Agonist-dependent modulation of G-protein coupling and transduction of 5-HT1A receptors in rat dorsal raphe nucleus

5-HT1A receptors couple to different Go/Gi proteins in order to mediate a wide range of physiological actions. While activation of post-synaptic 5-HT1A receptors is mainly related to inhibition of adenylyl cyclase activity, functionality of autoreceptors located in raphe nuclei has been classically ascribed to modifications of the activity of potassium and calcium channels. In order to evaluate the possible existence of agonist-directed trafficking for 5-HT1A autoreceptors in the rat dorsal raphe nucleus, we studied their activation by two agonists with a different profile of efficacy [(+)8-OH-DPAT and buspirone], addressing simultaneously the identification of the specific Gα subtypes ([35S]GTPγS labelling and immunoprecipitation) involved and the subsequent changes in cAMP formation. A significant increase (32%, p<0.05) in (+)8-OH-DPAT-induced [35S]GTPγS labelling of immunoprecipitates was obtained with anti-Gαi3 antibodies but not with anti-Gαo, anti-Gαi1, anti-Gαi2, anti-Gαz or anti-Gαs antibodies. In contrast, in the presence of buspirone, significant [35S]GTPγS labelling of immunoprecipitates was obtained with anti-Gαi3 (50%, p<0.01), anti-Gαo (32%, p<0.01) and anti-Gαi2 (29%, p<0.05) antibodies, without any labelling with anti-Gαi1, anti-Gαz or anti-Gαs. The selective 5-HT1A antagonist WAY 100635 blocked the labelling induced by both agonists. Furthermore, (+)8-OH-DPAT failed to modify forskolin-stimulated cAMP accumulation, while buspirone induced a dose-dependent, WAY 100635-sensitive, inhibition of this response (Imax 30.8±4.9, pIC50 5.95±0.46). These results demonstrate the existence of an agonist-dependency pattern of G-protein coupling and transduction for 5-HT1A autoreceptors in native brain tissue. These data also open new perspectives for the understanding of the differential profiles of agonist efficacy in pre- vs. post-synaptic 5-HT1A receptor-associated responses.This research was supported by Ministry of Science, SAF04–00941, SAF07-61862, Fundación Alicia Koplowitz, Fundación de Investigación Médica Mutua Madrileña, Instituto de Salud Carlos III and University of Cantabria-FAES research contract.Peer reviewe

Modulation of neuroplasticity pathways and antidepressant-like behavioural responses following the short-term (3 and 7 days) administration of the 5-HT4 receptor agonist RS67333

It has been recently suggested that activation of 5-HT4 receptors might exert antidepressant-like effects in rats after 3 d treatment, suggesting a new strategy for developing faster-acting antidepressants. We studied the effects of 3 d and 7 d treatment with the 5-HT4 receptor partial agonist RS67333 (1.5 mg/kg.d) in behavioural tests of chronic efficacy and on neuroplastic-associated changes, such as adult hippocampal neurogenesis, expression of CREB, BDNF, ß-catenin, AKT and 5-HT4 receptor functionality. RS67333 treatment up-regulated hippocampal cell proliferation, ß-catenin expression and pCREB/CREB ratio after 3 d treatment. This short-term treatment also reduced immobility time in the forced swim test (FST), together with a partial reversion of the anhedonic-like state (sucrose consumption after chronic corticosterone). Administration of RS67333 for 7 d resulted in a higher increase in the rate of hippocampal cell proliferation, a significant desensitization of 5-HT4 receptor-coupled adenylate cyclase activity and a more marked increase in the expression of neuroplasticity-related proteins (BDNF, CREB, AKT): these changes reached the same magnitude as those observed after 3 wk administration of classical antidepressants. Consistently, a positive behavioural response in the novelty suppressed feeding (NSF) test and a complete reversion of the anhedonic-like state (sucrose consumption) were also observed after 7 d treatment. These results support the antidepressant-like profile of RS67333 with a shorter onset of action and suggest that this time period of administration (3-7 d) could be a good approximation to experimentally predict the onset of action of this promising strategy.This work was supported by Ministerio de Ciencia e Innovación (SAF-07/61862), Fundación Alicia Koplowitz and Fundación de Investigación Médica Mutua Madrileña.Peer Reviewe

Association of gene-enviroment and age of pre-onset cannabis use with age at onset of psychosis in frist-episode patients

Trabajo presentado a la 10ª Reunión anual de la Sociedad Española de Investigación sobre Cannabinoides celebrada en Santander del 26 al 28 de noviembre de 2009.Es conocida la influencia negativa del uso de cannabis sobre el curso y pronóstico de la esquizofrenia. El cannabis es, además, la sustancia de abuso más utilizada en pacientes con esquizofrenia (15%-65%). Sin embargo sólo una pequeña proporción de consumidores de cannabis desarrollan psicosis. Varios estudios han demostrado que el uso de cannabis precede al debut de la psicosis en varios años (entre 4 y 5 ). El inicio precoz del consumo de cannabis en la adolescencia, puede por tanto, estar asociado a un debut precoz de la psicosis, con el consiguiente pronóstico negativo de la enfermedad. Varios polimorfismos de nucleótidoúnico (SNPs) del gen que codifica el receptor CB1 (CNR1; rs806379, rs1535255, rs2023239 y rs1049353) han sido asociados al consumo de drogas o alcohol (Zhang et al., Molecular Psychiatry, 9, 916–931. 2004, Schmidtetal., 2002 Drug and Alcohol Dependence, 65, 221–224) Variaciones en el gen de la triptófano hidroxilasa se han asociado con un mayor riesgo de psicosis (LiD, HeL: Hum Genet 2006). Finalmente, algunos de los SNPs en los receptores de la serotonina se han asociado con diversos trastornos psiquiátricos como la esquizofrenia o la depresión, sin embargo, los resultados de estos estudios de asociación genética han mostrado resultados conflictivos.Peer Reviewe

Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration

A Ferrés-Coy et al.Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, similar to 80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive -like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant -like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.This work was supported by grants from CDTI—Spanish Ministry of Science and Innovation—DENDRIA contribution, 'nLife all rights reserved' (to AB and FA); Instituto de Salud Carlos III PI10/00290 and PI13/01390 (to AB), PI/10/0123 (to JCL) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM); NARSAD Independent Investigator Grant from the Brain & Behavior Research Foundation Grant 20003 (to AB); Ministry of Economy and Competitiveness SAF2012-35183 (to FA) and SAF2011-25020 (to AP); and Generalitat de Catalunya, Secretaria d’Universitat i Recerca del Departament d’Economia i Coneixement (SGR2014) Catalan Government Grant 2009SGR220 (to FA). Some of these grants are co-financed by the European Regional Development Fund 'A way to build Europe'. AF-C is a recipient of a fellowship from Spanish Ministry of Education, Culture and Sport.Peer Reviewe

RNAi-mediated serotonin transporter suppression rapidly increases serotonergic neurotransmission and hippocampal neurogenesis

Current antidepressants, which inhibit the serotonin transporter (SERT), display limited efficacy and slow onset of action. Here, we show that partial reduction of SERT expression by small interference RNA (SERT-siRNA) decreased immobility in the tail suspension test, displaying an antidepressant potential. Moreover, short-term SERT-siRNA treatment modified mouse brain variables considered to be key markers of antidepressant action: reduced expression and function of 5-HT(1A)-autoreceptors, elevated extracellular serotonin in forebrain and increased neurogenesis and expression of plasticity-related genes (BDNF, VEGF, Arc) in hippocampus. Remarkably, these effects occurred much earlier and were of greater magnitude than those evoked by long-term fluoxetine treatment. These findings highlight the critical role of SERT in serotonergic function and show that the reduction of SERT expression regulates serotonergic neurotransmission more potently than pharmacological blockade of SERT. The use of siRNA-targeting genes in serotonin neurons (SERT, 5-HT(1A)-autoreceptor) may be a novel therapeutic strategy to develop fast-acting antidepressants

Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics

© The Author(s) 2019.A better understanding of the molecular mechanisms that participate in the development and clinical manifestations of schizophrenia can lead to improve our ability to diagnose and treat this disease. Previous data strongly associated the levels of deregulated ADAMTS2 expression in peripheral blood mononuclear cells (PBMCs) from patients at first episode of psychosis (up) as well as in clinical responders to treatment with antipsychotic drugs (down). In this current work, we performed an independent validation of such data and studied the mechanisms implicated in the control of ADAMTS2 gene expression. Using a new cohort of drug-naïve schizophrenia patients with clinical follow-up, we confirmed that the expression of ADAMTS2 was highly upregulated in PBMCs at the onset (drug-naïve patients) and downregulated, in clinical responders, after treatment with antipsychotics. Mechanistically, ADAMTS2 expression was activated by dopaminergic signalling (D1-class receptors) and downstream by cAMP/CREB and mitogen-activated protein kinase (MAPK)/ERK signalling. Incubation with antipsychotic drugs and selective PKA and MEK inhibitors abrogated D1-mediated activation of ADAMTS2 in neuronal-like cells. Thus, D1 receptors signalling towards CREB activation might participate in the onset and clinical responses to therapy in schizophrenia patients, by controlling ADAMTS2 expression and activity. The unbiased investigation of molecular mechanisms triggered by antipsychotic drugs may provide a new landscape of novel targets potentially associated with clinical efficacy.This work was supported by: SAF2016-76046-R and SAF2013-46292-R (MINECO and FEDER) to B.C.F., PI16/00156 (isciii and FEDER) to J.P.V., LUCHAMOS POR LA VIDA project to F.R.J. and J.P.V., SAF2017-83702-R (MINECO and FEDER), Red TERCEL RD12/0019/0024 (ISCIII) and GVA-PROMETEO 2018/041 (Generalitat Valenciana) to S.M. J.P.V. is supported by the RyC research programme (RYC-2013-14097) and F.R.J. by the predoctoral research programme (BES-2014-070615), from MINECO and FEDER

Evaluation of the endocannabinoid system in post-mortem human prefrontal cortex of alcoholic subjects

Trabajo presentado al "ECNP Workshop on Neuropsychopharmacology for Young Scientist in Europe" celebrado en Niza (Francia) del 4 al 7 de Marzo de 2010 y al "The 20th Annual Symposium on the Cannabinoids" celebrado en Suecia del 24 al 27 de julio de 2010.-- Trabajo publicado en la revista European Neuropsychopharmacology 20(Supl. 1): http://dx.doi.org/10.1016/S0924-977X(10)70011-0.-- et al.Peer reviewe

New antidepresant drugs

Trabajo presentado al "32 Congreso de la Sociedad Española de Farmacología" celebrado en León del 15 al 17 de septiembre de 2010.Depression constitutes an international public health problem. Major depression represents the fourth largest disease burden in 1990, and by 2020, it is estimated to be surpassed only by cardiovascular diseases. Depression exerts a considerable toll on the economy, via both direct and indirect costs. Despite the advances of the current medications, remission rates among depressed individuals are quite low, and rates of treatment resistance are high: 30% of depressed patients may be considered nonresponders. Indeed, current antidepressants have a delayed onset of action, requiring a minimum of 3 weeks before the full therapeutic effect occurs. Finally, side and adverse events, and drug drug interactions are common. Therefore, a need for new pharmacologic compounds with greater efficacy and tolerability in depressive disorders remains as a priority. For many years, antidepressant agents (ADs) that alter the availability of the so called classic neurotransmitters, serotonin, norepinephrine, and dopamine, have been a mainstay of therapeutic intervention in the treatment of depression. This is accomplished either by inhibiting the enzyme that breaks down these neurochemicals (ie, monoamine oxidase inhibitors) or by inhibiting the cellular transport protein that recycles the neurotransmitter after release into the synapse {tricyclic antidepressants (i.e. imipramine) and selective monoamine reuptake inhibitors (i.e. fluoxetine)}. Another approach that may extend the therapeutic action of monoamine based antidepressants is the use of compounds which exert a simultaneous action on 2 or more of the classic neurotransmitters (ie, venlafaxine). New molecules combining the effects on aminergic systems with new pharmacodynamic properties are being developed, trying to achieve rapid onset of action and low extent of adverse events: agomelatine, an agonist of melatonergic MT1 and MT2 receptors, serotonin 5 HT2C receptor antagonist and enhancer of the release of dopamine and noradrenaline is now available as sleep modulating antidepressant. Among the new drugs currently under investigation are those that target the corticotropin releasing hormone, dopamine and glutamate systems. Findings from animal models indicate that chronic stress and depression may be associated with morphologic changes in the brain. Specifically, it is believed that chronic stress leads to an increase in glucocorticoids and a decrease in protective neurotrophins. These changes, in conjunction with untreated depression, may result in lower levels of BDNF, ultimately leading to brain degeneration. Mood disorders seem to be associated with an impairment of the mechanisms controlling neuroplasticity and cell survival in brain. Supporting this, a reduction of the hippocampal volume in depressed patients and cortical neuronal loss has been reported. Chronic, but not acute antidepressant treatments and electroconvulsive shock increase neuronal proliferation in the dentate gyrus.The signaling systems appear to have a role in this response included: the cAMP CREB pathway, The BDNF TrkB receptors and the MAP kinase cascade, Wnt GSK3 β-catenin pathways. Even there is not yet new molecules, harmaceutical companies are investing in finding selective inhibitors of GSK3 as antidepressant drugs. Finally, Serotonin 5 HT4 agonist has been show to display an antidepressant like potential with a rapid onset of action. A 3 day regimen with such compounds modifies rat brain parameters considered to be key markers of antidepressant action as desensitization of 5 HT1A autoreceptors, increased tonus on hippocampal postsynaptic 5 HT1A receptors, enhanced phosphorylation of the CREB protein and neurogenesis in the hippocampus. On the other hand, the future development of pharmacogenetic studies with regard to ADs response would be a further step to the clinical use of gene profiles as predictors of therapeutic efficacy.Peer Reviewe

Genetic factors associated with drug-resistance of epilepsy: Relevance of stratification by patient age and aetiology of epilepsy

et al.Epilepsy drug-resistance may depend on the metabolism of antiepileptic drugs (AEDs), transport to the epileptic focus and/or target sensitivity. Furthermore, drug response depends on multiple characteristics of the patient, the epilepsy, and the antiepileptic drugs used. We have investigated the association between polymorphisms related to antiepileptic drug metabolism (CYP2C9, CYP2C19, and UGT), transport (ABCB1), and targets (SCN1A) both in a crude analysis and after adjusting by clinical factors associated with drug-resistance, and stratifying by patient age or aetiology of epilepsy. Caucasian outpatients (N = 289), children (N = 80) and adolescent-adults (N = 209), with idiopathic (N = 69), cryptogenic (N = 97) or symptomatic epilepsies (N = 123) were selected when they had either drug-resistance (with at least four seizures over the previous year after treatment with more than three appropriate AEDs at appropriate doses) or drug responsiveness (without seizures for at least a year). Samples were genotyped by allelic discrimination using TaqMan probes. No significant association between polymorphisms and drug-resistance was found either in the crude analysis or in the adjusted analysis. However, adults with the ABCB1_3435TT or 2677TT genotypes had a lower risk of drug-resistance than those with the CC or the GG genotypes. Furthermore, patients with symptomatic epilepsies with the ABCB1_3435CT or TT genotypes had a lower risk of drug-resistance than those with the CC genotype. An opposite but insignificant tendency was found in children and in idiopathic epilepsies. Although replication studies will be needed to confirm our results, they suggest that stratification by patient age and by the aetiology of epilepsy could contribute to unmask the association between ABCB1 polymorphisms and drug-resistance of epilepsy.The authors wish to thank the Marqués de Valdecilla Foundation (Santander, Spain, API05/02), the Spanish Ministry of Science (SAF07-61862) and the Spanish FISS (PI/05/0388) for their support of the study.Peer reviewe

Absence of core autophagy gene expression in an ex vivo central nervous system model infected with Listeria monocytogenes

et al.[EN]: Recent studies have suggested that autophagy can act as a protective immune mechanism against Listeria monocytogenes infection. L. monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive diseases in humans and animals, particularly in the central nervous system (CNS). Human listeriosis of the CNS can manifest in many ways, including meningitis and brain abscesses. The initial line of defence against bacterial colonisation is provided by microglia, resident phagocytes of the CNS parenchyma. Microglial cells are also well known for clearing dead and dying neural cells after injury, and therefore play a key role in infectious diseases and neurodegeneration. Little is known about the role of the autophagy pathway in host-pathogen interactions in the brain as most in vitro studies have used macrophages or epithelial cells to study this interaction. In the present work, a quantitative real time-PCR array analysis was performed to assess autophagy-related gene expression in a brain rat ex vivo organotypic nervous system model during L. monocytogenes infection. We found that, in brief, core autophagy gene expression is not modulated by the infection, despite the presence of intense microglial phagocytic activity on the brain tissue surface that can be seen by scanning electron microscopy. We conclude that, in our model, autophagy could play a role in homeostasis in the damaged brain tissue instead of an immune-relevant pathway.[ES]: Estudios recientes han evidenciado que la autofagia puede actuar como un mecanismo inmune protector frente a la infección con Listeria monocytogenes. L. monocytogenes es una bacteria grampositiva, intracelular facultativa, que causa enfermedades invasivas en humanos y animales, especialmente en el sistema nervioso central (SNC). La listeriosis humana en el SNC puede manifestarse de diferentes maneras, incluyendo meningitis y abscesos cerebrales. La línea principal de defensa frente a las infecciones bacterianas es proporcionada por la microglía, fagocitos residentes del parénquima del SNC. Las células de microglía son conocidas, también, por eliminar las células dañadas o muertas tras un daño cerebral, y por lo tanto desempeñan un papel clave en las enfermedades infecciosas y neurodegenerativas. Se sabe poco sobre el papel de la autofagia en las interacciones entre el hospedador y el patógeno, debido a que la mayoría de los estudios in vitro han usado macrófagos o células epiteliales. En el presente trabajo hemos utilizado matrices de PCR en tiempo real para analizar la expresión de genes de autofagia en un modelo organotípico de cerebro de rata infectado con L. monocytogenes. Hemos observado que, en general, la expresión de genes centrales de la autofagia no está modulada por la infección, a pesar de la presencia de una intensa actividad fagocítica de la microglía en la superficie del tejido cerebral, observada mediante microscopia electrónica de barrido. Concluimos que, en nuestro modelo, la autofagia podría desempeñar un papel clave en la homeostasis del tejido dañado en lugar de tener un papel inmune relevante.SRM holds a research contract from the Instituto de Formación e Investigación Marqués de Valdecilla (IFIMAV). JRV holds a Miguel Servet contract for Young Researchers from the Instituto de Salud Carlos III, Spain. The Instituto de Salud Carlos III (PS: CP08/100) and the Fondo de Investigaciones Sanitarias (FIS: PS09/00466) provide the financial support for this study and grants to JRV. EMV was supported by the Ministerio de Ciencia e Innovación (grants SAF07-61862 and SAF2011-25020). JMI was supported by the Spanish Ministerio de Economia y Competitividad (grant CGL2008-04559/BOS).Peer Reviewe