Melanocortins protect against progression of Alzheimer’s disease in triple-transgenic mice by targeting multiple pathophysiological pathways

Q5 Daniela Giuliani a, Alessandra Bitto b, Maria Galantucci a, Davide Zaffe c, Alessandra Ottani a,Natasha Irrera b, Laura Neri a, Gian Maria Cavallini d, Domenica Altavilla b, Annibale R. Botticelli e,Francesco Squadrito b, Salvatore Guarini a,*aDepartment of Biomedical, Metabolic and Neural Sciences, Section of Pharmacology and Molecular Medicine, University of Modena and Reggio Emilia, Modena, ItalybDepartment of Clinical and Experimental Medicine, Section of Pharmacology, University of Messina, Messina, ItalycDepartment of Biomedical, Metabolic and Neural Sciences, Section of Human Morphology, University of Modena and Reggio Emilia, Modena, ItalydDivision of Ophthalmology, University of Modena and Reggio Emilia, Modena, ItalyeDepartment of Molecular Medicine, University of Pavia, Pavia, Italy

a r t i c l e i n f o

Article history:Received 1 July 2013Received in revised form 20 August 2013Accepted 23 August 2013

Keywords:Alzheimer’s diseaseTriple-transgenic micePathophysiological pathwaysLearning and memoryMelanocortinsNeuroprotection

a b s t r a c t

Besides specific triggering causes, Alzheimer’s disease (AD) involves pathophysiological pathways thatare common to acute and chronic neurodegenerative disorders. Melanocortins induce neuroprotection inexperimental acute neurodegenerative conditions, and low melanocortin levels have been found inoccasional studies performed in AD-type dementia patients. Here we investigated the possible neuro-protective role of melanocortins in a chronic neurodegenerative disorder, AD, by using 12 week-old(at the start of the study) triple-transgenic (3xTg-AD) mice harboring human transgenes APPSwe,PS1M146V, and tauP301L. Treatment of 3xTg-ADmice, once daily until the end of the study (30 weeks ofage), with the melanocortin analog [Nle4,D-Phe7]-a-melanocyte-stimulating hormone (NDP-a-MSH)reduced cerebral cortex/hippocampus phosphorylation/level of all AD-related biomarkers investigated(mediators of amyloid/tau cascade, oxidative/nitrosative stress, inflammation, apoptosis), decreasedneuronal loss, induced over-expression of the synaptic activityedependent gene Zif268, and improvedcognitive functions, relative to saline-treated 3xTg-AD mice. Pharmacological blockade of melanocortinMC4 receptors prevented all neuroprotective effects of NDP-a-MSH. Our study identifies, for the firsttime, a class of drugs, MC4 receptor-stimulating melanocortins, that are able to counteract the pro-gression of experimental AD by targeting pathophysiological mechanisms up- and down-stream ofb-amyloid and tau. These data could have important clinical implications.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Alzheimer’s disease (AD) is the most common cause of de-mentia in elderly individuals, and a major cause of disability andmortality without an effective treatment (Galimberti et al., 2013;Iqbal and Grundke-Iqbal, 2010, 2011; Ittner and Götz, 2011;Sperling et al., 2013; Tayeb et al., 2012). Generally AD is diag-nosed in persons more than 65 years of age (sporadic AD),whereas the less prevalent form of the disease (familial or geneticAD) can occur much earlier. In some regions of AD brains, as thecortex and hippocampus, presenilin-1 (PS1) and presenilin-2

(PS2), which are 2 members of the g-secretase complex, andb-secretases process the amyloid precursor protein (APP) togenerate extra-cellular b-amyloid (Ab) fibrillar deposits (Ab pla-ques); also intraneuronal tau neurofibrillary tangles composed ofhyperphosphorylated tau protein develop, and both these hall-mark lesions trigger pathophysiological pathways that lead tosynaptic dysfunction, neurodegeneration and marked neuronalloss (Bagheri et al., 2011; Blennow, 2010; Giuliani et al., 2013;Iqbal and Grundke-Iqbal, 2010; Ittner and Götz, 2011; Kumaret al., 2013; Munoz and Ammit, 2010; Sanchez et al., 2012;Schellenberg and Montine, 2012; Sivanandam and Thakur, 2012;Sperling et al., 2013; Tayeb et al., 2012; Yu et al., 2009).

Interestingly, despite the different triggering causes, a numberof pathophysiological pathways/mechanisms leading to neuronaldamage and death are common to acute and chronic neurode-generative disorders including AD. Indeed, a common established

* Corresponding author at: Department of Biomedical, Metabolic and NeuralSciences, Section of Pharmacology and Molecular Medicine, University of Modenaand Reggio Emilia, via G. Campi 287, 41125 Modena, Italy. Tel.: þ39 59 2055371;fax: þ39 59 2055376.

E-mail address: salvatore.guarini@unimore.it (S. Guarini).

Contents lists available at ScienceDirect

Neurobiology of Aging

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0197-4580/$ e see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.neurobiolaging.2013.08.030

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role is played by excitotoxicity, mitochondrial dysfunction,oxidative and nitrosative stress, inflammatory response, defectiveautophagy, and apoptosis, as well as epigenetic mechanisms asmodifications of chromatin structure (Bagheri et al., 2011;Banerjee et al., 2010; Craig-Schapiro et al., 2009; Friedlander,2003; Gao et al., 2012; Giuliani et al., 2012, 2013; Jakovcevski andAkbarian, 2012; Karbowski and Neutzner, 2012; Lo, 2010; Munozand Ammit, 2010; Sivanandam and Thakur, 2012; Zipp and Aktas,2006). Pharmacological treatments successfully targeting thesepathways/mechanisms in some experimental neurodegenerativeconditions might also bring benefits in other neurodegenerativediseases.

