Nuevas aproximaciones terapéuticas mediante las vías de señalización del ácido lisofosfatídico: regulación farmacológica y trasplante neuronal

Los lisofosfolípidos son derivados de fosfolípidos de la membrana celular y uno de los más estudiados es el ácido lisofosfatídico (LPA), considerado como un importante regulador de diversas funciones biológicas. Hasta la fecha, se conocen seis receptores de LPA acoplados a proteínas G (LPA1-6), y entre sus funciones destaca la regulación de la plasticidad hipocampal adulta (Dash et al., 2004; Choi et al., 2010; Shin et al., 2012; revisado en Choi y Chun, 2013). Los ratones carentes del receptor LPA1 presentan déficits en la exploración, la memoria y la regulación emocional (Santín et al., 2009; Castilla-Ortega et al., 2010; Blanco et al., 2012; Castilla-Ortega et al., 2012; Pedraza et al., 2013; Castilla-Ortega et al., 2013) junto con alteraciones funcionales en el hipocampo y en la amígdala (Matas-Rico et al., 2008; Castilla-Ortega et al., 2011; Musazzi et al., 2011; Castilla-Ortega et al., 2012; García-Fernandez et al., 2012; Pedraza et al., 2013; Castilla-Ortega et al., 2013). Actualmente, por la importante participación en el correcto desarrollo del sistema nervioso central así como en numerosas patologías, el potencial clínico de los receptores de LPA está cobrando cada vez mayor interés, gracias a que se dispone de agonistas y antagonistas que pueden modificar la actividad de los receptores (revisado en Choi et al., 2010). Dentro de este contexto, uno de los objetivo de la Tesis Doctoral fue la modulación farmacológica del receptor LPA1, principalmente, mediante el uso de las minibombas osmóticas Alzet®, en condiciones in vivo durante 21 días determinando el papel del tratamiento a nivel fisiológico y conductual. En otro sentido, para un buen desarrollo del sistema nervioso central es imprescindible una correcta sintonía entre los sistemas neuronales activadores e inhibidores. Los ratones carentes del receptor LPA1, presentan pérdida de interneuronas GABAérgicas y un sistema GABAérgico alterado (Cunningham et al., 2006; Matas-Rico et al., 2009), además de tener déficits en la liberación de los neurotransmisores GABA y glutamato (Harrison et al., 2003; Roberts et al., 2005; Blanco et al., 2012). El segundo objetivo se centró, por ello, en el trasplante de precursores de interneuronas GABAérgicas derivados de la eminencia ganglionar medial en el hipocampo dorsal de ratones normales y nulos para el receptor LPA1 durante 31 días, a objeto de profundizar en los mecanismos dependientes del receptor y a su vez, desarrollar alternativas de rescate de fenotipo. Los resultados presentados en esta tesis avalan los tratamientos farmacológicos y la terapia celular en los modelos experimentales, que demuestran, por una parte, que el ácido lisofosfatídico impulsa la neurogénesis adulta y la maduración de las nuevas neuronas generadas en el hipocampo así como la regulación emocional, a través de la activación del receptor LPA1. Por otra parte, demuestran que la ausencia del receptor tiene lugar en un entorno neural que permite la integración de precursores de interneuronas para que, éstas, merced a su modulación GABAérgica potencien y mejoren la funcionalidad hipocampal tras la restauración de la red neuronal local, repercutiendo positivamente sobre la conducta relacionada con la ansiedad. Ambos experimentos, demuestran la validez de los tratamientos farmacológicos y la terapia celular para el estudio de alteraciones neurales dependientes de receptores de amplia distribución en el sistema nervioso

Effects of palmitoylethanolamide in cocaine-induced behaviours

Aims. Cocaine addiction is a chronically relapsing disorder characterized by the compulsion to seek and take the drug. Previous investigations have demonstrated that several drugs of abuse, as cocaine, can alter the levels of lipid-based signalling molecules such as the N-acylethanolamines (NAEs). In addition, NAEs levels in the brain are sensitive to cocaine self-administration and extinction training. In this context, this study aimed to investigate the effect of repeated and acute palmitoylethanolamide (PEA), an endogenous NAE, on the behavioural effects of cocaine using mouse models of conditioned reward and psychomotor activation. Methods. Using male C57BL/6J mice, the ability of repeated PEA injections (1 or 10 mg/kg i.p) to modulate the development of a conditioned place preference (CPP) and behavioural sensitization (BS) induced by cocaine (20 mg/kg i.p.) was evaluated. In addition, the expression of cocaine-induced CPP and BS after acute PEA administration was also studied. Results. PEA (1 and 10 mg/kg i.p) significantly reduced the development of cocaine-induced BS, but did not modify the acquisition of cocaine-induced CPP. Furthermore, both doses of PEA were able to reduce the expression of BS and CPP. Conclusions. Altogether, these findings show that exogenous administration of PEA attenuated psychomotor activation and impaired the expression of CPP induced by cocaine. Our results may be relevant in order to understand the role of NAEs in the development and treatment of cocaine addiction.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech. PSI2013-44901-P, AP2010-2044, FPU13/04819, CD12/0045

Cognitive impairment in a murine model of experimental autoimmune encephalomyelitis with relapsing-remitting course

Multiple sclerosis (MS) is a neuroinflammatory disorder characterized by demyelination and progressive axonal loss that affects the central nervous system. In addition of physical disability and the neurodegenerative process, MS associates with co-morbid behavioral, neuropsychiatric and cognitive impairment, including learning and memory deficits. The study of cognitive impairment in the currently most suitable experimental animal model of MS, experimental autoimmune encephalomyelitis (EAE), constitutes a very valuable tool to translate ultimately into clinical a better diagnosis and more effective treatment protocols. In our study, we analyzed the behavioral profile of a murine model of EAE induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55) which develops a relapsing-remitting course. In the early neuroinflammatory phase of the disease, i.e. 19-21 days post immunization (dpi), EAE mice exhibited deficits in motor coordination/skill learning (Rotarod test), and spatial working memory (spontaneous alternation in Y-maze), as well as depressive symptoms (tail suspension test) and anxiety-like behavior (elevated plus-maze). EAE mice did not yet show object recognition memory impairments, suggesting that reference memory was not altered in this phase. However, from 33-35 dpi until late phases (49-52 dpi), independently of clinical score, EAE mice exhibited a memory decline showing lower discrimination index in the object recognition test. EAE late phase was also characterized by motor coordination and spatial working memory impairments as well as higher anxiety-like behavior. Overall, these data demonstrates a differential pattern of gradual cognitive dysfunctions during the relapsing-remitting EAE course that could help to understand the development of progressive cognitive decline in MS patients. Funding: Andalusian Regional Ministries of Economy, Innovation, Science and Employment (SEJ-1863; CTS643) and of Health (PI-0234-2013; Nicola´s Monardes Programme).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Role of the LPA1 receptor in mood and emotional regulation

Depression is a debilitating psychiatric condition characterized by anhedonia and behavioural despair among others symptoms. Despite the high prevalence and devastating impact of depression, underlying neurobiological mechanisms of mood disorders are still not well known. Regardless its complexity, central features of this disease can be modelled in rodents in order to better understand the potential mechanisms underlying. On the other hand, the lack of LPA1 receptor compromises the morphological and functional integrity of the limbic circuit and the neurogenesis in hippocampus, induces cognitive alterations on hippocampal-dependent tasks and dysfunctional coping of chronic stress, provokes exaggerated endocrine responses to emotional stimuli and impairs adaptation of the hypothalamic-pituitary-adrenal axis after chronic stress. Factors, which all have been related with depression. Here, we sought to establish the involvement of the LPA1 receptor in regulation of mood and emotion. To this end, in wild-type and maLPA1-null mice active coping responses to stress were examined using the forced swimming test (FST). To assess hedonic behaviour saccharine preference test and female urine sniffing test were used. Our data indicated that the absence of the LPA1 receptor significantly affected to coping strategies. Thus, while null mice displayed less immobility than wt in FST, exhibited more climbing and less swimming behaviour, responses that could be interpreted as an emotional over-reaction (i.e., a panic-like response) to stress situations. Concerning hedonic behaviour, the lack of the LPA1 receptor diminished saccharin preference and female urine sniffing time. Overall, these data supports the role of LPA1 receptor in mood and emotional regulation. Specially, the lack of this receptor induced emotional dysregulation and anhedonic behaviour, a core symptom of depression.Universidad de Málaga, Campus de Excelencia Andalucía Tech. Andalusian Regional Ministries of Economy, Innovation, Science and Employment (SEJ-1863; CTS643) and of Health (PI-0234-2013; Nicolas Monardes Programme), MINECO (PSI2013-44901-P) and National Institute of Health Carlos III (Sara Borrel)

Enhancing adult hippocampal neurogenesis with lysophosphatidic acid: a proposal for erasing cocaine contextual memory

Stimulating adult hippocampal neurogenesis (AHN) has been uncovered as a promising approach in the manipulation of retrograde memories. This work aims to study whether increasing AHN with lysophosphatidic acid (LPA, an endogenous lysophospholipid with proneurogenic actions) promotes the forgetting of previously established cocaine-contextual associations. C57BL/6J mice previously trained in a cocaine-induced conditioned place preference (CPP) paradigm were submitted to 23 days of withdrawal, during which they received repeated intracerebroventricular infusions of LPA, ki16425 (a selective LPA1/3 receptors antagonist), or vehicle solution. Then, CPP maintenance was assessed, and the causal role of AHN in this process was evaluated using a mediation analysis. In a complementary experiment, wild-type and LPA1-null mice were acutely infused with LPA or ki16425 to determine the involvement of the LPA1 receptor in the in vivo proneurogenic actions of LPA. The chronic LPA treatment significantly weakened the long-term retention of a previously acquired cocaine-CPP memory, an effect clearly mediated by a LPA-induced increase in the number of adult-born dentate granule cells. In contrast, the ki16425-treated mice displayed aberrant responses of initially decreased CPP retention that progressively increased CPP across the extinction sessions, in absence of effects on AHN. The histological studies suggested that the proneurogenic actions of LPA were related to the enhancement of cell proliferation and critically depended on the LPA1 receptor function. Our results suggest that the LPA/LPA1-pathway acts as a potent in vivo modulator of AHN, and highlight the usefulness of a post-learning increase of adult-born hippocampal neurons as a strategy to promote the forgetting of cocaine-context associations.Plan Propio de Investigación y Transferencia. Campus de Excelencia Internacional Andalucía Tech. Spanish Ministry of Economy and Competitiveness (Agencia Estatal de Investigación), co‐funded by the European Research Development Fund (AEI/FEDER, UE) (PSI2013‐44901‐P and PSI2017‐82604‐R to L.J.S. and PSI2015‐73156‐JIN to E.C.O.); by the National System of Health‐Instituto de Salud Carlos III, which is co‐funded by AEI/FEDER, UE (Red de Trastornos Adictivos; RD16/0017/0001 to F.R.d.F.); and by the Andalusian R&D&I Programme, Regional Ministry of Economy and Knowledge (PAIDI CTS643 to G.E.T.). D.L.G.M. hold a FPU grant from the Spanish Ministry of Education, Culture and Sports (FPU13/04819 ). F.R.d.F. and G.E.T. are supported by Nicolas Monardes Programme, from the Andalusian Regional Ministry of Health. E.C.O. holds a ‘Jóvenes Investigadores’ grant (code: PSI2015‐73156‐JIN) from the Spanish Ministry of Economy and Competitiveness (Agencia Estatal de Investigación), which is co‐funded by the AEI/FEDER, UE

Palmitoylethanolamide attenuates cocaine-induced behavioral sensitization and conditioned place preference in mice

Cocaine addiction is a chronically relapsing disorder characterized by compulsive drug-seeking and drug-taking behaviors. Previous studies have demonstrated that cocaine, as well as other drugs of abuse, alters the levels of lipid-based signaling molecules, such as N-acylethanolamines (NAEs). Moreover, brain levels of NAEs have shown sensitivity to cocaine self-administration and extinction training in rodents. Given this background, the aim of this study was to investigate the effects of repeated or acute administration of palmitoylethanolamide (PEA), an endogenous NAE, on psychomotor sensitization and cocaine-induced contextual conditioning. To this end, the potential ability of repeated PEA administration (1 or 10 mg/kg, i.p.) to modulate the acquisition of cocaine-induced behavioral sensitization (BS) and conditioned place preference (CPP) was assessed in male C57BL/6J mice. In addition, the expression of cocaine-induced BS and CPP following acute PEA administration were also studied. Results showed that repeated administration of both doses of PEA were able to block the acquisition of cocaine-induced BS. Furthermore, acute administration of both doses of PEA was able to abolish the expression of BS, while the highest dose also abolished the expression of cocaine-induced CPP. Taken together, these results indicate that exogenous administration of PEA attenuated psychomotor sensitization, while the effect of PEA in cocaine-induced CPP depended on whether PEA was administered repeatedly or acutely. These findings could be relevant to understand the role that NAEs play in processes underlying the development and maintenance of cocaine addiction.Fil: Zambrana Infantes, Emma. Universidad de Málaga; EspañaFil: Rosell del Valle, Cristina. Universidad de Málaga; EspañaFil: Ladrón de Guevara Miranda, David. Universidad de Málaga; EspañaFil: Galeano, Pablo. Universidad de Málaga; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Castilla Ortega, Estela. Hospital Regional Universitario de Málaga; EspañaFil: Rodríguez De Fonseca, Fernando. Hospital Regional Universitario de Málaga; EspañaFil: Blanco, Eduardo. Universidad de Lleida; EspañaFil: Santín, Luis Javier. Universidad de Málaga; Españ

Social avoidance and altered stress axis in a mouse model of anxious depression

Prevalence of stress-related disorders, such as depression, is raising in modern societies. Indeed, current neurobiological research aims to elucidate the link between deregulation of the hypothalamic-pituitary-adrenal (HPA) axis among vulnerable individuals and the onset of depressive symptoms, such as social withdrawal. Herein, we seek to determine the role of LPA1 receptor in social behaviour and the performance of maLPA1-null mice, a model of anxious depression, in the dexamethasone (DEX) suppression test. For that purpose, we used the three-chamber test for social preference. Also, we administered vehicle or DEX 0.1mg/kg to wild-type (WT) mice and maLPA1-null mice, analysed corticosterone (CORT) response by ELISA method and determine glucocorticoid receptor (GR) expression and serum/glucocorticoid regulated kinase 1 (SGK1) in the hippocampus by Western-Blot analysis. We found that maLPA1-null mice lack preference for the social chamber as compared to WT animals. Additionally, mice lacking the LPA1 receptor did not suppress CORT after DEX treatment and increased significantly hippocampal SGK1 expression despite unaltered GR protein levels. These results provide further insight on the role of LPA1 receptors in depressive-like behavior and the pathological intracellular signals involved in stress regulation.Andalusian Regional Ministries of Economy, Innovation, Science and Employment (SEJ1863 to CP and CTS-643 to GE-T) and of Health (Nicolas Monardes Programme to GE-T). Spanish Ministry of Economy and Competitiveness (PSI2013-44901-P to LJS and CP; and co-financed with Funds of the European Commission “FEDER” PSI2017-83408-P to CP). Author AN-Q and RDM-F hold Grants of the Spanish Ministry of Education, Culture and Sports (FPU 16/05308; FPU14/01610, respectively). Author FJG-S held a Grant of the First Research and Transfer Plan of the University of Malaga. University of Malaga, Campus de Excelencia Internacional Andalucía Tech, and I Plan Propio de Investigación y Transferencia of the University of Malaga

Stress coping behaviour, brain connectivity and LPA1 receptor: similarities and differences between the genetic and the pharmacological approach

LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts as an intercellular signalling molecule. It has been recently proposed that this receptor has a key role in controlling depression-like behaviours and in the detrimental consequences of stress. Here, we sought to establish the involvement of the LPA1 receptor in brain activity after an acute stressor. To this end, we examined behavioural despair in mice with a constitutive depletion of the LPA1 receptor (maLPA1-null mice), wild-type mice and mice receiving one single icv dose of the LPA1 receptor antagonist Ki16425 or vehicle. Furthermore, the expression of c-Fos protein in stress-related brain areas and the corticosterone response following acute stress were examined. Our data indicated that, contrary to the knockout model, the antagonism of the LPA1 receptor significantly increased immobility in the Forced Swim Test. However, latency to first immobility was reduced in both experimental conditions. Immunohistochemistry studies revealed an increased in activity in key limbic structures such as medial prefrontal cortex in both the LPA1 antagonist-treated mice and maLPA1-null mice, with an interesting opposed effect on hippocampal activity. Following acute stress, the sole infusion of Ki16425 in the cerebral ventricle increased corticosterone levels. In conclusion, the alteration of LPA1 receptor function, through both genetic deletion or pharmacological antagonism, is involved in behavioural despair and hyperactivity of brain stress systems, thus contributing to explore specific susceptibility mechanisms of stress as targets for therapeutic recovery.Funding by the Andalusian Ministry of Economy, Innovation, Science and Employment (SEJ1863) and the Spanish Ministry of Education, Culture and Sports ( PSI2017 - 83408 - P). Authors RD. M-F and A. N-Q hold a Grant of the Spanish Ministry of Education, Culture and Sports (FPU14/01610 and FPU16/05308, respectively). Author S.T. holds a Grant of the Andalusian Ministry of Economy, Innovation, Science and Employment C. R. (FPDI 2016); Andalucía Tech. I Plan Propio de Investiga ción y Transferencia de la Universidad de Málaga. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

The absence of LPA1 receptor results in lipidome dysregulation and Neuropeptide-Y underexpression

LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts as an intercellular signaling molecule. It has been shown that the LPA1 receptor is involved in emotional regulation and, when depleted, has a key role in vulnerability to stress. In this sense, maLPA1-null mice, a knockout model for LPA1 receptor has been recently proposed as a model of anxious depression. Here, we sought to elucidate the effect of the genetic depletion of this receptor of LPA1 receptor in both lipidome and Neuropeptide-Y (NPY) signaling, two factors associated with adaptive stress regulation. For that purpose, we measured the lipidomic profile of wild-type mice and maLPA1-null mice in both hippocampus and serum. In addition, through immunohistochemical procedures we quantified NPY+ cells in hippocampus, basolateral amygdala (BLA) and central amygdala (CeA). Interestingly, the comparative lipidomics analysis revealed differences in certain subspecies which are related to LPA1 receptor functionality. Regarding NPY, we found a reduction in BLA, but not in hippocampus. Overall, both lipid abnormalities and amygdalar dysfunction of NPY can be related to lower resources in stress coping and, in turn, higher vulnerability to the noxious effect of stress that might lead to anxiety and depressive-like states.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

More adult-born dentate gyrus neurons to weaken cocaine-related retrograde memories: an in vivo strategy employing exogenous lysophosphatidic acid

The post-training enhancement of adult hippocampal neurogenesis (AHN) has been receiving growing interest as a potential method to manipulate retrograde memories. Recent hypothesis suggest that the addition of adult-born dentate granule cells might promotes remodeling of pre-existing hippocampal circuits, which might both clear cocaine-related memories and facilitate the learning of new adaptive information. Here, we study the effect of stimulating AHN in vivo with exogenous lysophosphatidic acid (LPA) on the maintenance of retrograde cocaine-contextual associative memories. Male C57BL/6J mice trained in a cocaine-induced Conditioned Place Preference (CPP) model were later submitted to repeated intracerebroventricular (i.c.v.) injections of LPA, Ki16425 or vehicle solution during withdrawal. Afterwards, the long-term persistence of the cocaine-CPP was assessed and the mediational role of AHN in this process was evaluated. In addition, wild-type and mice lacking the LPA1 receptor received a single i.c.v. injection of LPA, Ki16425 or vehicle to assess the role of the LPA1 receptor in the LPA-induced increase of AHN. Our results revealed that the chronic administration of LPA decreased the retention of a previously acquired cocaine-induced CPP. This effect was mediated by an LPA-induced increase of AHN. In contrast, mice treated with Ki16425 showed reduced cocaine-CPP retention, but they increased their preference for the cocaine-paired compartment throughout CPP extinction. Besides, no effects of Ki16425 on AHN were found. Immunohistochemical studies suggested that LPA stimulated cell proliferation and promoted neuronal maturation with a key role of the LPA1 receptor. These findings emphasize the relevance of LPA and its LPA1 receptor as an in vivo modulator of AHN and the utility of the post-training increase of adult-born hippocampal neurons to weaken cocaine-context associations.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec