Signaling transduction regulated by 5-hydroxytryptamine 1A receptor and orexin receptor 2 heterodimers

As G-protein-coupled receptors (GPCRs), 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 2 (OX2R) regulate the levels of the cellular downstream molecules. The heterodimers of different GPCRs play important roles in various of neurological diseases. Moreover, 5-HT1AR and OX2R are involved in the pathogenesis of neurological diseases such as depression with deficiency of hippocampus plasticity. However, the direct interaction of the two receptors remains elusive. In the present study, we firstly demonstrated the heterodimer formation of 5-HT1AR and OX2R. Exchange protein directly activated by cAMP (Epac) cAMP bioluminescence resonance energy transfer (BRET) biosensor analysis revealed that the expression levels of cellular cAMP significantly increased in HEK293T cells transfected with the two receptors compared with the 5-HT1AR group. Additionally, the cellular level of calcium was upregulated robustly in HEK293T cells co-transfected with 5-HT1AR and OX2R group after agonist treatment. Furthermore, western blotting data showed that 5-HT1AR and OX2R heterodimer decreased the levels of phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB). These results not only unraveled the formation of 5-HT1AR and OX2R heterodimer but also suggested that the heterodimer affected the downstream signaling pathway, which will provide new insights into the function of the two receptors in the brain

Synthesis and Evaluation of C-10 Nitrogenated Aporphine Alkaloids at Serotonin and Dopamine Receptors

Aporphine alkaloids, belonging to the isoquinoline class of compounds, have been investigated as a potential source of ligands for Central Nervous System (CNS) receptors. Previous research indicates that the aporphine scaffold may be manipulated to synthesize selective ligands for serotonin and dopamine receptors. Novel aporphine alkaloids containing C10 nitrogen substitutions were synthesized, and their affinities were evaluated at serotonin (5-HT1A, 5-HT1B, 5-HT2A, 5-HT7A) receptors and dopamine (D1, D2, D3, D4, and D5) receptors. Two series of racemic aporphine compounds with C10 nitrogenous functionalities were synthesized and analyzed at the aforementioned receptors. The first series of aporphine alkaloids contain C10 nitro, amine, amide, and methanesulfonamide motifs. Compounds in this C10 monosubstituted series displayed higher affinity at 5-HT1AR and 5-HT7AR and lacked affinity at 5-HT1BR and 5-HT2AR. This series contained compounds with an N6-methyl group and compounds with and N6-propyl group. The N6-methyl substituted C10 nitrogen functionalized aporphine analogs had higher binding affinities at 5-HT7AR versus 5-HT1AR. In contrast the N6-propyl sub-set of compounds exhibited a reversal of this selectivity. Compound 103a was the most potent compound and behaved as an antagonist at 5-HT7AR (Ki = 4.5 ± 0.6 nM, IC50 = 1.25 μM), with 10-fold selectivity over 5-HT1AR (Ki = 49 ± 6.3 nM). These monosubstituted analogs lacked significant binding among all dopamine receptor subtypes. C10 analogs with a benzofused aminothiazole moiety showed higher affinity and selectivity for serotonin receptors as compared to the C10 monosubstituted compounds. These compounds displayed high binding affinities for 5-HT1AR and 5-HT7AR; analogs containing an N6-methyl substitution favor binding at 5-HT7AR. Among the benzofused aminothiazole analogs compound 108a had the best binding affinity at 5-HT7AR (Ki = 6.5±0.8 nM) and functions as an antagonist (IC50 = 0.26 μM). These benzofused aminothiazole analogs also lacked affinity for dopamine receptors. Unlike analogs in the C10 monosubstituted subset, compounds with the benzofused aminothiazole moiety with an N6-methyl substitution displayed moderate affinity for 5-HT1BR. The second series of compounds contained a C1,2,10-trisusbtitution pattern on the aporphine core. The 1,2,10-trisusbtituted series of compounds as a group displayed weak binding affinity at 5-HT1AR and considerably higher binding affinity at 5-HT1BR. These compounds provided moderate affinity at 5-HT2AR and 5-HT7AR. At dopamine receptors, most of the trisubstituted series of compounds failed to show affinity towards D5 receptors suggesting a lack of tolerability at D5R receptors for C10 N substituted aporphines with moderate to low affinity at D1R, thus attaining D1R versus D5R selectivity. Compound 128e was the most potent D1R ligand (Ki = 58 nM) and lacked binding affinity at all other dopamine receptor subtypes. Compounds 103a, 108a, and 128e have been identified as three new lead compounds with promising pharmacodynamic properties for further tool and pharmaceutical optimization

Characterization of an animal model of cognitive impairment associated with schizophrenia. Effets of alpha2-adrenoceptor compounds.

382 p.Schizophrenia is a multi multifaceted, heterogeneous, chronic and debilitating disorder triggered by a series of interacting genetic, developmental and environmental factors. Cognitive impairment is considered a core feature of this disorder and highly dependent on the correct functioning of the prefrontal cortex, however, current antipsychotics lack efficacy for treating this condition. Prenatal exposure to infection is contemplated as one the most significant environmental risk factors for developing schizophrenia in the offspring. In this context, maternal immune activation animal models produce neurochemical and behavioral alterations considered relevant for the study of schizophrenia dysfunctions.The first part of this thesis consists of a neurochemical and behavioral characterization of an animal model of cognitive impairment associated with schizophrenia by the administration of the immunostimulant agent Poly (I:C). The second part involves the study of the effects of ¿2-adrenoceptor compounds on cognitive performance in the offspring. Finally, selective targeting of the locus coeruleus-prefrontal cortex circuit by different optogenetic approaches was performed.The Poly (I:C) animal model shows a catecholaminergic hypofunction in the prefrontal cortex with marked cognitive deficits, which are reversed by the administration of 2A-adrenoceptor agonist guanfacine and the 2C-adrenoceptor antagonist MK-912.These results support the important role of the noradrenergic system in the prefrontal cortex-dependent cognitive functions and the Poly (I:C) animal model as a promising translational model of cognitive impairment associated with schizophrenia

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Cannabidiol as a fast-acting antidepressant: identification of the molecular mechanisms implicated in its antidepressant effect

RESUMEN: La depresión mayor es una enfermedad mental común y severa. Dado que los tratamientos actuales poseen importantes limitaciones, es necesario encontrar otros fármacos más rápidos y eficaces. El cannabidiol, el principal componente no psicoactivo del Cannabis sativa, se postula como un posible candidato. En esta tesis, la infusión de cannabidiol en la corteza infralímbica produjo un efecto agudo de tipo antidepresivo en ratas, asociado a un incremento de marcadores de plasticidad sináptica en la corteza prefrontal. Dicho efecto no se observó tras 24 horas, pero se recuperó tras el bloqueo de los receptores 5-HT1A presinápticos, juntamente con la activación de ERK en la corteza prefrontal. En el modelo neuroinflamatorio inducido por lipopolisacárido, la pre-administración sistémica de cannabidiol produjo un efecto de tipo antidepresivo en ratones, en el cual la activación de los receptores 5-HT1A postsinápticos tuvo un papel clave. El efecto conductual se asoció a una disminución de la activación de las vías NF-ĸB y de la quinurenina en cerebro, de los niveles de interleucina-6 en cerebro y plasma, y a la modulación de los niveles de glutamato y GABA cerebrales.ABSTRACT: Major depression is a common and severe mental illness. Given that current antidepressant treatments have significant limitations, it is necessary to find other faster and more effective drugs. Cannabidiol, the main non-psychoactive component of Cannabis sativa, is being postulated as a possible candidate. In this thesis, cannabidiol infusion into the infralimbic cortex produced an acute antidepressant-like effect in rats, which was associated with an increase in synaptic plasticity markers in the prefrontal cortex. This effect was not observed after 24 hours, but it was recovered after blocking the presynaptic 5-HT1A receptors, together with the activation of ERK in the prefrontal cortex. In the lipopolysaccharide-induced neuroinflammatory model, the systemic pre-administration of cannabidiol produced an antidepressant-like effect in mice, in which the activation of postsynaptic 5-HT1A receptors played a key role. The behavioral effect was associated with a decrease in the activation of the NF-ĸB and kynurenine pathways in the brain and of interleukin-6 levels in the brain and plasma, and with the modulation of brain glutamate and GABA levels.Este trabajo ha sido financiado por: - Ministerio de Economía y Competitividad (SAF2015‐67457‐R MINECO/FEDER). - Ministerio de Ciencia, Innovación y Universidades (RTI2018‐097534‐B‐I00). - Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III