Constitutive activity and drug functional selectivity of 5-HT2A receptors in post-mortem brain of subjects with schizophrenia

385 p.La esquizofrenia es una enfermedad mental crónica e incapacitante de etiología aún desconocida. El receptor 2A de serotonina (5-HT2AR) es a la vez diana de compuestos alucinógenos y de antipsicóticos atípicos. Los estudios de funcionalidad del receptor en tejido cerebral humano post-mortem de sujetos con esquizofrenia sugieren una hiperactividad del 5-HT2AR. En este contexto se ha demostrado la existencia de una mayor activación de la proteína G¿i1 por el agonista del 5-HT2AR (±)DOI en cerebro humano post-mortem de sujetos con esquizofrenia, sin alteraciones en la vía canónica, es decir la hiperactividad muestra selectividad funcional. La presente tesis doctoral evalúa el hecho de si esta mayor activación se debe a una alteración en la actividad constitutiva del 5-HT2AR sobre la vía alucinógena dependiente de la vía G¿i1, que está aún por demostrar, y para hacerlo es necesario el uso de agonistas inversos. Entre ellos, pimavanerina y altaserina se comportaron como agonistas inversos de la vía G¿i1 pero comoantagonistas neutros sobre la vía canónica G¿q11. En cambio, volinanserina actúa como agonista inverso también sobre la vía G¿q11. Por ello, pimavanserina y volinanserina se utilizaron como herramientas farmacológicas para estudiar la existencia de una mayor actividad constitutiva en cerebro humano post-mortem de sujetos con esquizofrenia. Estos estudios confirmaron la existencia de una mayor actividad constitutiva del 5-HT2AR en sujetos con esquizofrenia frente a sujetos control, mediadaselectivamente por la vía pro-alucinógena G¿i1, mientras que no se observan cambios en la vía canónica G¿q/11 del 5-HT2AR. Además, fármacos antipsicóticos atípicos como clozapina y risperidona presentaron un perfil selectivo como agonistas inversos de la vía G¿i1 del 5-HT2AR. Estos hallazgos apoyan la hipótesis de que nuevos fármacos que actúen selectivamente como agonistas inversos del 5-HT2AR sobre la vía pro-alucinógena, podrán presentar una mayor eficacia

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

The Role of the Dopamine System in Post-Stroke Mood Disorders in Newborn Rats

Post-stroke mood disorders (PSMD) affect disease prognosis in adults. Adult rodent models underlie the importance of the dopamine (DA) system in PSMD pathophysiology. There are no studies on PSMD after neonatal stroke. We induced neonatal stroke in 7-day-old (P7) rats by temporal left middle cerebral artery occlusion (MCAO). Performance in the tail suspension test (TST) at P14 and the forced swimming test (FST) and open field test (OFT) at P37 were studied to assess PSMD. DA neuron density in the ventral tegmental area, brain DA concentration and DA transporter (DAT) expression as well as D2 receptor (D2R) expression and G-protein functional coupling were also studied. MCAO animals revealed depressive-like symptoms at P14 associated with decreased DA concentration and reduced DA neuron population and DAT expression. At P37, MCAO rats showed hyperactive behavior associated with increased DA concentration, normalization of DA neuron density and decreased DAT expression. MCAO did not modify D2R expression but reduced D2R functionality at P37. MCAO-induced depressive-like symptoms were reversed by the DA reuptake inhibitor GBR-12909. In conclusion, MCAO in newborn rats induced depressive-like symptoms and hyperactive behavior in the medium and long term, respectively, that were associated with alterations in the DA system.This research was funded by the PI19/00927, PID2019-106404RB-I00 and RD21/0012/0023 projects, integrated in the Spanish Plan for R + D + I, AES 2017–2020; funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) “A way to make Europe” (J.M.-O.) and the Irycis Chromatographic Services and Nervous System Markers Unit, UCS (2018/0135) (M.J.C.)

Exploring the reactivity of bicyclic α-iminophosphonates to access new imidazoline I<inf>2</inf> receptor ligands

Recent studies pointed out the modulation of imidazoline I2 receptors (I2-IR) by selective ligands as a putative strategy to face neurodegenerative diseases. Foregoing the classical 2-imidazoline/imidazole-containing I2-IR ligands, we report a family of bicyclic α-iminophosphonates endowed with high affinity and selectivity upon I2-IR and we advanced a representative compound B06 in preclinical phases. In this paper, we describe the synthetic possibilities of bicyclic α-iminophosphonates by exploring its ambivalent reactivity, leading to unprecedented molecules that showed promising activities as I2-IR ligands in human brain tissues and good BBB permeation capabilities. After in silico ADME prediction studies, we assessed the neuroprotective properties of selected compounds and beneficial effect in an in vitro model of Alzheimeŕs and Parkinson's disease. Along with their neuroprotective effect, compounds showed a potent anti-inflammatory response when evaluated in a neuroinflammation cellular model. Moreover, this is the first time that the neuroprotective effects of imidazoline I2-IR ligands in a transgenic Alzheimer's disease Caenorhabditis elegans strain are investigated. Using a thrashing assay, we found a significant cognition improvement in this in vivo model after treatment with the new bicyclic α-phosphoprolines. Therefore, our results confirmed the need of exploring structurally new I2-IR ligands and their potential for therapeutic strategies in neurodegeneration.This work was supported by Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (Spain, PID2019-107991RB-I00, PID2022-1380790B-I00), Basque Government (IT-1211-19 and 1512-22), Generalitat de Catalunya (GC) (2021 SGR 00357) and PDC2022-133441-I00 (MCIN/AEI/ 10.13039/501100011033 and by the “European Union NextGenerationEU/PRTR) and by UCM-Santander (PR44/21-29931 to J.A.M.-G.). The project leading to these results has received funding from “la Caixa” Foundation (ID 100010434) under agreement CI18-00002. This activity has received funding from the European Institute of Innovation and Technology (EIT). This body of the European Union receives support from the European Union’s Horizon 2020 research and innovation programme.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Exploring the reactivity of bicyclic α-iminophosphonates to access new imidazoline I2 receptor ligands

Recent studies pointed out the modulation of imidazoline I2 receptors (I2-IR) by selective ligands as a putative strategy to face neurodegenerative diseases. Foregoing the classical 2-imidazoline/imidazole-containing I2-IR ligands, we report a family of bicyclic α-iminophosphonates endowed with high affinity and selectivity upon I2-IR and we advanced a representative compound B06 in preclinical phases. In this paper, we describe the synthetic possibilities of bicyclic α-iminophosphonates by exploring its ambivalent reactivity, leading to unprecedented molecules that showed promising activities as I2-IR ligands in human brain tissues and good BBB permeation capabilities. After in silico ADME prediction studies, we assessed the neuroprotective properties of selected compounds and beneficial effect in an in vitro model of Alzheimeŕs and Parkinson’s disease. Along with their neuroprotective effect, compounds showed a potent anti-inflammatory response when evaluated in a neuroinflammation cellular model. Moreover, this is the first time that the neuroprotective effects of imidazoline I2-IR ligands in a transgenic Alzheimer’s disease Caenorhabditis elegans strain are investigated. Using a thrashing assay, we found a significant cognition improvement in this in vivo model after treatment with the new bicyclic α-phosphoprolines. Therefore, our results confirmed the need of exploring structurally new I2-IR ligands and their potential for therapeutic strategies in neurodegeneration.Depto. de Biología CelularFac. de MedicinaTRUEpu

The Role of the Dopamine System in Post-Stroke Mood Disorders in Newborn Rats

Post-stroke mood disorders (PSMD) affect disease prognosis in adults. Adult rodent models underlie the importance of the dopamine (DA) system in PSMD pathophysiology. There are no studies on PSMD after neonatal stroke. We induced neonatal stroke in 7-day-old (P7) rats by temporal left middle cerebral artery occlusion (MCAO). Performance in the tail suspension test (TST) at P14 and the forced swimming test (FST) and open field test (OFT) at P37 were studied to assess PSMD. DA neuron density in the ventral tegmental area, brain DA concentration and DA transporter (DAT) expression as well as D2 receptor (D2R) expression and G-protein functional coupling were also studied. MCAO animals revealed depressive-like symptoms at P14 associated with decreased DA concentration and reduced DA neuron population and DAT expression. At P37, MCAO rats showed hyperactive behavior associated with increased DA concentration, normalization of DA neuron density and decreased DAT expression. MCAO did not modify D2R expression but reduced D2R functionality at P37. MCAO-induced depressive-like symptoms were reversed by the DA reuptake inhibitor GBR-12909. In conclusion, MCAO in newborn rats induced depressive-like symptoms and hyperactive behavior in the medium and long term, respectively, that were associated with alterations in the DA system