[1] 21w Neuropharmacology and analgesia Melanocortins protect against brain damage and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer's disease

[1] 219w We previously reported that melanocortins induce neuroprotection in experimental acute and chronic neurodegenerative conditions, including Alzheimer's disease (AD) of mild severity. Here we investigated whether melanocortins afford neuroprotection and counteract cognitive decline in AD with a medium level of severity by using 24 week-old (at the start of the study) APP Swe transgenic mice (Tg2576). Salinetreated (days 1-50) control Tg2576 mice showed an impairment in spatial learning and memory, associated (at day 50, end of the study) with hippocampus at low levels of the synaptic activitydependent gene Zif268, relevant brain changes such as cerebral cortex/hippocampus increased level of β-amyloid (Aβ) deposit, and neuronal loss, in comparison with wild-type animals. Treatment of Tg2576 mice (once daily at days 1-50) with a nanomolar dose of the melanocortin analog [Nle 4 ,D-Phe 7 ]αmelanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aβ deposit, decreased neuronal loss, increased hippocampus Zif268 expression and improved cognitive functions, relative to saline-treated Tg2576 mice. Pharmacological blockade of melanocortin MC 4 receptors with the MC4 receptor antagonist HS024 prevented all favorable effects of NDP-α-MSH. Our data indicate that MC 4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD of medium severity through induction of neuroprotection and improvement of synaptic transmission. After further studies, these agents could gain a role as disease modifying therapeutics for AD.

[1] 162w Alzheimer's disease (AD) is a chronic disorder characterized by progressive neurodegeneration associated with cognitive decline and several behavioral deficits. Sporadic AD is generally diagnosed in people over 65 years of age, whereas familial or genetic AD (the less prevalent form of Alzheimer) is an early-onset autosomal dominant disease (Galimberti et al., 2013;Iqbal andGrundke-Iqbal, 2010, 2011;Ittner and Götz, 2011;Sperling et al., 2013;Tayeb et al., 2012). In some vulnerable regions of AD brains, βand γ-secretases process the amyloid precursor protein (APP) to generate extra-cellular β-amyloid (Aβ) deposits (Aβ plaques); in this disease, later, also intraneuronal tau neurofibrillary tangles (composed of hyperphosphorylated tau protein) develop. Subsequently, the amyloid/tau cascade triggers pathophysiological pathways (which are common to acute and chronic neurodegenerative disorders) that lead to synaptic dysfunction, neurodegeneration and marked neuronal loss (Alonso et al., 2008;Blennow, 2010;Giuliani et al. 2013Giuliani et al. , 2014;;Iqbal and Grundke-Iqbal, 2010;Ittner and Götz, 2011;Kim et al., 2013;Lilja et al., 2013;Lo, 2010;Martín-Moreno et al., 2012;Sperling et al., 2013;Tayeb et al., 2012).

[2] 71w Neuroprotective and neurorestorative strategies for acute and chronic neurodegenerative disorders, including AD, are under intensive studies, but effective and innovative approaches are still unavailable in clinical setting (Becker et al., 2007;Blennow, 2010;Galimberti et al., 2013;Giuliani et al., 2011Giuliani et al., , 2012Giuliani et al., , 2013Giuliani et al., , 2014;;Glat and Offen, 2013;Iqbal and Grundke-Iqbal, 2011;Lilja et al., 2013;Schiöth et al., 2012;Sun et al., 2003;Tayeb et al., 2012;Zhang and Chopp, 2009).

