Publications by authors named "Víctor Fernández-Dueñas"

71 Publications

Ecto-GPR37: a potential biomarker for Parkinson's disease.

Transl Neurodegener 2021 Feb 26;10(1). Epub 2021 Feb 26.

Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, L'Hospitalet de Llobregat, Spain.

Objective: α-Synuclein has been studied as a potential biomarker for Parkinson's disease (PD) with no concluding results. Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD.

Methods: GPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer's disease (AD) patients.

Results: GPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered.

Conclusion: GPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.
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http://dx.doi.org/10.1186/s40035-021-00232-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908677PMC
February 2021

Decreased striatal adenosine A-dopamine D receptor heteromerization in schizophrenia.

Neuropsychopharmacology 2021 02 3;46(3):665-672. Epub 2020 Oct 3.

Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, UB, L'Hospitalet de Llobregat, Barcelona, Spain.

According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A receptors (ARs) or their degree of functional heteromerization with dopamine D receptors (DR). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native AR-DR heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal DR without changes in AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal AR-DR heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of AR-DR heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both DR and AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal AR-DR heteromers. The degree of AR-DR heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.
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http://dx.doi.org/10.1038/s41386-020-00872-9DOI Listing
February 2021

Striatal Dopamine D-Muscarinic Acetylcholine M Receptor-Receptor Interaction in a Model of Movement Disorders.

Front Pharmacol 2020 13;11:194. Epub 2020 Mar 13.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

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 D and muscarinic acetylcholine M receptors (DR and MR, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of DR-MR complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET and NanoBiT) assays, performed in transiently transfected HEK293T cells. Subsequently, a DR-MR 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 DR-selective agonist (sumanirole) and/or an MR-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 DR/MR heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.
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http://dx.doi.org/10.3389/fphar.2020.00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083216PMC
March 2020

Design, Synthesis and Characterization of a New Series of Fluorescent Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulators.

Molecules 2020 Mar 27;25(7). Epub 2020 Mar 27.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat de Barcelona, 08907 L'Hospitalet de Llobregat, Spain.

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter' systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGluR) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGluRs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGluR allosteric modulators.
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http://dx.doi.org/10.3390/molecules25071532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180738PMC
March 2020

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BMC Biol 2020 01 23;18(1). Epub 2020 Jan 23.

Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD, 21224, USA.

Background: It has been hypothesized that heteromers of adenosine A receptors (A2AR) and cannabinoid CB receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals.

Results: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A receptors or dopamine D receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level.

Conclusions: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.
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http://dx.doi.org/10.1186/s12915-020-0739-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979073PMC
January 2020

Adenosine A-A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects.

Cells 2019 12 13;8(12). Epub 2019 Dec 13.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat de Barcelona, 08907 L'Hospitalet de Llobregat, Spain.

Guanosine, a guanine-based purine nucleoside, has been described as a neuromodulator that exerts neuroprotective effects in animal and cellular ischemia models. However, guanosine's exact mechanism of action and molecular targets have not yet been identified. Here, we aimed to elucidate a role of adenosine receptors (ARs) in mediating guanosine effects. We investigated the neuroprotective effects of guanosine in hippocampal slices from AR-deficient mice (AR) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective AR antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged AR. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of AR and AR co-expression in guanosine-mediated impedance responses in living cells. Guanosine prevented the reduction of cellular viability and increased reactive oxygen species generation induced by OGD in hippocampal slices from wild-type, but not from AR mice. Notably, while guanosine was not able to modify MRS7396 binding to AR-expressing cells, a partial blockade was observed in cells co-expressing AR and AR. The relevance of the AR and AR interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked AR agonist-mediated effects in doubly expressing AR and AR cells. Interestingly, while guanosine did not affect AR/AR heteromer formation, it reduced AR agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both AR and AR co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.
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http://dx.doi.org/10.3390/cells8121630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953045PMC
December 2019

Proximity Ligation Assay Image Analysis Protocol: Addressing Receptor-Receptor Interactions.

