Mechanisms of Hydrogen Sulfide against the Progression of Severe Alzheimer's Disease in Transgenic Mice at Different Ages.

Backgroud: Alzheimer disease is an age-related severe neurodegenerative pathology. The level of the third endogenous gas, hydrogen sulfide (H2S), is decreased in the brain of Alzheimer's disease (AD) patients compared with the brain of the age-matched normal individuals; also, plasma H2S levels are negatively correlated with the severity of AD. Recently, we have demonstrated that systemic H2S injections are neuroprotective in an early phase of preclinical AD. OBJECTIVES: This study focuses on the possible neuroprotection of a chronic treatment with an H2S donor and sulfurous water (rich of H2S) in a severe transgenic 3×Tg-AD mice model. METHOD: 3×Tg-AD mice at 2 different ages (6 and 12 months) were daily treated intraperitoneally with an H2S donor and sulfurous water (rich of H2S) for 3 months consecutively. We investigated the cognitive ability, brain morphological alterations, amyloid/tau cascade, excitotoxic, inflammatory and apoptotic responses. RESULTS: Three months of treatments with H2S significantly protected against impairment in learning and memory in a severe 3×Tg-AD mice model, at both ages studied, and reduced the size of Amyloid ß plaques with preservation of the morphological picture. This neuroprotection appeared mainly in the cortex and hippocampus, associated with reduction in activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in the phosphorylation of tau protein but also in the inflammatory and excitotoxic response. CONCLUSION: Our findings indicate that appropriate treatments with various sources of H2S, might represent an innovative approach to counteract early and severe AD progression in humans.

Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice.

OBJECTIVE AND DESIGN: Alzheimer's disease (AD) is associated with amyloid plaques (Aß) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice. SUBJECTS: Mice were 3 months at the beginning of the study. TREATMENT: Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip). METHODS: Morris water maze was used to assess learning and memory. Histology was performed to evidence Aß plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis. RESULTS: Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aß 1-42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aß plaques; and (4) neuronal loss, compared to saline-treated animals. CONCLUSIONS: Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aß and tau.

NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors.

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with the selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

Melanocortins as potential therapeutic agents in severe hypoxic conditions.

Melanocortin peptides with the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) sequences and synthetic analogs have protective and life-saving effects in experimental conditions of circulatory shock, myocardial ischemia, ischemic stroke, traumatic brain injury, respiratory arrest, renal ischemia, intestinal ischemia and testicular ischemia, as well as in experimental heart transplantation. Moreover, melanocortins improve functional recovery and stimulate neurogenesis in experimental models of cerebral ischemia. These beneficial effects of ACTH/MSH-like peptides are mostly mediated by brain melanocortin MC(3)/MC(4) receptors, whose activation triggers protective pathways that counteract the main ischemia/reperfusion-related mechanisms of damage. Induction of signaling pathways and other molecular regulators of neural stem/progenitor cell proliferation, differentiation and integration seems to be the key mechanism of neurogenesis stimulation. Synthesis of stable and highly selective agonists at MC(3) and MC(4) receptors could provide the potential for development of a new class of drugs for a novel approach to management of severe ischemic diseases.

Hydrogen sulfide slows down progression of experimental Alzheimer's disease by targeting multiple pathophysiological mechanisms.

