Pridopidine Reverses Phencyclidine-Induced Memory Impairment

Pridopidine is in clinical trials for Huntington's diseasetreatment. Originally developedas a dopamine D2receptor (D2R) ligand, pridopidine displays about 100-fold higheraffinity for the sigma-1 receptor (sigma-1R). Interestingly, pridopidine slows diseaseprogression and improves motor function in Huntington's disease model mice and,in preliminarily reports, Huntington's disease patients.The present study examinedthe anti-amnesic potential of pridopidine. Thus, memory impairment was produced inmice by administration of phencyclidine (PCP, 10 mg/kg/day) for 10 days, followedby 14 days' treatment with pridopidine (6 mg/kg/day), or saline. Finally, novel objectrecognition performance was assessed in the animals. Mice receiving PCP andsaline exhibited deficits in novel object recognition, as expected, while pridopidinetreatment counteracted PCP-induced memory impairment. The effect of pridopidine wasattenuated by co-administration of the sigma receptor antagonist, NE-100 (10 mg/kg).Our results suggest that pridopidine exerts anti-amnesic and potentially neuroprotectiveactions. These data provide new insights into the therapeutic potential of pridopidine asa pro-cognitive drug

Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson's Disease Post-Mortem Brain through a New AlphaScreen-Based Assay

Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity

Dopamine-induced arrestin recruitment and desensitization of the dopamine D4 receptor is regulated by G protein-coupled receptor kinase-2

The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor

Striatal dopamine D2-muscarinic acetylcholine M1 receptor-receptor interaction in a model of movement disorders

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic-cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic-cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D2 and muscarinic acetylcholine M1 receptors (D2R and M1R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D2R-M1R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET1 and NanoBiT®) assays, performed in transiently transfected HEK293T cells. Subsequently, a D2R-M1R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D2R-selective agonist (sumanirole) and/or an M1R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D2R/M1R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects

Development of a novel 1 receptor biosensor based on its heterodimerization with binding immunoglobulin protein in living cells

The σ1 receptor (S1R) is a ligand-regulated non-opioid intracellular receptor involved in several pathological conditions. The development of S1R-based drugs as therapeutic agents is a challenge due to the lack of simple functional assays to identify and classify S1R ligands. We have developed a novel nanoluciferase binary technology (NanoBiT) assay based on the ability of S1R to heteromerize with the binding immunoglobulin protein (BiP) in living cells. The S1R-BiP heterodimerization biosensor allows for rapid and accurate identification of S1R ligands by monitoring the dynamics of association-dissociation of S1R and BiP. Acute treatment of cells with the S1R agonist PRE-084 produced rapid and transient dissociation of the S1R-BiP heterodimer, which was blocked by haloperidol. The effect of PRE-084 was enhanced by calcium depletion, leading to a higher reduction in heterodimerization even in the presence of haloperidol. Prolonged incubation of cells with S1R antagonists (haloperidol, NE-100, BD-1047, and PD-144418) increased the formation of S1R-BiP heteromers, while agonists (PRE-084, 4-IBP, and pentazocine) did not alter heterodimerization under the same experimental conditions. The newly developed S1R-BiP biosensor is a simple and effective tool for exploring S1R pharmacology in an easy cellular setting. This biosensor is suitable for high-throughput applications and a valuable resource in the researcher's toolkit

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38β, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.We thank S Bartlett and F Chanut for English editing. We are grateful to RJ Davis, A Padmanabhan, M Costa and C López-Otín for critical reading of the manuscript. We thank Dr. RJ Davis for the MKK3 and MKK6 KO animals and Dr. Erwin F Wagner for the p38α flox mice. We thank AC Silva (ana@anasilva illustrations.com) for help with figure editing and design. This work was funded by a CNIC Intramural Project Severo Ochoa (Expediente 12–2016 IGP) to GS and JJ and PID2019-104399RB-I00 funded by MCIN/AEI/10.13039/501100011033 to GS. BGT was a fellow of FPI Severo Ochoa CNIC Program (SVP-2013-067639) and is an American Heart Association Postdoctoral Fellow (18POST34080175). RRB is a fellow of the FPU Program (FPU17/03847). The following grants provided additional funding: GS is granted by funds from European Regional Development Fund (ERDF): EFSD/Lilly European Diabetes Research Programme Dr Sabio, Fundación AECC PROYE19047SABI and Comunidad de Madrid IMMUNOTHERCAN-CM B2017/BMD-3733; US National Heart, Lung, and Blood Institute (R01 Grant HL122352), Fondos FEDER, Madrid, Spain, and Fundación Bancaria “La Caixa (project HR19/52160013); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); and Instituto de Salud Carlos III to JJ. IN was funded by EFSD/Lilly grants (2017 and 2019), the CNIC IPP FP7 Marie Curie Programme (PCOFUND-2012–600396), EFSD Rising Star award (2019), JDC-2018-Incorporación (MIN/JDC1802). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC FoundationS

p38γ is essential for cell cycle progression and liver tumorigenesis

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)–cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease

Acceptability and feasibility of a virtual community of practice to primary care professionals regarding patient empowerment : A qualitative pilot study

Background: Virtual communities of practice (vCoPs) facilitate online learning via the exchange of experiences and knowledge between interested participants. Compared to other communities, vCoPs need to overcome technological structures and specific barriers. Our objective was to pilot the acceptability and feasibility of a vCoP aimed at improving the attitudes of primary care professionals to the empowerment of patients with chronic conditions. Methods: We used a qualitative approach based on 2 focus groups: one composed of 6 general practitioners and the other of 6 practice nurses. Discussion guidelines on the topics to be investigated were provided to the moderator. Sessions were audio-recorded and transcribed verbatim. Thematic analysis was performed using the ATLAS-ti software. Results: The available operating systems and browsers and the lack of suitable spaces and time were reported as the main difficulties with the vCoP. The vCoP was perceived to be a flexible learning mode that provided up-to-date resources applicable to routine practice and offered a space for the exchange of experiences and approaches. Conclusions: The results from this pilot study show that the vCoP was considered useful for learning how to empower patients. However, while vCoPs have the potential to facilitate learning and as shown create professional awareness regarding patient empowerment, attention needs to be paid to technological and access issues and the time demands on professionals. We collected relevant inputs to improve the features, content and educational methods to be included in further vCoP implementation. Trial registration: ClinicalTrials.gov, NCT02757781. Registered on 25 April 2016

Acceptability and feasibility of a virtual community of practice to primary care professionals regarding patient empowerment: A qualitative pilot study

Background: Virtual communities of practice (vCoPs) facilitate online learning via the exchange of experiences and knowledge between interested participants. Compared to other communities, vCoPs need to overcome technological structures and specific barriers. Our objective was to pilot the acceptability and feasibility of a vCoP aimed at improving the attitudes of primary care professionals to the empowerment of patients with chronic conditions. Methods: We used a qualitative approach based on 2 focus groups: one composed of 6 general practitioners and the other of 6 practice nurses. Discussion guidelines on the topics to be investigated were provided to the moderator. Sessions were audio-recorded and transcribed verbatim. Thematic analysis was performed using the ATLAS-ti software. Results: The available operating systems and browsers and the lack of suitable spaces and time were reported as the main difficulties with the vCoP. The vCoP was perceived to be a flexible learning mode that provided up-to-date resources applicable to routine practice and offered a space for the exchange of experiences and approaches. Conclusions: The results from this pilot study show that the vCoP was considered useful for learning how to empower patients. However, while vCoPs have the potential to facilitate learning and as shown create professional awareness regarding patient empowerment, attention needs to be paid to technological and access issues and the time demands on professionals. We collected relevant inputs to improve the features, content and educational methods to be included in further vCoP implementation. Trial registration: ClinicalTrials.gov, NCT02757781. Registered on 25 April 2016.This study was financed by Instituto de Salud Carlos III and Cofinanced by Fondo Europeo de Desarrollo Regional (FEDER). Ministerio de Economía y Competitividad. Gobierno de España. (PI15/00164, PI15/00586, PI15/00566

Association Between Preexisting Versus Newly Identified Atrial Fibrillation and Outcomes of Patients With Acute Pulmonary Embolism

Background Atrial fibrillation (AF) may exist before or occur early in the course of pulmonary embolism (PE). We determined the PE outcomes based on the presence and timing of AF. Methods and Results Using the data from a multicenter PE registry, we identified 3 groups: (1) those with preexisting AF, (2) patients with new AF within 2 days from acute PE (incident AF), and (3) patients without AF. We assessed the 90-day and 1-year risk of mortality and stroke in patients with AF, compared with those without AF (reference group). Among 16 497 patients with PE, 792 had preexisting AF. These patients had increased odds of 90-day all-cause (odds ratio [OR], 2.81; 95% CI, 2.33-3.38) and PE-related mortality (OR, 2.38; 95% CI, 1.37-4.14) and increased 1-year hazard for ischemic stroke (hazard ratio, 5.48; 95% CI, 3.10-9.69) compared with those without AF. After multivariable adjustment, preexisting AF was associated with significantly increased odds of all-cause mortality (OR, 1.91; 95% CI, 1.57-2.32) but not PE-related mortality (OR, 1.50; 95% CI, 0.85-2.66). Among 16 497 patients with PE, 445 developed new incident AF within 2 days of acute PE. Incident AF was associated with increased odds of 90-day all-cause (OR, 2.28; 95% CI, 1.75-2.97) and PE-related (OR, 3.64; 95% CI, 2.01-6.59) mortality but not stroke. Findings were similar in multivariable analyses. Conclusions In patients with acute symptomatic PE, both preexisting AF and incident AF predict adverse clinical outcomes. The type of adverse outcomes may differ depending on the timing of AF onset.info:eu-repo/semantics/publishedVersio