Nicotinic receptor agonists as neuroprotective/neurotrophic drugs. Progress in molecular mechanisms

In the present work we reviewed recent advances concerning neuroprotective/neurotrophic effects of acute or chronic nicotine exposure, and the signalling pathways mediating these effects, including mechanisms implicated in nicotine addiction and nAChR desensitization. Experimental and clinical data largely indicate long-lasting effects of nicotine and nicotinic agonists that imply a neuroprotective/neurotrophic role of nAChR activation, involving mainly alpha 7 and alpha 4 beta 2 nAChR subtypes, as evidenced using selective nAChR agonists. Compounds interacting with neuronal nAChRs have the potential to be neuroprotective and treatment with nAChR agonists elicits long-lasting neurotrophic effects, e.g. improvement of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Nicotine addiction, which is mediated by interaction with nACh receptors, is believed to involve the modification of signalling cascades that modulate synaptic plasticity and gene expression. Desensitization, in addition to protecting cells from uncontrolled excitation, is recently considered as a form of signal plasticity. nAChR can generate these longe-lasting effects by elaboration of complex intracellular signals that mediate medium to long-term events crucial for neuronal maintenance, survival and regeneration. Although a comprehensive survey of the gene-based molecular mechanisms that underlie nicotine effects has yet not been performed a growing amount of data is beginning to improve our understanding of signalling mechanisms that lead to neurotrophic/neuroprotective responses. Evidence for an involvement of the fibroblast growth factor-2 gene in nAChR mechanisms mediating neuronal survival, trophism and plasticity has been obtained. However, more work is needed to establish the mechanisms involved in the effects of nicotinic receptor subtype activation from cognition-enhancing and neurotrophic effects to smoking behaviour and to determine more precisely the therapeutic objectives in potential nicotinic drug treatments of neurodegenerative diseases.In the present work we reviewed recent advances concerning neuroprotective/neurotrophic effects of acute or chronic nicotine exposure, and the signalling pathways mediating these effects, including mechanisms implicated in nicotine addiction and nAChR desensitization. Experimental and clinical data largely indicate long-lasting effects of nicotine and nicotinic agonists that imply a neuroprotective/neurotrophic role of nAChR activation, involving mainly alpha 7 and alpha 4 beta 2 nAChR subtypes, as evidenced using selective nAChR agonists. Compounds interacting with neuronal nAChRs have the potential to be neuroprotective and treatment with nAChR agonists elicits long-lasting neurotrophic effects, e.g. improvement of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Nicotine addiction, which is mediated by interaction with nACh receptors, is believed to involve the modification of signalling cascades that modulate synaptic plasticity and gene expression. Desensitization, in addition to protecting cells from uncontrolled excitation, is recently considered as a form of signal plasticity. nAChR can generate these longe-lasting effects by elaboration of complex intracellular signals that mediate medium to long-term events crucial for neuronal maintenance, survival and regeneration. Although a comprehensive survey of the gene-based molecular mechanisms that underlie nicotine effects has yet not been performed a growing amount of data is beginning to improve our understanding of signalling mechanisms that lead to neurotrophic/neuroprotective responses. Evidence for an involvement of the fibroblast growth factor-2 gene in nAChR mechanisms mediating neuronal survival, trophism and plasticity has been obtained. However, more work is needed to establish the mechanisms involved in the effects of nicotinic receptor subtype activation from cognition-enhancing and neurotrophic effects to smoking behaviour and to determine more precisely the therapeutic objectives in potential nicotinic drug treatments of neurodegenerative diseases

On the G-protein-coupled receptor heteromers and their allosteric receptor-receptor interactions in the central nervous system: focus on their role in pain modulation

The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via beta-arrestin 2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia

Understanding the role of adenosine A2AR heteroreceptor complexes in neurodegeneration and neuroinflammation

Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extracellularly, and acts as a neuromodulator. It operates as a volume transmission signal through diffusion and flow in the extracellular space to modulate the activity of both glial cells and neurons. The effects of adenosine are mediated via four adenosine receptor subtypes: A1R, A2AR, A2BR, A3R. The A2AR has a wide-spread distribution but it is especially enriched in the ventral and dorsal striatum where it is mainly located in the striato-pallidal GABA neurons at a synaptic and extrasynaptic location. A number of A2AR heteroreceptor complexes exist in the striatum. The existence of A2AR-D2R heteroreceptor complexes with antagonistic A2AR-D2R interactions in the striato-pallidal GABA neurons is well-known with A2AR activation inhibiting Gi/o mediated signaling of D2Rs. A2AR-mGluR5 heteroreceptor complexes were also found in with synergistic receptor-receptor interactions enhancing the inhibition of the D2R protomer signaling. They are located mainly in extrasynaptic regions of the striato-pallidal GABA neurons. Results recently demonstrated the existence of brain A2AR-A2BR heteroreceptor complexes, in which A2BR protomer constitutively inhibited the function of the A2AR protomer. These adenosine A2AR heteroreceptor complexes may modulate alpha-synuclein aggregation and toxicity through postulated bidirectional direct interactions leading to marked increases in A2AR signaling both in nerve cells and microglia. It is of high interest that formation of A2AR-A2ABR heteroreceptor complexes provides a brake on A2AR recognition and signaling opening up a novel strategy for treatment of A2AR mediated neurodegeneration. KEYWORDS: G protein-coupled receptor; Parkinson's diseases; adenosine A2A receptor; adenosine receptor; heteroreceptor complexes; neurodegeneration; neuroinflammation; oligomerizatio

Role of the 5-HT1A receptors in the effect of Galanin(1-15) on Fluoxetine-mediated action in the forced swimming test

Galanin N-terminal fragment (1-15) [GAL(1-15)] modulates the antidepressant effects induced by the 5-HT1A receptor (5-HT1AR) agonist in the forced swimming test (FST) and the binding characteristics and mRNA levels of 5-HT1AR in the dorsal hippocampus and dorsal raphe (DR). Recently, we observed that GAL(1-15) enhanced the antidepressant-like effects induced by Fluoxetine (FLX) in the FST. In this work, we have studied whether the effects of GAL(1–15) on FLX action were mediated via 5-HT1AR, analyzing the effect of the 5-HT1AR antagonist WAY100635 in this effect and if the binding characteristics and mRNA levels of 5-HT1AR in the DR and dorsal hippocampus are modified by GAL(1-15)+FLX. Groups of rats (n=6-8) received three injections of sc FLX(10mg/kg) and 15 minutes before the FST a single icv injection of GAL(1-15) (1nmol) and 5HT1AR antagonist WAY100635(6nmol) icv alone or in combination. We also analyzed the effects of GAL(1-15)+FLX in the binding characteristics of the 5-HT1AR agonist [H3]-8-OH-DPAT and 5-HT1A mRNA levels in the DR, CA1 and Dentate Gyrus (DG). WAY100635 significantly blocked the reduction in immobility time (p<0.05), and the increase in swimming time (p<0.01) induced by GAL(1-15)+FLX in the FST. GAL(1-15)+FLX produced a significant increase in the 5HT1AR mRNA levels in CA1 (p<0.05) and DG (p<0.05). This effect was not observed in the DR. Moreover, GAL(1-15)+FLX produced a significant decrease in the Kd value (p<0.01) and in the Bmax value (p<0.05) of [3H]-8-OH-DPAT in the DG. These effects were not observed in the CA1 or in the DR. These results indicate that 5HT1AR participates in the GAL(1-15)/FLX interactions in the FST and the mechanism underlying affected the binding characteristics and the mRNA levels of 5-HT1AR specifically in the dorsal hippocampus. The heteroreceptor 5-HT1AR-GALR1-GALR2 located in the dorsal hippocampus may be the target for GAL(1-15). This work was supported by SAF2016-79008-P; PSI2013-44901-P.SAF2016-79008-P; PSI2013-44901-P. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A Role For Galanin N-Terminal Fragment (1-15) In Anxiety And Depression in rats

Ponencia InvitadaGalanin (GAL) is involved in several functions including mood regulation. The GAL N-terminal fragment (1-15) [GAL(1-15)] also participates at central level and a differential role of GAL(1-15) compared with GAL has been proposed. In this work we have analysed if GAL(1-15) contributes to depression- and anxiety -related behaviours using the forced swimming test, tail suspension test, open field and light/dark test. We tested the involvement of the GAL receptor 2 (GALR2) in GAL(1-15) effects with the GAL receptor antagonist M871 and with an in vivo model of siRNA GALR2 knockdown rats. The proximity of GALR1 and GALR1 was also examined with the proximity ligation assay (PLA). GAL(1-15) induced strong depression-like and anxiogenic-like effects in all the tests. The involvement of the GALR2 was demonstrated with M871 and with the siRNA GALR2 knockdown rats. The PLA indicated the existence of GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. Our results indicate that GAL(1-15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GALR1-GALR2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety. This study was supported by Junta de Andalucía CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Antidepressant-like effects induced by galanin 2/neuropeptide Y Y1 heterodimers in the dentate gyrus of the hippocampus