Methods Mol Biol 2019 ;2040:41-50

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Proximity ligation assay (PLA) is an antibody-based method that permits studying protein-protein interactions with high specificity and sensitivity. In brief, when a pair of specific antibodies is in close proximity, the complementary DNA strands they bear engage into a rolling circle amplification and generate, in situ, a single fluorescent signal, which indicates the presence of a protein-protein interaction. Proper image analysis methods are needed to provide accurate quantitative assessment of the obtained fluorescent signals, namely, PLA data. In this chapter, we outline basic aspects of image analysis (including software, data import, image processing functions, and analytical tools) that can be used to extract PLA data from confocal microscopy images using ImageJ. A step-by-step protocol to determine and quantify PLA fluorescence signals is included. Overall, the accurate capture and subsequent analysis of PLA confocal images constitutes a crucial step to properly interpret data obtained with this powerful experimental approach.
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http://dx.doi.org/10.1007/978-1-4939-9686-5_3DOI Listing
April 2020

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

Int J Mol Sci 2019 Jul 23;20(14). Epub 2019 Jul 23.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat de Barcelona, 08907 L'Hospitalet de Llobregat, Spain.

: 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. : Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. : 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. : The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.
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http://dx.doi.org/10.3390/ijms20143600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678849PMC
July 2019

L-Serine dietary supplementation is associated with clinical improvement of loss-of-function -related pediatric encephalopathy.

Sci Signal 2019 06 18;12(586). Epub 2019 Jun 18.

Bellvitge Biomedical Research Institute (IDIBELL)-Unit of Neuropharmacology and Pain, University of Barcelona, Barcelona 08908, Spain.

Autosomal dominant mutations in are associated with severe encephalopathy, but little is known about the pathophysiological outcomes and any potential therapeutic interventions. Genetic studies have described the association between de novo mutations of genes encoding the subunits of the -methyl-d-aspartate receptor (NMDAR) and severe neurological conditions. Here, we evaluated a missense mutation in , causing a proline-to-threonine switch (P553T) in the GluN2B subunit of NMDAR, which was found in a 5-year-old patient with Rett-like syndrome with severe encephalopathy. Structural molecular modeling predicted a reduced pore size of the mutant GluN2B-containing NMDARs. Electrophysiological recordings in a HEK-293T cell line expressing the mutated subunit confirmed this prediction and showed an associated reduced glutamate affinity. Moreover, GluN2B(P553T)-expressing primary murine hippocampal neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of the AMPA receptor subunit GluA1 at stimulated synapses. Furthermore, the naturally occurring coagonist d-serine restored function to GluN2B(P553T)-containing NMDARs. l-Serine dietary supplementation of the patient was hence initiated, resulting in the increased abundance of d-serine in the plasma and brain. The patient has shown notable improvements in motor and cognitive performance and communication after 11 and 17 months of l-serine dietary supplementation. Our data suggest that l-serine supplementation might ameliorate -related severe encephalopathy and other neurological conditions caused by glutamatergic signaling deficiency.
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http://dx.doi.org/10.1126/scisignal.aaw0936DOI Listing
June 2019

Optical Modulation of Metabotropic Glutamate Receptor Type 5 In Vivo Using a Photoactive Drug.

Methods Mol Biol 2019 ;1947:351-359

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Barcelona, Spain.

Optopharmacology is a very promising approach based on the use of light-deliverable drugs, which allows manipulating physiological processes with high spatiotemporal resolution. Light-dependent drugs (i.e. caged-compounds) targeting G protein-coupled receptors (GPCRs) have been developed to provide great pharmacological precision on the control of pain. Metabotropic glutamate type 5 (mGlu) receptors are widely expressed through the pain neuraxis and play a key role in pain transmission. In line with this, selective mGlu receptor negative allosteric modulators (NAMs) have consistently shown analgesic activity in experimental animal models of inflammatory pain. Accordingly, we synthesized a light-deliverable drug (i.e. caged compound) using the chemical structure of raseglurant, a mGlu receptor NAM, as a molecular scaffold. And thereafter, we evaluated the analgesic activity of the caged compound in formalin-injected (hind paw) mice upon light irradiation (405 nm). Of note, light was both delivered at the peripheral (i.e. hind paw) and central level (i.e. thalamus), by means of brain-implanted fiber-optics. The novel light-deliverable drug, JF-NP-26, showed antinociceptive activity upon violet light irradiation either of the hind paw or the thalamus, demonstrating the ability of precisely activating, in time and space, the caged compound. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin nociception test in mice using an optopharmacology approach (i.e. light-deliverable compounds).
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http://dx.doi.org/10.1007/978-1-4939-9121-1_20DOI Listing
August 2019

