-MSH modulate the apoptotic process

The activation of the MAPK members JNK1/2 and ERK1/2 isalso involved in the apoptotic machinery occurring after brain is-chemia (Cuny, 2009; Giuliani et al., 2006b; Leker and Shohami,2002; Subramaniam and Unsicker, 2010; Sugino et al., 2000). Asshown above, in our experimental model of brain ischemia both

Fig. 2. RO27-3225 and NDP-α-MSH improve learning and memory in gerbils subjected to transient global brain ischemia. Histograms' height indicates latency to escape onto thehidden platform (Morris water-maze test; mean values±S.E.M.; n=12–16 animals per group). The first session (A) started at day 4 after the ischemic episode; the second session(B) took place at day 3 after the end of the first session (that is, at day 11 after stroke). The effect of RO27-3225 (45–180 μg/kg) and NDP-α-MSH (340 μg/kg), i.p. injected twice dailyfor 11 days following brain ischemia, was prevented by pretreatment with the MC4 receptor antagonist HS024 (130 μg/kg i.p., before each administration of RO27-3225 and NDP-α-MSH). Pretreatment with saline did not alter the outcomes of RO27-3225, NDP-α-MSH or saline treatment (not shown). Sham= sham ischemic; Isch= ischemic; S = saline; RO=RO27-3225; NDP= NDP-α-MSH; HS= HS024. *Pb0.05, at least, versus the corresponding value of ischemic gerbils treated with saline (one-way ANOVA followed by Tukey's test).

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RO27-3225 and NDP-α-MSH blunted the activation of JNK1/2 andERK1/2 (Fig. 3A and B).

To gain insight into the role of melanocortin MC4 receptor stimu-lation in the apoptotic process following ischemic stroke, after thesecond session of behavioral study we investigated BAX and Bcl-2,the caspase pathway, and the possible influence of RO27-3225 orNDP-α-MSH. We chose to study the activity of the downstream exe-cutioner caspase-3, for its specific involvement in DNA damage andconsequent cell death (Friedlander, 2003; Leker and Shohami, 2002;Moskowitz et al., 2010). In control stroke gerbils, at day 11 after theischemic insult the hippocampal expression of the pro-apoptotic pro-teins BAX and active caspase-3 was significantly higher, whereas thatof the anti-apoptotic protein Bcl-2 lowered, as compared with shamischemic animals (Fig. 4A,B and C). In gerbils treated with eitherRO27-3225 or NDP-α-MSH, BAX and active caspase-3 expressionwas dramatically reduced, while Bcl-2 expression increased (Fig. 4A,B and C). As expected, such effect of RO27-3225 and NDP-α-MSHwas again abated by gerbil pretreatment with the MC4 receptor an-tagonist HS024 (Fig. 4A,B and C).

3.4. RO27-3225 and NDP-α-MSH trigger repair mechanisms

Next, we investigated Zif268 protein, which is rapidly induced byseveral stimuli including ischemia. This immediate early gene, besidesbeing required for memory formation, is involved in injury repair andis considered an indicator of recently activated neurons (Beckmannand Wilce, 1997; Giuliani et al., 2009; Tashiro et al., 2007).

Immunoblot analysis of the hippocampus showed that the ische-mic insult caused a significant increase in Zif268 levels, relative tosham ischemic animals (Fig. 5). Delayed treatment for 11 days with

both RO27-3225 and NDP-α-MSH induced a further significant in-crease in the protein expression of this activity-dependent gene(Fig. 5). This effect, as well, was counteracted by pharmacologicalblockade of MC4 receptors with HS024 (Fig. 5).