Previously, we have described the Galanin (GAL) and Neuropeptide Y Y1 (NPYY1) interactions through GAL receptor 2 and NPYY1 receptor 1 (GALR2/NPYY1R) heterodimers in the Dentate Gyrus (DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA) (1,2). The current work is to evaluate GALR2 and NPYY1R interactions in relation to depression-like behaviour and c-Fos expression in the Hippocampal DG. Rats (n=6-8) were forced to swim for a 15-min period (pre-test) and 24 h later were subjected to a 5-min swimming session (test) 15 min after the administration alone or in combination of GAL, the NPYY1R agonist [Leu31,Pro34]NPY and the GALR2 antagonist M871. The total duration of immobility, swimming, and climbing periods were scored during the test. For c-Fos immunohistochemistry, experimental groups of rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused with 4 % Paraformaldehyde 90 min after icv injections. Then, brains were coronally sliced and immunostained. As primary antibodies, an antibody against c-Fos protein (1:5000, sc- 52, Santa Cruz Biotechnology, CA, USA), revealed with 3,3´-Diaminobenzidine (DAB) plus nickel, was used as an indirect marker of neural activity. The antibody to Calbindin-D28 k (1:1000, Santa Cruz Biotecnology, CA, USA), revealed with DAB, was used to outline the granular region since it marks mainly hippocampal granule cells. Sections were analyzed using the optical fractionator stereological method. We observed that icv injection of GAL and NPYY1R agonist significantly enhanced the decrease in the immobility (p<0,001) and the increase in the swimming behaviour (p<0,001) compared with the NPYY1R agonist alone. Moreover, a significant enhancement of the decrease in climbing behavior (p<0.05) was also observed. Furthermore, GALR2 is involved in this GALR/NPYY1R interaction, since the presence of the GALR2 antagonist M871 counteracted the enhancement of the decrease in immobility (p<0.01) and in climbing behavior (p<0.05) as well as the increase in swimming time (p<0.001) induced by the coadministration of GAL and NPYY1R agonist in the FST. Specific cells populations within DG subregions may be involved in this behavioural effect since the coadministration of GAL and NPYY1 agonist enhances the NPYY1R-mediated reduction (p<0.05) in the number of c-Fos immunoreactive nuclei in the polymorphic region. In this region, the GABA interneurons could be involved in the interaction since c-Fos IR colocalized with a GABAergic marker (GAD65/67) after NPYY1R agonist injection. Moreover, within the granular cells layer, GAL and NPYY1 agonist coadministration significantly increased c-Fos IR expression in the entire granular cell layer compared with GAL (p<0.05) and [Leu31,Pro34]NPY (p<0.01) alone. Again, the co-treatment with the GALR2 antagonist M871 completely reversed the GAL contribution to the responses in both regions, the polymorphic and the granular layer of the DG, demonstrating the involvement of GALR2 in the GAL actions. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative mechanism in DG in depression-related behavior and may give the basis for the development of drugs targeting GALR2/NPYY1R heteroreceptor complexes in the DG of the hippocampus for the treatment of depression. Study supported by Junta de Andalucia CVI6476 and Proyecto Puente-Universidad de Málaga. 1. Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the dentate gyrus are related with antidepressant-like effects. Narváez M, Borroto-Escuela DO, Millón C, Gago B, Flores-Burgess A, Santín L, Fuxe K, Narváez JA, Díaz-Cabiale Z. Brain Struct Funct 2016 Nov. 221(8):4129-4139. 2. Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the amygdala lead to increased anxiolytic actions. Narváez M, Millón C, Borroto-Escuela D, Flores-Burgess A, Santín L, Parrado C, Gago B, Puigcerver A, Fuxe K, Narváez JA, Díaz-Cabiale Z. Brain Struct Funct. 2015 Jul;220(4):2289-301.Study supported by Junta de Andalucia CVI6476 and Proyecto Puente. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