New ionic targets of 3,3',5'-triiodothyronine at the plasma membrane of rat Sertoli cells.

Biochim Biophys Acta Biomembr 2019 04 9;1861(4):748-759. Epub 2019 Jan 9.

Departamento de Bioquímica - Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Cx. Postal 5069, CEP: 88040-970 Florianópolis, SC, Brazil; Núcleo de Bioeletricidade Celular (NUBIOCEL) - Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Cx. Postal 5069, CEP: 88040-970 Florianópolis, SC, Brazil. Electronic address:

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT on calcium (Ca) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT. Our results indicate that rT induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT occurred in <5 min in Sertoli cells. In addition, the rT metabolite, T, also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na/K ATPase pump. The rapid response of rT is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells.
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http://dx.doi.org/10.1016/j.bbamem.2019.01.002DOI Listing
April 2019

Adenosine A-Cannabinoid CB Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ-Tetrahydrocannabinol-Induced Cognitive Impairment.

Mol Neurobiol 2019 Aug 4;56(8):5382-5391. Epub 2019 Jan 4.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-Universitat de Barcelona, C/Feixa Llarga s/n, 08907, L'Hospitalet de Llobregat, Spain.

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ-tetrahydrocannabinol (Δ-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ-THC-induced cognitive impairment in an adenosine A receptor (AR)-dependent manner. Furthermore, we reveal the existence of AR and cannabinoid CB receptor (CBR) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent AR-CBR functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal AR/CBR complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of AR-CBR interactions in the brain.
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http://dx.doi.org/10.1007/s12035-018-1456-3DOI Listing
August 2019

Chronic adenosine A receptor blockade induces locomotor sensitization and potentiates striatal LTD IN GPR37-deficient mice.

J Neurochem 2019 03 28;148(6):796-809. Epub 2019 Jan 28.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Adenosine A receptors (A R) play a key role in modulating dopamine-dependent locomotor activity, as heralded by the sensitization of locomotor activity upon chronic A R blockade, which is associated with elevated dopamine levels and altered corticostriatal synaptic plasticity. Since the orphan receptor GPR37 has been shown to modulate A R function in vivo, we aimed to test whether the A R-mediated sensitization of locomotor activity is GPR37-dependent and involves adaptations of synaptic plasticity. To this end, we administered a selective A R antagonist, SCH58261 (1 mg/kg, i.p.), daily for 14 days, and the locomotor sensitization, striatum-dependent cued learning, and corticostriatal synaptic plasticity (i.e., long-term depression) were compared in wild-type and GPR37 mice. Notably, GPR37 deletion promoted A R-associated locomotor sensitization but not striatum-dependent cued learning revealed upon chronic SCH58261 treatment of mice. Furthermore, chronic A R blockade potentiated striatal long-term depression in corticostriatal synapses of GPR37 but not of wild-type mice, thus correlating well with neurochemical alterations of the adenosinergic system. Overall, these results revealed the importance of GPR37 regulating A R-dependent locomotor sensitization and synaptic plasticity in the basal ganglia circuitry. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
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http://dx.doi.org/10.1111/jnc.14653DOI Listing
March 2019

Dopamine receptor heteromers: biasing antipsychotics.

Future Med Chem 2018 12 6;10(23):2675-2677. Epub 2018 Dec 6.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

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http://dx.doi.org/10.4155/fmc-2018-0335DOI Listing
December 2018

Neuromodulatory Effects of Guanine-Based Purines in Health and Disease.

