NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC 4 receptors

NDP-α-MSH

3 -The number of BrdU/NeuN in Fig 4 is not exactly the same as the number of BrdU/NeuN/Zif268 triple-stained cells.Anyway, surprisingly, in our experimental conditions the number of triple stained cells is very high, and with enthusiasm in Fig. 5 of the previous version (now Fig. 6) we decided to show a field were all BrdU-labeled cells are also colocalized with NeuN and Zif268 (this is specified in the figure caption of the previous and revised version of our manuscript).We agree with the Reviewer that newborn cells usually requires a month or more to migrate; indeed, as stated in methods, results and figure caption, we counted newborn cells at day 50 of the study, that is, 39 days after the last BrdU injection.Anyway, in the previous Fig 5 (now Fig. 6) there are no newborn cells in the outer parts of DG: the 2 right arrowheads in the previous Fig.5F (now Fig. 6 C, without arrows) belong to DG (see red box in panel A of the new Fig. 5).
4 -As stated in methods and shown in Fig. 1, in our experimental model BrdU treatment was performed on days 1-11 of the study,: therefore, younger cells BrdU positive are 39day old, and younger cells not BrdU-labeled obviously are not visible.In the previous Fig.5G (now Fig. 6B) a high number of cells (old and newborn neurons) -but not almost allis Zif positive (please compare panel A with panel B of Fig. 6).In summary, in the previous Fig.5H (now Fig. 6F) there are 3 types of cells: a few white cells (BrdU/NeuN/Zif triplestained, 12%, active neurons born on days 1-11 of the study ), cyan cells (NeuN/Zif double-stained, 60%, active neurons born before and after days 1-11 of the study) and green cells (NeuN-stained, 28%, no active neurons born before and after days 1-11 of the study).Anti-apoptotic immunoreactions have not be done, so we are unsure if Zif stained also such cells.All appropriate controls have been done for immunocytochemistry (see methods).. 5 --See answer to the 1 st question of Reviewer 1. Cell number per volume was calculated as indicated in Endocrinology, 147, 1126Endocrinology, 147, -1135Endocrinology, 147, , 2006.We did not perform stereological evaluation, but only a cell count to allow roughly comparison among experimental groups.Immunocytochemistry for Amyloid was not performed since we preferred the histochemical method that in combination with the polarized light examination prevents false positives.We added further details on histological methods (page 8, lines 10-14).The Reviewer's suggestion "a more convincing morphological (cytoarchitectual) analysis would be welcome including the distribution of amyloid beta deposits over the different hippocampal subregions" was not the goal of this study but it will be in the future.6 -The figure 3 (now modified) is a representative image depicting the hippocampus situation.As reported in the caption, the Hematoxylin-Eosin staining (C), particularly in the saline treated mice, highlighted nucleus degeneration, pyknosis, cellular shrinkage and pericellular vacuolization.7 -Our experience indicates that 50 days of ip injection does not cause suffering or appreciable stress.This has not interfered with results, because all groups (including controls) have been ip injected for 50 days.This long-term treatment schedule has been chosen to allow maturation of newborn neurons.8 -Our very interesting and exciting results on neuroprotection published in 2014 on EJP and NBA encouraged us to investigate also neurogenesis, because (from a scientific and practical point of view) very important and of great novelty.9 -The possible mechanism of the neurogenic effect of melanocortins was discussed in pages 14-15 of the previous version.In the revised manuscript we discussed some more about MC receptors and AD (page 15, lines 10-20).
10 -Taking into account the Reviewer's comments, in the introduction of the revised manuscript we deleted the reference by Giuliani et al. (2013) about neuroprotection and added Franco and Cedazo-Minguez (2014), and we cited all the relevant key papers related to neurogenesis suggested by the Reviewer.
We thank you for your statement "I like their work and this approach very much" and for your useful criticism.Our work has been now improved as suggested.As stated in the above point 8, we investigated neurogenesis after the important results obtained on neuroprotection.In our opinion, the neurogenic effect is a relevant novelty, because it constitutes a robust framework of the potential therapeutic value of melanocortins against AD (obviously after further studies).
Minor comments 1 -OK: so far, the most appropriate reference is that by Ben Menachem-Zidon et al., 2014 (page 15, line 1).
2 -As stated at page 16, lines 2-6) melanocortins are devoid of appreciable toxicity .Unfortunately, until now we are unable to give further explanations (besides those reported in page 15, lines 10-20) on how melanocortins protect against amyloid overexpression.Further studies are needed.
3 -For many years our results on Morris Water Maze (MWM) have been depicted in the same manner (and published in many important Journals, please see references of our group), because (in our opinion) of easier understanding for readers.4 -This was probably due to the repeated confocal acquisition to have a well-defined image, without background, that increases contrast.
5 -Microglia increase was seen in isocortex and rarely in the hippocampus.In the revised manuscript, reference to Fig. 3 has been deleted (page 10, line 10).
6 -See answer to the 1 st question of Reviewer 1. 7 -OK, the order of figures has been corrected.

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The excitation/emission wavelengths have been added in Material and Methods (page 8, line 6).9 -The figure 5 has been split (Figures 5 & 6) to show better overlap.10 -As suggested, English has been examined by a native speaker.
We recently discovered that melanocortins, in addition to their neuroprotective action, strongly stimulate neurogenesis in gerbils affected by acute neurodegeneration caused by transient global cerebral ischemia; the data showed that the newborn cells developed properties of mature and functional neurons (Giuliani et al., 2011;Spaccapelo et al., 2013).The availability of drugs that combine both neuroprotective and neuroregenerative influences could be of great clinical relevance for innovative therapeutic approaches in acute and chronic neurodegenerative disorders.Therefore, in order to further assess the potential beneficial effects of melanocortins in AD, we investigated influences of a melanocortin analog on the neurogenic process in APP Swe transgenic mice.

Animals
Twenty-four week-old (at the start of the study) male Tg2576 mice and their wild-type littermates (Taconic; Hudson, NY) were used.These mice, which harbor human transgene APP Swe , overexpress the human APP695 isoform with the Swedish double mutations K670N/M671L (Hsiao et al.,1996;Lilja et al., 2013).Animals were kept in air-conditioned colony rooms (temperature 21  1°C, humidity 60%) on a natural light/dark cycle, with food in pellets and tap water available ad libitum.General health conditions and body weight were recorded throughout the observation period, and rectal temperature was maintained close to 37°C by means of heating lamps; the latter procedure was adopted to avoid an hypothermia-mediated neuroprotection potentially induced by melanocortins (Giuliani et al., 2006(Giuliani et al., , 2012) that could interfere with data interpretation.At the end of the study, animal sacrifice was performed under general anesthesia with i.p. injection of 50 mg/kg sodium pentobarbital (Sigma-Aldrich, St. Louis, MO).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.

Assessment of spatial learning and memory
The mouse ability to learn and recall was evaluated by means of the Morris water-maze test with minor modifications, as previously described in our papers (Bitto et al., 2012;Giuliani et al., 2006Giuliani et al., , 2007Giuliani et al., , 2009Giuliani et al., , 2011Giuliani et al., , 2013Giuliani et al., , 2014aGiuliani et al., , 2014b;;Spaccapelo et al., 2011Spaccapelo et al., , 2013)).Of note, mice used in the present study were 6-8 month old, that is, an age characterized by mild/moderate cognitive decline and AD (Hsiao et al.,1996;Lilja et al., 2013).Briefly, 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 °C) rendered opaque with milk.Mice (10 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 random sequence: 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 twenty-seventh week (starting 14 days after the first BrdU injection) and thirty-first week of age.During the twenty-seventh week of age (study performed mainly for an experiencedependent recruitment of newborn cells into spatial memory networks during the critical period of maturation) (Veyrac et al,2013), mice were subjected to a first 5-day training sequence followed by a second 1-day training 3 days later.Two other similar training sessions of the Morris test were repeated during the thirty-first week of age (last week of the study).

Histology
Following the last behavioral test (day 50 of the study; 31 week-old mice) and within 90 min after the last behavioral test (to detect Zif268 positivity, indicator of recently activated neurons) (Giuliani et al., 2011;Tashiro et al., 2007), transcardial perfusion of ice-cold 4% paraformaldehyde (phosphate-buffered) was performed and the brains were removed and processed for histologic examination and fluorescence immunohistochemistry.
Histometry was performed by using an image analyzer and software (analySIS, Soft Imaging System GmbH, Münster, Germany).Morphology and cells positive to BrdU, NeuN and Zif268, were estimated in five randomly selected fields per slide.The density of cells was estimated in a 100 µm-thick band overlapping the pyramidal cell layer of the DG.Extracellular Aβ deposits were estimated on the whole slide.

Statistical analysis
All data were collected and analyzed by an observer blind to the treatment; they are shown as mean ± SEM.Statistical analysis was performed using either two-way repeated measures ANOVA (behavioral data) or one-way ANOVA (all other data), followed by the Student-Newman-Keuls' test.A value of p < 0.05 was considered significant.

NDP--MSH improves learning and memory
To investigate the typical AD impairment of learning and memory (Galimberti et al., 2013;Tayeb et al., 2012), the ability of Tg2576 mice to find the spatial location of an hidden platform in the Morris apparatus was performed in animals prepared for the neurogenesis study (that was executed on day 50).Saline-treated control Tg2576 mice showed impaired ability in platform finding both during the first and second training sessions (days 14-21 of the study; carried out mainly for an experience-dependent recruitment of newborn cells into spatial memory networks during the critical period of maturation; data not shown), as well as during the third and fourth sessions (days 43-50 of the study), relative to wild-type mice (Fig. 2A,B).Conversely, NDP-α-MSH-treated (for 50 days) Tg2576 mice displayed significantly better performance in learning and memory in all sessions of the Morris test, as compared with saline-treated control Tg2576 mice (Fig. 2A,B).
Based on our previous observations, we investigated the role of melanocortin MC 4 receptors mainly expressed in the CNS (Mountjoy, 2010), whose activation induced neuroprotection in acute brain injury and AD (Bitto et al., 2012;Giuliani et al., 2006Giuliani et al., , 2014aGiuliani et al., , 2014b) ) and promoted neurogenesis in stroke (Giuliani et al., 2011).Consistent with our previous findings, the favourable effects of NDP-α-MSH on learning and memory performance in Tg2576 mice was totally prevented by pretreatment with the selective MC 4 receptor antagonist HS024 (Fig. 2A,B).
Of note, neither NDP-α-MSH alone nor HS024 alone significantly affected learning and memory in wild-type mice (not shown).Furthermore, no signs of toxicity were recorded in wild-type and AD animals (ruffled fur, diarrhea, lethargy, aggressiveness, hypothermia, alterations of spontaneous locomotor exploration and grooming), and body weight variations throughout the study were similar in all experimental groups (not shown).
These data confirm our original findings and further suggest that a chronic treatment with melanocortin MC 4 receptor agonists represents a safe strategy to counteract cognitive decline in AD mice.

NDP--MSH preserves histological integrity of the isocortex and hippocampus
Generally, Aβ plaques and morphological alterations first appear in the frontal isocortex and extend thereafter to other isocortex regions and to hippocampus (Giuliani et al., 2014a(Giuliani et al., , 2014b;;Oddo et al., 2003).Therefore, to verify existence of brain damage in the mice over the course of AD development, histological examination was performed in the isocortex and hippocampus after the last session of the Morris test (day 50 of the study).In our experimental conditions, in saline-treated Tg2576 mice the histological picture was characterized by significant extracellular Aβ deposits and neurons showing pyknosis, swollen perikaryon and cellular shrinkage, with appreciable neuronal loss, mainly in the isocortex and, in lesser extent, in the hippocampus (Fig. 3), relative to wild-type animals.Microglia increase was also seen in the isocortex and rarely in the hippocampus.Treatment of Tg2576 mice with NDP-α-MSH likewise improved the general morphological picture of the isocortex and hippocampus, with a reduction in Aβ deposits and a greater number of viable neurons, in comparison with saline treatment (Fig. 3).As expected, the favourable effects of NDP-α-MSH on the isocortex and hippocampus were reversed by Tg2576 mouse pretreatment with the MC 4 receptor antagonist HS024 (Fig. 3).
These data provide evidence for a brain damage during AD progression in mice prepared for neurogenesis investigation, and confirm our previous findings that melanocortin MC 4 receptor activation reduces brain histological alterations (Giuliani et al., 2014a(Giuliani et al., , 2014b)).

NDP--MSH stimulates neural progenitor proliferation in the dentate gyrus
In subsequent experiments aimed at studying neurogenesis, which was the main aim in the present research , we investigated neural stem/progenitor cell proliferation in the hippocampus.As described in Methods, mice were previously treated with BrdU throughout days 1-11 of the study and sacrificed on day 50 (90 min after the last behavioral test); we chose this time-point as the newborn cells need several weeks to develop into mature and functional neurons (Arvidsson et al., 2002;Becker et al., 2007;Giuliani et al., 2011;Veyrac et al., 2013).In our study, few BrdUlabeled, newly formed cells were detected on day 50 in the hippocampus DG of saline-treated Tg2576 mice and saline-treated wild-type animals (Fig. 4A).Conversely, NDP-α-MSH treatment of Tg2576 mice was associated with a very high number of BrdU-labeled cells, as compared with saline-treated animals (Fig. 4A and Fig. 6C).The neuroproliferative effects of NDP-α-MSH were prevented by pretreatment with the MC 4 receptor antagonist HS024 (Fig. 4A).
The present data demonstrate that melanocortin MC 4 receptor activation induces neural stem/progenitor cell proliferation in AD.

Newly generated cells develop properties of functionally integrated neurons
In order to characterize the phenotype of BrdU positive cells, immunoreactivity for the mature neuronal marker NeuN and the astrocyte marker GFAP was evaluated by double-labeling.At confocal microscopy examination, a great number of double-labeled BrdU-NeuN cells, but no double-labeled BrdU-GFAP cells (that is, newly formed astrocytes) was found within the DG of NDP-α-MSH-treated Tg2576 mice (Fig. 4B,C, Fig. 5, Fig. 6).
Colocalization of BrdU incorporation with the neuronal marker NeuN and the early functional gene Zif268 was then examined in the same sections.Zif268 (also known as Egr-1, Krox-24, etc.) is primarily expressed after synaptic activation and is used as an indicator of recently activated neurons (Becker et al., 2007;Giuliani et al., 2011;Tashiro et al., 2007).Impressively, in the DG of NDP-α-MSH-treated Tg2576 mice we counted a number of triple-labeled BrdU-NeuN-Zif268 cells very high and strictly close to that of double-labeled BrdU-NeuN cells, that is, almost all BrdU-NeuN immunoreactive cells colocalized with Zif268 (Fig. 4D, Fig. 6C).
Consistent with a primary role of MC 4 receptors in the neurogenic process, pretreatment of Tg2576 mice with the MC 4 receptors antagonist HS024 prevented the favourable effects of NDP-α-MSH on double (BrdU-NeuN)-and triple (BrdU-NeuN-Zif268)-labeled cell density (Fig. 4C,D).
These data indicate that treatment of AD mice with melanocortin MC 4 receptor agonists shifts newborn cells toward the neuronal phenotype and provides a favourable microenvironment for functional integration.