3.5. RO27-3225 and NDP-α-MSH reduce neuronal lossin the hippocampus

A brief episode of global cerebral ischemia causes selective loss ofhippocampus CA1 pyramidal cells, with consequent cognitive decline(Giuliani et al., 2006a,b, 2009). After the end of the second session ofbehavioral study (day 11 after stroke), therefore, we processed thehippocampus for histology. In saline-treated stroke gerbils we founda diffuse damage, e.g. several neurons of the pyramidal layer withpyknosis, nuclear dust, swollen perikaryon, cellular shrinkage and ab-sence of Nissl substance, which may account for the molecularchanges shown above (Fig. 6A and B). Treatment with RO27-3225and NDP-α-MSH for 11 days preserved morphological picture of thehippocampus, and in these melanocortin-treated animals a numberof viable neurons significantly higher were detected, as comparedwith saline-treated ischemic ones (Fig. 6A and C). Pretreatmentwith the MC4 receptor antagonist HS024 halted such favorable effectsof RO27-3225 and NDP-α-MSH (Fig. 6A).

4. Discussion

Ischemic stroke remains a major cause of adult disability anddeath worldwide and, therefore, novel effective therapeutic interven-tions are needed. Recently, we demonstrated that the melanocortinanalog NDP-α-MSH induces a strong neuroprotection in global and

Fig. 3. RO27-3225 and NDP-α-MSH inhibit MAPK activation and TNF-α overexpressionin the hippocampus of gerbils subjected to transient global brain ischemia. Histograms'height indicates mean values±S.E.M. (n=6–8 animals per group) obtained at day 11after stroke. The effect of RO27-3225 (180 μg/kg) and NDP-α-MSH (340 μg/kg), i.p.injected twice daily for 11 days following brain ischemia, on ERK1/2 (A), JNK1/2 (B) andTNF-α (C) was prevented by gerbil pretreatment with the MC4 receptor antagonistHS024 (130 μg/kg i.p., before each administration of RO27-3225 and NDP-α-MSH). Pre-treatment with saline did not alter the outcomes of RO27-3225, NDP-α-MSH or salinetreatment (not shown). The top of each panel shows representative immunoblotshighlighting activated JNK1/2, ERK 1/2 and TNF-α expression; β-actin expression is alsoshown. Sham = sham ischemic; Isch = ischemic; S = saline; RO = RO27-3225; NDP =NDP-α-MSH; HS = HS024. *Pb0.001 versus value of ischemic gerbils treated with saline(one-way ANOVA followed by Tukey's test).

Fig. 4. RO27-3225 andNDP-α-MSH inhibit BAX and active caspase-3 expression, and increasesthat of Bcl-2, in the hippocampus of gerbils subjected to transient global brain ischemia. Histo-grams' height indicates mean values±S.E.M. (n=6–8 animals per group) obtained at day 11after stroke. The effect of RO27-3225 (180 μg/kg) and NDP-α-MSH (340 μg/kg), i.p. injectedtwice daily for 11 days following brain ischemia, on Bcl-2 (A), BAX (B) and active caspase-3(C) was prevented by gerbil pretreatment with the MC4 receptor antagonist HS024(130 μg/kg i.p., before each administration of RO27-3225 and NDP-α-MSH). Pretreatmentwith saline did not alter the outcomes of RO27-3225, NDP-α-MSH or saline treatment (notshown). The top of each panel shows representative immunoblots highlighting BAX, Bcl-2 andactivated caspase-3 expressions; β-actin expression is also shown. Sham = sham ischemic;Isch = ischemic; S = saline; RO=RO27-3225; NDP = NDP-α-MSH; HS = HS024. *Pb0.001versus value of ischemic gerbils treatedwith saline (one-way ANOVA followed by Tukey's test).

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Fig. 5. RO27-3225 and NDP-α-MSH induce Zif268 overexpression in the hippocampusof gerbils subjected to transient global brain ischemia. Histograms' height indicatesmean values±S.E.M. (n=6–8 animals per group) obtained at day 11 after stroke.The effect of RO27-3225 (180 μg/kg) and NDP-α-MSH (340 μg/kg), i.p. injected twicedaily for 11 days following brain ischemia, was prevented by pretreatment with theMC4 receptor antagonist HS024 (130 μg/kg i.p., before each administration of RO27-3225 and NDP-α-MSH). Pretreatment with saline did not alter the outcomes ofRO27-3225, NDP-α-MSH or saline treatment (not shown). The top of the figureshows representative immunoblots highlighting Zif268 and β-actin expressions.Sham = sham ischemic; Isch = ischemic; S = saline; RO = RO27-3225; NDP =NDP-α-MSH; HS = HS024. *Pb0.05, at least, versus value of ischemic gerbils treatedwith saline (one-way ANOVA followed by Tukey's test).