It has been previously reported that brain hydrogen sulfide (H2S) synthesis is severely decreased in Alzheimer's disease (AD) patients, and plasma H2S levels are negatively correlated with the severity of AD. Here we extensively investigated whether treatment with a H2S donor and spa-waters rich in H2S induces neuroprotection and slows down progression of AD. Studies with sodium hydrosulfide (a H2S donor) and Tabiano's spa-water were carried out in three experimental models of AD. Short-term and long-term treatments with sodium hydrosulfide and/or Tabiano's spa-water significantly protected against impairment in learning and memory in rat models of AD induced by brain injection of ß-amyloid1-40 (Aß) or streptozotocin, and in an AD mouse model harboring human transgenes APPSwe, PS1M146V and tauP301L (3xTg-AD mice). The improvement in behavioral performance was associated with hippocampus was size of Aß plaques and preservation of the morphological picture, as found in AD rats. Further, lowered concentration/phosphorylation levels of proteins thought to be the central events in AD pathophysiology, namely amyloid precursor protein, presenilin-1, Aß1-42 and tau phosphorylated at Thr181, Ser396 and Ser202, were detected in 3xTg-AD mice treated with spa-water. The excitotoxicity-triggered oxidative and nitrosative stress was counteracted in 3xTg-AD mice, as indicated by the decreased levels of malondialdehyde and nitrites in the cerebral cortex. Hippocampus reduced activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in phosphorylation of tau protein but also in inflammation and apoptosis, was also found. Consistently, decrease in tumor necrosis factor-α level, up-regulation of Bcl-2, and down-regulation of BAX and the downstream executioner caspase-3, also occurred in the hippocampus of 3xTg-AD mice after treatment with Tabiano's spa-water, thus suggesting that it is also able to modulate inflammation and apoptosis. Our findings indicate that appropriate treatments with H2S donors and Tabiano's spa-waters, and may be other spa-waters rich in H2S content, might represent an innovative approach to slow down AD progression in humans by targeting multiple pathophysiological mechanisms.

Modulation of the JAK/ERK/STAT signaling in melanocortin-induced inhibition of local and systemic responses to myocardial ischemia/reperfusion.

The janus kinases (JAK), extracellular signal-regulated kinases (ERK) and signal transducers and activators of transcription (STAT) pathways have been shown to play a cardioprotective role. We previously gave evidence that melanocortins afford cardioprotection in conditions of myocardial ischemia/reperfusion. Here we aimed to investigate the influence of melanocortins on the JAK/ERK/STAT signaling in cardiac and systemic responses to prolonged myocardial ischemia/reperfusion. Ischemia was produced in rats by ligature of the left anterior descending coronary artery for 30 min. At the end of the 2-h reperfusion, western blot analysis of the cardioprotective transcription factors pJAK2, pERK1/2, pTyr-STAT3 and pSer-STAT3, the inflammatory mediator tumor necrosis factor-α (TNF-α), the pro-apoptotic factors BAX and c-jun N-terminal kinases (pJNK), the anti-apoptotic protein Bcl-XL, as well as of the cardioprotective enzyme heme oxygenase-1 (HO-1), was performed in the left ventricle and spleen. Intravenous treatment, during coronary artery occlusion, with the melanocortin analogs [Nle(4), D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) and adrenocorticotropic hormone 1-24 [ACTH-(1-24)], induced a left ventricle up-regulation of pJAK2, pERK1/2 and pTyr-STAT3 (JAK-dependent), and a reduction in pJNK and TNF-α levels; these effects of NDP-α-MSH and ACTH-(1-24) were associated with over-expression of the pro-survival proteins HO-1 and Bcl-XL, and marked decrease of the myocardial infarct size. Melanocortin treatment did not affect left ventricle pSer-STAT3 (ERK1/2-dependent) and BAX levels. In the spleen, NDP-α-MSH and ACTH-(1-24) induced similar effects on the expression of the above transcription factors/proteins, except for pERK1/2 (down-regulated) and HO-1 (unaffected). Blockade of JAK and ERK pathways with AG490 and U0126, respectively, abrogated the myocardial infarct size reduction by NDP-α-MSH. These results indicate that melanocortins inhibit local and systemic inflammatory and apoptotic cascades triggered by prolonged myocardial ischemia/reperfusion, with consequent reduction in myocardium infarct size, seemingly via activation of the JAK/STAT signaling and with modulation of an ERK (STAT unrelated) signaling pathway.

Protective effects of melanocortins on short-term changes in a rat model of traumatic brain injury*.

