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The dopamine D 4 receptor (D 4 R) 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. D 4 R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D 2 receptor (D 2 R), dopamine-induced arrestin recruitment and desensitization at the D 4 R 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 D 4 R in HEK293T cells. We also studied desensitization of D 4 R-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 D 4 R and accelerated the rate of desensitization of D 4 R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D 4 R and thus increases our understanding of the signaling and desensitization of this receptor.

Pridopidine is in clinical trials for Huntington's disease treatment. Originally developed as a dopamine D receptor (D R) 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.

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 D2 receptor (D2R) activation triggers both G protein- and β-arrestin-dependent signaling. Biased D2R ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of D2R to heteromerize with adenosine A2A receptor (A2AR) has been associated to D2R agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the A2AR dependence of D2R/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 D2R/A2AR homomers and heteromers, we demonstrated that the D2R agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when A2AR/D2R heteromers were expressed. Subsequently, the role of A2AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and A2AR−/− mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in A2AR−/− 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 D2R/A2AR heteromerization facilitates D2R β-arrestin recruitment, and furthermore, reveal a pivotal role for A2AR in the antipsychotic-like activity of the β-arrestin-biased D2R ligand, UNC9994.

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 (D R and M R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D R-M R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET and NanoBiT ) assays, performed in transiently transfected HEK293T cells. Subsequently, a D R-M R 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 D R-selective agonist (sumanirole) and/or an M R-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 D R/M R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.

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 2 and muscarinic acetylcholine M 1 receptors (D 2 R and M 1 R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D 2 R–M 1 R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET 1 and NanoBiT ® ) assays, performed in transiently transfected HEK293T cells. Subsequently, a D 2 R–M 1 R 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 D 2 R-selective agonist (sumanirole) and/or an M 1 R-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 D 2 R/M 1 R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.

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 A2A receptors (A2ARs) or their degree of functional heteromerization with dopamine D2 receptors (D2R). 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 A2AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A2AR 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 D2R without changes in A2AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers. The degree of A2AR-D2R 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.

[This corrects the article DOI: 10.3389/fphar.2020.00194.].[This corrects the article DOI: 10.3389/fphar.2020.00194.].

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.

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.