Fig. 6. RO27-3225 and NDP-α-MSH protect against neuronal death in the hippocampus CA(A) indicates mean values±S.E.M. (n=6–8 animals per group) obtained 11 days after sttwice daily for 11 days following brain ischemia, was prevented by pretreatment withRO27-3225 and NDP-α-MSH). Pretreatment with saline did not alter the outcomes of RO27the morphology of the CA1 pyramidal layer after Nissl stain (bar=25 μm): the saline-treat(C) shows several neurons with pyknosis (a), nuclear dust (b), swollen perikaryon (c), cellugerbil (C) shows a lower extent of damage with a higher number of viable neurons. Sham=HS = HS024. *Pb0.01 versus value of ischemic gerbils treated with saline (one-way ANO

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focal brain ischemia in gerbils and rats. The protective effect ofNDP-α-MSH occurs also when treatment starts several hours after is-chemia, and includes a suppression of the main ischemia-related,early developed mechanisms of damage, such as the excitotoxic, in-flammatory and apoptotic responses, as found throughout the 24 hfollowing the ischemic insult. Moreover, this is followed by a reduc-tion in the brain morphological alterations, and improvement in func-tional recovery, as cognitive processes, sensory-motor orientationand limb use (Giuliani et al., 2006a,b, 2007b, 2009; Ottani et al.,2009).

The present data expand our previous observations, giving directevidence that selective stimulation of the melanocortin MC4 receptorsubtype can help treatment of cerebral ischemia. Here we show, infact, for the first time that short-term treatment of stroke gerbilswith nanomolar amounts of the selective MC4 receptor agonistRO27-3225 counteracts the prolonged and/or recurrent inflammatoryand apoptotic responses following the ischemic episode, as detectedat day 11 after stroke. Indeed, at this late time point we recorded amelanocortin-induced favorable influence on the hippocampal ex-pression of TNF-α, BAX, ERK1/2, JNK1/2, caspase-3 and Bcl-2, withconsequent reduction in neuronal loss and dose-dependent improve-ment in learning and memory. Moreover, here we show that RO27-3225 induces overexpression of Zif268, a member of the early growthresponse gene family that could have a key role in learning and mem-ory recovery after stroke, as well as in triggering early repair mecha-nisms. In our present investigation, similar effects were obtained withthe non-selective NDP-α-MSH, used as a reference melanocortin.

Melanocortins act by stimulation of G protein-coupled, seven-transmembrane receptors, and five melanocortin receptor subtypes(MC1 to MC5) have been so far recognized (Catania, 2008; Cataniaet al., 2004; Holloway et al., 2011; Mountjoy, 2010; Patel et al.,

1 subfield of gerbils subjected to transient global brain ischemia. Histograms' heightroke. The effect of RO27-3225 (180 μg/kg) and NDP-α-MSH (340 μg/kg), i.p. injectedthe MC4 receptor antagonist HS024 (130 μg/kg i.p., before each administration of-3225, NDP-α-MSH or saline treatment (not shown). Representative images showinged sham ischemic gerbil (B) shows typical neurons; the saline-treated ischemic gerbillar shrinkage (d) and absence of Nissl substance (e); the RO27-3225-treated ischemicsham ischemic; Isch= ischemic; S = saline; RO= RO27-3225; NDP=NDP-α-MSH;

VA followed by Tukey's test).

