Pimavanserin exhibits serotonin 5-HT 2A receptor inverse agonism for G αi1 - and neutral antagonism for G αq/11 -proteins in human brain cortex

[EN] Pimavanserin is claimed as the first antipsychotic drug that shows selectivity for serotonin 5- HT 2 receptors (5-HT 2 Rs) and lacks of affinity for dopamine D 2 receptors (D 2 Rs). Cell-based func- tional assays suggest that pimavanserin and antipsychotics with D 2 R/5-HT 2 R affinity could act as inverse agonists of 5-HT 2A Rs. However, there is not evidence of such pharmacological profile in native brain tissue. 5-HT 2A Rs are able to engage both canonical G αq/11 - and non-canonical G αi1 -proteins. In the present study, the response to pimavanserin of the 5-HT 2A R coupling to G αq/11 - and G αi1 -proteins was measured in membranes of postmortem human prefrontal cortex by antibody-capture [ 35 S]GTP γS binding scintillation proximity assays. Pimavanserin promoted a concentration-dependant inhibition of the 5-HT 2A R coupling to G αi1 -proteins whereas the re- sponse of G αq/11 -proteins was unaltered, suggesting inverse agonism and neutral antagonism properties, respectively. The inhibition was abolished in the presence of the selective 5-HT 2A R antagonist MDL-11,939 and was absent in brain cortex of 5-HT 2A R knock-out mice when com- pared to respective 5-HT 2A R wild-type animals. In conclusion, the results demonstrate the ex- istence of constitutive 5-HT 2A R activity in human brain for the signalling pathway mediated by G αi1 -proteins. Pimavanserin demonstrates 5-HT 2A R functional selectivity and exhibits inverse agonist profile towards G αi1 -proteins, which is considered the effector pathway promoting hal- lucinogenic responses. In contrast, pimavanserin behaves as neutral antagonist on the 5-HT 2A R coupling to the canonical G αq/11 -protein pathway. The results strengthen the relevance of inverse agonism as potential mechanism of antipsychotic activity. Moreover, the existence of functional selectivity of 5-HT 2A Rs for different G α-proteins could contribute to better design of 5-HT 2A R-related antipsychotic drugs.Spanish Ministry of Science, Innovation and Universities and European ERDF Funds (SAF2009-08,460 and 2017–88,126-R), and the Basque Government (IT-1211–19 and KK-2019/00- 49). The authors would like to thank the staffmembers of the Basque Institute of Legal Medicine for their cooperation in the study. IM-A was recipient of a predoctoral fellowships from the Basque Government

A novel double-hit animal model of schizophrenia: behavioural assessment in male and female mice

Background: A growing body of evidence support that maternal prenatal infections represent a risk factor for schizophrenia in offspring. Moreover, stressful events during critical neurodevelopmental periods, such as adolescence, may trigger the onset of the disease in predisposed individuals. Thus, a prenatal priming event (i.e. maternal infection during pregnancy) that would induce vulnerability, followed by a second stressful hit in peripuberty may lead to the onset of schizophreniaDisclosure statement: Supported by the Basque Government (IT1211-19), the European Union's Horizon 2020 research and innovation programme (Marie Sklodowska-Curie grant agreement No 747487) and SAF 2017-88126-R. NC is recipient of a FPI fellowship (PRE2018-084002) from MICINN

Requirement of JNK-Mediated Phosphorylation for Translocation of Group IVA Phospholipase A2 to Phagosomes in Human Macrophages

Producción CientíficaEicosanoids are a broad family of lipids that play a critical role in host defense against bacterial and fungal infections. The first enzyme in the metabolic pathway for the generation of eicosanoids is group IVA phospholipase A(2), also known as cytosolic phospholipase A(2)alpha (cPLA(2)alpha). During phagocytosis, cPLA(2)alpha has been found to translocate to the phagosome, although the molecular mechanism involved in such a translocation has not been elucidated. By using enhanced GFP-tagged proteins we show in this work that a nonphosphorylatable cPLA(2)alpha mutant (S505A) does not translocate to the phagosomes, but a mutant that mimics phosphorylation on Ser(505) (S505E) does it so readily. During phagocytosis, endogenous cPLA(2)alpha is phosphorylated at Ser(505), and inhibitors of JNK, but not of other related kinases such as p38 or the extracellular-regulated kinases 1 and 2, completely block such a phosphorylation. Inhibition of JNK activity also inhibits the translocation of cPLA(2)alpha to phagosomal membranes, as well as arachidonic acid release to the extracellular medium. Moreover, the S505E mutant makes the enzyme refractory to JNK inhibition, translocating normally to phagosomal membranes. Collectively, these data support a key role for JNK-mediated cPLA(2)alpha phosphorylation at Ser(505) in the sequence of events leading to translocation and activation of the enzyme to phagosomal membranes in human macrophage

Paliperidone Reversion of Maternal Immune Activation-Induced Changes on Brain Serotonin and Kynurenine Pathways

Emerging evidence indicates that early-life exposure to environmental factors may increase the risk for schizophrenia via inflammatory mechanisms. Inflammation can alter the metabolism of tryptophan through the oxidative kynurenine pathway to compounds with neurotoxic and neuroprotective activity and compromise serotonin (5-HT) synthesis. Here we investigate the role of serotonergic and kynurenine pathways in the maternal immune activation (MIA) animal model of schizophrenia. The potential reversion exerted by long-term antipsychotic treatment was also evaluated. MIA was induced by prenatal administration of polyinosinic:polycytidylic acid (poly (I:C)) in mice. Expression of different proteins and the content of different metabolites involved in the function of serotonergic and kynurenine pathways was assessed by RT-PCR, immunoblot and ELISA analyses in frontal cortex of the offspring after puberty. MIA decreased tissue 5-HT content and promoted changes in the expression of serotonin transporter, 5-HT2A and 5-HT2C receptors. Expression of indoleamine 2,3-dioxygenase 2 (IDO2) and kynurenine 3-monooxygenase (KMO) was increased by poly (I:C) whereas kynurenine aminotransferase II and its metabolite kynurenic acid were not altered. Long-term paliperidone was able to counteract MIA-induced changes in 5-HT and KMO, and to increase tryptophan availability and tryptophan hydroxylase-2 expression in poly (I:C) mice but not in controls. MIA-induced increase of the cytotoxic risk ratio of kynurenine metabolites (quinolinic/kynurenic acid) was also reversed by paliperidone. MIA induces specific long-term brain effects on serotonergic activity. Such effects seem to be related with alternative activation of the kynurenine metabolic pathway towards a cytotoxic status. Atypical antipsychotic paliperodine partially remediates abnormalities observed after MIA.This work was supported by MINECO-FEDER Funds (SAF201675500-R to JL; SAF2017-88126-R to JM); Centro de Investigacion en Red de Salud Mental, CIBERSAM; and the Basque Government (IT1211-19). Editoria

Calpains as Potential Therapeutic Targets for Myocardial Hypertrophy

Calpain; Calpastatin; Myocardial hypertrophyCalpaína; Calpastatina; Hipertrofia miocárdicaCalpaïna; Calpastatina; Hipertròfia miocàrdicaDespite advances in its treatment, heart failure remains a major cause of morbidity and mortality, evidencing an urgent need for novel mechanism-based targets and strategies. Myocardial hypertrophy, caused by a wide variety of chronic stress stimuli, represents an independent risk factor for the development of heart failure, and its prevention constitutes a clinical objective. Recent studies performed in preclinical animal models support the contribution of the Ca2+-dependent cysteine proteases calpains in regulating the hypertrophic process and highlight the feasibility of their long-term inhibition as a pharmacological strategy. In this review, we discuss the existing evidence implicating calpains in the development of cardiac hypertrophy, as well as the latest advances in unraveling the underlying mechanisms. Finally, we provide an updated overview of calpain inhibitors that have been explored in preclinical models of cardiac hypertrophy and the progress made in developing new compounds that may serve for testing the efficacy of calpain inhibition in the treatment of pathological cardiac hypertrophy.This study was funded by the Instituto de Salud Carlos III of the Spanish Ministry of Health FIS-PI20/01681) and the research network CIBERCV (CB16/11/00479)

Characterization of dopamine D2 receptor coupling to G proteins in postmortem brain of subjects with schizophrenia

Background: Alterations of dopamine D-1 (D1R) and D-2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects. Methods: G-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [S-35]GTP gamma S-binding stimulation induced by increasing concentrations (10(-10)-10(-3) M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration-response curves to NPA stimulation of [S-35]GTP gamma S binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17). Results: In caudate, [S-35]GTP gamma S-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [S-35]GTP gamma S-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 mu M) and control (EC50H = 7.24 nM; EC50L = 15.14 mu M) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters. Conclusions: Feasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [S-35]GTP gamma S-binding assays appears to be restricted to signalling through inhibitory G(i/o) proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.This work was supported by the Spanish State Research Agency and EDR Funds (SAF-2017-88126-R to JJM;PID2019-106404RB-100 to LFC), the Basque Government (IT1211/19 to JJM ; ELKARTEK Programme KK-2019/00049 to RD-A), and the National Institutes of Health (R01MH084894 & NIH-R01MH111940 to JG-M)

Serotonin 5-HT2A, 5-HT2c and 5-HT1A receptor involvement in the acute effects of psilocybin in mice. In vitro pharmacological profile and modulation of thermoregulation and head-twich response

The psychedelic 5-HT2A receptor (5HT2AR) agonist psilocybin (or the active metabolite psilocin) has emerged as potential useful drug for various neuropsychiatric diseases, with a rapid onset of therapeutic activity. However, the mechanisms responsible for such effects remain incompletely characterized. We aimed to study in vitro pharmacological profile and in vivo acute mechanism of psilocin/psilocybin. Competition binding studies with psilocin were performed in brain and cell cultures. The role of 5HT2AR, 5-HT2C receptors (5HT2CR) and 5-HT1A receptors (5HT1AR) on the psychosis-like head-twitch response (HTR) and on body temperature in mice after psilocybin administration were evaluated. Psilocin showed similar affinities for 5HT2AR (Ki: 120-173 nM), 5HT2CR (Ki: 79-311 nM) and 5-HT1AR (Ki: 152-146 nM) in human and mice brain. Psilocybin induced a dosedependent HTR (maximal effect 17.07 +/- 1.31 at 1 mg/kg i.p.) that was completely suppressed by the 5HT2AR antagonist MDL11939 (1 mg/kg). Higher doses of psilocybin (3 mg/kg) induced lower HTR (9.00 +/- 0.53). The 5HT2CR antagonist SB242084 (0.1 mg/kg) increased HTR exerted by psilocybin (3 mg/kg). Psilocybin significantly raised core body temperature at low dose (0.125 mg/kg) (Emax=0.67 +/- 0.15 degrees C), whereas a significant decrease was induced by doses over 1 mg/kg (Emax = -1.31 +/- 0.16 degrees C). Pre-treatment with the 5HT1AR antagonist WAY100635 reversed the decrease of body temperature after psilocybin (1 mg/kg), causing hyperthermia (Emax = 0.94 +/- 0.26 degrees C). The present work provides key findings on the 5HT2AR, 5-HT2CR and 5HT1AR involvement in the acute central effects of psilocybin. The results may be relevant for understanding the mechanism of action underlying the therapeutic effects and side effects of this psychedelic drug.This work was supported by Grant PID2021-123508OB-I00, funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe, by the Basque Government (IT-1211-19; IT-1512-22) , by CIBER-Consorcio Centro de Investigacion Biomedica en Red- (CB/07/09/0008) , Instituto de Salud Carlos III, and by Fundacion Vital Funda-zioa (VITAL21/17) . IE-S received a predoctoral fellowship from the UPV/EHU

Intracellular inflammatory and antioxidant pathways in postmortem frontal cortex of subjects with major depression: effect of antidepressants

Background: Studies show that Toll-like receptors (TLRs), members of the innate immune system, might participate in the pathogenesis of the major depressive disorder (MDD). However, evidence of this participation in the brain of patients with MDD has been elusive. Methods: This work explores whether the protein expression by immunodetection assays (Western blot) of elements of TLR-4 pathways controlling inflammation and the oxidative/nitrosative stress are altered in postmortem dorsolateral prefrontal cortex of subjects with MDD. The potential modulation induced by the antidepressant treatment on these parameters was also assessed. Thirty MDD subjects (15 antidepressant-free and 15 under antidepressant treatment) were matched for gender and age to 30 controls in a paired design. Results: No significant changes in TLR-4 expression were detected. An increased expression of the TLR-4 endogenous ligand Hsp70 (+ 33%), but not of Hsp60, and the activated forms of mitogen-activated protein kinases (MAPKs) p38 (+ 47%) and JNK (+ 56%) was observed in MDD. Concomitantly, MDD subjects present a 45% decreased expression of DUSP2 (a regulator of MAPKs) and reduced (- 21%) expression of the antioxidant nuclear factor Nrf2. Antidepressant treatment did not modify the changes detected in the group with MDD and actually increased (+ 25%) the expression of p11, a protein linked with the transport of neurotransmitters and depression. Conclusion: Data indicate an altered TLR-4 immune response in the brain of subjects with MDD. Additional research focused on the mechanisms contributing to the antidepressant-induced TLR-4 pathway modulation is warranted and could help to develop new treatment strategies for MDD.This work was supported by the Instituto de Salud Carlos III and Spanish Ministry of Economy, Industry and Competitiveness (MINECO) through the Plan Estatal de I+D+i 2013-2016 (FIS-PI13/01102 and SAF2016-75500-R to JCL), the Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2017-83053-R to JRC), the Basque Government (IT-616-13), CIBERSAM and the EDR Funds. JRC and BGB are Ramon y Cajal fellows (MINECO)

Glucocorticoid receptor dysregulation underlies 5-HT2AR-dependent synaptic and behavioral deficits in a mouse neurodevelopmental disorder model.

Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with polyinosinic-polycytidylic acid potassium salt as a mouse MIA model, we show here that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulation of GR translocation in frontal cortex of schizophrenia subjects. We also found that repeated corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (adeno-associated virus) mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in the mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders.National Institutes of Health R01MH084894 (to J. G.-M.), R01MH111940 (to J. G.-M.), NIH-N01DA-17-8932 (to P. M. B.), NIH-N01DA-19-8949 (to P. M. B.), and F30MH116550 (to J. M. S.), and Basque Government IT1211-19 (to J. J. M.) participated in the funding of this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health