Front Cell Neurosci 2018 23;12:376. Epub 2018 Oct 23.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

The function of guanine-based purines (GBPs) is mostly attributed to the intracellular modulation of heteromeric and monomeric G proteins. However, extracellular effects of guanine derivatives have also been recognized. Thus, in the central nervous system (CNS), a guanine-based purinergic system that exerts neuromodulator effects, has been postulated. The thesis that GBPs are neuromodulators emerged from and studies, in which neurotrophic and neuroprotective effects of these kinds of molecules (i.e., guanosine) were demonstrated. GBPs induce several important biological effects in rodent models and have been shown to reduce seizures and pain, stabilize mood disorder behavior and protect against gliomas and diseases related with aging, such as ischemia or Parkinson and Alzheimer diseases. studies to evaluate the protective and trophic effects of guanosine, and of the nitrogenous base guanine, have been fundamental for understanding the mechanisms of action of GBPs, as well as the signaling pathways involved in their biological roles. Conversely, although selective binding sites for guanosine have been identified in the rat brain, GBP receptors have not been still described. In addition, GBP neuromodulation may depend on the capacity of GBPs to interact with well-known membrane proteins in glutamatergic and adenosinergic systems. Overall, in this review article, we present up-to-date GBP biology, focusing mainly on the mechanisms of action that may lead to the neuromodulator role of GBPs observed in neurological disorders.
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http://dx.doi.org/10.3389/fncel.2018.00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232889PMC
October 2018

PBF509, an Adenosine A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders.

Front Pharmacol 2018 19;9:1200. Epub 2018 Oct 19.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Adenosine A receptor (AR) antagonists have emerged as complementary non-dopaminergic drugs to alleviate Parkinson's disease (PD) symptomatology. Here, we characterize a novel non-xhantine non-furan AR antagonist, PBF509, as a potential pro-dopaminergic drug for PD management. First, PBF509 was shown to be a highly potent ligand at the human AR, since it antagonized AR agonist-mediated cAMP accumulation and impedance responses with K values of 72.8 ± 17.4 and 8.2 ± 4.2 nM, respectively. Notably, these results validated our new AR-based label-free assay as a robust and sensitive approach to characterize AR ligands. Next, we evaluated the efficacy of PBF509 reversing motor impairments in several rat models of movement disorders, including catalepsy, tremor, and hemiparkinsonism. Thus, PBF509 (orally) antagonized haloperidol-mediated catalepsy, reduced pilocarpine-induced tremulous jaw movements and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in unilaterally 6-OHDA-lesioned rats. Moreover, PBF509 (3 mg/kg) inhibited L-DOPA-induced dyskinesia (LID), showing not only its efficacy on reversing parkinsonian motor impairments but also acting as antidyskinetic agent. Overall, here we describe a new orally selective AR antagonist with potential utility for PD treatment, and for some of the side effects associated to the current pharmacotherapy (i.e., dyskinesia).
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http://dx.doi.org/10.3389/fphar.2018.01200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202948PMC
October 2018

Triglyceride Form of Docosahexaenoic Acid Mediates Neuroprotection in Experimental Parkinsonism.

Front Neurosci 2018 28;12:604. Epub 2018 Aug 28.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina, Institut d'Investigació Biomèdica de Bellvitge, Universitat de Barcelona, Barcelona, Spain.

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. The main treatment of PD consists of medication with dopamine-based drugs, which palliate the symptoms but may produce adverse effects after chronic administration. Accordingly, there is a need to develop novel neuroprotective therapies. Several studies suggest that omega-3 polyunsaturated fatty acids (-3 PUFA) might provide protection against brain damage. Here, we studied several experimental models of PD, using striatal neuronal cultures, striatal slices, and mice, to assess the neuroprotective effects of docosahexaenoic acid (DHA), the main -3 PUFA in the brain, administered in its triglyceride form (TG-DHA). Hence, we determined the beneficial effects of TG-DHA on neural viability following 6-hydroxydopamine (6-OHDA)-induced neurotoxicity, a well-established PD model. We also implemented a novel mouse behavioral test, the beam walking test, to finely assess mouse motor skills following dopaminergic denervation. This test showed potential as a useful behavioral tool to assess novel PD treatments. Our results indicated that TG-DHA-mediated neuroprotection was independent of the net incorporation of PUFA into the striatum, thus suggesting a tight control of brain lipid homeostasis both in normal and pathological conditions.
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http://dx.doi.org/10.3389/fnins.2018.00604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127646PMC
August 2018

Functional coupling of GABA receptors and the channel TRPV4 mediates rapid progesterone signaling in the oviduct.

