Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression.

gma σ1 and σ2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ1R interacting with dopamine D1 and D2 receptors, the potential regulation of dopaminergic transmission by σ2R is also unknown. We here demonstrate that σ2R may form heteroreceptor complexes with D1 but not with D2 receptors. Remarkably σ1, σ2, and D1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ2R induces bias in signal transduction as σ2R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ1R, suggest that the D1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ1, σ2, and D1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit. KEYWORDS: ERK1/2 phosphorylation; acute; addiction; cAMP; chronic; dopamine D1 and D2 receptors; label-free; signalin

Differential effect of amphetamine over the corticotropin-releasing factor CRF2 receptor, the orexin OX1 receptor and the CRF2-OX1 heteroreceptor complex

Stress is one of the factors underlying drug seeking behavior that often goes in parallel with loss of appetite. We here demonstrate that orexin 1 receptors (OX1R) may form complexes with the corticotropin releasing factor CRF2 receptor. Two specific features of the heteromer were a cross-antagonism and a blockade by CRF2 of OX1R signaling. In cells expressing one of the receptors, agonist-mediated signal transduction mechanisms were potentiated by amphetamine. Sigma 1 (σ1) and 2 (σ2) receptors are targets of drugs of abuse and, despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is not known. We here show that σ1 receptors interact with CRF2 receptors and that σ2 receptors interact with OX1R. Moreover, we show that amphetamine effect on CRF2 receptors was mediated by σ1R whereas the effect on OX1 receptors was mediated by σ2R. Amphetamine did potentiate the negative cross-talk occurring within the CRF2-OX1 receptor heteromer context, likely by a macromolecular complex involving the two sigma receptors and the two GPCRs. Finally, in vivo microdialysis experiments showed that amphetamine potentiated orexin A-induced dopamine and glutamate release in the ventral tegmental area (VTA). Remarkably, the in vivo orexin A effects were blocked by a selective CRF2R antagonist. These results show that amphetamine impacts on the OX1R-, CRF2R- and OX1R/CRF2R-mediated signaling and that cross-antagonism is instrumental for in vivo detection of GPCR heteromers

Implicacion estructural y funcional de heterómeros de receptores acoplados a proteínas G de relevancia en adicción, enfermedad de Huntington y cáncer