Melanocortins are endogenous peptides of theadrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) family (Brzoska et al., 2008; Vergoni and Bertolini, 2000;Wikberg and Mutulis, 2008), derived by the post-translationalcleavage of proopiomelanocortin and sharing the core sequence(6e9) of MSH. This sequence is necessary to bind all melano-cortin receptors and for agonist activity, the ligand selectivitybeing determined by residues outside the core region: indeed,these neuropeptides act via 5 melanocortin receptor subtypes(MC1eMC5) expressed on the cell surface with different tissuedistribution (Brzoska et al., 2008; Catania et al., 2004; Giulianiet al., 2012; Mountjoy, 2010; Wikberg and Mutulis, 2008). Mel-anocortins including synthetic analogs have been shown toinduce neuroprotection in acute experimental neurodegenerativeconditions such as ischemic stroke, subarachnoid hemorrhage,traumatic brain injury, and spinal cord injury, with long-lastingfunctional recovery (Bharne et al., 2011; Bitto et al., 2012; Chenet al., 2008; Gatti et al., 2012; Giuliani et al., 2006, 2007, 2009;Huang and Tatro, 2002; Ottani et al., 2009; Savos et al., 2011;Sharma et al., 2006; Spaccapelo et al., 2011). The neuro-protective effects of melanocortins, as found in some of theseinvestigations, are mediated by central nervous system (CNS)melanocortin MC4 receptors; they occur in a broad therapeutictreatment window and through an inhibition of the mainmechanisms of brain damage, for example, the excitotoxic, in-flammatory and apoptotic responses, and an improvement ofsynaptic transmission and plasticity (for review, see Giulianiet al., 2012).

On the basis of the above background we hypothesized thatmelanocortins could be able to counteract the progression of achronic neurodegenerative disease as AD. This hypothesis alsowas supported by sporadic studies performed a lot of years ago,where low ACTH/a-MSH levels were found in the cerebrospinalfluid/brain of patients with AD-type dementia (Arai et al., 1986;Facchinetti et al., 1984; Rainero et al., 1988). In the presentresearch, therefore, we investigated the possible neuroprotectiverole of melanocortins in a triple-transgenic mouse model of AD(3xTg-AD) that most closely mimics human AD features.

2. Methods

2.1. Animals

For these investigations, male 12-week-old (at the start of thestudy) 3xTg-AD mice and their wild-type littermates (JacksonLaboratories, Bar Harbor, ME) were used. These mice harbor hu-man transgenes APPSwe, PS1M146V and tauP301L (that is, they co-express mutant human APP, PS1, and tau protein, respectively)(Giuliani et al., 2013; Oddo et al., 2003; Wang et al., 2010). Theywere kept in air-conditioned colony rooms (temperature 21 �C �1 �C, humidity 60%) on a natural light/dark cycle, with foodin pellets and tap water available ad libitum. Body weightwas recorded throughout the observation period, and rectal

temperature was maintained close to 37 �C by means of heatinglamps. Animal sacrifice at the end of the study was performedunder general anesthesia with sodium pentobarbital (50 mg/kg)intraperitoneally (i.p.) (Sigma-Aldrich, Milan, Italy). Housing con-ditions and experimental procedures were in strict accordancewith the “Principles of Laboratory Animal Care Q1” (National In-stitutes of Health [NIH] publication no. 86-23, revised 1985) andthe European Community regulations on the use and care of ani-mals for scientific purposes (CEE Council 89/609; Italian D.L. 22-1-92 No. 116), and were approved by the Committee on AnimalHealth and Care of Modena and Reggio Emilia University.

2.2. Drugs and treatment schedules

[Nle4, D-Phe7]-a-MSH (NDP-a-MSH; agonist at melanocortinMC1, MC3, MC4, and MC5 receptors), synthetic melanocortinanalog with long-lasting biological activity (Giuliani et al., 2006,2007, 2011; Minutoli et al., 2011) kindly provided by Prof PaoloGrieco (University of Naples Federico II), and the selectivemelanocortin MC4 receptor antagonist HS024 (Giuliani et al.,2006, 2007, 2011; Minutoli et al., 2011), cyclic MSH analogpurchased from Tocris (Bristol, UK), were used. NDP-a-MSH wasdissolved in saline solution (1 mL/kg) and i.p. administered(340 mg/kg, once daily for 18 weeks). Pretreatment with HS024(130 mg/kg, dissolved in saline solution 1 mL/kg), when done,was performed i.p. once daily, 20 minutes before eachadministration of NDP-a-MSH. Control animals (3xTg-AD andwild-type mice) received an equal volume of saline solution bythe same route of administration; as additional control animals,wild-type mice treated with NDP-a-MSH alone and HS024alone, and 3xTg-AD mice treated with HS024 alone, were alsoused. The doses of NDP-a-MSH and HS024 were chosen on thebasis of our previous studies performed in other experimentaldegenerative conditions, including neurodegeneration (Bittoet al., 2012; Giuliani et al., 2006, 2007, 2009, 2011; Minutoliet al., 2011).

2.3. Assessment of spatial learning and memory

We used the Morris Water Maze test with minor modifica-tions, as previously described (Bitto et al., 2012; Giuliani et al.,2006, 2007, 2009, 2011, 2013). This test measures animal’s abil-ity to learn, to remember, and to go to a place in space definedonly by its position relative to distal extramaze cues. The appa-ratus consisted of a circular white pool (80 cm in diameter and 55cm in height) filled to a depth of 15 cm with water (27 �C)rendered opaque with milk powder. Mice (12 per group) weretrained to find the spatial location of a platform of clear perspexhidden by arranging for its top surface (7 cm in diameter) to be 1cm below the water level. To this end, 4 points on the apparatuswall (North, South, East, West) were defined by means ofdifferent geometrical figures, and conspicuous cues were placedin a fixed position around the pool. In each daily training, latencyto escape onto the hidden platform was recorded. Each mousereceived 4 daily trials starting each time from a different cardinalpoint in a random succession: in each trial, if the animal failed tolocate the platform within 60 seconds, escape latency wasconsidered same as 60 seconds (therefore, the daily maximallypossible total escape latency was 240 seconds). During the 17thweek of age, mice were subjected to a first 5-day trainingsequence (to assay learning), followed 3 days later by a second1-day training (to assay memory). Two other sessions (like pre-vious ones) of the Morris Water Maze test were undertaken againduring the 30th week of age (last week of the study, lasted 18weeks). Tests were performed in a sound-proof room.