GALR2/NPYY1R heterodimers interact at receptor level in the dentate gyrus of the hippocampus in rats

Previously, we have described Galanin(GAL) and Neuropeptide Y Y1(NPYY1) interactions at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers in the amygdala. The aim of this work was to study GAL/NPYY1R interactions in the Dentate Gyrus (DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay (PLA). Rats (n=6) were sacrificed 15 minutes or 5 hours after icv injections of GAL (3nmol) and DG sections were incubated with NPYY1R agonist [I125]-[Leu31,Pro34]PYY (25 pM) or NPYY1R-33PdATP specific probe, for autoradiography and in situ hybridization respectively. Autoradiograms were analyzed using NIH image analysis system and Student’s unpaired t-test was used. For PLA, DG sections were incubated with anti-GALR2 Rabbit (1:100) and anti-NPYY1R Goat (1:200). PLA signals were detected with PLA PLUS or MINUS probes for rabbit or goat/mouse antibodies. PLA signals were visualized by using a confocal microscope Leica TCS-SL confocal microscope (Leica). We observed that GAL significant increased the NPYY1R agonist [I125]-[Leu31,Pro34]PYY binding in the DG by 20% (p<0,05) and the NPYY1R mRNA expression in the granular layer of DG by 31% (p<0,001). Moreover, PLA-positive red clusters were found specifically in the polymorphic layer and subgranular zone of the DG. No PLA clusters were observed neither in the molecular layer of the DG nor in the corpus callosum, an area that seems to lack of GALR2 receptor. These results demonstrate a novel mechanism of interaction between GAL and NPY1R in the DG at receptor level, probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. Study supported by Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Study supported by Junta de Andalucia CVI6476

Galanin interacts with Neuropeptide Y Y1 receptor in the dentate gyrus of the hippocampus through GALR2/NPYY1R heterodimers

We have shown Galanin(GAL) and Neuropeptide Y Y1(NPYY1) interactions at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers in the amygdala. The aim of this work was to analyze GAL/NPYY1R interactions in the Dentate Gyrus(DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA). Rats(n=6) were sacrificed 15 minutes or 5 hours after icv injections of GAL(3nmol) and DG sections were incubated with NPYY1R agonist [I125]-[Leu31,Pro34]PYY(25 pM) or NPYY1R-33PdATP specific probe, for autoradiography and in situ hybridization respectively. Autoradiograms were analyzed using NIH image analysis system and Student’s unpaired t-test was used. For PLA, DG sections were incubated with anti-GALR2 Rabbit(1:100) and anti-NPYY1R Goat(1:200). PLA signals were detected with PLA PLUS or MINUS probes for rabbit or goat/mouse antibodies. PLA signals were visualized by using a confocal microscope Leica TCS-SL confocal microscope(Leica). We observed that GAL significant increased the NPYY1R agonist [I125]-[Leu31,Pro34]PYY binding in the DG by 20% (p<0,05) and the NPYY1R mRNA expression in the granular layer of DG by 31% (p<0,001). Moreover, PLA-positive red clusters were found specifically in the polymorphic layer and subgranular zone of the DG. No PLA clusters were observed neither in the molecular layer of the DG nor in the corpus callosum, an area that seems to lack of GALR2 receptor. These results demonstrate a novel mechanism of interaction between GAL and NPY1R in the DG at receptor level, probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. Study supported by Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Galanin receptor 2 modifies neuropeptide Y Y1 receptor internalization and β-Arrestin recruitment