Discussion
An estimated 35.6 million people worldwide were affected by AD in 2012, and this number may triple by 2050.Therefore the aging of human population worldwide and the consequent increase in AD incidence represent a real social and economic alarm (Freiherr et al., 2013;Gustavsson et al., 2011;Ittner and Götz, 2011;Sperling et al., 2013).Indeed, AD remains a major cause of disability and mortality without effective treatment, as current approved therapies (drugs that activate antiglutamatergic and pro-cholinergic mechanisms) only induce modest and transient improvement of the main symptoms (Galimberti et al., 2013;Tayeb et al., 2012).Novel neuroprotective and neurorestorative strategies for AD, by means of pharmacological therapy, immunotherapy against Aβ, and cell and gene therapy, are under investigation in animals and humans (Bayer and Wirths, 2014;Becker et al., 2007;Ben Menachem-Zidon et al., 2014;Freiherr et al., 2013;Galimberti et al., 2013;Giuliani et al.,.2013Giuliani et al.,. , 2014aGiuliani et al.,. , 2014b;;Glat and Offen, 2013;Iqbal and Grundke-Iqbal, 2011;Lilja et al., 2013;Tayeb et al., 2012;Wang et al., 2010).However, disappointing results are arising from these novel studies (Salloway et al., 2014), and, therefore, effective approaches to slow down AD progression are still unavailable in clinical practice.
Here we report that a 50-day treatment with the melanocortin peptide NDP-α-MSH counteracts learning and memory decline in Tg2576 mice with mild/moderate AD.The NDP-α-MSH-induced improvement in cognitive performance was associated with an improved histological picture within the isocortex and hippocampus, with decreased Aβ deposits, and a very high number of BrdU immunoreactive newly generated cells in the hippocampus DG; few BrdU-labeled cells were detected in saline-treated Tg2576 mice, and in saline-treated wild-type animals (whose good cognitive performance likely depends on an unaltered brain histological picture).The present data also show that, in NDP-α-MSH-treated Tg2576 mice, almost all BrdU positive cells on day 50 were also NeuN immunoreactive and expressed Zif268, an indicator of functionally integrated neurons.
Therefore, melanocortin treatment appear to shift cells toward a neuronal phenotype and to promote functional integration.All these beneficial events occurred at nanomolar doses and seemingly with an involvement of central MC 4 receptors, as NDP-α-MSH failed to protect Tg2576 mice pretreated with the selective MC 4 receptor antagonist HS024.
In our previous studies that investigated neuroprotective effects of melanocortins in transgenic mouse models of mild/moderate AD, APP Swe /PS1 M146V /tau P301L mice and Tg2576 mice (Giuliani et al., 2014a(Giuliani et al., , 2014b)), we found that melanocortin MC 4 receptor stimulation inhibits the amyloid/tau cascade, oxidative and nitrosative stress, inflammatory and apoptotic responses, with consequent protection against brain morphological alterations and cognitive decline.Our previous results indicate that MC 4 receptor stimulation leads to improved synaptic transmission and plasticity, as strongly suggested by hippocampus overexpression of Zif268, a transcription factor that controls major processes of synaptic activity (Giuliani et al., 2014a(Giuliani et al., , 2014b)).Consistently, a full and elegant demonstration that melanocortin MC 4 receptors regulate hippocampal synaptic plasticity has been recently provided by Shen and coworkers (2013).
Our present data demonstrate for the first time that melanocortins, acting at MC 4 receptors, also induce neurogenesis in a transgenic mouse model of AD.This finding strengthens the potential therapeutic value of these endogenous agents.In the present study we investigated the hippocampus neurogenesis, and the present results are consistent with a melanocortin-induced amplification of the neurogenic process in the SGZ; obviously, other established germinal zones may be targeted by melanocortins as well.
Very recently we found that melanocortin treatment of gerbils subjected to experimental stroke strongly stimulates brain generation of new cells, which develop properties of mature and seemingly functionally integrated neurons, with a high proportion of long-term survival (Giuliani et al., 2011;Spaccapelo et al., 2013).Our biomolecular studies on the neurogenic process in stroke gerbils showed that treatment with the melanocortin NDP-α-MSH induces neural stem/progenitor cell proliferation in the DG.This action was associated with activation of MC 4 receptors and triggering of the canonical Wnt-3A/-catenin and Sonic hedgehog signaling pathways (Giuliani et al., 2011;Spaccapelo et al., 2013), which play a key role in maintenance and proliferation of stem/progenitor cells and in neuronal fate determination (Benarroch, 2013;Suh et al., 2009).