Sci Signal 2018 08 14;11(543). Epub 2018 Aug 14.

Laboratory of Molecular Physiology, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona 08003, Spain.

The molecular mechanism by which progesterone (P4) modulates the transport of ova and embryos along the oviduct is not fully resolved. We report a rapid response to P4 and agonists of γ-aminobutyric acid receptors A and B (GABA) in the mouse oviduct that was characterized by oscillatory Ca signals and increased ciliary beat frequency (CBF). Pharmacological manipulation, genetic ablation, and siRNA-mediated knockdown in oviductal cells, as well as overexpression experiments in HEK 293T cells, confirmed the participation of the cationic channel TRPV4, different subunits of GABA (α1 to α3, β2, and β3), and GABA in P4-induced responses. TRPV4-mediated Ca entry in close proximity to the inositol trisphosphate receptor was required to initiate and maintain Ca oscillations after P4 binding to GABA and transactivation of G protein-coupled GABA receptors. Coimmunoprecipitation experiments and imaging of native tissue and HEK 293T cells demonstrated the close association of GABA and GABA receptors and the activation of G proteins in response to P4 and GABA receptor agonists, confirming a molecular mechanism in which P4 and GABAergic agonists cooperatively stimulate cilial beating.
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http://dx.doi.org/10.1126/scisignal.aam6558DOI Listing
August 2018

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.

Hum Mol Genet 2018 10;27(20):3528-3541

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.
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http://dx.doi.org/10.1093/hmg/ddy264DOI Listing
October 2018

Effects of the Dopamine Stabilizer, Pridopidine, on Basal and Phencyclidine-Induced Locomotion: Role of Dopamine D2 and Sigma-1 Receptors.

CNS Neurol Disord Drug Targets 2018 ;17(7):522-527

Unitat de Farmacologia, Departament Patologia i Terapeutica Experimental, Facultat de Medicina i Ciencies de la Salut, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Background: Pridopidine, a compound in clinical trials for Huntington's disease treatment, was originally synthesized as a dopamine D2 receptor (D2R) ligand, but later found to possess higher affinity for the sigma-1 receptor (S1R). However, the putative contributions of D2R and S1R to the behavioral profile of acutely administered pridopidine have not been investigated.

Objective: The present study sought to compare the effects of acute pridopidine on wild-type vs. D2R and S1R knockout mice, at high (60 mg/kg) and low (6 mg/kg) doses.

Method: Pridopidine effects on basal and phencyclidine-induced locomotor activity was measured in the open field test. Additionally, the actions of pridopidine on prepulse inhibition was measured in animals treated with saline or phencyclidine.

Results: Whereas inhibition of spontaneous and phencyclidine-induced locomotion was readily observed at 60 mg/kg pridopidine, neither locomotor stimulation in habituated mice, nor any effects on prepulse inhibition were detected upon pridopidine treatment. Surprisingly, inhibition of spontaneous locomotion was unaffected by both D2R and S1R deletion.

Conclusion: The present results suggest the involvement of additional targets, besides D2R and S1R, in mediating locomotor inhibition by pridopidine.
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http://dx.doi.org/10.2174/1871527317666180627103337DOI Listing
October 2019

Remote control of movement disorders using a photoactive adenosine A receptor antagonist.

J Control Release 2018 08 31;283:135-142. Epub 2018 May 31.