We have recently described a Galanin receptor 2(GALR2) and Neuropeptide Y Y1 receptor(NPYY1R) interaction at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers. The aim of this work was to study if GALR2 and NPYY1R costimulation modified NPYY1R internalization and β-Arrestin recruitment after in HEK293T cells. HEK293T cells were transfected with NPYY1REGFPor β-Arrestin2GFP2 cloned with standard molecular biology techniques employing PCR and fragment replacement strategies. NPYY1REGFP/GALR2 and NPYY1R/GALR2 with β- Arrestin2GFP2 HEK293T coexpressing cells were incubated with NPY 1μM and/or GAL1μM, at different times. Antagonist studies were performed 15 min prior to the addition of agonist with NPYY1R antagonist BIBP3226 10μM or GALR2 antagonist M871 10 μM. Timed-interval images of NPYY1REGFP or β-Arrestin2GFP2 endosomes in different cell groups were acquired using a confocal microscope following agonist addition. Percentage of internalization was determined by Leica software analysis of total membrane fluorescence compared to total internal compartment fluorescence at the various time points. We observed that addition of NPY induced a rapid decrease in the cell surface expression of NPYY1REGFP and a redistribution of β-Arrestin2GFP2. In fact, we observed a maximum of internalization of 80% three minutes after the NPY stimulation. However, combined treatment with GAL and NPY induced a delay in the internalization of NPYY1REGFP, with a maximum of internalization thirty minutes after the co-stimulation. Moreover, a delay in the β-Arrestin2GFP2 redistribution was observed. The specific GALR2 antagonist M871 abolished these delays in internalization of NPYY1REGFP and β-Arrestin2GFP2 redistribution, suggesting that this effect was mediated through the coactivation of GALR2 and NPYY1R. These results demonstrate that costimulation with GAL and NPY delays the internalization of  NPYY1REGFP by decreasing recruitment of β-Arrestin2GFP2 and probably could change intracellular signaling. This study was supported by Junta de Andalucia CVI6476.Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Galanin (1-15) enhances the behavioral effects of fluoxetine in the forced swimming test: a new therapeutic strategy against depression

The selective serotonergic (5- HT) reuptake inhibitors, including Fluoxetine (FLX), are the most commonly used for treatment of major depression. However, the understanding of the mechanism of action of FLX beyond its effect of elevating 5-HT is limited. The interaction between 5-HT system and neuropeptides signaling could be a key aspect. The neuropeptide Galanin(1-15) [GAL(1-15)], induced a strong depression-like and anxiogenic-like effects in the forced swimming test (FST), the tail suspension test, the open field and the light/dark test. The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and in the dorsal raphe were involved in these effects. We have analyzed the effect of GAL(1–15) on FLX-mediated responses in the FST. We tested the involvement of GALR in the GAL(1–15) effect with the selective GALR2 antagonist M871 and using siRNA GALR2 or GALR1 knockdown rats. Groups of rats received three injections of sc FLX(2.5mg/Kg) or FLX(10mg/Kg) and a single icv injection of a threshold dose of GAL(1-15)(1nmol) 15 minutes before the FST. In a second set of experiments, we determined the involvement of GALR1 and GALR2 in the effect of GAL(1-15) on FLX-mediated action. Groups of rats received three injections of sc FLX(10mg/kg), a single icv injection of GAL(1-15) (1nmol) and the GALR2 antagonist M871 (3nmol) icv alone or in combination. Also, in siRNA GALR1 or GALR2 knockdown rats we coadministered FLX(10mg/Kg) and GAL(1-15)(1nmol). The coadministration of sc FLX(2.5mg/Kg) and icv injection of GAL(1-15)(1nmol) induced antidepressant-like effects with a significant decrease in the immobility (p<0.05). Moreover, an increase in the swimming time (p <0.05) was also observed. The strong enhancement by GAL(1-15) of the antidepressant-like effects mediated by FLX was validated using the effective dose of FLX 10mg/kg. Icv GAL(1-15) significantly decreased the immobility time induced by the effective dose of FLX(10mg/kg) by 50% in the FST (p<0.05). Moreover, an increase of the swimming time by about 40% versus FLX(10mg/kg) group was also observed (p<0.01). The GALR2 antagonist M871 3nmol significantly blocked the GAL(1–15)-induced reduction of the immobility time (p<0.05), and increase in the swimming time (p<0.01) found after coadministration of icv injection of GAL(1-15) and sc FLX(10mg/kg) in the FST. The coadministration of sc FLX(10mg/kg) and icv injection of GAL(1-15) in siRNA GALR1 or GALR2 knockdown animals did not produce a further reduction of the immobility time and a further increase in the swimming time compared to FLX alone. In the current study we describe for the first time that GAL(1-15) enhances the antidepressant-like effects induced by FLX in the FST. Indications were also obtained for the involvement of a GALR1/GALR2 heteroreceptor complex in the GAL (1-15)-mediated actions based on the use of the specific GALR2 antagonist M871 and icv injections of GALR1 siRNA or GALR2 siRNA producing a reduction of GALR1 or GALR2, respectively. The results open up the possibility to use GAL(1-15) as for a combination therapy with FLX as a novel strategy for treatment of depression. This work was supported by grants awarded by Spanish Ministry of Economy (SAF2016-79008-P),Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech