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    • 专利摘要:
    Combination comprising galanin and [leu31, pro34] npy; uses and methods. The invention relates to a combination comprising galanin and [leu31, pro34] npy. Additionally, the invention relates to uses of said combination and medicaments comprising it, directed to therapeutic use against a disease of the nervous system, particularly depression. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2589165A1
    申请号:ES201500842
    申请日:2015-11-20
    公开日:2016-11-10
    发明作者:Manuel NARVÁEZ PELÁEZ；Carmelo MILLÓN PEÑUELA；Belén GAGO CALDERÓN；Antonio FLORES BURGESS；Luis Javier SANTÍN NÚÑEZ；José Angel NARVÁEZ BUENO；Zaida DÍAZ CABIALE
    申请人:Universidad de Malaga；
    IPC主号:
    专利说明:

    lOa said diseases of the nervous system, particularly depression. COMBINATION THAT INCLUDES GALANINA AND [Leu31, Pro34lNPY; USES AND METHODS
    INVENTION SECTOR
    The invention generally relates to combinations comprising galanin receptor 2 agonists (GALR2), or derivatives or functional fragments of said agonists, and agonists of Y1 receptor of neuropeptide Y (NPYY1 R), or derivatives or functional fragments thereof ; and its therapeutic use against a disease of the nervous system, particularly depression, or in the preparation of drugs or pharmaceuticals for therapy against
    BACKGROUND OF THE INVENTION
    Hippocampal formation is critically involved in different brain functions such as spatial, contextual and episodic memory (Burgess et al. 2002) and in the stress response (McEwen and Magarinos 1997); but also in neuropsychiatric conditions such as depression (Campbell and Macqueen 2004). In addition, different neurotransmitters and substances related to neurotransmitters that modulate depressive actions in the short-term hippocampus have recently been described (Hiroaki-Sato et al. 2014; BeUio et al. 2014). Within the hippocampal subregions, the dentate gyrus (DG) is a key region in the regulation of these functions and is formed by the molecular and granular cell layers and the pOlimorphic region (or hilus) (Amaral et al. 2007). Although the DG receives projections primarily of the entorinal cortex, the polymorphic layer of the DG receives noradrenergic projections of the locus coeruleus (Blackstad et al. 1967) and serotonergic rafe nuclei containing cells positive for calbindin-, calretinin-, somatostatin-, and Neuropeptide Y (NPY) (Calena-Dell'Osso on al. 2013; Sperk on al. 2007).
    The novel strategies in psychiatric disorders are focused on the DG as a potential brain target (Hagihara et al. 2013). Neuropeptides and their receptors have received special attention in this area, especially NPY, which is a therapeutic target in emotional disorders, including depressive behaviors (Holmes et al. 2003; Kormos and Gaszner 2013).
    Neuropetid Y (NPY) is widely distributed within the nervous system, with high concentrations in different cortical and limbic regions. In the hippocampus, the largest population of NPY immunoreactive cells are found in the polymorphic layer of the DG, all of them being GABAergic neurons (Kohler et al. 1986; Sperk et al. 2007). Y1 receptors
    2
    and Y2 of the NPY neuropeptide (NPYY1 R and NPYY2R, respectively) are also expressed in the hippocampus, although NPYY1 R is abundantly distributed throughout the DG (Dumont et al. 1996; Paredes et al. 2003). In addition, the increase in NPY and in the expression of NPYY1 R in the DG has been directly related to the antidepressant effect of NPY in rats, suggesting a role of the NPY-NPVY1R system in the patho-physiology of depression (Jimenez-Vasquez et al. 2007; Bjornebekk et al. 2010; Catena-Dell'Osso et al. 2013). Thus, intracerebroventricular (icv) administration of NPY or [LeuJ Pro34] NPY (NPVY1R agonist) induces an antidepressant effect on the forced swimming test (FST), which is blocked by co-administration of an NPYY1R antagonist (Redrobe et al. 2002; Stogner and Holmes 2000). Thus, the potentiation of NPY transmission through the NPYY1 receptor is one of the mechanisms underlying antidepressants and electroconvulsive therapies (Caberlotto et al. 1999; Madsen et al. 2000). On the other hand, genetic and environmental models of depression in rats show a decreased level of NPY and NPYY R levels in the DG (Malhe el al. 1998: Jimenez-Vasquez el al. 2007).
    Galanin (GAL) is also a neuropeptide widely distributed in the central nervous system (Jacobowitz et al. 2004). Three LAG receptors (GALRs) are involved in behaviors related to depression through modulation of the neuroendocrine and monoaminergic systems, with a different action depending on the subtype of GALR (Fuxe et al. 2012; Wrenn and Holmes 2006). Thus, the icv infusion of GAL or the stimulation of GALR1 and GALR3 produces a depression-type phenotype, while the activation of GALR2, which is expressed mainly in the DG (O'Donnell et al. 1999), induces an anlidepressive behavior in the FST (Barr el al. 2006; Kuleeva el al. 2007; Kuteeva et al. 2008; Borroto-Escuela et al. 2014; Millon et al. 2014).
    It has been shown that GAL interacts with NPYY1R in different regions including the nuclei of the tonsil, a key region in behaviors related to stress in rats (Diaz-Cabiale et aL, 2011; Narvaez et aL, 2014). It has been described as GAL, through GALR2, it enhances the anxiolytic actions mediated by NPYY1 R. This facilitating interaction GALR2 / NPYY1 R takes place in the tonsil at the cellular and receptor level involving the formation of GALR2 / NPYY1R heteroreceptor complexes (Narvaez al. 2014). In addition, studies of intracellular signals indicated that GALR2 in the GALR2 / NPYY1 R heteroreceptor complex changes the coupling of Gq to Gi / o in both receptors, which would both operate through Gi / o, producing an additive inhibitory effect on effects through adenyl cyclase (Narvaez et aL, 2014).
    BRIEF DESCRIPTION OF THE INVENTION
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    The interactions between GALR2 and NPYY1 R in the DG have been evaluated by quantitative autoradiography and in situ hybridization. In addition, the presence of GALR2 / NPYY1 R heteroreceptor complexes has been analyzed in DG subregions by in situ proximity linkage assay (Borroto-Escuela et al. 2013). A GALR2 / NPYY1 R interaction has been found at the level of depressive behavior in the forced swimming test in rats. In addition, a specific cell activation using immunohistochemistry has been found in
    DG subregions induced by the GALR2 / NPYY1R interaction.
    Galanin (GAL) and the NPYY1 R agonists participate in the regulation of behavior and both peptides interact in different functions at the central level. The interaction between the GAL receptor 2 (GALR2) and the Y1 receptor of the neuropeptide Y (NPYY1 R) in the dentate gyrus (OG) of the hippocampus in relation to the behavior related to depression has been analyzed. Using autoradiography, in situ hybridization, and in situ proximity bonding, the interaction between GALR2 and NPYY1 R in the DG has probably been shown to involve the formation of GALR2 / NPYY1 R heteroreceptor complexes. These complexes have been specifically observed in polymorphic subregions and DG subgranular, where both receptors colocalize. In addition, this GALR2 / NPYV1 R interaction has been related to an increase in the antidepressant effect mediated by NPYY1R in the forced swimming test. Specific cell populations within the DG subregions may be involved in this effect at the behavioral level since co-activation between GALR2 and NPYV1R enhances the c-Fos reduction mediated by NPYY1R in the polymorphic region. These results indicate that the interaction between GALR2 / NPYY1 R may indicate a novel mechanism in the DG in depression-related behavior and may lay the groundwork for the development of drugs that act on the GALR21NPYY1 R heteroreceptor complexes in the hippocampus DG for the treatment of depression.
    Accordingly, the present invention generally relates to a combination comprising gala nina and [Leu31, Pro34] NPY, galanin receptor 2 agonists (GALR2), and Y1 receptor of neuropeptide Y (NPYY1 R) respectively.
    The invention also relates to the use of the aforementioned combination in therapy (including prevention or treatment) against a disease of the nervous system, or in the preparation of drugs or pharmaceuticals for therapies against diseases of the nervous system. In an embodiment of the invention, said nervous system disease is depression.
    Additionally, the invention relates to the aforementioned medicaments or pharmaceutical products, for example in the form of a pharmaceutical composition comprising a pharmaceutically acceptable card, diluent or excipient; and its use in therapy against a disease of the nervous system. In an embodiment of the invention, said nervous system disease is depression.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1. Effects of galanin after icv administration on the ligand of
    125 31 34
    [I] Leu, Pro PYY, agonist of the NPYY1 receptor (NPYY1 R), with the NPYY1 R receptor in the dentate gyrus of the hippocampus. Graph showing the specific values of the linked
    125 31 34
    [I] Leu, Pro PYY (25 pM), expressed as mean ± SEM (Standard Error of the Mean, standard error of the mean). Non-specific binding (in the presence of NPY 1 IJM) was digitally subtracted from the measurements. * P <0.05 versus control group according to Student's t-test (n = 6 per group). Abbreviations: aCSF = cerebrospinal fluid, which indicates the baseline levels of
    125 31 34
    [I] -Leu Pro PYY. GAL = galanina.
    Figure 2. Effect of the icv administration of galanin on the mRNA expression of the NPYY1 receptor (NPYY1 R) in the dentate gyrus of the hippocampus. Graph showing the values, expressed as mean ± SEM, of the NPYY1 R mRNA optical density (concentration
    probe 2.4 pmoll lJl, marked with P-dATP). u * p <0.001 versus control group according to the
    Student's t-test (n = 6 per group). Abbreviations: aCSF = cerebrospinal fluid, which indicates the basal levels of optical density of the mRNA of NPYY1 R. GAL = galanina.
    Figure 3. Detection of heteroreceptor complexes between galanin receptor 2 (GALR2) and NPY receptor Y1 (NPYY1 R) by proximity linkage (PLA). White circles indicate positive PLA regions mainly in the polymorphic layer, but also in the subgranular zone of the dentate gyrus (Bregma -3.1 mm). Black circles indicate a negative PLA signal in the molecular layer of the dentate gyrus and in the corpus callosum.
    31 34
    Figure 4. Effect of galanin and [Leu-Pro] NPY, agonist of Y1 receptor of neuropeptide Y (NPYY1R), on behavior related to depression in the forced swimming test. Galanin potentiation of the action related to depressive behavior mediated by NPYV1 R is blocked by M871, a galanin receptor 2 antagonist (GALR2). The graphs show the data, expressed as mean ±
    SEM, obtained in the forced swimming test: (a) immobility time, (b) time dedicated to swimming, (e) time dedicated to climbing. ANOVA of a factor followed by the Newman-Keuls multiple comparisons test (n = 6-8 animals in each group). In (a): '*' * '* P <0.001 versus the rest of the groups; = F # #P <0.001 versus aCSF and GAL + Y1 3 nmol; aP <0.001 versus aCSF; '*' * P <0.01 versus GAL + Y1 3 nmol. In (b): # ## P <0.001 versus aCSF, GAL 3 nmol, GAL + Y1 3 nmol; '*' * '* P <0.001 versus the rest of the groups. In (e): '*' * '* P <0.001 versus aCSF; '* P <0.05 versus Y1 3 nmol and GAL + Y1 + M871 3 nmol. Abbreviations: GAL = galanina; Y1 =
    31 34 31
    [Leu -Pro] NPY, NPYY1 receptor agonist; GAL + Y1 = co-administration of GAL and [Leu
    34 31 34
    Pro) NPY; GAL + Y1 + M871 = co-administration of GAL, [Leu -Pro) NPY and M871, antagonist of GALR2.
    Figure 5. Effects of galanin and the NPYY1 receptor agonist (NPYY1 R), alone or in combination with M871, galanin receptor 2 antagonist (GALR2), on the expression of cFas in the polymorphic and granular layers of the dentate gyrus. (a-b) Quantification of the total number of c-Fos IR nuclei (that is, c-Fos detected by immunohistochemistry) within the polymorphic and granular layers. The data, expressed as mean ± SEM, show the differences between the groups after intracerebroventricular injection of aCSF, GAL, ", ..
    [Leu -Pro] NPY, or the co-administration of both peptides and M871. (a) The co-administration of ", ..
    GAL Y [Leu -Pro] NPY decreases c-Fos expression compared to both peptides alone ", ..
    and the aCSF group. In addition the effect of the co-administration of GAL and [Leu -Pro) NPY is blocked with M871. '* P <0.05 versus aCSF; '* P <O, 05 versus CSF, Y1 and GAL + Y1 + M871; '' 'P <O, 001 versus Y1 and GAL + Y1 + M871; '<' <'<P <O, 001 versus aCSF and GAL. (b) The ", ..
    Co-administration of GAL and [Leu -Pro] NPY increases e-Fas expression compared to both peptides alone and the aCSF group. In addition the effect of the co-administration of GAL and ", ..
    [Leu -Pro] NPY crashes with M871. '* P <O, 05 versus GAL + Y1 3 nmol; '*' * P <O, 01 versus GAL, Y1 and GAL + Y1 + M871; '*' * '* P <O, 001 versus aCSF; , *, * P <O, 01 versus Y1 and GAL + Y1 + M871. ANOVA of a factor followed by the Newman-Keuls multiple comparisons test (n = 4 in each ", ..
    group). Abbreviations: aCSF = cerebrospinal fluid; GAL = galanina; Y1 = [Leu -Pro] NPY, ", ..
    NPYY1 receptor agonist; GAL + Y1 = co-administration of GAL and [Leu -Pro] NPY; ", ..
    GAL + Y1 + M871 = co-administration of GAL, [Leu -Pro] NPY and the GALR2 antagonist M871.
    EMBODIMENTS OF THE INVENTION
    Throughout this application several publications are referenced. The contents of all these publications and those referenced within them are incorporated by reference in their entirety in this application in order to describe in a more complete manner the state of the art to which this invention belongs. The terminology used here is for the purpose of describing specific embodiments only and is not intended to be limiting.
    Materials and methods
    Animals
    Male Sprague-Dawley rats from CRIFFA (Barcelona; 200-250 gr) were used that had free access to food and water. They were kept under the standard conditions of 12-hour light / dark cycles, at controlled temperature (22 ± 2 OC) and relative humidity (55-60%). The experimental procedures were approved by the Ethics Committee of the University of Malaga, in accordance with the European Directive (86 / 609fEEC) and Spanish (Royal Decree 53/2013).
    Detailed descriptions of intracerebral cannulas and administration of the peptides to animals are provided as complementary information.
    Quantitative Autoradiography
    The procedure and doses of LAG are based on previous work (Diaz-Cabiale et al. 2011; Narvaez et al. 2014). Fifteen minutes after the icv injections of aCSF or GAL (3 nmol) (n = 6 per group) the rats were sacrificed by decapitation and their brains were quickly removed from the bone and frozen at -40 oC in isopentane. Coronal sections (14 IJm thick) were cut in a cryostat (HM550, Microm International) at bregma levels according to the Paxinos and Watson atlas (Paxinos G. 1986) (Bregma hippocampal DG levels of -2.12 mm to -4.52 mm) and mounted on gelatinized slides. Sections were pre-incubated for one hour at room temperature in a Krebs-Ringer phosphate buffer (KRP) at pH 7.4 and then incubated for 2 h in KRP buffer with 0.1% BSA, 0.05% bacitracin, 25 pM NPYY1R agonist ['' 'I) - [Leu' ', Pro' ') PYY (Perkin-Elmer, USA) (Dumont et al. 1996). Non-specific binding was defined as binding in the presence of NPY 1 IJM. After incubation, the sections were washed four times (2 min each time) in cold KRP buffer, immersed in deionized water to remove salts, and quickly dried in a cold air stream. Sections were placed on X-ray cassettes and exposed to Hyperfilms films, (Kodak Biomax MR film, Kodak, Rochester, NY) for 6 days together with 1251 microscales (Amersham International) as reference standards.
     09-20-201 6
    Autoradiograms were analyzed in the hippocampus DG (0.15 mm2 square) as previously described (Parrado et al. 2007).
    In situ hybridization
    In situ hybridization was carried out following previously described protocols (Bjornebekk et al. 2010).
    Briefly, 5 hours after the icv injection of aCSF or GAL (3 nmol) (n = 6 per group) the coronal sections of the brain (14 μm) were cut in cryostat and mounted on slides. The hybridization cocktail contained 50% formamide, 4x SSC (1x SSC, 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 1x Denhardt solution, 1% Sarcosyl, 0.02 M Na3P04, pH 7, 0, 10% dextran sulfate, 0.06 MY dithiothreitol, 0.1 mg / ml salmon sperm DNA. Specific oligonucleotide probes for NPYY1R were used. The probes were labeled with 33P-dATP (PerkinElmer, Boston, MA). Hybridization was performed for 18 hours in a humid chamber at 42DC. After hybridization, the sections were washed 4 times 20 min each time in 1 x SSC at 60DC. Finally, the sections were washed for 10 seconds in autoclaved water, dehydrated in alcohol and air dried. The sections were then exposed in one mm (Kodak Biomax MR film, Kodak, Rochester, NY) for 7 days before development. The films were scanned and the values in optical density were quantified in the different regions marked in the DG.
    In situ proximity bonding test
    The in situ proximity bonding (PLA) test was performed as previously described (Borroto-Escuela et al. 2013; Narvaez et al. 2014). Untreated animals (n = 3) were perfused with 4% paraformaldehyde, brains and sections at the DG level were obtained. Sections were incubated with blocking solutions (5% goat serum) and permeabilizers (0.3% Triton X100 in PBS) for 60 minutes each time. Primary antibodies directed against GALR2 and NPYY1R obtained by different hosts (rabbit, Alomone Lab, 1: 100; and goat, Santa Cruz Biotechnology INC, CA, 1: 200; respectively) were incubated for 24 hours at 4DC. The PLA signal was detected following the instructions of the supplier (Duolink PLA in situ detection kit; Olink, Sweden) with PLA with or without probes for rabbit or goat antibodies. The sections were mounted on slides with a fluorescence mounting medium (Oako) containing 4 ', 6-diamidino-2-phenylindole (DAPI) (1: 200), which marks the nuclei with blue color. For the control experiments, only one primary antibody was used. PLA signals were visualized using a TCS-SL confocal microscope (Leica).
     09-20-201 6
    Forced swimming test
    Depression-like behavior was analyzed by FST, originally proposed as a model of stress-induced depression (Porsolt et al. 1977). Importantly, the immobility response in FST can be prevented by different types of antidepressant treatments, including tricyclic antidepressants, monoamine oxidase inhibitors, SSRls, and NA reuptake inhibitors (Petit-Demouliere et al. 2005; Kuteeva al. 2008).
    The animals adapted to the manipulation and were taken to the experiment room (8090 lux) for at least one hour to get used before the icv administration of the peptides. The rats were individually placed in cylindrical tanks with a height of 50 cm and a width of 20 cm, containing water (25 ± 0.5 ° C) with a height of 30 cm. The animals were forced to swim for a period of 15 minutes (pre-test) and 24 hours later they were subjected to a 5-minute session (test) 15 minutes after the administration of GAL, [Leu31, Pro34) NPY (agonist of NPYY1R) and M871 (GALR2 antagonist), alone or in combination (n = 6-8 animals in each group). The effective doses for GAL, [Leu3 Pro34) NPY and M871 were selected based on dose-response curves performed previously (DiazCabiale on al. 2011; Millon on al. 2014; Narvaez on al. 2014). The lolal hare dedicated to flolar (immobility), swimming, and climbing was recorded during the 5-minute test. Rats are considered to be immobile when they float without difficulty, performing only those movements necessary to keep their heads above the water. The rats were considered to be in a swimming situation when they actively swam around the cylinder, while the rats were considered to be in a climbing situation when performing a vigorous movement on the cylinder walls. After the swimming sessions, the rats were removed from the tank, carefully dried in heated cages and returned to the original cages. Behavioral experiments were always performed between 09:00 and 14:00 hours.
    C-Fos immunohistochemistry
    The animals were divided into five experimental groups: (1) aCSF: control group; (2) 3 nmol GAL: group treated with 3 nmol galanin; (3) Y1 2.5nmol: group that received [Leu3 Pro34] NPY 2.5 nmol; (4) GAL 3 nmol + Y1 2.5nmol: group administered with both substances; (5) GAL + Y1 + M871 3 nmol: group injected with GAL, [Leu31, Pro34] NPY and M871 3 nmol (n = 4 in each group). All doses are based on previously published protocols (Parrado et al. 2007; Kuteeva et al. 2008; Diaz-Cabiale et al. 2011).