Unitat de Farmacologia, Departament Patologia i Terapéutica Experimental, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; Institut de Neurociències, Universitat de Barcelona, Spain. Electronic address:

G protein-coupled adenosine receptors are promising therapeutic targets for a wide range of neuropathological conditions, including Parkinson's disease (PD). However, the ubiquity of adenosine receptors and the ultimate lack of selectivity of certain adenosine-based drugs have frequently diminished their therapeutic use. Photopharmacology is a novel approach that allows the spatiotemporal control of receptor function, thus circumventing some of these limitations. Here, we aimed to develop a light-sensitive caged adenosine A receptor (AR) antagonist to photocontrol movement disorders. We synthesized MRS7145 by blocking with coumarin the 5-amino position of the selective AR antagonist SCH442416, which could be photoreleased upon violet light illumination (405 nm). First, the light-dependent pharmacological profile of MRS7145 was determined in AR-expressing cells. Upon photoactivation, MRS7145 precluded AR ligand binding and agonist-induced cAMP accumulation. Next, the ability of MRS7145 to block AR in a light-dependent manner was assessed in vivo. To this end, AR antagonist-mediated locomotor activity potentiation was evaluated in brain (striatum) fiber-optic implanted mice. Upon irradiation (405 nm) of the dorsal striatum, MRS7145 induced significant hyperlocomotion and counteracted haloperidol-induced catalepsy and pilocarpine-induced tremor. Finally, its efficacy in reversing motor impairment was evaluated in a PD animal model, namely the hemiparkinsonian 6-hydroxydopamine (6-OHDA)-lesioned mouse. Photo-activated MRS7145 was able to potentiate the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (l-DOPA). Overall, MRS7145 is a new light-operated AR antagonist with potential utility to manage movement disorders, including PD.
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http://dx.doi.org/10.1016/j.jconrel.2018.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098950PMC
August 2018

Pridopidine Reverses Phencyclidine-Induced Memory Impairment.

Front Pharmacol 2018 10;9:338. Epub 2018 Apr 10.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

Pridopidine is in clinical trials for Huntington's disease treatment. Originally developed as a dopamine D receptor (DR) ligand, pridopidine displays about 100-fold higher affinity for the sigma-1 receptor (sigma-1R). Interestingly, pridopidine slows disease progression and improves motor function in Huntington's disease model mice and, in preliminarily reports, Huntington's disease patients. The present study examined the anti-amnesic potential of pridopidine. Thus, memory impairment was produced in mice by administration of phencyclidine (PCP, 10 mg/kg/day) for 10 days, followed by 14 days' treatment with pridopidine (6 mg/kg/day), or saline. Finally, novel object recognition performance was assessed in the animals. Mice receiving PCP and saline exhibited deficits in novel object recognition, as expected, while pridopidine treatment counteracted PCP-induced memory impairment. The effect of pridopidine was attenuated by co-administration of the sigma receptor antagonist, NE-100 (10 mg/kg). Our results suggest that pridopidine exerts anti-amnesic and potentially neuroprotective actions. These data provide new insights into the therapeutic potential of pridopidine as a pro-cognitive drug.
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http://dx.doi.org/10.3389/fphar.2018.00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902730PMC
April 2018

Adenosine A-dopamine D receptor heteromers operate striatal function: impact on Parkinson's disease pharmacotherapeutics.

Neural Regen Res 2018 Feb;13(2):241-243

Unitat de Farmacologia, DepartamentPatologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat; Institut de Neurociències, Universitat de Barcelona, Barcelona, Spain.

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http://dx.doi.org/10.4103/1673-5374.226388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879894PMC
February 2018

Adenosine A-A Receptor Heteromer as a Possible Target for Early-Onset Parkinson's Disease.

Front Neurosci 2017 22;11:652. Epub 2017 Nov 22.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

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http://dx.doi.org/10.3389/fnins.2017.00652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702635PMC
November 2017

Antiparkinsonian Efficacy of Guanosine in Rodent Models of Movement Disorder.

Front Pharmacol 2017 4;8:700. Epub 2017 Oct 4.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina, Bellvitge Institute for Biomedical Research, Universitat de Barcelona, Barcelona, Spain.