[3] 209w Melanocortins are endogenous peptides of the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) family, and acting via five different melanocortin G protein-linked, seven transmembrane receptor subtypes (MC 1 -MC 5 ) (Brzoska et al., 2008;Catania et al., 2004;Giuliani et al., 2012;Mountjoy, 2010;Wikberg and Mutulis, 2008). Melanocortins and their synthetic analogs have been reported to possess a multitude of protective actions (Bazzani et al., 2001;Bertolini et al., 1989;Brzoska et al., 2008;Catania et al., 2004;Corander et al., 2009;Giuliani et al., 2012;Guarini et al., 1996Guarini et al., , 1997;;Minutoli et al., 2011;Mioni et al., 2003;Wikberg and Mutulis, 2008), including neuroprotective actions associated with long-lasting functional recovery, as found in acute experimental neurodegenerative conditions (Bharne et al., 2011;Bitto et al., 2012;Chen et al., 2008;Gatti et al., 2012;Giuliani et al., 2006Giuliani et al., , 2007bGiuliani et al., , 2009;;Savos et al., 2011;Spaccapelo et al., 2011). The neuroprotective effects of melanocortins are mediated by CNS melanocortin MC 4 receptors and occur through an inhibition of the main mechanisms of brain damage (for review see: Giuliani et al., 2012). Recently we found that MC 4 receptorstimulating melanocortins are also able to induce neuroprotection in a triple-transgenic mouse model of AD at a low level of severity, by targeting pathophysiological mechanisms up-and downstream of Aβ and tau (Giuliani et al., 2014).

[4] 49w To gain insight into the potential therapeutic value of melanocortins against AD progression, in the present research we investigated melanocortins in another transgenic mouse model of AD, APP Swe mice, and at a medium level of disease severity, by studying their influence on cognitive processes and brain histological alterations.

[1] 108w To investigate a common feature of AD, severe cognitive decline (Galimberti et al., 2013;Tayeb et al., 2012), we studied the ability of Tg2576 mice to find the spatial location of an hidden platform in the Morris apparatus. Control Tg2576 mice treated with saline for 50 days showed impaired ability (as compared with wild-type mice) in platform finding both during the first and second training session (31 week-old mice) (Fig. 1). On the contrary, in Tg2576 animals treated for 50 days with the melanocortin NDP-α-MSH, a significant improvement in learning and memory performance occurred in both sessions of the Morris test, compared with salinetreated control Tg2576 mice (Fig. 1).

[2] 100w Melanocortin MC 4 receptors are largely expressed in CNS, and activation of this receptor subtype has been demonstrated to induce neuroprotection in neurodegenerative conditions (Giuliani et al., 2006, 2012, 2013, Mountjoy, 2010). We investigated, therefore, the role of MC 4 receptors in Tg2576 mice. As expected, the favorable effect of NDP-α-MSHwhich activates MC 1 , MC 3 , MC 4 and MC 5 receptors (Giuliani et al., 2006(Giuliani et al., , 2007a(Giuliani et al., , 2007b(Giuliani et al., , 2012) ) on learning and memory performance was completely prevented by pretreatment with the MC 4 receptor antagonist HS024 (Fig. 1).

[3] 69w At the here used nanomolar doses, neither NDP-α-MSH alone nor HS024 alone significantly affected learning and memory in wild-type mice (not shown). No signs of toxicity were recorded in wild-type and Tg2576 mice during treatments: indeed, the general conditions appeared close to normality, as reflected by spontaneous locomotor exploration, grooming and smooth fur. Moreover, body weight variations throughout the 50-day observation period in all experimental groups overlapped (Fig. 2).

[4] 101w The expression of the immediate early gene Zif268 is dependent on synaptic activity, and detection of Zif268 protein is used for investigating recently activated neurons and synaptic plasticity (Giuliani et al., 2009(Giuliani et al., , 2011(Giuliani et al., , 2014;;Tashiro et al., 2007;Veyrac et al., 2013). Western blot analysis of Zif268 protein in the hippocampus of the above mice, killed within 90 min after the end of the last session of behavioral study, showed a very low Zif268 expression in saline-treated Tg2576 mice, relative to wild-type animals. On the contrary, treatment with NDP-α-MSH significantly upregulated the expression of Zif268 (Fig. 3).

[5] 25w The ability of NDP-α-MSH to induce Zif268 over-expression in the hippocampus was counteracted by animal pretreatment with the MC 4 receptor antagonist HS024 (Fig. 3).