OBJECTIVE: Treatment for traumatic brain injury remains elusive despite compelling evidence from animal models for a variety of therapeutic targets. Melanocortins have established neuroprotective effects against experimental ischemic stroke. We investigated whether melanocortin treatment of traumatic brain injury induces neuroprotection and promotes functional recovery. DESIGN: Randomized experiment. SETTING: Research laboratory at a university hospital. SUBJECTS: Male Sprague-Dawley rats (n = 215). INTERVENTIONS: Experimental rat model of diffuse traumatic brain injury, the impact-acceleration model. MEASUREMENT AND MAIN RESULTS: Brain tissue nitrites, phosphorylation level of extracellular signal-regulated kinases, and c-jun N-terminal kinases; and expression of active caspase-3, tumor necrosis factor-α, BAX, and Bcl-2 as well as serum levels of interleukin-6, high mobility group box-1, interleukin-10, and brain histologic damage were evaluated 24 or 48 hrs after the insult. Sensorimotor orientation and limb use were evaluated at day 7 and learning and memory at days 23-30 after injury. Posttraumatic treatment every 12 hrs with the melanocortin analog [Nle, D-Phe]-α-melanocyte-stimulating hormone (starting 3 or 6 hrs after injury) inhibited traumatic brain injury-induced upregulation of nitric oxide synthesis, phosphorylation level of extracellular signal-regulated kinases, phosphorylation level of c-jun N-terminal kinases, and active caspase-3; reduced expressions/levels of tumor necrosis factor-α, BAX, interleukin-6, and high mobility group box-1; and increased those of Bcl-2 and interleukin-10. These molecular changes were associated with a reduction in brain tissue damage, as highlighted by histopathological findings and improved functional recovery. Pretreatment with the melanocortin MC4 receptor antagonist HS024 abated the positive effects of [Nle, D-Phe]-α-melanocyte-stimulating hormone. CONCLUSIONS: Our data indicate that melanocortins protect against traumatic brain injury, in a broad time window and through activation of MC4 receptors, by counteracting the main traumatic brain injury-related mechanisms of damage. These findings could have major clinical implications.

Molecular changes induced in rat liver by hemorrhage and effects of melanocortin treatment.

BACKGROUND: Melanocortin peptides improve hemodynamic parameters and prevent death during severe hemorrhagic shock. In the present research we determined influences of a synthetic melanocortin 1/4 receptor agonist on the molecular changes that occur in rat liver during hemorrhage. METHODS: Controlled-volume hemorrhage was performed in adult rats under general anesthesia by a stepwise blood withdrawal until mean arterial pressure fell to 40 mmHg. Then rats received either saline or the synthetic melanocortin 1/4 receptor agonist Butir-His-D-Phe-Arg-Trp-Sar-NH2 (Ro27-3225; n = 6-8 per group). Hemogasanalysis was performed throughout a 60-min period. Gene expression in liver samples was determined at 1 or 3 h using quantitative real-time polymerase chain reaction. RESULTS: At 1 h, in saline-treated shocked rats, there were significant increases in activating transcription factor 3 (Atf3), early growth response 1 (Egr1), heme oxygenase (decycling) 1 (Hmox1), FBJ murine osteosarcoma viral oncogene homolog (Fos), and jun oncogene (Jun). These changes were prevented by Ro27-3225 (mean ± SEM: Atf3 152.83 ± 58.62 vs. 579.00 ± 124.13, P = 0.002; Egr1 13.21 ± 1.28 vs. 26.63 ± 1.02, P = 0.001; Hmox1 3.28 ± 0.31 vs. 166.54 ± 35.03, P = 0.002; Fos 4.36 ± 1.03 vs. 14.90 ± 3.44, P < 0.001; Jun 6.62 ± 1.93 vs. 15.07 ± 2.09, P = 0.005; respectively). Increases in alpha-2-macroglobulin (A2m), heat shock 70kD protein 1A (Hspa1a), erythropoietin (Epo), and interleukin-6 (Il6) occurred at 3 h in shocked rats and were prevented by Ro27-3225 treatment (A2m 6.90 ± 0.82 vs. 36.73 ± 4.00, P < 0.001; Hspa1a 10.34 ± 3.28 vs. 25.72 ± 3.64, P = 0.001; Epo 0.49 ± 0.13 vs. 2.37 ± 0.73, P = 0.002; Il6 1.05 ± 0.15 vs. 1.88 ± 0.23, P < 0.001; respectively). Further, at 3 h in shocked rats treated with Ro27-3225 there were significant increases in tight junction protein 1 (Tjp1; 27.30 ± 2.43 vs. 5.03 ± 1.68, P < 0.001) and nuclear receptor subfamily 4, group A, member 1 (Nr4a1; 91.03 ± 16.20 vs. 30.43 ± 11.0, P = 0.01) relative to sham animals. Treatment with Ro27-3225 rapidly restored blood pressure, hemogasanalysis parameters, and lactate blood levels. CONCLUSIONS: Melanocortin treatment significantly prevents most of the systemic and hepatic detrimental changes induced by hemorrhage.

Beneficial Effects of Polydeoxyribonucleotide (PDRN) in an In Vitro Model of Fuchs Endothelial Corneal Dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a bilateral, hereditary syndrome characterized by progressive irreversible injury in the corneal endothelium; it is the most frequent cause for corneal transplantation worldwide. Oxidative stress induces the apoptosis of corneal endothelial cells (CECs), and has a crucial function in FECD pathogenesis. The stimulation of the adenosine A2A receptor (A2Ar) inhibits oxidative stress, reduces inflammation and modulates apoptosis. Polydeoxyribonucleotide (PDRN) is a registered drug that acts through adenosine A2Ar. Thus, the goal of this study was to assess the effect of PDRN in an in vitro FECD model. Human Corneal Endothelial Cells (IHCE) were challenged with H2O2 (200 µM) alone or in combination with PDRN (100 µg/mL), PDRN plus ZM241385 (1 µM) as an A2Ar antagonist, and CGS21680 (1 µM) as a well-known A2Ar agonist. H2O2 reduced the cells' viability and increased the expression of the pro-inflammatory markers NF-κB, IL-6, IL-1ß, and TNF-α; by contrast, it decreased the expression of the anti-inflammatory IL-10. Moreover, the pro-apoptotic genes Bax, Caspase-3 and Caspase-8 were concurrently upregulated with a decrease of Bcl-2 expression. PDRN and CGS21680 reverted the negative effects of H2O2. Co-incubation with ZM241385 abolished the effects of PDRN, indicating that A2Ar is involved in the mode of action of PDRN. These data suggest that PDRN defends IHCE cells against H2O2-induced damage, potentially as a result of its antioxidant, anti-inflammatory and antiapoptotic properties, suggesting that PDRN could be used as an FECD therapy.

Melanocortin 4 receptor stimulation decreases pancreatitis severity in rats by activation of the cholinergic anti-inflammatory pathway.