[spa] La oligomerización es un proceso común en la biología de los receptores de membrana, compartida por los GPCRs. Los GPCRs constituyen aproximadamente un 45% de las dianas de los fármacos comercializados en la actualidad y siguen siendo el foco principal de la investigación biomédica y de los programas de desarrollo de nuevos fármacos. Aún así, el problema clave sigue siendo la falta de selectividad de los ligandos ortostéricos y la gran variedad de procesos que se ven modulados por la estimulación de un mismo GPCR dependiendo de su localización o interacción con otros receptores o proteínas. Los heterómeros de receptores se consideran un nuevo tipo de diana farmacológica por su posible especificidad de localización y por sus características bioquímicas y funcionales demostrablemente diferentes a las de sus componentes individuales, entre ellas las modulaciones alostéricas. El alosterismo puede ser debido a varias circunstancias entre las que destacan interacciones entre los protómeros del complejo y entre éstos y las proteínas G. Las consecuencias a nivel farmacológico y funcional son el reconocimiento diferencial de ligandos y la modulación del tráfico intracelular y del acoplamiento a proteínas G. El sistema orexinérgico es un buen ejemplo de sistema implicado en un amplio abanico de procesos fisiológicos y fisiopatológicos, desde el control de la ingesta y la regulación de los estados de sueño/vigilia a la regulación neuroendocrina y la participación en los mecanismos moleculares de adicción a drogas. La oligomerización de GPCRs constituye un proceso de regulación de orden superior poco explorado en el caso del receptor OX1 pero que se sugiere de gran relevancia para explicar los efectos moduladores de las orexinas. En esta Tesis se ha descrito por primera vez la formación de heterodímeros entre los receptores OX1, GHS-R1a y LEP y del heterotrímero LEP-GHS-R1a-OX1 en un sistema de expresión heteróloga, y se ha obtenido evidencia de la expresión del complejo heterotrimérico en neuronas hipotalámicas de rata. Estos resultados sugieren un papel importante de la oligomerización en la mediación de la respuesta combinada a señales orexigénicas y anorexigénicas originadas tanto a nivel central como periférico. En esta Tesis se describe también por primera vez la formación del heterómero de receptores CRF2-OX1 y su interacción con los receptores σ. El balance σ1/σ2 y la expresión de los heterómeros OX1/CRF2 en una célula determinada condicionan el efecto de la anfetamina y la señalización inducida por orexina y CRF. El sistema cannabinoide desempeña, asimismo, un marcado papel modulador en múltiples procesos fisiológicos y su desregulación se asocia con alteraciones patológicas, desde enfermedades neurodegenerativas a procesos de inflamación, desórdenes cardiovasculares y cáncer. La actividad neuroprotectora de los cannabinoides se ha descrito tanto in vivo como in vitro y hoy en día se investiga su gran potencial terapéutico en enfermedades neurodegenerativas. En esta Tesis se ha identificado una localización preferente del heterómero de receptores A2A-CB1 en proyecciones GABAérgicas del estriado dorsal, neuronas afectadas de manera paradigmática en la enfermedad de Huntington, y una disminución de la expresión del complejo en estadios avanzados de la enfermedad. Los resultados sugieren la importancia de funciones específicas asociadas a la formación de dicho complejo que se ven afectadas negativamente en estadios tardíos de la progresión de la enfermedad. La capacidad protectora de los cannabinoides se observa también en procesos oncogénicos. Los receptores CB2 y GPR55 se encuentran sobre-expresados en diversos tipos de tumores humanos y se ha descrito su implicación en el control del destino de las células tumorales. En esta Tesis se ha descrito la formación de heterodímeros de receptores CB2-GPR55 en células cancerígenas y cómo la modulación de estos complejos puede modificar la actividad anti-tumoral de los cannabinoides in vivo.[eng] The ‘receptor heteromer’ concept, in which receptors of the same and different gene families can combine among themselves to generate new and unique biochemical and functional characteristics, is becoming widely accepted for GPCRs and constitutes an emerging area in the field of GPCR cell signaling. Receptor heteromers must be understood as dimeric or higher order molecular entities that are the result of combinatorial evolution and they constitute novel therapeutic targets due to their possible specific location and their unique biochemical and functional properties, such as the allosteric modulations. Allosterism, as a result of interactions between individual protomers in the oligomer or between them and G proteins, can cause differential ligand binding and/or the modulation of intracellular trafficking and G protein coupling. In this context, the results here presented show the relevant role of GPCR oligomerization in different pathological processes such as addiction, Huntington’s disease and cancer, focusing on orexinergic and cannabinoid systems as modulatory systems involved in a wide range of physiological and pathophysiological processes. Here we describe for the first time the formation of heterodimers between LEP, GHS- R1A and OX1 receptors in a heterologous system, and the formation of heterotrimers involving the three receptors in hypothalamic primary cultures, suggesting an important role of these complexes in the response to a combination of different orexigenic and anorexigenic signals originated both centrally and peripherally. We demonstrate the formation of CRF2-OX1 receptor heteromers in a heterologous system and in the rat VTA. These receptors can interact also with sigma receptors, and the final balance between σ1/σ2 receptors and the expression of CRF2-OX1 receptor heteromers in a particular cell determines the effect of amphetamine and the signaling induced by orexin and CRF. Here we also identified the precise location of CB1-A2A receptor heteromers in the GABAergic projections in the dorsal striatum, that these complexes possess unique signaling properties and undergo an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Finally, we show that CB2R and GPR55 form receptor heteromers in cancer cells, that these structures possess unique signaling properties and that the modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo

Contemporary use of cefazolin for MSSA infective endocarditis: analysis of a national prospective cohort

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P <0.01) and previous renal failure (57.9%, P <0.01). Patients treated with cloxacillin presented higher rates of septic shock (25%, P = 0.033) and new-onset or worsening renal failure (47.3%, P = 0.024) with significantly higher rates of in-hospital mortality (38.5%, P = 0.017). One-year IE-related mortality and rate of relapses were similar between treatment groups. None of the treatments were identified as risk or protective factors. Conclusion: Our results suggest that cefazolin is a valuable option for the treatment of MSSA IE, without differences in 1-year mortality or relapses compared with cloxacillin, and might be considered equally effective