     09-20-201 6
    Rats anesthetized with sodium pentobarbital (Mebumal; 100 mg / kg, Lp.) Were perfused with 4% paraformaldehyde (pesolvolumen, Sigma) 90 minutes after icv injections and brains were cut into coronal sections and immunohistochemicals were performed based on previously published protocols (Oiaz-Cabiale et al. 2011). The primary antibody, an antibody against the c-Fos protein (1: 5000, se-52, Santa Cruz Siotechnology. CA), developed with DAS + nickel, was used as an indirect marker of neuronal activity. Calbindin-D28k antibody (1: 1000, Santa Cruz Biotechnology, CA), developed with DAB, was used to label the granular region since it exclusively marks granular hippocampal cells, although there are some interneurons in the hilus that are immunoreactive to the Calbindin (Scharfman et al. 2002). Biotinylated specific secondary antibodies were used. The sections were mounted on glass slides and the different layers of the dentate gyrus were analyzed using an optical fractionator method based on a stereological microscope (BX51 microscope, Olympus. Denmark) previously used (Diaz-Cabiale etaI.2011).
    Statistic analysis
    The data are presented as the mean ± SEM and the number of samples (n) is indicated in the description of the figures. All data were analyzed using GraphPad PRISM 6.0 (GraphPad Software, La Jolla, CA).
    To compare two experimental conditions, the statistical analysis of the unpaired Student's t-test was used. In all other cases, the analysis of the variance (ANOVA) of a factor followed by the Newman-Keuls test was used. The differences were considered significant at p <0.05 ('p <0.05 "p <0.01 ·" p <0.001).
    Additional information
    Intracerebral cannulas
    Rats anesthetized intraperitoneally with Equitesin (3.3 ml / Kg) were implanted with a stainless steel guide cannula with a 22 gauge needle (Plastics One In) in the right lateral ventricle with the following stereotactic coordinates i: +1.4 lateral mm, -1 mm posterior to bregma, and 3.6 mm below the surface of the bone (Paxinos el al. 1985). After surgery, the animals were located individually and recovery was allowed for 7 days. This method of cannulation and post-surgical care has been previously standardized (Diaz-Cabiale et al. 2011; Narvaez et al. 2014).
    Intracerebroventricular administration of peptides
    Cannulated rats were arbitrarily assigned to different groups. The peptides were prepared again for each administration by dissolving them in aCSF and injected into the lateral ventricle. The total volume was 5 lJl per injection with an infusion time of 1 min Galanin, [Leu31, Pro34] NPY (Ki = 0.39 nM for NPYY1R) and M871 (Ki = 13.1 and 420 nM for GALR2 and GAlR1 respectively) were obtained from Tocris Bioscience (Bristol, UK). The experimental group and the size of each group are indicated in the different procedures. After the experiments, the injection site was checked using sections obtained in cryostat (HM550, Microm International). The procedures of intracerebroventricular injections (icv) and the preparation of cerebrospinal fluid (aCSF) have been previously standardized in the inventors' laboratory (Diaz-Cabiale et al. 2011; Narvaez al. 2014).
    Analysis of the images of autoradiography and in situ hybridization
    Autoradiograms of the autoradiography and in situ hybridization experiments were analyzed as previously described (Parrado et al. 2007; Razani et al. 2001) using a computer image analysis system. Briefly, the measurements were performed with the ImageJ system (NIH, USA) bilaterally in the different regions marked in the OG of the hippocampus (0.15 mm2). One measure was obtained by region and rat, since the average of the measurements was calculated. Duplicate sections for autoradiography and triplicate for in situ hybridization were measured for each treatment. Prefabricated polymers marked 1251_ (Amersham Microscale, Amersham, Little Chalfont, UK) were used to convert the gray values to phentomol / milligram protein. Semi-quantitative measurements of autoradiograms of in situ hybridization were analyzed from the specific optical density (OD) values measured.
    Stereological analysis of the immunohistoguimic c-Fos
    An Olympus BX51 microscope (Olympus, Denmark) connected to a computer and a JVC digital video camera was used. Stereological analysis of the positive samples for c-Fos was performed in the region of the dentate gyrus (DG) at the face-caudal level using an optical fractionator. This method combines optical dissection with a sample fractionator system to exclude divergent volumes (Gundersen et al. 1988). Use a phase

    contrast and the immunoreactivity of the calbindin of the granular cell layer allowed to delineate the area of the DG of each section (Paxinos et al, 1985). Sections were taken every 150 µm, starting with the ventral part of the DG (approximately 2.12 mm after Bregma). Approximately 4.52 mm after Bregma was completed in the dorsal part of the DG. The number of c-Fos IR was quantified in at least five representative sections at 150 IJm per animal (4 rats per group). Using C.A.S.T. Grid (Olympus; Albertslund, Denmark) generated a random sample of samples with a known area (plot a) for each section. After the object was counted (LQ-) the total number of positive cells was estimated as: N = LQ-x fs x fax fh (Gundersen et al. 1988), where fs is the numerical fraction of the section used, fa is the fraction of the area and fh is the linear fraction of the thickness of the section. The error coefficient (EC) for each estimate and animal was between 0.05 and 0.1. The total CE of each group was between 0.07 and 0.08. The count of the marked neurons began 5 IJm below the surface and centered through a 20 IJm section of the optical plane. At least five sections were counted for each region per animal. The number of frames used per animal in the DG was 90-110, representing 20% of the total volume analyzed.
    Double immunofluorescence
    Previously published protocols were used (Narvaez et al. 2014). Rats without treatment (n = 3) were perfused with 4% paraformaldehyde, brains and sections were obtained at the DG level. An initial incubation was performed in solutions with blockers (5% goat serum) and permeabilizers (0.3% Triton X100 in PBS) for 60 minutes each time. The primary rabbit antiGALR2 antibody (Alomone Lab, 1: 1DO) was incubated for 4872 hours at 4 ° C and detected with a DyLight 549 red rabbit anti-rabbit secondary antibody (Jackson immunoResearch Laboratories, 1: 100). Sections were also incubated with a goat anti-NPYY1R antibody (Santa Cruz Biotechnology INC, USA, 1: 200) in a manner similar to that described, and was detected in this case with a DyLight green goat anti-goat mouse secondary antibody 488 (Jackson Laboratories ImmunoResearch, 1: 100). The nuclei were detected with 4 ', 6-diamidino-2-phenylindole (DAPI) (1: 200). Sections were mounted on slides with a fluorescent mounting medium (Dako) and visualized using a TCS-SL confocal microscope (Leica).
    C-Fos / GAD double immunomarking
    The protocols for double immunohistochemistry of c-Fos and glutamate decarboxylase (GAD) 65/67 have been previously described (Narvaez et aL, 2014). The primary antibody used GAD65 / 67 has been validated to detect GABAergic neurons (1: 1000, sc-7513; Santa Cruz Biotechnology, CA) (Papay, 2006). In fact, GAD 65/67 has been used to label GABAergic neurons in the hilus and subgranular area (Muller et al. 2001). Chromogen 3-3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma, Spain) was used with the GAD 65/67 antibody to obtain a brown reaction. The c-Fos immunohistochemistry (revealed with DAB + nickel) has been described earlier in this document. The sections were mounted on glass slides and the photomicrographs were obtained (BX51 microscope, Olympus, Denmark).
    Results
    Galanin increases the bound to NPYY1 R and mRNA expression of NPVY1R in DG
    The icv injection of galanin 3 nmol produces an increase of 20% (t = 1, 924, p <O, 05, df = 10) in the bound of [1 12'l_ [Leu31, Pro34] pyy at 25 pM ( NPYY1R agonist) in the DG (Figure 1) suggesting that galanin increases the recognition of NPYV1R in this area. The binding of C25I] _ [leu31, Pro34] PYY is mainly observed in the molecular and granular layers.
    In addition, galanin modifies not only the bound to NPYY1 R but also the mRNA expression of NPYY1 R in the granular layer of the DG (Figure 2). Thus, 5 hours after icv administration of 3 nmol galanin an increase of 31% (t = 5,327, p <O, 001, df = 10) is detected in the mRNA expression of NPYY1 R in the granular layer of DG .
    GALR2 AND NPYV1R form positive PLA clusters in specific subregions of the DG
    Within the DG, PLA-positive clusters are found specifically in the polymorphic layer and subgranular area of the DG (Figure 3, white circles). These PLA clusters indicate that GALR2 and NPYY1R are very close and may form GALR2fNPYY1 R heteroreceptor complexes in these regions. The specificity of the signal is confirmed by the fact that no PLA clusters are observed neither in the molecular layer of the DG, nor in the corpus callosum, an area in which there is no GAlR2 (O'Donnell et al. 1999) (Figure 3, circulate black).
    In the same direction of these results, an abundant colocalization of GALR2 and NPYY1R is observed specifically in the polymorphic layer and the subgranular zone of the hilus.
    Galanin potentiates NPYY1R-mediated antidepressant behavior
    In the FST, the rats were exposed to water for 15 minutes. Twenty-four hours later, immobility, swimming and climbing were recorded during a second exposure to water
     09-20-201 6
    5 minutes to study behaviors related to depression. The icv administration of the NPYY1R agonist (3 nmol) produces an antidepressant effect on the FST, since it significantly decreases immobility (ANOVA of a factor, F4,30 = 44,81, p <O, 001, Newman-Keuls test : p <O, 001; Figure 4a) and increased swimming (one-way ANOVA, F 4.30 = 48.25, P <0.001, Newman-Keuls test: p <0.001; Figure 4b). In addition, there is a significant decrease in the escalation (one-way ANOVA, F4.30 = 14.42, p <0.001, Newman-Keuls test: p <0.001; Figure 4c).
    On the contrary, the iclan of galanin (3 nmol) significantly increases the immobility time (Newman-Keuls test: p <0.001; Figure 4a) and decreases the climbing behavior (Newman-Keuls test: p <O, 001 ; Figure 4c). These behavior patterns indicate that galanin produces a depressive response.
    However, it was observed that galanin causes a strong increase in the antidepressant action of the NPYY1 R agonist after co-administration of the nina gala and said NPYY1 agonist R. The icv injection of both substances significantly increases the decrease in immobility (test of Newman-Keuls: p <0.001; Figure 4a) and increases swimming (Newman-Keuls test: p <0.001; Figure 4b) compared to the NPYY1R agonist alone. In addition, there is also a significant enhancement of the decrease in climbing (Newman-Keuls test: p <0.05; Figure 4c).
    GALR2 is involved in this interaction since the presence of M871 blocks the increase in the decrease in immobility (Newman-Keuls test: p <0.01; Figure 4a) and the escalation (Newman-Keuls test: p < 0.05; Figure 4c) as well as the increase in swimming (Newman-Keuls test: p <0.001; Figure 4b) induced by co-administration of galanin and the NPYY1R agonist in the FST.
    M871, alone, at the dose of 3 nmol, has no effect on immobility (t = 1, 398, P 0.096, df = 10, mean ± SEM: 86.9 ± 7), climbing (t = 0.475, p 0.322, df = 10, mean ± SEM: 44.4 ± 6) or swimming (t = 0.695, p 0.251, df = 10, mean ± SEM: 146 ± 15) compared to the control.
    Activation pattern of c-Fos and cell populations involved after co-administration of galanin and [Leu31, Pro34] NPY in the OG subregions
    As seen in Figure 5, the icv injection of [Leu31, Pro34] NPY (2.5 nmol) produces a decrease in the number of c-Fas IR in the polymorphic region (one-factor ANOVA, F4.15 = 20, 81, p <0.001, Newman-Keuls test: p <O, 05; Figure 5a), while a
     09-20-201 6
    increase of e-Fas IR in the granular layer of the DG (one-way ANOVA, F4, 15 = 15.08, p <0.001, Newman-Keuls test: p <0.01; Figure 5b). On the other hand, galanin (3 nmol) significantly increases the number of e-Fas IR in both areas, the polymorphic (NewmanKeuls test: p <O, 05) and the granular layer of the DG (Newman-Keuls test: p <O, 01) (Figure 5a, b). I dont know
    observe c-Fos IR in the molecular layer after administration of galanin or the NPYY1R agonist in isolation.
    However, the cayjection of the NPYVR1 agonist and galanin significantly modify the number of c-Fos IR compared to the effect of the galanin or NPYY1R agonist in isolation in both DG subregions. Within the polymorphic zone, the co-administration of galanin and [Leu31, Pro34] NPY significantly decreases the c-Fos IR compared with the agonist of the NPYY1R (Newman-Keuls test: p <0.05), the galanin (Newman test -Keuls: p <O, 001) and the control group (Newman-Keuls test: p <O, 001) (Figure 5a). In this region, GABA interneurons could be involved in the interaction since c-Fos IR collocates with the GABAergic marker (GAD65f67) after injection of the agonist of the
    NPYY1R.
    Within the granular layer, the co-administration of galanin and the NPYY1 agonist significantly increases the expression of c-Fos IR in the granular layer (Figure 5a) compared to the administration of galanin (Newman-Keuls test: p <O, 05 ) and [Leu31, Pro34] NPY (Newman-Keuls test: p <O, 01) in isolation.
    Co-treatment with M871 completely blocks the effect of galanin in both regions, the polymorphic and granular layer of the DG (Figure 5a, b), demonstrating that GALR2 is involved in the actions of the galanin.
    Discussion
    The present invention demonstrates for the first time the existence of an interaction between GALR2 and NPYY1 R in the DG at the receptor level, probably involving the formation of GALR2 / NPYY1 R heteroreceptor complexes based on the proximity binding assay. These complexes are specifically observed in the polymorphic and subgranular subregions of the DG, where the expression of both receptors colocalize. In addition, this GALR2 / NPYV1 R interaction is associated with an antidepressant increase that involves a specific subpopulation within the DG subregions. Thus, co-activation of GALR2 and NPYY1 R enhances the reduction of e-Fas IR in the polymorphic region mediated by NPYY1 R. This response may be linked to the increase in e-Fas IR observed in granular cells,
     09-20-201 6
    and can give a mechanism at the circuit level that would explain the increase in activity
    antidepressant observed after galanin ca-treatment with [Leu 31, Pro34) NPY.
    In autoradiography experiments it is observed that galnin interacts at the membrane level with NPYV1R, since galanin produces an increase in the binding of C251] _ [Leu31, Pro34) pYY in DG. This modification of the NPYY1 R agonist ligand indicates an increase in the affinity of NPYY1 R in the DG, since the NPYY1 R agonist concentration
    used (25pM) is in the range of the value of Kd, where the main changes are at affinity level (Dumont et al. 1996). This effect could be involved in the increase in antidepressant activity observed after co-treatment with galanin and with [Leu31, Pro34) NPY in the forced swimming test through an increase in the recognition and signal of NPYY1R in the GALR2 heteroreceptor complex -NPYY1R. GALR2 can also change signals mediated by Gq to Gilo in this receptor complex (Narvaez et al. 2014). An increase in the binding of the NPYY1 R agonist in the tonsil induced by the administration of galanin and in turn associated with an increase in the anxiolytic effect induced by NPYY1R (Narvaez et al. 2014) has been previously described.
    In addition, it has been observed that GAL also increases mRNA expression of NPYY 1 R in DG, an effect that has been related to antidepressant treatments. Thus, electroconvulsive therapy (ECT) in rats (Madsen et al. 2000) and ECT (Jimenez-Vasquez et al. 2007), and administration of escitalopram (Bjornebekk et al. 2010) in Flinders rats, a genetic model of depression, increases the mRNA of NPYV1 R in the DG, and correlates with the antidepressant effect in the forced swimming test. Based on these studies, the increase in mRNA of galanin-induced NPYY1 R in DG may be relevant for depressive disorders and their treatment.
    Current behavioral results demonstrate that galanin potentiates the antidepressant effects mediated by the agonist [Leu31, Pro34] NPY, decreasing immobility and increasing swimming in the FST. The sharp increase in swimming observed may involve changes in extracellular levels of serotonin. In fact, it is known that selective SSRls produce an increase in swimming in the FST (Kuteeva et al. 2008). Since galanin modulates serotonergic transmission in the hippocampus (Yoshitake et al. 2014), it cannot be excluded that serotonin could be involved in antidepressant actions mediated by GAL_ [Leu31, Pro34] NPY. In addition, GALR2 is involved in the GALRINPYY1 R interaction, since M871 blocks the observed response and in addition GALR2 has been proposed in previous work that measures the antidepressant effects of galanin in vivo (Saar et al. 2013; Kuteeva et al. 2008). The results obtained after the administration of galanin and [Leu3 ProJ4) NPY independently coincide with previous studies confirming the pro-depressive role of GAL and the antidepressant action of the agonist of NPYY1R (Redrobe et al. 2002; Kotagale et al. 2013; Kuteeva et al. 2008). The behavioral effects observed in the FST are independent of the motor activity, since neither the galanin, nor the NPYY1 R agonist nor its co-administration produced motor alteration in different behavioral tests (Narvaez et al. 2014).
    Since the hippocampus participates in depressive behaviors in the FST (Hiroaki-Sato et al. 2014; Bettio et al. 2014), our results indicate the specific participation of the DG. Within the subregions of the DG, colocalization of GALR2 and NPYV1R and the presence of GALR2 / NPVY1 R heteroreceptor complexes in the pOlimorphic and subgranular layers, where both receptors have been previously described (Depczynski et al. 1998; Paredes et al. 2003). GALR2 / NPYY1 R heteroreceptor complexes exist in different regions, including the amygdala (Narvaez et al. 2014). Thus, the integrated signal of the GALR2 / NPYY1R heteroreceptor complex in the paracapsular medial nucleus of the tonsil enhances the anxiolytic effects induced by [Leu3 Pro34) NPY and is related to a decrease in c-Fos IR at the cellular level. (Narvaez et al. 2014). Importantly, the GALR2 / NPYY1 R heteroreceptor complexes in the DG correlate with a decrease in c-Fos in the polymorphic layer. The mechanism for this interaction could be that coactivation of the protomers of the GALR2 / NPYY1 R heteroreceptor complexes changes the GALR2 coupling from Gq to Gi / o together with an increase in the affinity of NPYY1R (Narvaez et al. 2014). In this way, both GALR2 and NPYY1R would be coupled to Gi / o and produce an increase in AC-PKA-CREB inhibition (Narvaez et al. 2014) and the decrease in c-Fos IR observed in the hilus after co-administration of galanin and the NPYY1R agonist.
    The increase in the number of c-Fos IR observed in the granular cell layer after co-administration of GAL and the NPYY1R agonist may be mediated by the decrease in activity of polymorphic GABAergic interneurons in the hilus. In fact, the NPYY1R agonist produces an inhibition in polymorphic GABAergic neurons (Paredes et al. 2003), allowing disinhibition and increased c-Fos expression in DG granular cells (Andrews-Zwilling et al. 2012; Ledri et al. 2012). However, it cannot be excluded that projections of the mosy cells of the hilus (Month) to the granular cells are also involved in this effect, in fact, the axons of the mosy cells in vivo produce the excitation of the inhibitory interneurons that inhibit the activity of DG cells and an inverse correlation at the level of c-Fos IR profiles between MCs cells and granular cells has been described (Jinde et al. 2012; Duffy et al. 2013). In addition, indirect mechanisms may also be involved: For example, corticosterone induces a different decrease in the transcription of c-fos, fos8 and fra-1 in DG compared to other hippocampal subregions (Hansson and Fuxe 2008).
    5 Previous work shows an increase in the expression of c-Fos in DG granular cells after antidepressant therapies such as acute administration of imipramine (li et al. 2013), reboxetine and the combination with mirtazapine (Masana et al. 2012 ) and exercise (Clark on al. 2011). In fact, the increase in the activity of granular cells in the DG has been related to the decrease in immobility in the FST (Jinde et al. 2012; Masana et al. 10 2012). This specific activation within the DG could explain the antidepressant effect
    observed after the co-administration of GAL and [Leu31, Pro34] NPY.
    Based on all of the above, the existence of an interaction between GALR2 and NPYY1 R at the receptor level is demonstrated, probably involving the formation of GALR2fNPYY1 R heteroreceptor complexes 15 in the DG, specifically in the polymorphic and subgranular subregions. This GALR2fNPYY1 R interaction is associated with an increase in antidepressant action involving specific cell populations within DG subregions. Thus, the data contained in this document offers a new anti-depressive integrative mechanism based on the potentiation of the GALR2 interaction
    20 NPYY1R allowing an increase in the signal mediated by Gi / o and an increase in the recognition of NPYY1 R in the DG.
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    权利要求:
    Claims (3)
    [1]
    1. Combination comprising galanin and [Leu31, Pro34] NPY.
    [2]
    2. Use of a combination according to the preceding claim in the preparation of a medicament or pharmaceutical product for depression therapy.
    [3]
    3. A medicament comprising a combination according to claim 1 characterized in that it comprises a pharmaceutically acceptable carrier, diluent or excipient.
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    ES2644978B1|2018-09-25|
    WO2017085350A1|2017-05-26|
    ES2589165B2|2018-11-15|
    ES2644978A1|2017-12-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2707210B2|2017-10-02|2020-09-30|Univ Malaga|GALand analogs thereof for use in the prevention and / or treatment of alcohol-related disorders and effects.|
    ES2883838A1|2020-06-04|2021-12-09|Univ Malaga|PREVENTION AND / OR TREATMENT OF COGNITIVE IMPAIRMENT ASSOCIATED WITH DEMENTIA SYNDROMES|
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