OBJECTIVE: Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure. Melanocortin peptides have been successfully used in experimental models of organ failure and shock, and their protective effect occurs through the activation of a vagus nerve-mediated cholinergic anti-inflammatory pathway by acting at brain melanocortin 4 receptors. In the light of these observations, we studied the effects of the selective melanocortin 4 receptor agonist RO27-3225 in an experimental model of cerulein-induced pancreatitis. DESIGN: Randomized experiment. SETTING: Research laboratory at a university hospital. SUBJECT: Experimental pancreatitis in rats. INTERVENTIONS: Acute pancreatitis was induced in male Sprague-Dawley rats by intraperitoneal injections of cerulein (80 µg/kg, four injections at hourly intervals). Before pancreatitis induction, groups of animals were subjected to bilateral cervical vagotomy, pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective melanocortin 4 receptor antagonist HS024, or not pretreated. Thirty minutes after the first cerulein injection, rats were intraperitoneally treated with a nanomolar dose of RO27-3225 or vehicle. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 mins after treatment with RO27-3225 or vehicle, and for a 30-min period. MEASUREMENTS AND MAIN RESULTS: Serum lipase and amylase activity, tumor necrosis factor-α and interleukin-6 expression, pancreatic myeloperoxidase activity, and histologic damage were evaluated; neural efferent activity of vagal fibers was also assessed. RO27-3225 reduced cerulein-induced serum lipase and amylase activity, blunted the expression of tumor necrosis factor-α and interleukin-6, abated the increase in pancreatic myeloperoxidase activity, and protected against histologic damage. Furthermore, RO27-3225 markedly increased neural efferent activity along the vagus nerve. Vagotomy, chlorisondamine, and HS024 abated these protective effects of RO27-3225. CONCLUSIONS: Our data show that melanocortin 4 receptor agonists reduce pancreatitis severity through the activation of the cholinergic anti-inflammatory pathway. These findings could be of particular interest in the clinical setting.

Treatment of cerebral ischemia with melanocortins acting at MC4 receptors induces marked neurogenesis and long-lasting functional recovery.

Melanocortins produce neuroprotection against ischemic stroke with subsequent long-lasting functional recovery, through melanocortin MC(4) receptor activation. Here we investigated whether the long-lasting beneficial effect of melanocortins in stroke conditions is associated with a stimulation of neurogenesis. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10 min; then, they were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells. Delayed treatment (up to 9 h after the ischemic injury) for 11 days with the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) improved learning and memory throughout the 50-day observation period. Immunohistochemical examination of the hippocampus on day 50 showed, in the dentate gyrus, an elevated number of BrdU immunoreactive cells colocalized with NeuN (used as indicator of mature neurons) and Zif268 (used as indicator of functionally integrated neurons). Retrospective analysis during the early stage of neural stem/progenitor cell development (days 3 and 4 after stroke) showed, in NDP-α-MSH-treated gerbils, a high degree of daily cell proliferation and revealed that NDP-α-MSH favorably affects Wnt-3A signaling pathways and doublecortin expression. Pharmacologic blockade of MC(4) receptors prevented all effects of NDP-α-MSH. These data indicate that treatment of cerebral ischemia with MC(4) receptor agonists induces, with a broad window of therapeutic opportunity, long-lasting functional recovery associated with a large number of mature and likely functional newborn neurons in brain injured areas. Our findings reveal previously undescribed effects of melanocortins which might have major clinical implications.

High mobility group box-1 expression correlates with poor outcome in lung injury patients.

Chest trauma is frequently followed by pulmonary contusion and sepsis. High mobility group box-1 (HMGB-1) is a late mediator of severe sepsis that has been associated with mortality under experimental conditions. We studied HMGB-1 mRNA expression in patients with lung injury and its relationship with the severity of trauma and survival. A total of 24 consecutive patients with chest trauma referring to the Intensive Care Unit of Messina University Hospital, were enrolled. Lung trauma was established on the basis of chest X-ray and computed tomography. Injury Severity Score (ISS), Revised Trauma Score (RTS) and Glasgow Coma Scale (GCS) were also assessed. Accordingly to these results 6 patients were considered as controls because of no penetrating trauma and low ISS. Blood and broncho-alveolar lavage fluid (BALF) from chest trauma patients were withdrawn at admission and 24h after the beginning of the standard therapeutic protocol. HMGB-1 mRNA increased significantly in blood (r=0.84) and BALF (r=0.87) from patients with trauma and pulmonary contusion and positively correlated with the severity of trauma (based on ISS and RTS) and the final outcome. HMGB-1 protein levels were also elevated in BALF macrophages from severe trauma patients compared to control subjects, furthermore TNF-alpha and its receptor TNFR-1 mRNA levels were also markedly increased in patients with a poor outcome respect to other subjects. Our study suggests that HMGB-1 may be an early indicator of poor clinical outcome in patients with chest trauma.