Chasing Gravitational Waves with the Chereknov Telescope Array

Presented at the 38th International Cosmic Ray Conference (ICRC 2023), 2023 (arXiv:2309.08219)2310.07413International audienceThe detection of gravitational waves from a binary neutron star merger by Advanced LIGO and Advanced Virgo (GW170817), along with the discovery of the electromagnetic counterparts of this gravitational wave event, ushered in a new era of multimessenger astronomy, providing the first direct evidence that BNS mergers are progenitors of short gamma-ray bursts (GRBs). Such events may also produce very-high-energy (VHE, > 100GeV) photons which have yet to be detected in coincidence with a gravitational wave signal. The Cherenkov Telescope Array (CTA) is a next-generation VHE observatory which aims to be indispensable in this search, with an unparalleled sensitivity and ability to slew anywhere on the sky within a few tens of seconds. New observing modes and follow-up strategies are being developed for CTA to rapidly cover localization areas of gravitational wave events that are typically larger than the CTA field of view. This work will evaluate and provide estimations on the expected number of of gravitational wave events that will be observable with CTA, considering both on- and off-axis emission. In addition, we will present and discuss the prospects of potential follow-up strategies with CTA

Chasing Gravitational Waves with the Chereknov Telescope Array

Presented at the 38th International Cosmic Ray Conference (ICRC 2023), 2023 (arXiv:2309.08219)2310.07413International audienceThe detection of gravitational waves from a binary neutron star merger by Advanced LIGO and Advanced Virgo (GW170817), along with the discovery of the electromagnetic counterparts of this gravitational wave event, ushered in a new era of multimessenger astronomy, providing the first direct evidence that BNS mergers are progenitors of short gamma-ray bursts (GRBs). Such events may also produce very-high-energy (VHE, > 100GeV) photons which have yet to be detected in coincidence with a gravitational wave signal. The Cherenkov Telescope Array (CTA) is a next-generation VHE observatory which aims to be indispensable in this search, with an unparalleled sensitivity and ability to slew anywhere on the sky within a few tens of seconds. New observing modes and follow-up strategies are being developed for CTA to rapidly cover localization areas of gravitational wave events that are typically larger than the CTA field of view. This work will evaluate and provide estimations on the expected number of of gravitational wave events that will be observable with CTA, considering both on- and off-axis emission. In addition, we will present and discuss the prospects of potential follow-up strategies with CTA

Performance of a proposed event-type based analysis for the Cherenkov Telescope Array

The Cherenkov Telescope Array (CTA) will be the next-generation observatory in the field of very-high-energy (20 GeV to 300 TeV) gamma-ray astroparticle physics. Classically, data analysis in the field maximizes sensitivity by applying quality cuts on the data acquired. These cuts, optimized using Monte Carlo simulations, select higher quality events from the initial dataset. Subsequent steps of the analysis typically use the surviving events to calculate one set of instrument response functions (IRFs). An alternative approach is the use of event types, as implemented in experiments such as the Fermi-LAT. In this approach, events are divided into sub-samples based on their reconstruction quality, and a set of IRFs is calculated for each sub-sample. The sub-samples are then combined in a joint analysis, treating them as independent observations. This leads to an improvement in performance parameters such as sensitivity, angular and energy resolution. Data loss is reduced since lower quality events are included in the analysis as well, rather than discarded. In this study, machine learning methods will be used to classify events according to their expected angular reconstruction quality. We will report the impact on CTA high-level performance when applying such an event-type classification, compared to the classical procedure

Chasing Gravitational Waves with the Chereknov Telescope Array

Presented at the 38th International Cosmic Ray Conference (ICRC 2023), 2023 (arXiv:2309.08219)2310.07413International audienceThe detection of gravitational waves from a binary neutron star merger by Advanced LIGO and Advanced Virgo (GW170817), along with the discovery of the electromagnetic counterparts of this gravitational wave event, ushered in a new era of multimessenger astronomy, providing the first direct evidence that BNS mergers are progenitors of short gamma-ray bursts (GRBs). Such events may also produce very-high-energy (VHE, > 100GeV) photons which have yet to be detected in coincidence with a gravitational wave signal. The Cherenkov Telescope Array (CTA) is a next-generation VHE observatory which aims to be indispensable in this search, with an unparalleled sensitivity and ability to slew anywhere on the sky within a few tens of seconds. New observing modes and follow-up strategies are being developed for CTA to rapidly cover localization areas of gravitational wave events that are typically larger than the CTA field of view. This work will evaluate and provide estimations on the expected number of of gravitational wave events that will be observable with CTA, considering both on- and off-axis emission. In addition, we will present and discuss the prospects of potential follow-up strategies with CTA