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We applied the group-I metabotropic glutamate (mGlu) receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), to neonatal or adult rat hippocampal slices at concentrations (10 μm) that induced a short-term depression (STD) of excitatory synaptic transmission at the Schaffer collateral/CA1 synapses. DHPG-induced STD was entirely mediated by the activation of mGlu5 receptors because it was abrogated by the mGlu5 receptor antagonist, MPEP [2-methyl-6-(phenylethynyl)pyridine], but not by the mGlu1 receptor antagonist, CPCCOEt [7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester]. Knowing that ephrin-Bs functionally interact with group-I mGlu receptors (Calò et al., 2005), we examined whether pharmacological activation of ephrin-Bs could affect DHPG-induced STD. We activated ephrin-Bs using their cognate receptor, EphB1, under the form of a preclustered EphB1/Fc chimera. Addition of clustered EphB1/Fc alone to the slices induced a small but nondecremental depression of excitatory synaptic transmission, which differed from the depression induced by 10 μmDHPG. Surprisingly, EphB1/Fc-induced synaptic depression was abolished by MPEP (but not by CPCCOEt) suggesting that it required the endogenous activation of mGlu5 receptors. In addition, coapplication of DHPG and EphB1/Fc, resulted in a large and nondecremental long-term depression. The effect of clustered EphB1/Fc was specific because it was not mimicked by unclustered EphB1/Fc or clustered EphA1/Fc. These findings raise the intriguing possibility that changes in synaptic efficacy mediated by mGlu5 receptors are under the control of the ephrin/Eph receptor system, and that the neuronal actions of ephrins can be targeted by drugs that attenuate mGlu5 receptor signaling.

Spontaneously depressed flinders sensitive line (FSL) rats showed a reduced expression of mGlu2/3 metabotropic glutamate receptors in the hippocampus, as compared to "non-depressed" flinders resistant line (FRL) rats. No changes in mGlu2/3 receptor protein levels were found in other brain regions, including the amygdala, hypothalamus, and cerebral cortex. Biochemical analysis of receptor signalling supported the reduction of mGlu2/3 receptors in the hippocampus of FSL rats. Accordingly, the selective mGlu2/3 receptor agonist, LY379268 (1microM) reduced forskolin-stimulated cAMP formation by 56% and 32% in hippocampal slices from FRL and FSL rats, respectively. In addition, LY379268 enhanced 3,5-dihydroxyphenylglycine-stimulated inositol phospholipid hydrolysis from 65% to 215% in hippocampal slices from FRL rats, whereas it was inactive in slices from FRL rats. We also examined the behavioural response of FSL rats to systemic injection of LY379268 (0.5mg/kg, i.p., once a day for 1-21 days) by measuring the immobility time in the forced swim test, which is known to be increased in these rats. LY379268 was administered alone or combined with the classical antidepressant, chlorimipramine (10mg/kg, i.p.). LY379268 alone had no effect at any of the selected time-points, whereas chlorimipramine alone reduced the immobility time only after 21 days of treatment. In contrast, when combined with LY379268, chlorimipramine reduced the immobility time during the first 14 days of treatment. These data support the view that mGlu2/3 receptors might be involved in the pathophysiology of depressive disorders, and that pharmacological activation of these receptors may shorten the latency of antidepressant medication.

We examined the interaction between the selective serotonin reuptake inhibitor, fluoxetine, and group-II metabotropic glutamate (mGlu) receptors using progenitor cells isolated from cultured cerebellar granule cells, considered as an in vitro model of antidepressant-drug induced neurogenesis. These cells expressed mGlu3 receptors negatively coupled to adenylyl cyclase. A 72-h treatment with either fluoxetine or low concentrations of mGlu2/3 receptor agonists (LY379268 or 2R,4R-APDC) enhanced cell proliferation. The action of fluoxetine was mediated by the activation of 5-HT(1A) receptors. We found a strong synergism between fluoxetine and LY379268 in enhancing cell proliferation and inhibiting cAMP formation. The increased cell proliferation induced by fluoxetine+LY379268 was abrogated by the cAMP analogue, 8-Br-cAMP, as well as by drugs that inhibit the mitogen-activated protein kinase and phosphatidyilinositol-3-kinase pathways. Interestingly, fluoxetine and LY379268 also acted synergistically in promoting neuronal differentiation when progenitor cells were incubated in the presence of serum. These data support the hypothesis that a combination between classical antidepressants and mGlu2/3 receptor agonists may be helpful in the experimental treatment of depression.

Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.

Functional mGlu receptor subtypes are found in stem/progenitor cells, and regulate proliferation, differentiation, and survival of these cells. Activation of mGlu5 receptors supports self-renewal of embryonic stem cells, which are pluripotent cells isolated from the blastocyst capable of generating all the body's cell lineages, including germ cells. Differentiation of embryonic stem cells into embryoid bodies is associated with the induction of mGlu4 receptors, the activation of which drives cell differentiation towards the mesoderm and endoderm lineages. Different mGlu receptor subtypes, mGlu3 and mGlu5 receptors in particular, are found in neural stem cells (stem cells resident in the CNS that give rise to neurons, astrocytes or oligodendrocytes) isolated from the developing brain or from regions of persistent neurogenesis of the adult brain (e.g. the subventricular zone lining the wall of the lateral ventricles). The evidence that activation of mGlu3 and mGlu5 receptors stimulates proliferation of these cells is particularly interesting because of the similarities between neural stem cells and putative cancer stem cells that support the growth of malignant gliomas. A link among mGlu receptors, stem cells and cancer is supported by the finding that mGlu4 receptors are expressed by cerebellar granule cell neuroprogenitors, which are the putative cells of origin of medulloblastomas. The study of mGlu receptors in stem/progenitor cells has potential applications in the optimisation of protocols of cell expansion and differentiation aimed at cell replacement strategies, and may gain new insights into the pathophysiology of neurodevelopmental disorders and brain tumours.

The use of anabolic-androgenic steroids (AASs) in the world of sport has raised a major concern for the serious, sometimes life-threatening, side effects associated with these drugs. Most of the CNS effects are of psychiatric origin, and whether or not AASs are toxic to neurons is yet unknown. We compared the effect of testosterone with that of the AASs, 19-nortestosterone (nandrolone), stanozolol, and gestrinone, on excitotoxic neuronal death induced by N-methyl-d-aspartate (NMDA) in primary cultures of mouse cortical cells. In the most relevant experiments, steroids were applied to the cultures once daily during the 4 days preceding the NMDA pulse. Under these conditions, testosterone amplified excitotoxic neuronal death only at very high concentrations (10 muM), whereas it was protective at concentrations of 10 nM and inactive at intermediate concentrations. Low concentrations of testosterone became neurotoxic in the presence of the aromatase inhibitors, i.e. anastrozole and aminoglutethimide, suggesting that the intrinsic toxicity of testosterone was counterbalanced by its aromatization into 17beta-estradiol. As opposed to testosterone, nortestosterone, stanozolol and gestrinone amplified NMDA toxicity at nanomolar concentrations; their action was insensitive to aromatase inhibitors, but was abrogated by the androgen receptor antagonist, flutamide. None of the AASs were toxic in the absence of NMDA. These data suggest that AASs increase neuronal vulnerability to an excitotoxic insult and may therefore facilitate neuronal death associated with acute or chronic CNS disorders.

Background: Anorexia is frequently found in end-stage renal disease and is a reliable predictor of morbidity and mortality in hemodialysis (HD) patients. The pathogenesis of anorexia is complex and the appetite-modulating hormone ghrelin could be involved. Two forms of circulating ghrelin have been described: acylated ghrelin (Methods: 34 HD patients and 15 healthy controls were studied. The presence of anorexia was assessed by a questionnaire. Serum des-acyl ghrelin was measured in HD patients and in 15 body mass index-, sex- and age-matched controls by ELISA. Energy intake was assessed by a 3-day dietary diary, and fat-free mass (FFM) was evaluated by body impedance analysis. Data have been statistically analyzed and are presented as mean ± SD. Results: 14 patients (41%) were found to be anorexic, and 20 patients (59%) non-anorexic. Energy intake (kcal/day) was significantly lower in anorexic than in non-anorexic patients (1,682 ± 241 vs. 1,972.50 ± 490; p < 0.05). FFM (%) was lower in anorexic than in non-anorexic patients (65.8 ± 4.4 vs. 70.9 ± 8.7; p = 0.05). Plasma des-acyl ghrelin levels (fmol/ml) were significantly higher in HD patients than in controls (214.88 ± 154.24 vs. 128.93 ± 51.07; p < 0.05), and in anorexic HD patients than in non-anorexic (301.7 ± 162.4 vs. 159.1 ± 115.5; p < 0.01). Conclusion: Anorexia is highly prevalent among HD patients and des-acyl ghrelin could be involved in its pathogenesis.

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.

The mGlu5 receptor is the only metabotropic glutamate receptor subtype expressed by mouse embryonic stem (ES) cells grown under non-differentiating conditions [Cappuccio, I., Spinanti, P. Porcellini, A., Desiderati, F., De Vita, T., Storto, M., Capobianco, L., Battaglia, G., Nicoletti, F., Melchiorri, D., 2005. Endogenous activation of mGlu5 metabotropic glutamate receptors supports self-renewal of cultured mouse embryonic stem cells. Neuropharmacology 1, 196–205]. We now report that ES cells differentiating into embryoid bodies (EBs) progressively lose mGlu5 receptors and begin to express mGlu4 receptors at both mRNA and proteinc level. A 4-day treatment of EBs with the mGlu4 receptor agonist, l -2-amino-4-phosphonobutanoate ( l -AP4), increased mRNA levels of the mesoderm marker, brachyury and the endoderm marker, H19, and decreased the expression of the transcript for the primitive ectoderm marker, fibroblast-growth factor-5 (FGF-5). These effects were prevented by the mGlu4 receptor antagonists, α-methylserine-O-phosphate (MSOP). Plating of EBs for 4 days in vitro in ITSFn medium induced cell differentiation towards a neural lineage, as reflected by the expression of the intermediate filament protein, nestin, and the homeobox protein, Dlx-2. Pharmacological activation of mGlu4 receptors during cell incubation in ITSFn medium increased the expression of both neural markers. Similar results were obtained when neural differentiation was induced by exposure of EBs to retinoic acid. These data suggest that differentiation of cultured ES cells is associated with changes in the expression pattern of mGlu receptors and that activation of mGlu4 receptors affects cell differentiation in a context-dependent manner.

We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with mGlu5 receptors in the corpus striatum. In striatal slices, activation of ephrinB2 by a clustered form of its target receptor, EphB1, amplified the mGlu receptor-mediated stimulation of polyphosphoinositide (PI) hydrolysis. This effect was abolished in slices treated with mGlu1 or NMDA receptor antagonists but was not affected by pharmacological blockade of mGlu5 receptors. An interaction among ephrinB2, mGlu1 receptor, and NMDA was supported by the following observations: (1) the NR1 subunit of NMDA receptors coimmunoprecipitated with mGlu1a receptors and ephrinB2 in striatal lysates; (2) clustered EphB1 amplified excitatory amino acid-stimulated PI hydrolysis in cultured granule cells grown under conditions that favored the expression of mGlu1a receptors; and (3) clustered EphB1 amplified the enhancing effect of mGlu receptor agonists on NMDA toxicity in cortical cultures, and its action was sensitive to mGlu1 receptor antagonists. Finally, fluorescence resonance energy transfer and coclustering analysis in human embryonic kidney 293 cells excluded a physical interaction between ephrinB2 and mGlu1a (or mGlu5 receptors). A functional interaction between ephrinB and mGlu1 receptors, which likely involves adaptor or scaffolding proteins, might have an important role in the regulation of developmental plasticity.

An overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors has been implicated in the pathophysiology of oligodendrocyte damage in demyelinating disorders of the CNS. We decided to examine the effect of testosterone on excitotoxic death of oligodendrocytes because a gender difference exists in the incidence and disease course of multiple sclerosis. Short-term pure cultures of oligodendrocytes (4 days in vitro) were exposed to a brief pulse with kainate or AMPA + cyclothiazide for the induction of excitotoxicity. Exposure to testosterone enantate was slightly toxic per se and amplified both AMPA and kainate toxicity. Testosterone treatment induced all gene targets of p53, and amplified the induction of these genes induced by kainate. The effect of testosterone was mediated by the activation of androgen receptors and was resistant to the aromatase inhibitors, dl-aminoglutethimide and 4-hydroxyandrost-4-ene-3,17-dione. Testosterone treatment also potentiated the stimulation of 45Ca2+ influx induced by AMPA + cyclothiazide or kainate without changing the expression of the glutamate receptor (GluR) 1, -2/3, and -4 subunits of AMPA receptors or the GluR6/7 subunits of kainate receptors. We conclude that testosterone amplifies excitotoxic damage of oligodendrocytes acting at an early step of the death cascade triggered by AMPA/kainate receptors.

Following the hypothesis of the “signal-to-noise” ratio (Riedel, 1996) we examined whether changes in the activity of group-I metabotropic glutamate (mGlu) receptors in the hippocampus are associated with a condition that specifically enhances the learning capacity in rats. As a model, we used rats that had been nursed by mothers drinking a solution of corticosterone (13.5 mg of daily intake of corticosterone hemisuccinate) during the lactation period. These rats were prone to learn, as indicated by a better performance in a passive avoidance test. Stimulation of polyphosphoinositide (PI) hydrolysis by the mGlu receptor agonist, 1S,3R-1-amino-cyclopentan-1,3-dicarboxylic acid (1S,3R-ACPD), was attenuated in hippocampal slices prepared from corticosterone-nursed male and female rats at 30 or 60 days of postnatal life, an age at which an increased learning capacity could be demonstrated. This effect was specific because the PI response to carbamylcholine was unchanged. A reduced PI hydrolysis in corticosterone-nursed rats was also observed when group-I mGlu receptors (i.e. mGlu1 and -5 receptors) were selectively activated using 3,5-dihydroxyphenylglycine or 1S,3R-APCD combined with the selective group-II mGlu receptor antagonist, 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionate. Western blot analysis showed a selective reduction in the expression of mGlu1a receptor protein in the hippocampus of corticosterone-nursed rats, whereas expression of mGlu5 and mGlu2/3 receptors was unchanged. The reduction in mGlu-receptor mediated PI hydrolysis in the hippocampus may contribute to the greater learning capacity of corticosterone-nursed rats by reducing the background noise over which a specific signal must be superimposed during learning. This hypothesis was supported by the evidence that mGlu-receptor stimulated PI hydrolysis was amplified in hippocampal slices from rats subjected to a passive avoidance learning paradigm, and that this amplification was greater in slices from corticosterone-nursed rats of both sexes.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.

We used cultured cerebellar granule cells to examine whether native group-III metabotropic glutamate (mGlu) receptors are coupled to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways. Cultured granule cells responded to the group-III mGlu receptor agonist, L-2-amino-4-phosphonobutanoate (l-AP4), with an increased phosphorylation and activity of MAPKs (ERK-1 and -2) and an increased phosphorylation of the PI-3-K target, protein kinase B (PKB/AKT). These effects were attenuated by the group-III antagonists, alpha-methyl-serine-O -phosphate (MSOP) and (R,S )-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l-AP4 also induced the nuclear translocation of beta-catenin, a downstream effector of the PI-3-K pathway. To assess the functional relevance of these mechanisms we examined the ability of l-AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K(+) from 25 to 10 mm. Neuroprotection by l-AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI-3-K pathway. Taken together, these results show for the first time that native group-III mGlu receptors are coupled to MAPK and PI-3-K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.