[6] 99w After the last session of behavioral tests we studied AD-related brain istological alterations (Blennow, 2010;Iqbal and Grundke-Iqbal, 2010;Lilja et al., 2013). Examination of the hippocampus and WT+S WT+NDP Tg+S WT+HS Body weight (g) days Tg+NDP 50 Tg+HS+NDP 1 10 20 30 40 Fig. 2. Time-course of mouse body weight during the 50-day observation period. Histograms' height indicates mean values-S.E.M. (n ¼12 mice per group). WT¼ wild-type mice; Tg¼ Tg2576 mice; S¼ saline; NDP ¼NDP-α-MSH; HS ¼ HS024. At each time-point there was not statistically significant difference among groups (P 40.05) (two-way repeated measures ANOVA followed by the Student-Newman-Keuls' test).

[7] 117w isocortex of saline-treated 31 week-old Tg2576 mice revealed a substantial extracellular amyloid deposit (Fig. 4A, B and C) and neurons showing nucleus degeneration, nuclear dust, pyknosis, cellular shrinkage, pericellular vacuolization and swollen perikaryon, with considerable neuronal loss and increase of microglia cells, inside the isocortex (Fig. 5) and in a lesser extent in the hippocampus. Conversely, in Tg2576 mice treated with NDP-α-MSH we observed a significantly improved morphological picture, with a marked reduction in amyloid plaque deposit (Fig. 4A, B and D), and a greater number of viable neurons (Fig. 5), relative to saline-treated Tg2576 animals. Also these neuroprotective effects of NDP-α-MSH were prevented by pretreatment with the MC 4 receptor antagonist HS024 (Fig. 4A and B).

[1] 106w AD is a major cause of disability and mortality without an effective treatment. Current approved therapy for AD relies on activation of anti-glutamatergic and pro-cholinergic mechanisms ─ whose alteration underlies the main symptomatology of AD ─ but with only modest and transient symptom improvement (Galimberti et al., 2013;Tayeb et al., 2012). The rapid aging of populations worldwide, with consequent increase in AD incidence ─ an estimated 35.6 million people worldwide had AD in 2012, and this number may triple by 2050 ─ therefore represents a real social and economic problem (Freiherr et al., 2013;Gustavsson et al., 2011;Iqbal andGrundke-Iqbal, 2010, 2011;Ittner and Götz, 2011;Sperling et al., 2013).

[2] 316w Here we show that the melanocortin peptide NDP-α-MSH significantly protects against impairment in learning and memory in Tg2576 mice with moderate AD. The NDP-α-MSH-induced improvement in behavioral performance is associated with hippocampus overexpression of the synaptic activity-dependent gene Zif268, and decreased Aβ deposits and neuronal loss in the isocortex and hippocampus, in comparison with saline-treated Tg2576 animals. All these protective events occurred at nanomolar doses and seem to be mediated by central MC 4 receptors, since NDP-α-MSH failed to protect Tg2576 mice pretreated with the rather selective MC 4 receptor antagonist HS024. A deposit/slide ( m 2 ) * * # # A deposit/slide ( m 2 ) Hippocampus Isocortex * * Tg+S Tg+NDP Hippocampus 4. NDP-α-MSH reduces Aβ deposit in the hippocampus and isocortex of Tg2576 mice. Histograms' height indicates mean values7 S.E.M. at 31 weeks of age (day 50 of the study; n¼ 6 mice per group). (A and B) The beneficial effects of NDP-α-MSH (340 mg/kg i.p., once daily for 50 days) were prevented by pretreatment with the MC 4 receptor antagonist HS024 (130 mg/kg i.p., before each administration of NDP-α-MSH). (C and D) Representative histological pictures of the hippocampus (CA1/CA2): notice the lower amount of Aβ deposit in the NDP-α-MSH-treated Tg2576 mouse (D) at ordinary (image on the left, brown plaques), polarized (center, birefringent image) and fluorescent (right) light, as compared with saline-treated one (C: a massive Aβ deposit is present in this field) (ethanol-Congo red/Weigert hematoxylin stainingthioflavin S). WT¼wild-type mice; Tg ¼ Tg2576 mice; S¼ saline; NDP ¼NDP-α-MSH; HS¼ HS024. n P o 0.001 versus the corresponding value of Tg2576 mice treated with saline; # Po 0.001 versus the corresponding value of Tg2576 mice treated with NDP-α-MSH alone (one-way ANOVA followed by the Student-Newman-Keuls' test). Field wide ¼205 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