Melanocortins and the cholinergic anti-inflammatory pathway.

Experimental evidence indicates that small concentrations of inflammatory molecules produced by damaged tissues activate afferent signals through ascending vagus nerve fibers, that act as the sensory arm of an "inflammatory reflex". The subsequent activation of vagal efferent fibers, which represent the motor arm of the inflammatory reflex, rapidly leads to acetylcholine release in organs of the reticuloendothelial system. Acetylcholine interacts with α7 subunit-containing nicotinic receptors in tissue macrophages and other immune cells and rapidly inhibits the synthesis/release of tumor necrosis factor-α and other inflammatory cytokines. This neural anti-inflammatory response called "cholinergic anti-inflammatory pathway" is fast and integrated through the central nervous system. Preclinical studies are in progress, with the aim to develop therapeutic agents able to activate the cholinergic anti-inflammatory pathway. Melanocortin peptides bearing the adrenocorticotropin/α-melanocyte-stimulating hormone sequences exert a protective and life-saving effect in animals and humans in conditions of circulatory shock. These neuropeptides are likewise protective in other severe hypoxic conditions, such as prolonged respiratory arrest, myocardial ischemia, renal ischemia and ischemic stroke, as well as in experimental heart transplantation. Moreover, experimental evidence indicates that melanocortins reverse circulatory shock, prevent myocardial ischemia/reperfusion damage and exert neuroprotection against ischemic stroke through activation of the cholinergic anti-inflammatory pathway. This action occurs via stimulation of brain melanocortin MC3/MC4 receptors. Investigations that determine the molecular mechanisms of the cholinergic anti-inflammatory pathway activation could help design of superselective activators of this pathway.

Functional recovery after delayed treatment of ischemic stroke with melanocortins is associated with overexpression of the activity-dependent gene Zif268.

Melanocortin peptides afford strong neuroprotection and improve functional recovery in experimental ischemic stroke; they also have established neurotrophic actions. The expression of the immediate early gene Zif268 is dependent on synaptic activity and is involved in injury repair and memory formation. Here, we investigated the role of Zif268 in learning and memory recovery after delayed treatment of ischemic stroke with the melanocortin analog [Nle(4), D-Phe(7)]alpha-MSH (NDP-alpha-MSH). A 10-min period of global cerebral ischemia was induced by occluding both common carotid arteries in gerbils. Treatment with a nanomolar dose of NDP-alpha-MSH (every 12h for 11 days) was performed starting 3h or 9h after stroke induction; where indicated, gerbils were pretreated with the melanocortin MC(4) receptor antagonist HS024. Animals were subjected to the Morris water-maze test (four sessions from 4 to 50 days after the ischemic episode). Fifty days after stroke, histological damage and Zif268 expression were investigated in the hippocampus. Treatment with NDP-alpha-MSH significantly reduced hippocampal damage, including neuronal death, and improved learning and memory recovery. This protective effect was long-lasting (50 days, at least) and associated with Zif268 overexpression, with both schedules of NDP-alpha-MSH treatment. Pharmacological blockade of MC(4) receptors prevented these effects. Our data indicate that MC(4) receptor-mediated actions of melanocortins could trigger repair mechanisms able to improve neuronal functionality and synaptic plasticity, and to promote long-lasting functional recovery from ischemic stroke with Zif268 gene involvement.

Melanocortin Receptor-4 and Glioblastoma Cells: Effects of the Selective Antagonist ML00253764 Alone and in Combination with Temozolomide In Vitro and In Vivo.

Currently, no description of melanocortin receptor-4 (MC4R) expression or activity is available in human cancer cells, including glioblastoma (GBM). The aim of this study is to evaluate the presence of MC4Rs in GBM cells and the selective inhibition of their activity through the MC4R antagonist ML00253764 alone and in association with temozolomide in vitro and in vivo. MC4R genotyping and gene expression were performed on human GBM cells (U-87 and U-118) with real-time PCR. MC4R western blotting, immunohistochemistry, and immunofluorescence were obtained in both cell lines and in human tissues. Proliferation, cell cycle, and apoptotic assays were performed with ML00253764, whereas the synergism of the simultaneous combination with temozolomide was evaluated by the combination index method. ERK1/2 and Akt phosphorylation were quantified by ELISA. In vivo experiments were performed in U-87 xenografted nude mice. Both GBM cell lines and tumor tissues expressed MC4R receptors. The selective antagonist ML00253764 determined an antiproliferative and proapoptotic activity through the inhibition of the phosphorylation of ERK1/2 and Akt. Moreover, the simultaneous combination of temozolomide and ML00253764 determined a highly synergistic effect on GBM cells. The same combination in vivo showed a strong and significant decrease of GBM tumor volumes if compared to the single drug treatments, with an excellent tolerability profile. In conclusion, MC4R is present in GBM cells and its selective inhibition determined antiproliferative and proapoptotic effects, through the inhibition of ERK1/2 and Akt phosphorylation, and the synergistic enhancement of temozolomide effects in vitro and in vivo.

Melanocortin Receptor-4 Gene Polymorphisms in Glioblastoma Patients Treated with Concomitant Radio-Chemotherapy.

Melanocortins are peptides with well-recognized antiinflammatory and neuroprotective activity. No data are currently available on melanocortin receptor-4 (MC4R) gene polymorphisms and tumors, including glioblastomas (GBMs), or their relationship with radiotherapy or chemotherapy. The aim of this study was to evaluate the possible predictive/prognostic role of the MC4R SNPs on GBM patients. Fifty-five patients with a proven diagnosis of GBM, treated with radiotherapy and temozolomide, were consecutively enrolled. MC4R gene SNPs (rs17782313, rs489693, rs8087522, rs17700633) were analyzed by a validated TaqMan® SNP genotyping assays. Univariate and multivariate analyses were performed. A P < 0.0125 (Bonferroni's correction) was considered significant ( Clinicaltrial.gov identifier NCT02458508). The median progression-free survival (PFS) and median overall survival (OS) of these patients were 9.54 (95% CI 5.4-14.3) months and 24.9 (95% CI 17.8-34.6) months, respectively. The MC4R rs489693 AA genotype was significantly associated with a shorter PFS and OS. Indeed, with regard to PFS, patients harboring the rs489693 AA genotype had a median PFS of 2.99 months whereas patients with AC/CC genotypes had a median PFS of 10.82 months (P = 0.009). Interestingly, the rs489693 AA patients also had a lower median OS as compared with the median OS of the AC/CC genotypes (10.75 vs. 29.5 months, respectively, P = 0.0001). This study suggests that the MC4R rs489693 AA genotype is significantly associated with a shorter PFS and OS in patients treated with radiotherapy and temozolomide. These findings represent a relevant effort to identify novel clinical markers for RT-CT therapy in GBM to be validated in future pharmacogenetic clinical trials.

The disaccharide trehalose inhibits proinflammatory phenotype activation in macrophages and prevents mortality in experimental septic shock.

Proinflammatory phenotype activation in macrophages (MPhis) after sepsis orchestrates an inflammatory response leading to multiple organ dysfunction. Trehalose preserves cell viability during exposure to a range of environmental stresses. We investigated whether trehalose may inhibit endotoxin-induced activation of the inflammatory phenotype in MPhis. Rat peritoneal MPhis were stimulated with 50 microg/mL of Salmonella enteritidis lipopolysaccharide (LPS). Stimulated MPhis were coincubated with trehalose (25, 50, and 100 mmol), sucrose (100 mmol), or RPMI alone. Macrophages cultures were used for Western blot analysis of extracellular-regulated kinase, c-jun-N terminal kinase, and inducible nitric oxide synthase; interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) gene expression by real-time reverse transcriptase-polymerase chain reaction, and supernatants for measuring the release of inflammatory cytokines and nitrite content. In vitro trehalose significantly blunted LPS-induced extracellular-regulated kinase (LPS = 21 +/- 6 integrated intensity; LPS + trehalose 100 mmol = 2 +/- 0.3 integrated intensity), c-jun-N terminal kinase (LPS = 15 +/- 5 integrated intensity; LPS + trehalose 100 mmol = 3.5 +/- 0.9 integrated intensity), and inducible nitric oxide synthase activation (LPS = 12 +/- 3 integrated intensity; LPS + trehalose 100 mmol = 1 +/- 0.09 integrated intensity), blunted IL-1beta (LPS = 5 +/- 1.9 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.5 +/- 0.8 n-folds/beta-actin), IL-6 (LPS = 4 +/- 1.5 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.4 +/- 0.5 n-folds/beta-actin), and TNF-alpha (LPS = 4.2 +/- 1.6 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.1 +/- 0.7 n-folds/beta-actin) gene expression, and markedly reduced the release of inflammatory cytokines and nitrite content. Furthermore, in vivo trehalose prevented mortality in rats challenged with a lethal dose (20 mg/kg; LD90) of LPS (80% survival rate and 70% survival rate 24 and 72 h after LPS injection, respectively) and reduced serum TNF-alpha. Sucrose did not modified inflammatory phenotype in vitro nor in vivo protected against endotoxin-induced mortality. Our study suggests that trehalose inhibits proinflammatory phenotype activation in MPhis and prevents endotoxin-induced mortality.

Neuroprotection in focal cerebral ischemia owing to delayed treatment with melanocortins.

In gerbils subjected to transient global cerebral ischemia, melanocortin peptides produce long-lasting protection with a broad time window, and through the activation of central nervous system melanocortin MC(4) receptors. Here we aimed to investigate whether melanocortins are neuroprotective also in a rat model of focal cerebral ischemia induced by intrastriatal microinjection of endothelin-1. The vasoconstrictor agent endothelin-1 caused a significant impairment in spatial learning and memory, as well as in sensory-motor orientation and limb use, associated with severe striatal morphological damage including intense neuronal death and an almost complete myelin degradation. Treatment of ischemic rats with a nanomolar dose (340 microg/kg/day i.p. for 11 days, beginning 3 h or 9 h after endothelin-1 microinjection) of the melanocortin analog [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) significantly reduced striatal damage, and improved subsequent functional recovery, with all scheduled NDP-alpha-MSH treatments. Pharmacological blockade of melanocortin MC(4) receptors prevented the protective effect of NDP-alpha-MSH. Our findings give evidence that melanocortins are neuroprotective, with a broad time window, also in a severe model of focal cerebral ischemia, and suggest that melanocortin MC(4) receptor agonists could produce neuroprotection in different experimental models of ischemic stroke.

Multiple beneficial effects of melanocortin MC4 receptor agonists in experimental neurodegenerative disorders: Therapeutic perspectives.

Melanocortin peptides induce neuroprotection in acute and chronic experimental neurodegenerative conditions. Melanocortins likewise counteract systemic responses to brain injuries. Furthermore, they promote neurogenesis by activating critical signaling pathways. Melanocortin-induced long-lasting improvement in synaptic activity and neurological performance, including learning and memory, sensory-motor orientation and coordinated limb use, has been consistently observed in experimental models of acute and chronic neurodegeneration. Evidence indicates that the neuroprotective and neurogenic effects of melanocortins, as well as the protection against systemic responses to a brain injury, are mediated by brain melanocortin 4 (MC4) receptors, through an involvement of the vagus nerve. Here we discuss the targets and mechanisms underlying the multiple beneficial effects recently observed in animal models of neurodegeneration. We comment on the potential clinical usefulness of melanocortin MC4 receptor agonists as neuroprotective and neuroregenerative agents in ischemic stroke, subarachnoid hemorrhage, traumatic brain injury, spinal cord injury, and Alzheimer's disease.