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2010; Wikberg and Mutulis, 2008). MC1 is expressed in melanocytes,melanoma cells and cells involved in the immune/inflammatory re-sponse; MC2, the ACTH receptor, is mainly expressed in the adrenalglands but also in white adipose tissue and in the skin; MC3 isexpressed in the brain, placenta, gut, and heart; MC4 occurs in variousbrain areas, but it has been recently recognized also in peripheral or-gans in the rat; the MC5 receptor, initially recognized in the brain, wassubsequently found to be ubiquitous, but mainly in the periphery.MC3 and MC4 are the predominant melanocortin receptor subtypesexpressed in the CNS, being expression of MC4 broader than that ofMC3 receptors, with a relatively high expression of MC4 receptors inthe hippocampus. Notably, melanocortins including NDP-α-MSHand the pentapeptide RO27-3225 reach the CNS after systemic injec-tion, and also experimental evidence points in this direction (Banksand Kastin, 1995; Benoit et al., 2000; Catania et al., 2004; Giuliani etal., 2007a; Catania, 2008; Wikberg and Mutulis, 2008; Corander etal., 2009; Mountjoy, 2010; Holloway et al., 2011). Moreover, perme-ability of the blood–brain barrier significantly increases after stroke(Michalski et al., 2010). The present investigation shows that the neu-roprotective effect of the non-selective melanocortin agonist NDP-α-MSH –which activates the MC1, MC3, MC4 and MC5 receptor subtypes(Catania et al., 2004; Giuliani et al., 2007a) – and of the selective MC4receptor agonist RO27-3225 (Benoit et al., 2000; Giuliani et al., 2007a;Grieco et al., 2003) is totally prevented by the selective MC4 receptorantagonist HS024, giving evidence that such effect is mediated bymelanocortin MC4 receptors. Indeed, HS024 has an approximately65, 20 and 12-fold higher affinity for the MC4 receptors relative toMC1, MC3 and MC5 receptors, respectively (Giuliani et al., 2007a;Kask et al., 1998). Our findings are contrasting with those reportedby Regan et al. (2009) who did not observe neuroprotection againststroke by using a novel MC4 receptor agonist. But our experimentalconditions, as well as assessment procedure of neuroprotection,were completely different from those by Regan et al. (2009).

In this study we chose to start treatment 3 h after the ischemicinsult without exploring the therapeutic treatment window, sinceit was beyond the scope of our investigation. Of note, our previousresearches showed that NDP-α-MSH time window for a successfulapproach to ischemic stroke is very broad (up to 9–12 h) (Giulianiet al., 2006a,b, 2007b, 2009; Ottani et al., 2009).

Experimental evidence indicates that melanocortin peptides couldbe physiologically involved in neuroprotection against brain injuries,as ischemic stroke (Giuliani et al., 2006a,b, 2007b, 2009, 2010) andtrauma (Bitto et al., submitted), which share many pathophysiologicalpathways that lead to secondary cellular death (Leker and Shohami,2002). Consistently, it has been shown that patients with acute trau-matic brain injury and very low circulating levels of α-MSH havemore unfavorable outcome, as compared with patients with higher α-MSH plasma levels (Magnoni et al., 2003); accordingly, here we showthat MC4 receptor blockade worsens memory in animals subjected totransient global cerebral ischemia, and this indicates that MC4 blockaderestrains endogenous melanocortins from exerting some physiologicalprotective effect. Low plasma levels of interleukin-10 (IL-10) are associ-ated with early worsening of neurological symptoms in patients withacute stroke (Vila et al., 2003), and evidence was given that brain IL-10is necessary for neuroprotection (Xin et al., 2011); interestingly,melanocortins induce IL-10 overexpression in regulating inflam-matory response following injuries including traumatic brain inju-ry (Catania, 2008; Bitto et al., submitted).