[3] 131w It is well known that extracellular Aβ deposits and intraneuronal tau hyperphosphorylation play a crucial role in AD pathophysiology (Alonso et al., 2008;Blennow, 2010;Galimberti et al., 2013;Iqbal andGrundke-Iqbal, 2010, 2011;Ittner and Götz, 2011;Kim et al., 2013;Martín-Moreno et al., 2012;Oddo et al., 2003;Tayeb et al., 2012), and evidence indicates that Aβ and tau exert neuronal toxicity not only through separate mechanisms but also by reciprocal interactions (Ittner and Götz, 2011). In our previous study performed in a triple-transgenic mouse model with mild AD severity (Giuliani et al., 2014) we found that melanocortin MC 4 receptor stimulation: (a) reduces brain Aβ generation and tau phosphorylation successfully targeting some proteins/sites of the amyloid/tau cascade; (b) blunts oxidative and nitrosative stress; (c) decreases levels of inflammatory and apoptotic mediators; and (d) protects against cognitive decline.

[4] 217w Our data, obtained in another transgenic mouse model of AD that mimics human AD features of medium severity, confirm and extend our previous findings that melanocortins acting at MC 4 receptors are able to slow down AD progression. The here provided evidence that melanocortins induce Zif268 overexpression in Tg2576 mice ─ according to our previous findings in different conditions of neurodegeneration (Giuliani et al., 2009(Giuliani et al., , 2011(Giuliani et al., , 2014) ) ─ suggests that NDP-α-MSH-induced Zif268 upregulation might have been of key importance in the improvement of synaptic transmission and cognitive processes detected in the present investigation: indeed, Zif268 is rapidly induced as transcription factor not only by a variety of physiological stimuli (including learning and memory), but also by pathological stimuli (as a mediator of compensation and repair mechanisms), and the essential role of Zif268 in synaptic plasticity is well established (Beckmann and Wilce, 1997;Tashiro et al., 2007;Veyrac et al., 2013). Notably, AD synaptic dysfunction seems to start before the occurrence of typical brain histological alterations (Oddo et al., 2003). Consistent with our interpretation of the present results, a full demonstration that melanocortin MC 4 receptors play a crucial role in the regulation of hippocampal synaptic plasticity has been recently provided by means of electrophysiological, immunocytochemical and biomolecular studies (Shen et al., 2013).

[5] 312w The unavailability into clinical setting, so far, of novel pharmacological and non-pharmacological strategies to slow down AD progression may be mainly due to the fact that most studies aimed to block just one of the AD pathophysiological mechanisms and also to 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). It is worth noting, therefore, that our previous and present studies found that a class of endogenous agents, melanocortins acting at MC 4 receptors, slow down AD progression by favorably affecting several pathophysiological pathways and repair mechanisms, up-and down-stream of Aβ and tau (Giuliani et al., 2014;present data). From a practical point of view, it is relevant that a considerable improvement in learning and memory, and a marked reduction in Aβ accumulation and morphological alterations, also occur in the brain of the present AD model of medium severity. Further, the potential therapeutic value of melanocortins in AD is strengthened by the lack of signs of toxicity throughout the 50-day treatment period: accordingly, melanocortins including synthetic analogs have been repeatedly reported to be devoid of appreciable toxicity also in long-term treatments (reviewed by : Brzoska et al., 2008;Catania et al., 2004;Giuliani et al., 2012;Wikberg and Mutulis, 2008). Finally, neither NDP-α-MSH nor the MC 4 receptor antagonist HS024 affected animal body weight during the treatment period. Mice used in the present study were freely feeding and treated with NDP-α-MSH and HS024 during the daytime: of note, the established ability of melanocortin MC 4 receptor agonists to decrease food intake and body weight is very evident in animals deprived of food for many hours, and also in freely feeding animals treated during the nocturnal phase (reviewed by : Schiöth, 2001;Vergoni and Bertolini, 2000); further, long-term treatment with MC 4 receptor agonists or antagonists only transiently affected food conversion in rats (Jonsson et al., 2002).