Previous studies have identified the mammalian homologue of Bv8 (mBv8), a small protein originally isolated from skin secretions of the frog, Bombina variegata. In situ hybridization showed that mBv8 RNA was widely expressed in the rodent CNS, with high levels being detected in layer II of the cerebral cortex, limbic regions, cerebellar Purkinje cells, and dorsal and ventral horns of the spinal cord. A similar pattern of distribution was found by examining the presence of mBv8 protein by immunocytochemistry. Addition of frog Bv8 to cultured cerebellar granule cells reduced the extent of apoptotic death induced by switching the growing medium from 25 to 5 mM K+. Bv8 could also protect cultured cortical neurons against excitotoxic death. Both effects were prevented by PD98059 and LY294002, which inhibit the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways, respectively. In cultured cerebellar granule cells, Bv8 stimulated both the MAPK and the PI-3-K pathways, as revealed by Western blot analysis of phosphorylated p44/p42 MAPKs and phosphorylated Akt, respectively. We conclude that mBv8 acts as an endogenous neurotrophic factor and supports neuronal survival through the activation of the MAPK/PI-3-K pathways.

Melatonin protection against ethanol-induced gastroduodenal injury was investigated in duodenum-ligated rats. Melatonin, injected i.p. 30 min before administration of 1 ml of absolute ethanol, given by gavage, significantly decreased ethanol-induced macroscopic, histological and biochemical changes in the gastroduodenal mucosa. Ethanol-induced lesions were detectable as haemorrhagic streaks. Ethanol administration damaged 36% and 25% of the total gastric and duodenal surface, respectively. Melatonin treatment reduced ethanol-induced gastric and duodenal damage to 14% and 8%, respectively. When indomethacin was given together with ethanol, the gastric damaged area was 44% of the total surface, while the duodenal damaged area was 35%; melatonin administration reduced the damage to only 13% of the total gastric surface and to 12% of total duodenal surface. Both stomach and duodenum of ethanol-treated animals showed polymorphonuclear leukocyte (PMN) infiltration. The number of PMN increased more than 600 and 200 times in stomach and duodenum, respectively, following ethanol administration. Melatonin treatment reduced ethanol-induced PMN infiltration by 38% in the stomach and 20% in the duodenum. In indomethacin-ethanol-treated rats, the number of PMN increased by 875% compared to control group in the stomach and by 264% in duodenum. Melatonin administration reduced the indomethacin-ethanol-induced PMN rise by 57% in the stomach and 40% in the duodenum. Gastroduodenal total glutathione (tGSH) concentration and glutathione reductase (GSSG-Rd) activity were significantly reduced following ethanol and indomethacin-ethanol administration. Melatonin ameliorated both the decrease in tGSH concentration as well as the reduction of GSSG-Rd activity elicited by ethanol both in the stomach and duodenum; melatonin was effective against indomethacin-ethanol-induced damage only in the stomach. Ethanol-induced gastroduodenal damage is believed to be mediated by the generation of free radicals. Recently, a number of in vivo and in vitro experiments have shown melatonin to be an effective antioxidant and free radical scavenger; thus, we conclude that the protection by melatonin against ethanol-induced gastroduodenal injury is due, at least in part, to its radical scavenging activity. British Journal of Pharmacology (1997) 121, 264–270; doi:10.1038/sj.bjp.0701104

Representing the first gene therapy to be approved in the Western world, alipogene tiparvovec (Glybera; Uniqure) has recently been said to have had a “substantial impact from a regulatory perspective” (Nature Rev. Drug Discov. 11, 664; 2012) 1 . The therapy was granted marketing authorization in the European Union for the treatment of lipoprotein lipase deficiency, which results in a clinically heterogeneous condition with a risk of potentially life-threatening pancreatitis 2 , at the end of 2012. The decision followed a positive opinion by the European Medicines Agency (EMA)’s Committee for Medicinal Products for Human Use (CHMP) 3