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

Long-lasting memory deficits in mice withdrawn from cocaine are concomitant with neuroadaptations in hippocampal basal activity, GABAergic interneurons and adult neurogenesis

Cocaine addiction disorder is notably aggravated by concomitant cognitive and emotional pathology that impedes recovery. We studied whether a persistent cognitive/emotional dysregulation in mice withdrawn from cocaine holds a neurobiological correlate within the hippocampus, a limbic region with a key role in anxiety and memory but that has been scarcely investigated in cocaine addiction research. Mice were submitted to a chronic cocaine (20 mg/kg/day for 12 days) or vehicle treatment followed by 44 drug-free days. Some mice were then assessed on a battery of emotional (elevated plus-maze, light/dark box, open field, forced swimming) and cognitive (object and place recognition memory, cocaine-induced conditioned place preference, continuous spontaneous alternation) behavioral tests, while other mice remained in their home cage. Relevant hippocampal features [basal c-Fos activity, GABA+, parvalbumin (PV)+ and neuropeptide Y (NPY)+ interneurons and adult neurogenesis (cell proliferation and immature neurons)] were immunohistochemically assessed 73 days after the chronic cocaine or vehicle protocol. The cocaine-withdrawn mice showed no remarkable exploratory or emotional alterations but were consistently impaired in all the cognitive tasks. All the cocaine-withdrawn groups, independent of whether they were submitted to behavioral assessment or not, showed enhanced basal c-Fos expression and an increased number of GABA+ cells in the dentate gyrus. Moreover, the cocaine-withdrawn mice previously submitted to behavioral training displayed a blunted experience-dependent regulation of PV+ and NPY+ neurons in the dentate gyrus, and neurogenesis in the hippocampus. Results highlight the importance of hippocampal neuroplasticity for the ingrained cognitive deficits present during chronic cocaine withdrawal

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

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)

Inactivation of human plasma alters the structure and biomechanical properties of engineered tissues

Acknowledgments The authors are grateful to Echevarne Laboratories for the coagulation experiments and to Gloria Carmona and Rosario Sánchez Pernaute as well as all members of the Unidad de Producción y Reprogramación Celular (UPRC) for technical help and support. All figures were created through BioRender.com accessed on 1 September 2021.Funding This research was funded by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III), grants FIS PI17/0391, RTC-2017- 6658-1, PI20/0317 and ICI19/00024 (BIOCLEFT), co-financed by Fondo Europeo de Desarrollo Regional ERDF-FEDER, European Union and PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain.Fibrin is widely used for tissue engineering applications. The use of blood derivatives, however, carries a high risk of transmission of infectious agents, necessitating the application of pathogen reduction technology (PRT). The impact of this process on the structural and biomechanical properties of the final products is unknown. We used normal plasma (PLc) and plasma inactivated by riboflavin and ultraviolet light exposure (PLi) to manufacture nanostructured cellularized fibrin-agarose hydrogels (NFAHs), and then compared their structural and biomechanical properties. We also measured functional protein C, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and coagulation factors [fibrinogen, Factor (F) V, FVIII, FX, FXI, FXIII] in plasma samples before and after inactivation. The use of PLi to manufacture cellularized NFAHs increased the interfibrillar spacing and modified their biomechanical properties as compared with cellularized NFAH manufactured with PLc. PLi was also associated with a significant reduction in functional protein C, FV, FX, and FXI, and an increase in the international normalized ratio (derived from the PT), APTT, and TT. Our findings demonstrate that the use of PRT for fibrin-agarose bioartificial tissue manufacturing does not adequately preserve the structural and biomechanical properties of the product. Further investigations into PRT-induced changes are warranted to determine the applications of NFAH manufactured with inactivated plasma as a medicinal product.Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III), grants FIS PI17/0391, RTC-2017- 6658-1, PI20/0317 and ICI19/00024 (BIOCLEFT)Fondo Europeo de Desarrollo Regional ERDF-FEDEREuropean UnionPE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spai

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

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

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

What role does the LPA1 receptor play in regulating emotional-like behaviours?

The 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 proposed that this receptor has a role in controlling anxiety-like behaviours and in the detrimental consequences of stress. In general, the neurobiological mechanism of fear extinction is strikingly similar to that of the adaptative stress response (distress regulation), sharing similar neuroanatomical, neuroendocrine, and neurochemical basis. Inadequate control of the stress response could precipitate or provoke anxiety disorders. In this context, we tried to elucidate the LPA1 receptor involvement in emotional regulation. For this purpose, we first examined fear extinction, a type of emotional regulation, in normal wild-type (wt) and maLPA1-null mice using two different extinction procedures (cued fear extinction and contextual fear extinction). Additionally, to study the role of the LPA1 receptor in the absence of developmental abnormalities induced by its permanent loss, the effect of the LPA1 antagonist Ki16425 administration was examined in contextual fear extinction on wild-type mice. Next, we studied the consequences of the absence of the LPA1 receptor in two key areas involved in emotional regulation, characterizing the structure and GABAergic composition of the medial prefrontal cortex (mPFC) and the amygdala by immunohistochemical detection of neuron specific nuclear protein (NeuN), GABA-positive cells and calcium-binding proteins (calretinin (CR), parvalbumin (PV), and calbindin (CB)). Lastly, we examined the corticosterone response and the expression of a marker of neuronal activity, c-Fos protein, in the amygdala and the mPFC after acute stress. Our results revealed that lack of the LPA1-receptor induces exaggerated amygdala reactivity and endocrine responses to emotional stimuli (e.g., an acute episode of stress), revealing a role of the LPA1 receptor in regulating emotional-like behaviours. Considering that a reduction of GABAergic inhibitory control in the amygdala may be a common mechanism to generate a heightened emotional state, the abnormal emotional response reported in LPA1-null mice could be explained, at least in part, by a significant reduction of GABAérgic composition of the amygdala observed in these animals. Taking together, the LPA1 receptor is involved in emotional behaviours and in the anatomical integrity of the corticolimbic circuit, the deregulation of which may be a susceptibility factor for anxiety disorders and a potential therapeutic target for the treatment of these diseases.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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