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2.4. Histology

At the end of the last behavioral test (30-week-oldmice), transcardial perfusion with ice-cold 4% paraformaldehyde(phosphate-buffered) was performed in 5 animals per group(randomly taken), then brains were removed and processed forhistological examination, as previously described (Giuliani et al.,2006, 2007, 2009; Spaccapelo et al., 2011). Hippocampus and iso-cortex morphology was studied in 7-mm-thick paraffin-embeddedsections, hematoxylin-and-eosin stained; the extent of Ab plaqueswas assessed after ethanoleCongo red/Weigert hematoxylinstaining or aqueous thioflavin-S (Giuliani et al., 2013; Salkovic-Petrisic et al., 2011). Morphological analyses were performed byusing an Axiophot photomicroscope (Carl Zeiss, Jena, Germany),under ordinary, polarized, and fluorescent light. Histometry wasperformed by using an image system (analySIS, Soft Imaging Sys-tem GmbH, Münster, Germany). Extracellular Ab deposits (Ab pla-ques) were estimated, on the whole slide stained by Congo red, in 5seriated sections per animal taken every 100 mm starting �1 mm(frontal isocortex) and þ2 mm (hippocampus) from the bregma0 coordinate.

2.5. Assay of malondialdehyde concentration

To assess oxidative stress, concentration of malondialdehyde(MDA), used asmarker of lipid peroxidation index, wasmeasured inthe cerebral cortex of mice using a colorimetric commercial kit(Calbiochem, San Diego, CA) (Bagheri et al., 2011; Giuliani et al.,2013). We chose this brain region because the hippocampal mate-rial was used for all the other biological assessments scheduled inthe present study. Briefly, after the last behavioral test, theremaining animals (7 per group, 30 weeks old) were sacrificed andthe cortex was dissected from the brain, blotted dry, and thenweighed. After centrifugation of the cortex lysate, 0.65 mL of 10.3mmol/L N-methyl-2phenyl-indole in acetonitrile were added to 0.2mL of the supernatant. After vortexing for 3 to 4 seconds and adding0.15 mL of HCl 37%, the samples were mixed well, closed with atight stopperQ2 , and incubated at 45 �C for 60 minutes. The sampleswere then cooled on ice, and optical density was spectrophoto-metrically measured at 532 nm (340 ATTC; SLT Lab Instruments,Grödig, Austria). MDA concentrationwas calculated from a standardcurve established with serial dilutions of tetraethoxypropane, andexpressed as mmol/g of tissue.

2.6. Assay of nitrite concentration

To assess nitrosative stress, concentration of nitrites (as NOproduct) was alsomeasured in the cerebral cortex of the abovemice(7 per group) sacrificed after the last behavioral test, as previouslydescribed (Bagheri et al., 2011; Giuliani et al., 2013). Briefly, thecortex was dissected from the brain, blotted dry, and thenweighed.After centrifugation of the cortex lysate, 100 mL of the supernatantwere incubated with an equal volume of Griess reagent (1% sul-phanilamide and 0.1% naphthyl-ethylenediamine dihydrocloride in2.5% phosphoric acid). After 10 minute of incubation at roomtemperature, optical density of the cromophore so formed wasspectrophotometrically measured at 540 nm (340 ATTC; SLT LabInstruments). Nitrite concentration was calculated from a standardcurve established with serial dilutions of sodium nitrite, andexpressed as nmol/g of tissue.

2.7. Enzyme-linked immunosorbent assay

Interleukin-1b (IL-1b) was measured in the cerebral cortex witha specific enzyme-linked immunosorbent assay (ELISA) kit (Abcam,

Cambridge, UK). Brain cortex lysates were obtained by using adounce homogenizer and lysis buffer (0.1 mol/L Tris-HOCl, pH 7.3).After centrifugation at 15,000 rpm, supernatants were assayed forIL-1b following the manufacturer’s instructions. Intensity of thecolor was spectrophotometrically measured at 450 nm (340 ATTC;SLT Lab Instruments), and lysate levels were compared to a stan-dard curve and expressed as pg/mL.

2.8. Isolation of cytoplasmic and nuclear proteins and Western blotanalysis

The whole hippocampi were also dissected from the abovemouse brains (7 animals per group; mice were sacrificed within 90minutes after the end of the last behavioral test to detect, as well,Zif268 expression: Giuliani et al., 2009, 2011; Tashiro et al., 2007).The hippocampi were used to measure phosphorylation state/level of PS1, APP, Ab, tau, Zif268, p38, tumor necrosis factorea(TNF-a), and caspase-3. After extraction of nuclear (for Zif268analysis) and cytoplasmatic (for analysis of all the other markers)proteins, as previously described (Bitto et al., 2012; Giuliani et al.,2006, 2009, 2013; Ottani et al., 2009), proteins (30 mg for eachsample) were denatured, electrophoretically separated, andtransferred onto nitrocellulose membranes. Staining of the blotswith Ponceau’s solution showed that total protein amount wasequal in each lane. The blots were then blocked and incubatedovernight at 4 �C with the following primary antibodies: phospho(p)-tau Thr181, p-tau Ser396, p-tau Ser202, p-APP Thr668, Ab(1-42 specific), PS1 and p-p38 (Cell Signaling, Charlottesville, VA),active caspase-3 (Bio-Vision, San Francisco, CA), TNF-a (Millipore,Billerica, MA), Zif268 (Abcam). The day after, the membranes wereincubated with a specific secondary antibody peroxidase-conjugated for 1 hour at room temperature. To prove equalloading, the blots were analyzed for b-actin expression (house-keeping gene) using an antieb-actin antibody (Cell Signaling). Themembranes were analyzed by the enhanced chemiluminescencesystem according to the manufacturer’s protocol (Millipore). Theprotein signals were quantified by scanning densitometry using abio-image analysis system (Bio-Profil, Celbio, Italy) and expressedas relative integrated intensity in comparison with those of naïvewild-type animals (Bitto et al., 2012; Giuliani et al., 2006, 2009,2013; Ottani et al., 2009).

2.9. Statistical analysis

All data were detected blind to the treatment and are shown asmean � SEM. Values were analyzed by means of 2-way repeated-measures analysis of variance (ANOVA; behavioral data) or 1-wayANOVA (all other data) followed by the StudenteNewmaneKeuls’test. A value of p < 0.05 was considered significant.

3. Results

3.1. NDP-a-MSH improves learning, memory, and synaptic activity

Progressive memory loss and severe cognitive decline charac-terize AD (Galimberti et al., 2013; Tayeb et al., 2012). We investi-gated, therefore, the ability of 3xTg-ADmice to learn, to remember,and to go to a place in space defined only by their position relativeto distal extramaze cues (Morris Water Maze test). Saline-treatedcontrol 3xTg-AD mice showed impaired ability (as comparedwith wild-type mice) in place finding both during the first andsecond training sessions (assay of learning and memory, respec-tively; 17-week-oldmice), and during the third and fourth sessions(assay of learning/memory; 30-week-old mice) (Fig. 1AeD).Conversely, in 3xTg-AD animals treated with the melanocortin

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NDP-a-MSH, a significant improvement in learning and memoryperformance occurred from the first training day, compared withsaline solutionetreated control 3xTg-AD mice (Fig. 1AeD); inter-estingly, during the first and second training sessions behavioralperformance in NDP-a-MSH-treated 3xTg-AD animals was evensignificantly better than that of wild-type mice treated with salinesolution (Fig. 1A and B).

Zif268 is an immediate early gene whose expression isdependent on synaptic activity and, like other immediate earlygenes, it is used as indicator of recently activated neurons, syn-aptic transmission, and plasticity (Giuliani et al., 2009, 2011;Tashiro et al., 2007; Sanchez et al., 2012). Accordingly, wemeasuredZif268 protein levels in the hippocampus of the abovemice sacrificed within 90 minutes after the end of the fourthsession of behavioral study. Immunoblot analysis showed a verylow Zif268 expression in saline solutionetreated 3xTg-AD mice,relative to wild-type animals. In contrast, treatment of 3xTg-ADmice with NDP-a-MSH produced a significant increase in theexpression of this activity-dependent gene (Fig. 1E).

MC3 and MC4 are the predominant melanocortin receptorsubtypes expressed in CNS, and MC4 activation has been found to

induce neuroprotection in acute neurodegenerative conditions(for reviews, see Giuliani et al., 2012; Mountjoy, 2010). Therefore,we investigated the role of this receptor subtype. The favorableeffect of NDP-a-MSHdwhich activates MC1, MC3, MC4, and MC5receptors (Giuliani et al., 2006, 2007, 2009, 2011) don thebehavioral performance and hippocampus Zif268 expression of3xTg-AD mice was completely prevented by pretreatment withthe selective MC4 receptor antagonist HS024 (Fig. 1AeE).

Of note, at the nanomolar doses used here, neither NDP-a-MSHalone nor HS024 alone significantly affected learning, memory, orZif268 expression inwild-typemice (not shown). Body weight wasequivalent in all experimental groups throughout the 18-weekobservation period (not shown), and no signs of toxicity wererecorded in mice treated with NDP-a-MSH and HS024.

3.2. NDP-a-MSH preserves brain morphological picture

To study the AD-related brain morphological alterations(Blennow, 2010; Iqbal and Grundke-Iqbal, 2010, 2011; Oddo et al.,2003), after the fourth session of behavioral tests, in some ani-mals (randomly taken from each experimental group) we

Fig. 1. NDP-a-MSH improves learning and memory in 3xTg-AD mice; this effect is associated with hippocampus Zif268 over-expression. Histograms’ height indicates mean values �standard error of the mean of (AeD) latency to escape onto the hidden platform (Morris Water Maze test; n ¼ 12 mice per group), and (E) Zif268 expression assessed within 90minutes after the last session of the Morris test (n ¼ 7). The first session (A) started at the beginning of the 17th week of age; the second session (B) took place 2 days after the end ofthe first session; the third session (C) started at the beginning of the 30th week of age; the fourth session (D) took place 2 days after the end of the third session. The effects of NDP-a-MSH (340 mg/kg i.p., once daily for 18 weeks, starting treatment at 12 weeks of age) were prevented by pretreatment with the MC4 receptor antagonist HS024 (130 mg/kg i.p.,before each administration of NDP-a-MSH). (E) Top shows representative immunoblots highlighting Zif268 expression; b-actin expression is also shown. Abbreviations: WT, wild-type mice; 3xTg, 3xTg-AD mice; S, saline solution; NDP, NDP-a-MSH; HS, HS024. * p < 0.001 versus the corresponding value of 3xTg-AD mice treated with saline solution;# p < 0.001 versus the corresponding value of 3xTg-AD mice treated with NDP-a-MSH alone.

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processed the hippocampus and cerebral cortex (vulnerableregions of the AD brain) for histology. Inside the frontal isocortexof saline solutionetreated 30-week-old 3xTg-AD mice, we founda number of neurons showing irreversible (pyknosis, nucleardust) or reversible (swollen perikaryon, cellular shrinkage)alterations, with considerable neuronal loss and increase ofmicroglia cells. In the hippocampus and frontal isocortex of salinesolutionetreated 30-week-old 3xTg-AD mice, we also observed asubstantial extracellular amyloid deposit (Fig. 2AeC). On thecontrary, in 3xTg-AD mice treated with NDP-a-MSH, we detecteda significant preservation of the morphological picture, with amarked reduction in amyloid deposit (hippocampus and frontalisocortex), and a greater number of viable neurons (frontal iso-cortex), if compared with saline-treated 3xTg-AD animals(Fig. 2A, B, and D). The beneficial effects of NDP-a-MSH onmorphological picture, amyloid deposit and neuron viability wereprevented by pretreatment with the selective MC4 receptorantagonist HS024 (Fig. 2A and B).

3.3. NDP-a-MSH reduces level/phosphorylation state of amyloid/taucascade proteins

In the hippocampus of the above-mentioned 30-week-old3xTg-AD mice, we also investigated, by Western blot analysis, themain proteins of the amyloid cascade and tau protein (Craig-Schapiro et al., 2009; Giuliani et al., 2013; Iqbal and Grundke-Iqbal, 2010; Oddo et al., 2003; Yu et al., 2009). In 3xTg-AD micetreated with saline solution, we detected hyperphosphorylationof APP at Thr668 and tau protein at Thr181, Ser396 and Ser202, aswell as over-expression of PS1 and Ab (Fig. 3AeF). On the con-trary, in 3xTg-AD mice treated with NDP-a-MSH, the phosphor-ylation/expression level of the amyloid/tau cascade proteins

markedly decreased, relative to that of saline-treated 3xTg-ADmice (Fig. 3AeF). This protective effect was inhibited by pre-treatment with the MC4 receptor antagonist HS024 (Fig. 3AeF).

3.4. NDP-a-MSH inhibits p38 activation

The mitogen-activated protein kinases (MAPK) members,particularly p38, play an important role in phosphorylation of tauprotein and synaptic dysfunction (Munoz and Ammit, 2010; Yuet al., 2009). Furthermore, in neurodegenerative diseasesincluding AD, p38 and the other MAPK are involved as signaltransducers in excitotoxicity and inflammation, as well as in theapoptotic process (Giuliani et al., 2012, 2013; Munoz and Ammit,2010). We therefore investigated p38, and Western blot analysisshowed an increased activity of this kinase in the hippocampus ofsaline solutionetreated 30-week-old 3xTg-AD mice, as comparedwith wild-type animals (Fig. 4). In 3xTg-AD mice treated withNDP-a-MSH, there was a significant reduction of p38 activity, andthis protective effect was inhibited by pretreatment with the MC4receptor antagonist HS024 (Fig. 4).

3.5. NDP-a-MSH attenuates oxidative and nitrosative stress

To explore another feature of AD, namely excitotoxicity-triggered oxidative and nitrosative stress (Bagheri et al., 2011;Galimberti et al., 2013; Gao et al., 2012; Giuliani et al., 2013;Tayeb et al., 2012), at the end of the fourth session of the Mor-ris Water Maze test we evaluated MDA concentration (as an indexof oxidative stress-induced lipid peroxidation) and nitrite con-centration (nitric oxide breakdown products, as an index ofnitrosative stress) in the cerebral cortex, another vulnerableregion of the AD brain. In saline-treated 3xTg-AD mice we

Fig. 2. NDP-a-MSH reduces histological damage and Ab deposit in the hippocampus and isocortex of 3xTg-AD mice. Histograms’ height indicates mean values � standard error ofthe mean at 30 weeks of age (n ¼ 5 mice per group). The beneficial effects of NDP-a-MSH (340 mg/kg i.p., once daily for 18 weeks) (A and B) were prevented by pretreatment with theMC4 receptor antagonist HS024 (130 mg/kg i.p., before each administration of NDP-a-MSH). (C and D) Representative histological pictures of the isocortex. Note the lessermorphological damage and lower amount of Ab deposit in the NDP-a-MSH-treated 3xTg-AD mouse (D) at ordinary (image on the left, brown plaques), polarized (center, birefringentimage), and fluorescent (right) light, as compared with saline solutionetreated one (C: a massive Ab deposit is present in this field) (ethanoleCongo red/Weigert hematoxylinstainingethioflavin S). Abbreviations: WT, wild-type mice; 3xTg, 3xTg-AD mice; S, saline solution; NDP, NDP-a-MSH; HS, HS024. * p < 0.001 versus the corresponding value of 3xTg-AD mice treated with saline solution; # p < 0.001 versus the corresponding value of 3xTg-AD mice treated with NDP-a-MSH alone. Field wide ¼ 205 mm.

web4C/FPO

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detected a higher concentration of MDA and nitrites, as comparedwith wild-type animals (Fig. 5A and B). In 3xTg-AD mice treatedwith NDP-a-MSH, there was a significant decrease of MDA andnitrite levels, and this favorable effect was abated by pretreat-ment with the MC4 receptor antagonist HS024 (Fig. 5A and B).

3.6. NDP-a-MSH blunts inflammation and apoptosis

Finally, to gain insights into the neuroprotective mechanismsof NDP-a-MSH, in 30-week-old 3xTg-AD mice, we also explored,

by Western blot analysis and ELISA, the inflammatory andapoptotic cascades, which have an established pathophysiologicalrole in AD (Friedlander, 2003; Ghosh et al., 2013; Tayeb et al.,2012; Zipp and Aktas, 2006).

Assay of hippocampus TNF-a and cortex IL-1b showed highlevels of these pro-inflammatory cytokines in saline-treated3xTg-AD mice, but low levels in those treated with NDP-a-MSH(Fig. 6A and B). In addition, this neuroprotective effect of NDP-a-MSH was blunted by pretreatment with the MC4 receptor antag-onist HS024 (Fig. 6A and B).

Fig. 3. NDP-a-MSH reduces APP, PS1, Ab, and tau level/phosphorylation state in the hippocampus of 3xTg-AD mice. Histograms’ height indicates mean values � standard error of themean at 30 weeks of age (n ¼ 7 mice per group). The effect of NDP-a-MSH (340 mg/kg i.p., once daily for 18 weeks) on p-APP Thr668 (A), PS1 (B), Ab1e42 (C), p-tau Thr181 (D), p-tauSer396 (E), and p-tau Ser202 (F) expression was prevented by pretreatment with the MC4 receptor antagonist HS024 (130 mg/kg i.p., before each administration of NDP-a-MSH). Topof each panel shows representative immunoblots highlighting p-APP Thr668, PS1, Ab1e42, p-tau Thr181, p-tau Ser396, and p-tau Ser202 expression; b-actin expression is also shown.Abbreviations: WT, wild-type mice; 3xTg, 3xTg-AD mice; S, saline solution; NDP, NDP-a-MSH; HS, HS024. * p < 0.001 versus the corresponding value of 3xTg-AD mice treated withsaline; # p < 0.001 versus the corresponding value of 3xTg-AD mice treated with NDP-a-MSH alone.

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Next, to investigate apoptosis, we assessed expression of thedownstream executioner caspase-3, for its specific involvementin DNA fragmentation. In the hippocampus of saline-treated3xTg-AD mice, caspase-3 level was significantly higher thanthat of wild-type animals, whereas in 3xTg-AD mice treated withNDP-a-MSH it was very much reduced (Fig. 6C). As expected, theeffect of NDP-a-MSH on caspase-3 expression was as wellinhibited by pretreatment with the MC4receptor antagonistHS024 (Fig. 6C).

4. Discussion

AD is a fatal disease characterized by progressive neurode-generative disorders associated with cognitive decline andbehavioral deficits; among the various disturbances, visionalterationsddue not only to pathological changes in the visualcortical areas, but also to retinal degenerationdand impairmentin certain immune responses can also occur (Iqbal and Grundke-Iqbal, 2010; Ittner and Götz, 2011; Schellenberg and Montine,2012; Sivak, 2013; Sperling et al., 2013; Sy et al., 2011). Highglutamate activity and impaired cholinergic transmission un-derlie the major symptomatology of AD; therefore, currentapproved therapy relies on activation of anti-glutamatergic andcholinergic mechanisms, but it induces only modest and tran-sient symptom improvement (Galimberti et al., 2013; Tayeb et al.,2012). The rapid aging of populations worldwide, with theconsequent increase in AD incidence, represents a real social andeconomic alarm (Gustavsson et al., 2011); thus, innovative phar-macological approaches are required.

Here we show that the melanocortin peptide NDP-a-MSHsignificantly protects against impairment in learning and memoryin a 3xTg-AD mouse model that most closely mimics human AD,such effect being accompanied by a hippocampus significant in-crease in the expression of the synaptic activityedependent geneZif268. The NDP-a-MSHeinduced improvement in behavioralperformance is also associated with isocortex and hippocampusreduced Ab deposits, APP and tau phosphorylation levels, anddecreased neuronal loss. Moreover, hippocampus activation of theMAPK member p38 is counteracted. It worth noting that, understress conditions, the MAPK members play an important role inphosphorylation of tau protein at multiple sites (Munoz andAmmit, 2010; Yu et al., 2009). Also, as transcription factors,MAPK are involved in excitotoxicity, inflammation and apoptosis(Beug et al., 2012; Giuliani et al., 2012; Munoz and Ammit, 2010).Accordingly, we show that under NDP-a-MSH treatment theoxidative and nitrosative stress is diminished by melanocortintreatment, as indicated by the reduced levels of MDA and nitritesin the cerebral cortex. Inflammatory cascade is too attenuated, asproved by the decrease in the hippocampus TNF-a and cortexIL-1b levels: notably, IL-1b, a major pro-inflammatory cytokine inthe brain, is thought to play a pivotal role in AD (Ghosh et al.,2013). Furthermore, apoptosis is modulated, as indicated bycounteraction of caspase-3 activation. All of these protectiveevents occurred at nanomolar doses and seem to be mediated bycentral MC4 receptors, as NDP-a-MSH failed to protect 3xTg-ADmice pretreated with the selectiveMC4 receptor antagonist HS024.

Fig. 4. NDP-a-MSH inhibits p38 activation in the hippocampus of 3xTg-AD mice.Histograms’ height indicates mean values � SEM at 30 weeks of age (n ¼ 7 mice pergroup). The effect of NDP-a-MSH (340 mg/kg i.p., once daily for 18 weeks) on p-p38 wasprevented by pretreatment with the MC4 receptor antagonist HS024 (130 mg/kg i.p.,before each administration of NDP-a-MSH). Top of the panel shows representativeimmunoblots highlighting p-p38 expression; b-actin expression is also shown.Abbreviations: WT, wild-type mice; 3xTg, 3xTg-AD mice; S, saline solution; NDP, NDP-a-MSH; HS, HS024. * p< 0.001 versus the corresponding value of 3xTg-ADmice treatedwith saline; # p < 0.001 versus the corresponding value of 3xTg-AD mice treated withNDP-a-MSH alone.

Fig. 5. NDP-a-MSH reduces malondialdeyde (MDA) and nitrite levels in the cerebral cortex of 3xTg-AD mice. Histograms’ height indicates mean values � SEM at 30 weeks of age(n ¼ 7 mice per group). The effect of NDP-a-MSH (340 mg/kg i.p., once daily for 18 weeks) on (A) MDA and (B) nitrite levels was prevented by pretreatment with the MC4 receptorantagonist HS024 (130 mg/kg i.p., before each administration of NDP-a-MSH). Abbreviations: WT, wild-type mice; 3xTg, 3xTg-AD mice; S, saline solution; NDP, NDP-a-MSH; HS,HS024. * p < 0.001 versus the corresponding value of 3xTg-AD mice treated with saline; # p < 0.001 versus the corresponding value of 3xTg-AD mice treated with NDP-a-MSH alone.

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Extracellular Ab depositsdthe major component of the Abplaque being Ab1e42, but shorter Ab peptides also being pre-sentdand intraneuronal tau hyperphosphorylation play a crucialrole in AD pathophysiology (Blennow, 2010; Galimberti et al.,2013; Iqbal and Grundke-Iqbal, 2010, 2011; Ittner and Götz,2011; Kumar et al., 2013; Oddo et al., 2003; Tayeb et al., 2012).Evidence indicates that Ab and tau exert neuronal toxicity notonly through separate mechanisms but also by reciprocal in-teractions, and among the 6 tau isoforms found in the humanbrain both 3R and 4R forms appear to be involved in AD (Hanischet al., 2010; Ittner and Götz, 2011; Wolfe, 2009). The 3xTg-ADmice used in the present studydwhere Ab pathology developsearlier than tau pathology as it occurs in human AD, consistentwith the amyloid cascade hypothesisdencode human APPSwe,PS1M146V, and tauP301L protein hyperphosphorylated at multiplesites, tau being the 0N/4R isoform (Giuliani et al., 2013; Oddoet al., 2003): interestingly, our present data provide evidencethat melanocortins reduce Ab generation and tau phosphoryla-tion successfully targeting APP Thr668, PS1, and tau protein atsites Thr181, Ser396, and Ser202. It is worth noting that amongthe several potential phosphorylation sites of APP and tau, APPThr668, as well as tau Thr181, tau Ser396, and tau Ser202, play arelevant role in AD pathology: indeed, hyperphosphorylation ofAPP Thr668 is essential for increasing Ab generation, andhyperphosphorylation of tau Thr181, tau Ser396, and tau Ser202plays a key role in converting tau into a molecule that inhibitsmicrotubule assembly and polymerizes into intraneuronalneurofibrillary tangles (Blennow, 2010; Chang et al., 2006; Craig-Schapiro et al., 2009; Hanisch et al., 2010; Iqbal and Grundke-Iqbal, 2010, 2011; Ittner and Götz, 2011; Oddo et al., 2003; Yuet al., 2009).

Nevertheless, there is awareness of the involvement of severalother etiopathogenic mechanisms in AD, and determining thesequence of pathophysiological events that occur in this disorderis of key importance for the identification of appropriate phar-macological approaches; thus far, however, this sequence has notbeen well defined. Interestingly, the earliest clinical signsobserved in AD patientsdepisodic memory loss and cognitivedeclinedare thought to be a result of synaptic dysfunction ratherthan generation of Ab plaques or tau neurofibrillary tangles, orneuronal loss: indeed, hippocampus intraneuronal accumulationof Ab (Oddo et al., 2003), as well as increased activity of the pro-apoptotic protein caspase-3 (D’Amelio et al., 2011), seem to play arole in triggering early synaptic dysfunction in AD before thebrain occurrence of typical histopathological features. Of note,our results provide evidence that NDP-a-MSH is also able toinduce down-regulation of caspase-3 activity, and up-regulationof Zif268, a gene (also known as Egr-1, Krox-24, etc.) essentialfor synaptic transmission and plasticity and involved in manyprocesses related to injury repair (Giuliani et al., 2009, 2011;Tashiro et al., 2007). Furthermore, NDP-a-MSH counteractedactivation of p38, a MAPK member involved in several patho-physiological processes of AD including depression of synaptictransmission (Munoz and Ammit, 2010).

Another AD pathophysiological event, glutamate-inducedexcitotoxicity (partially mediated by p38 down-stream of

Fig. 6. NDP-a-MSH modulates inflammatory and apoptotic cascades in 3xTg-AD mice.Histograms’ height indicates mean values � standard error of the mean at 30 weeks ofage (n ¼ 7 mice per group). The effect of NDP-a-MSH (340 mg/kg i.p., once daily for 18weeks) on the levels of TNF-a (A) and caspase-3 (C) in the hippocampus and IL-1b (B)in the cerebral cortex was prevented by pretreatment with the MC4 receptor antagonist

HS024 (130 mg/kg i.p., before each administration of NDP-a-MSH). (A and C) Tops ofpanels show representative immunoblots highlighting tumor necrosis factorea andcaspase-3 expression; b-actin expression is also shown; interleukin-1b was assessedby enzyme-linked immunosorbent assay. Abbreviations: WT, wild-type mice; 3xTg,3xTg-AD mice; S, saline solution; NDP, NDP-a-MSH; HS, HS024. * p < 0.01, at least,versus the corresponding value of 3xTg-AD mice treated with saline solution; # p <

0.01 versus the corresponding value of 3xTg-AD mice treated with NDP-a-MSHalone.

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glutamate receptor), causes generation of large amounts ofradical species (Bagheri et al., 2011; Galimberti et al., 2013; Gaoet al., 2012; Giuliani et al., 2013; Karbowski and Neutzner,2012; Munoz and Ammit, 2010; Tayeb et al., 2012), and a pro-tective effect of NDP-a-MSH against oxidative and nitrosativestress also occurred in the AD brain of 3xTg-AD mice employed inthe present study. Of interest, melanocortins have been reportedto counteract excitotoxicity and to reduce free radical discharge,including nitric oxide, in severe hypoxic/traumatic conditions,effects that cannot be ascribed to a direct radical scavengingactivity of melanocortins (for review, see Giuliani et al., 2012).

Furthermore, our data showing that melanocortin-inducedcognitive improvement is likewise associated with brainreduced levels of pro-inflammatory cytokines and the apoptosisdown-stream executioner caspase-3, as well as with inhibitedactivation of p38 MAPK, involved in these processes as a regu-latory factor, support the idea that, for an effective neuro-protection against AD, counteracting these mechanisms is alsosuitable (Friedlander, 2003; Ghosh et al., 2013; Munoz andAmmit, 2010; Tayeb et al., 2012; Zipp and Aktas, 2006). It isrelevant to note that melanocortins have established, adrenal-independent, anti-inflammatory effects (Catania et al., 2004;Giuliani et al., 2012), and our present data are consistent withthis notion: indeed, NDP-a-MSH (unlike ACTH) does not stimu-late adrenal glands, because it does not bind MC2 receptors,which are expressed in adrenal cortex and mediate glucocorticoidrelease.

Although many innovative neuroprotective strategiesdinclud-ing pharmacological therapy, immunotherapy against Ab and cell/gene therapydhave been shown to attenuate AD pathology inanimal models, clinical usefulness of these approaches has notbeen confirmed thus far, mainly because most studies aimed toblock just 1 of the AD pathophysiological mechanisms and alsobecause of toxic side effects (Blennow, 2010; Galimberti et al.,2013; Glat and Offen, 2013; Iqbal and Grundke-Iqbal, 2011;Schiöth et al., 2012; Tayeb et al., 2012). Theoretically, effectivepharmacological neuroprotection for AD may require poly-drugtherapy or a single drug active against several mechanisms ofbrain damage. It is worth noting that our data show, for the firsttime, that a class of endogenous agents, melanocortins acting atMC4 receptors, favorably affect several AD-related pathophysio-logical mechanisms, up- and down-stream of Ab and tau. Inter-estingly, no signs of toxicity were recorded in the presentinvestigation throughout the 18 weeks of melanocortin treatment;indeed, NDP-a-MSH and the MC4 receptor antagonist HS024 wereadministered at nanomolar doses, and melanocortins are devoid ofappreciable toxicity in long-term treatments as well (Brzoska et al.,2008; Catania et al., 2004; Minutoli et al., 2011; Wikberg andMutulis, 2008).

Melanocortins are known to reach the CNS in pharmacologi-cally relevant concentrations after systemic administration, andexperimental evidence, as well, points in this direction (Banks andKastin, 1995; Catania et al., 2004; Corander et al., 2009; Giulianiet al., 2012; Vergoni and Bertolini, 2000). Endogenous melano-cortins are largely distributed in the CNS, and MC4 receptors arehighly expressed in various brain areas including the cortex andhippocampus (Catania et al., 2004; Giuliani et al., 2012; Mountjoy,2010; Wikberg and Mutulis, 2008): melanocortins and these re-ceptors may play a physiological neuroprotective role not only inacute neurodegenerative disorders (reviewed by Giuliani et al.,2012) but also in AD. Consistently, low ACTH/a-MSH levels in thecerebrospinal fluid and some brain areas were noticed in patientswith dementia likely related to AD (Arai et al., 1986; Facchinettiet al., 1984) and in the cerebrospinal fluid of patients with lateonset of AD-type dementia (Rainero et al., 1988). Furthermore, a-

MSH produces neurotrophic effects on central cholinergic neuronsthat can be useful in AD (Anderson, 1986). Again, a-MSH has beenshown to modulate production of inflammatory mediators byAb-stimulated cultured murine microglial cells (Galimberti et al.,1999). Finally, MC4 receptor activation induced brain-derivedneurotrophic factor (BDNF) expression in cultured rat astrocytes(Caruso et al., 2012), and BDNF has proved to be neuroprotective inseveral experimental models of neurodegenerative diseasesincluding AD (Nagahara and Tuszynski, 2011). The multiplebeneficial effects against AD found in our present study, therefore,might be due to a physiologically arranged self-defence machinerythat is activated after the MC4 receptor-mediated signal trans-duction of NDP-a-MSH, and that targets multiple AD-relatedpathophysiological pathways; notably, learning was faster inNDP-a-MSH-treated 3xTg-AD animals than in wild-type onestreated with saline or NDP-a-MSH, likely because of a protectiveAD-induced sensitization of MC4 receptors.

It is noteworthy that, very recently, we provided evidence thattreatment of experimental stroke with melanocortins alsostrongly stimulates the brain generation of new cells, whichdevelop properties of mature and seemingly functionally inte-grated neurons (Giuliani et al., 2011; Spaccapelo et al., 2013);melanocortins could be physiologically involved in neurogenesisas well, and such an effect would deserve to be investigated inAD also.

Interestingly, infectious agents may contribute to the onsetand progression of AD (reviewed by Sy et al., 2011), and mela-nocortin peptides have been shown to enhance microbial killingactivity of neutrophils, and even exert direct broad-spectrumantimicrobial activity (Catania et al., 2004; Grieco et al., 2013);these effects could be relevant in AD as well.

In conclusion, in the present study, we found that consider-able neuroprotection can be obtained with melanocortins actingat MC4 receptors administered in the course of AD. However,investigations by beginning treatment when AD is at more severestages are also needed: such investigations could allow furtherinsights into the neuroprotective role of these endogenous pep-tides, and could provide the potential for developing a noveland more physiological approach to the management of ADprogression.

Disclosure statement

The authors state no conflicts of interest.

Acknowledgements

This work was supported in part by grants from Ministero del-l’Istruzione, dell’Università e della Ricerca (MIUR), Roma, and Uni-versity of Modena and Reggio Emilia, Italy.

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