Further, activation of these pathways was associated with early up-regulation of the repair factor Zif268 and the neurogenesis facilitating factor interleukin-10 (IL-10) in the DG (Spaccapelo et al., 2013).It is reasonable to hypothesize that the same molecular mechanisms underlay the neurogenic effects of melanocortins found in AD animals in the present study.In particular, the known ability of melanocortins to induce Zif268 overexpression (Giuliani et al., 2009(Giuliani et al., , 2011(Giuliani et al., , 2014a(Giuliani et al., , 2014b) ) could have contributed to brain injury repair and improvement of cognitive processes.Indeed, Zif268 is rapidly induced as transcription factor by a variety of physiological and pathological stimuli (such as ischemia, neurodegeneration, learning and memory, etc.), the natural induction of adequate expression level being limited by various causes including the disease severity.The essential role of Zif268 in synaptic plasticity, as well as in long-term survival, maturation and functional integration of newborn neurons, is well established (Giuliani et al., 2009;Tashiro et al., 2007;Veyrac et al., 2013).To allow the Zif268-mediated activity-dependent long-term survival and functional integration of newborn cells, in the present research we subjected mice to two preparatory spatial learning and memory sessions starting 14 days after the first BrdU injection, that is, during the critical period of selection and maturation of a number of new neurons (Veyrac et al.,2013).
Impairment of neurogenesis in AD has been attributed to brain overexpression of the proinflammatory cytokine IL-1β signaling.Indeed this cytokine appears to be involved in the generation of AD hallmark lesions (such as increased expression of amyloid precursor protein, tau hyperphosphorylation, over-activity of acetylcholinesterase, etc.).Further, IL-1β may cause an unfavourable microenvironment by reducing expression of neurogenetic factors such as brain-derived neurotrophic factor (BDNF) (Ben Menachem-Zidon et al., 2014).Interestingly, we previously showed that melanocortin MC 4 receptor agonists inhibit IL-1β expression in the isocortex of AD mice (Giuliani et al., 2014a), and MC 4 receptor activation has been reported to increase brain expression of BDNF (Caruso et al., 2012(Caruso et al., , 2014)).Thus, also these mechanisms may have contributed to the neurogenic effect of melanocortins in AD mice reported in the present research.
Melanocortin MC 4 receptors are mainly expressed in various brain areas including the cortex and hippocampus (Caruso et al., 2014;Catania et al., 2004;Giuliani et al., 2012;Mountjoy, 2010;Wikberg and Mutulis, 2008), and are thought to play a physiological protective role against different types of brain injury.Specifically, experimental evidence suggests that the MC 4 receptormediated signal transduction of melanocortins activates pathways that promote the expression of several salutary transcription factors/signaling molecules, including those involved in synaptic plasticity, neuroprotection, neural progenitor cell proliferation and differentiation (Caruso et al., 2014;Giuliani et al., 2012Giuliani et al., , 2014aGiuliani et al., , 2014b;;Holloway et al., 2011).The possibility of a physiological protective role of melanocortin MC 4 receptor agonists also against AD progression is supported by the following observations: (i) α-MSH produces established neurotrophic effects on central cholinergic neurons, whose loss is a feature of AD (Anderson, 1986); (ii) low ACTH/α-MSH levels were detected in the cerebrospinal fluid/brain of patients with AD-type dementia (Arai et al.,1986;Facchinetti et al.,1984;Rainero et al.,1988).It is presently unknown whether number/activity of brain MC receptors is changed in AD.
It is worth noting that we previously found that melanocortins acting at MC 4 receptors are able to counteract AD progression by inhibiting several AD-related pathophysiological mechanisms upand down-stream Aβ and tau (Giuliani et al., 2014a(Giuliani et al., , 2014b)).The present data also show that this class of endogenous agents induces neurogenesis; further, neuroprotection could ameliorate neurogenesis by providing a favorable microenvironment, also advantageous for survival of newly generated cells.Finally, the potential therapeutic value of melanocortins for AD treatment is strengthened by the lack of signs of toxicity throughout the treatment period, both in the present (7 weeks) and previous (up to 18 weeks; Giuliani et al., 2014aGiuliani et al., , 2014b) ) research.Accordingly, melanocortins have been repeatedly reported to be devoid of appreciable toxicity also in long-term treatments, both in animals and humans, and some melanocortin compounds have already been successfully tested in clinical conditions different from neurodegeneration, with evidence of safety in humans (reviewed by : Brzoska et al., 2008;Catania et al., 2004;Corander et al., 2009;Giuliani et al., 2012;Wikberg and Mutulis, 2008).
In conclusion, the present study demonstrates that melanocortins induce intense neurogenesis via activation of MC 4 receptors in AD transgenic mice when treatment is started at a mild/moderate level of disease severity.The neurogenic effect, together with the neuroprotective action previously demonstrated, represent a robust framework of the potential therapeutic value of melanocortins against AD including cognitive impairment: however, specific correlation and/or link between cognitive performance and neurogenesis should be studied.After further investigations in AD animals ─ also at older ages with progressive severity of AD ─ favourable results would suggest that melanocortins could be an innovative neuroprotective and neuroregenerative strategy to reduce AD progression, as well as to improve aging-impaired neurogenesis in humans.

Fig. 4 .
Fig. 4. NDP--MSH induces generation of new cells which develop properties of mature and

Fig. 6 .
Fig. 6.Representative confocal images of the experiments of Fig. 4. NDP--MSH-treated Tg2576 Fig 1 Click here to download high resolution image .