Guanosine (GUO) is a guanine-based purine nucleoside with important trophic functions and promising neuroprotective properties. Although the neuroprotective effects of GUO have been corroborated in cellular models of Parkinson's disease (PD), its efficacy as an antiparkinsonian agent has not been fully explored in PD animal models. Accordingly, we evaluated the effectiveness of GUO in reversing motor impairments in several rodent movement disorder models, including catalepsy, tremor, and hemiparkinsonism. Our results showed that orally administered GUO antagonized reserpine-mediated catalepsy, reduced reserpine-induced tremulous jaw movements, and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine in unilaterally 6-hydroxidopamine-lesioned rats. In addition, at 5 and 7.5 mg/kg, GUO inhibited L-DOPA-induced dyskinesia in rats chronically treated with a pro-dopaminergic agent. Overall, we describe the therapeutic potential of GUO, which may be effective not only for reversing parkinsonian motor impairments but also for reducing dyskinesia induced by treatment for PD.
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http://dx.doi.org/10.3389/fphar.2017.00700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632808PMC
October 2017

The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A receptor controlling its cell surface expression and function in vivo.

Sci Rep 2017 08 25;7(1):9452. Epub 2017 Aug 25.

Unitat de Farmacologia, Departament Patologia i Terapéutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A receptor (AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and AR at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal AR cell surface expression that correlated well with an increased AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced AR agonist-induced catalepsy and an increased response to AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling AR biology in the striatum, which may be relevant for PD management.
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http://dx.doi.org/10.1038/s41598-017-10147-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573386PMC
August 2017

Antipsychotic-Like Efficacy of Dopamine D Receptor-Biased Ligands is Dependent on Adenosine A Receptor Expression.

Mol Neurobiol 2018 Jun 5;55(6):4952-4958. Epub 2017 Aug 5.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, 08907, Barcelona, Spain.

Dopamine D receptor (DR) activation triggers both G protein- and β-arrestin-dependent signaling. Biased DR ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of DR to heteromerize with adenosine A receptor (AR) has been associated to DR agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the AR dependence of DR/β-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between β-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of DR/AR homomers and heteromers, we demonstrated that the DR agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when AR/DR heteromers were expressed. Subsequently, the role of AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and AR mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in AR mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that DR/AR heteromerization facilitates DR β-arrestin recruitment, and furthermore, reveal a pivotal role for AR in the antipsychotic-like activity of the β-arrestin-biased DR ligand, UNC9994.
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http://dx.doi.org/10.1007/s12035-017-0696-yDOI Listing
June 2018

Rett-like Severe Encephalopathy Caused by a De Novo GRIN2B Mutation Is Attenuated by D-serine Dietary Supplement.

Biol Psychiatry 2018 Jan 16;83(2):160-172. Epub 2017 Jun 16.

Bellvitge Biomedical Research Institute-Unit of Neuropharmacology and Pain Group, University of Barcelona, Barcelona, Spain. Electronic address:

Background: N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity, neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR.

Methods: We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and translated it to the clinical practice.

Results: Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated. Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after 17 months of D-serine dietary supplementation.

Conclusions: Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical improvement.
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http://dx.doi.org/10.1016/j.biopsych.2017.05.028DOI Listing
January 2018

Angiotensin II type 1/adenosine A receptor oligomers: a novel target for tardive dyskinesia.

Sci Rep 2017 05 12;7(1):1857. Epub 2017 May 12.

Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D receptors (DRs). Striatal DR functioning may be finely regulated by either adenosine A receptor (AR) or angiotensin receptor type 1 (ATR) through putative receptor heteromers. Here, we examined whether AR and ATR may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical ATR-AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between ATR and AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of ATR/AR heteromers in the striatum by proximity ligation in situ assay. Finally, we took advantage of a TD animal model, namely the reserpine-induced vacuous chewing movement (VCM), to evaluate a novel multimodal pharmacological TD treatment approach based on targeting the ATR/AR complex. Thus, reserpinized mice were co-treated with sub-effective losartan and istradefylline doses, which prompted a synergistic reduction in VCM. Overall, our results demonstrated the existence of striatal ATR/AR oligomers with potential usefulness for the therapeutic management of TD.
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http://dx.doi.org/10.1038/s41598-017-02037-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431979PMC
May 2017