[6] 607w Endogenous melanocortins are largely distributed in the CNS, and MC 4 receptors are highly expressed in various brain areas including the cortex and hippocampus (Catania et al., 2004;Giuliani et al., 2012;Mountjoy, 2010;Wikberg and Mutulis, 2008). The MC 4 receptor-mediated signal transduction of melanocortins could activate pathways that play a physiological protective role by affecting the expression of several transcription factors/ signaling molecules involved in neuroprotection and repair, not only in acute neurodegenerative diseases (Giuliani et al., 2006(Giuliani et al., , 2009(Giuliani et al., , 2011(Giuliani et al., , 2012;;Spaccapelo et al., 2011Spaccapelo et al., , 2013) ) but also in AD, as suggested by the present results. Notably, observational studies from different laboratories support the hypothesis that melanocortins could be physiologically involved in neuroprotection against AD: (a) loss of cholinergic neurons is a feature of AD, and α-MSH produces established neurotrophic effects on central cholinergic neurons (Anderson, 1986); (b) low ACTH/α-MSH levels were found in the cerebrospinal fluid and some brain areas in sporadic studies carried out in patients with AD-type dementia (Arai et al., 1986;Facchinetti et al., 1984;Rainero et al., 1988); (c) brain-derived neurotrophic factor (BDNF) induces protective effects in several experimental models of neurodegenerative diseases including AD (Nagahara, Tuszynski 2011), and MC 4 receptor activation has been reported to increase the expression of BDNF in rat astrocytes (Caruso et al., 2012). A limitation of the present study could be represented by the use of HS024 to block melanocortin MC 4 receptors, because this cyclic MSH analog binds mainly but not exclusively the MC 4 receptors [Ki values (nmol/liter, mean 7S.E.M.) obtained from competition curves on human MC 1 , MC 3 , MC 4 and MC 5 receptors: 18.6 73.3, 5.45 0.29 70.14 and 3.297 1.15, respectively (Kask et al., 1998)]. Kask et al. (1998) also conducted functional studies in rats, however in the present research we used mice and, obviously, difference among animal species exists. Anyway, evidence suggests that the gene encoding for the MC 4 receptor is highly conserved in mammals ─ both rat and mouse homologous genes are 94% identical to the human gene (for BLASTs analysis see: http://www.ncbi.nlm.nih.gov/homologene/4320) ─ and the dose used in our present study is within the concentration range for the proved selectivity at MC 4 receptors (Caruso et al., 2012;Getting et al., 2006;Giuliani et al., 2006Giuliani et al., , 2007aGiuliani et al., , 2007bGiuliani et al., , 2014;;Kask et al., 1998;Minutoli et al., 2011;Spaccapelo et al., 2011). Consistent with the idea that the neuroprotective effects of melanocortins do not occur through stimulation of central MC 3 receptors À the other melanocortin receptor subtype predominant within the brain (Catania et al., 2004;Giuliani et al., 2012;Mountjoy, 2010;Wikberg and Mutulis, 2008) À administration of γ 2 -MSH, a selective agonist at MC 3 receptors, failed to induce neuroprotection in stroke conditions (Giuliani et al., 2006). With regard to MC 1 receptors, only limited expression has been demonstrated in the CNS (neurons of the periaqueductal gray matter of the midbrain) (Catania et al., 2004;Wikberg, and Mutulis, 2008;Holloway et al., 2011); furthermore, the fact that the MC receptor antagonist HS024 has an approximately 65-fold higher affinity for MC 4 receptors relative to MC 1 receptors (Kask et al. 1998) also argues against an involvement of MC 1 receptors in the here shown neuroprotective effects of melanocortins. Finally, melanocortin MC 5 receptors are ubiquitous mainly in the peripheral tissues (Catania et al., 2004;Giuliani et al., 2012;Holloway et al., 2011;Wikberg, and Mutulis, 2008), and the very low affinity of HS024 and NDP-α-MSH for MC 5 receptors (Kask et al., 1998) argues, as well, against a possible neuroprotective role for these receptors in our study.