It has been repeatedly reported that the anti-inflammatory effectsof melanocortins are adrenal-independent, and NDP-α-MSH andRO27-3225 do not stimulate adrenal glands, because they do notbind MC2 receptors (expressed in adrenal cortex and mediating glu-cocorticoid release) (Benoit et al., 2000; Catania et al., 2004; Coranderet al., 2009; Patel et al., 2010; Wikberg and Mutulis, 2008). Therefore,although the involvement of glucocorticoid release from the adrenalglands was not studied in the present experiments, the possibility

that the here reported anti-inflammatory effects of melanocortinsmay be an indirect consequence of glucocorticoid release should beruled out. We previously reported that, in rats subjected to focal cere-bral ischemia, melanocortins protect against both cerebral and sys-temic damage through the activation, at least in part, of a vagusnerve-mediated “cholinergic anti-inflammatory pathway”, as detected10–20 h after stroke (Giuliani et al., 2010; Ottani et al., 2009). It hasbeen established that the cholinergic anti-inflammatory pathwayis involved in the defense mechanisms (Guarini et al., 2003; Thayerand Sternberg, 2010; Tracey, 2002, 2007), and evidence indicatesthat melanocortins trigger this cholinergic protective pathway, likelythrough activation of brain MC3/MC4 receptors (Giuliani et al., 2010;Guarini et al., 2004; Mioni et al., 2005). In our present study we did notinvestigate the vagus nerve-mediated cholinergic anti-inflammatorypathway, but the here reported data give direct evidence that brainmelanocortinMC4 receptor stimulation induces neuroprotection againstdamage consequent to a brain ischemic insult through a blockade ofthe main ischemia-related mechanisms of damage, namely the in-flammatory response and the following apoptotic machinery: theseare established targets of the cholinergic anti-inflammatory pathwaythrough acetylcholine, the major vagus nerve neurotransmitter(Guarini et al., 2003; Thayer and Sternberg, 2010; Tracey, 2002, 2007).A melanocortin MC4 receptor-mediated activation of the cholinergicanti-inflammatory pathway also in our present experimental condition,therefore, cannot be ruled out.

Our present data also showing, at day 11 after stroke, an over-expression of Zif268 during treatmentwithmelanocortinMC4 receptoragonists, suggest that another physiological protective mechanismagainst stroke is likely activated. Zif268 is rapidly induced by severalstimuli including ischemia, and it is used as a marker of recentlyactivated neurons (Beckmann and Wilce, 1997; Tashiro et al., 2007).After melanocortin treatment, overexpressed Zif268 could be involvedas transcription factor in the regulation of target gene expressionrequired for injury repair, and simply as protein it could also im-prove activated neuron functionality. In concrete terms, our datashowing a significant increase in Zif268 levels in the whole hippo-campus of melanocortin-treated animals point to a gain in the num-ber of activated/functional neurons. Indeed, in both RO27-3225- andNDP-α-MSH-treated gerbils we found a significantly higher numberof viable neurons, relative to saline-treated stroke animals. Thesefindings are in agreement with our previous data showing a long-lasting NDP-α-MSH-induced functional recovery, associated with ahigh number of viable neurons and overexpression of Zif268 in thehippocampus, as assessed 50 days after stroke (Giuliani et al., 2009).

In conclusion, the present data give direct evidence for the firsttime of the neuroprotective effect of melanocortin MC4 receptoragonists. The experimental model of hypoxic–ischemic brain injurywe used in the present study represents human stroke conditionsdue, for example, to atherosclerotic involvement of the commoncarotid arteries, respiratory arrest, cardiac arrest, choking and othercauses (Woodruff et al., 2011). Our previous and present findings onthe protective effects of melanocortins in experimental ischemic condi-tions suggest a physiological protective role of these endogenouspeptides (Catania, 2008; Catania et al., 2004; Corander et al., 2009;Holloway et al., 2011; Mountjoy, 2010; Patel et al., 2010; Wikbergand Mutulis, 2008). If these findings will be confirmed in other ani-mal models of cerebral ischemia, melanocortin analogs selectivelyacting at MC4 receptors could provide potential for a physiologicaland innovative approach to treatment of ischemic stroke caused bydifferent kinds of diseases, in humans.

Acknowledgments

This work was supported in part by grants from Ministerodell'Istruzione, dell'Università e della Ricerca (MIUR), Roma, andUniversity of Modena and Reggio Emilia, Italy.

486 L. Spaccapelo et al. / European Journal of Pharmacology 670 (2011) 479–486

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