[7] 146w In conclusion, overall our previous (Giuliani et al., 2014) and present data give evidence that melanocortins acting at MC 4 receptors induce neuroprotection and counteract cognitive decline in AD transgenic mice when treatment starts at a mild-tomoderate level of disease severity, and an important role for these beneficial effects is likely played by the improvement in synaptic transmission. However, another weakness arises from these results, as compared with other studies (Martín-Moreno et al., 2012), because we planned our research in young Tg2576 mice. Indeed, investigations by beginning melanocortin treatment at other ages that reflect the progressive severity of AD, and aimed at thoroughly assessing their influence on amyloid cascade and tau hyperphosphorylation, as well as on cognitive performance, are again needed. New favorable results could strengthen the idea that melanocortins are of potential clinical relevance for an innovative, may be physiological, neuroprotective-based management of AD progression.

[1] 173w For these investigations, male 24 week-old (at the start of the study) Tg2576 mice and their wild-type littermates (Taconic; Hudson, NY) were used. These mice harbor human transgene APP Swe , that is, they overexpress the human APP695 isoform with the Swedish double mutations K670N/M671L (Hsiao et al.,1996;Lilja et al., 2013;Martín-Moreno et al., 2012). Animals were kept in air-conditioned colony rooms (temperature 217 1 1C, humidity 60%) on a natural light/dark cycle, with food in pellets and tap water available ad libitum. Body weight was recorded throughout the observation period, and rectal temperature was maintained close to 37 1C by means of heating lamps. Animal killing at the end of the study was performed under general anesthesia with sodium pentobarbital (50 mg/kg) intraperitoneally (i.p.) (Sigma-Aldrich, Milan, Italy). Housing conditions and experimental procedures were in strict accordance with the European Community regulations on the use and care of animals for scientific purposes (CEE Council 89/609; Italian D.L. 22-1-92 No. 116), and were approved by the Animal Ethics Committee of Modena and Reggio Emilia University.

[2] 158w The whole hippocampi were dissected from brains of some animals (6 per group). Mice were killed within 90 min after the end of the last behavioral test (day 50 of the study; 31 week-old mice) to detect Zif268 expression (Zif268 protein is transiently expressed after synaptic activation: Giuliani et al., 2009Giuliani et al., , 2011;;Tashiro et al., 2007;Veyrac et al., 2013). After extraction of nuclear proteins, as previously described (Giuliani et al., 2009(Giuliani et al., , 2014)), proteins (30 μg for each sample) were denatured, electrophoretically separated and transferred onto nitrocellulose membranes. Staining of the blots with Ponceau's solution showed that total protein amount was equal in each lane. The blots were then blocked and incubated overnight at 4 1C with a mouse monoclonal primary antibody for Zif268 (Abcam, Cambridge, UK). The day after, the membranes were incubated with a specific secondary antibody peroxidase-conjugated, goat anti-mouse immunoglobulin G (Thermo Scientific, Rockford, IL) for 1 h at room temperature.

[3] 87w To prove equal loading, the blots were analyzed for β-actin expression (house-keeping gene) using an anti-β-actin antibody (Cell Signaling, Charlottesville, VA). The membranes were analyzed by the enhanced chemiluminescence system according to the manufacturer's protocol (Millipore, Billerica, MA). The protein signals were quantified by scanning densitometry using a bioimage analysis system (Bio-Profil, Celbio, Italy) and expressed as relative integrated intensity in comparison with those of naïve wild-type animals (Bitto et al., 2012;Giuliani et al., 2006Giuliani et al., , 2009Giuliani et al., , 2013Giuliani et al., , 2014)).

[4] 180w At the end of the last behavioral test, transcardial perfusion with ice-cold 4% paraformaldehyde (phosphate-buffered) was performed in the remaining animals (6 per group), then mouse brains were removed and processed for histological examination. Isocortex and hippocampus morphologies were studied in 7-mm thick paraffin-embedded sections, hematoxylin-eosin stained, as previously described (Giuliani et al., 2006(Giuliani et al., , 2007b(Giuliani et al., , 2009;;Spaccapelo et al., 2011); the extent of Aβ plaques was assessed in 7-mm thick paraffin-embedded sections after ethanol-Congo red/Weigert hematoxylin staining or aqueous thioflavin-S (Giuliani et al., 2013(Giuliani et al., , 2014;;Salkovic-Petrisic et al., 2011). Histological analyses were performed by using an Axiophot photomicroscope (Carl Zeiss, Jena, Germany), under ordinary, polarized and fluorescent light. Histometry was performed by using an image analyzer and software (analySIS, Soft Imaging System GmbH, Münster, Germany). Morphology was estimated in five randomly selected fields per slide, and extracellular Aβ deposit (Aβ plaques) on the whole slide; in all cases, five seriated sections per animal, taken every 100 μm starting À 1 mm (frontal isocortex) and þ2 mm (hippocampus) from bregma zero coordinate, were used.

[5] 45w All data were detected blind to the treatment and are shown as mean 7S.E.M. Values were analyzed by means of two-way repeated measures ANOVA (behavioral data and body weight) followed by the Student-Newman-Keuls' test, or one-way ANOVA (all other data) followed by the Student-Newman-Keuls' test.

[6] 6w P value o0.05 was considered significant.

[1] 232w [Nle 4 ,D-Phe 7 ]α-melanocyte-stimulating hormone (NDP-α-MSH), synthetic melanocortin analog with long-lasting biological activity (Giuliani et al., 2006(Giuliani et al., , 2007a(Giuliani et al., , 2007b(Giuliani et al., , 2009(Giuliani et al., , 2011(Giuliani et al., , 2014) ) (kindly provided by Prof. Paolo Grieco, University of Naples Federico II), was dissolved in saline (1 ml/kg) and i.p. administered (340 μg/kg once daily for 50 days, starting from 24th and until 31st week of age). Pretreatment with the selective melanocortin MC 4 receptor antagonist HS024 (Cys-Nle-Arg-His-D-Nal-Arg-Trp-Gly-Cys, cyclic MSH analog with S-S bridge between two Cys; Tocris, Bristol, UK), when done, was performed i.p. (130 mg/kg, dissolved in saline 1 ml/kg) 20 min before each administration of NDP-α-MSH (Giuliani et al., 2006(Giuliani et al., , 2007a(Giuliani et al., , 2007b(Giuliani et al., , 2009(Giuliani et al., , 2011(Giuliani et al., , 2014)). Control animals (Tg2576 and wild-type mice) received an equal volume of saline by the same route of administration; as further control animals, wild-type mice treated with NDP-α-MSH alone and HS024 alone were also used. All drugs and saline were administered during the daytime (8:00-9:00 a.m.). The doses of NDP-α-MSH and HS024 were chosen on the basis of our previous studies performed in experimental acute and chronic neurodegenerative conditions (Bitto et al., 2012;Giuliani et al., 2006Giuliani et al., , 2007bGiuliani et al., , 2009Giuliani et al., , 2011Giuliani et al., , 2014)).

[2] 263w We used the Morris water-maze test with minor modifications, as repeatedly described (Bitto et al., 2012;Giuliani et al., 2006Giuliani et al., , 2007bGiuliani et al., , 2009Giuliani et al., , 2011Giuliani et al., , 2013Giuliani et al., , 2014;;Spaccapelo et al., 2011Spaccapelo et al., , 2013)). Briefly, this test measures animal's ability to learn, remember and go to a place in space defined only by its position relative to distal extramaze cues. The apparatus consisted of a circular white pool (80 cm in diameter and 55 cm in height) filled to a depth of 15 cm with water (27 1C) rendered opaque with milk. Mice (12 per group) were trained to find the spatial location of a platform of clear perspex hidden by arranging for its top surface (7 cm in diameter) to be 1 cm below the water level. To this end, four cardinal points on the apparatus wall were defined by means of different geometrical figures, and conspicuous cues were placed in a fixed position around the pool. In each daily training, latency to escape onto the hidden platform was recorded. Each mouse received four daily trials starting each time from a different cardinal point in a random succession: in each trial, if the animal failed to locate the platform within 60 s, escape latency was considered same as 60 s (therefore, the daily maximally possible total escape latency was 240 s). The study was carried out during the 31st week of age: mice were subjected to a first five-day training sequence followed three days later by a second one-day training.