The mGlu5 Receptor Protomer-Mediated Dopamine D2 Receptor Trans-Inhibition Is Dependent on the Adenosine A2A Receptor Protomer: Implications for Parkinson's Disease.

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

Optical Control of Adenosine-Mediated Pain Modulation.

Adenosine receptors (ARs) play many important roles in physiology and have been recognized as potential targets for pain relief. Here, we introduce three photoswitchable adenosine derivatives that function as light-dependent agonists for ARs and confer optical control to these G protein-coupled receptors. One of our compounds, AzoAdenosine-3, was evaluated in the classical formalin model of pain. The molecule, active in the dark, was not metabolized by adenosine deaminase and effectively reduced pain perception in a light-dependent manner. These antinociceptive effects suggested a major role for A1R and A3R in peripheral-mediated pain sensitization, whereas an average adenosine-mediated antinociceptive effect will be facilitated by A2AR and A2BR. Our results demonstrate that a photoswitchable adenosine derivative can be used to map the contribution of ARs mediating analgesia in vivo.

Optical control of adenosine A3 receptor function in psoriasis.

Psoriasis is a chronic and relapsing inflammatory skin disease lacking a cure that affects approximately 2% of the population. Defective keratinocyte proliferation and differentiation, and aberrant immune responses are major factors in its pathogenesis. Available treatments for moderate to severe psoriasis are directed to immune system causing systemic immunosuppression over time, and thus concomitant serious side effects (i.e. infections and cancer) may appear. In recent years, the Gi protein-coupled A3 receptor (A3R) for adenosine has been suggested as a novel and very promising therapeutic target for psoriasis. Accordingly, selective, and high affinity A3R agonists are known to induce robust anti-inflammatory effects in animal models of autoimmune inflammatory diseases. Here, we demonstrated the efficacy of a selective A3R agonist, namely MRS5698, in preventing the psoriatic-like phenotype in the IL-23 mouse model of psoriasis. Subsequently, we photocaged this molecule with a coumarin moiety to yield the first photosensitive A3R agonist, MRS7344, which in photopharmacological experiments prevented the psoriatic-like phenotype in the IL-23 animal model. Thus, we have demonstrated the feasibility of using a non-invasive, site-specific, light-directed approach to psoriasis treatment.

Decreased striatal adenosine A2A-dopamine D2 receptor heteromerization in schizophrenia.

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.

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

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 (mGlu5) receptors are widely expressed through the pain neuraxis and play a key role in pain transmission. In line with this, selective mGlu5 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 mGlu5 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).

Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment.

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 Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-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 Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R 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 A2AR-CB1R interactions in the brain.

PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders.

Adenosine A2A receptor (A2AR) antagonists have emerged as complementary non-dopaminergic drugs to alleviate Parkinson's disease (PD) symptomatology. Here, we characterize a novel non-xhantine non-furan A2AR antagonist, PBF509, as a potential pro-dopaminergic drug for PD management. First, PBF509 was shown to be a highly potent ligand at the human A2AR, since it antagonized A2AR agonist-mediated cAMP accumulation and impedance responses with KB values of 72.8 ± 17.4 and 8.2 ± 4.2 nM, respectively. Notably, these results validated our new A2AR-based label-free assay as a robust and sensitive approach to characterize A2AR 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 A2AR antagonist with potential utility for PD treatment, and for some of the side effects associated to the current pharmacotherapy (i.e., dyskinesia).

Remote control of movement disorders using a photoactive adenosine A2A receptor antagonist.

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 A2A receptor (A2AR) antagonist to photocontrol movement disorders. We synthesized MRS7145 by blocking with coumarin the 5-amino position of the selective A2AR antagonist SCH442416, which could be photoreleased upon violet light illumination (405 nm). First, the light-dependent pharmacological profile of MRS7145 was determined in A2AR-expressing cells. Upon photoactivation, MRS7145 precluded A2AR ligand binding and agonist-induced cAMP accumulation. Next, the ability of MRS7145 to block A2AR in a light-dependent manner was assessed in vivo. To this end, A2AR 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 A2AR antagonist with potential utility to manage movement disorders, including PD.

Antipsychotic-Like Efficacy of Dopamine D2 Receptor-Biased Ligands is Dependent on Adenosine A2A Receptor Expression.

Dopamine D2 receptor (D2R) activation triggers both G protein- and ß-arrestin-dependent signaling. Biased D2R ligands activating ß-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 ß-arrestin recruitment. Accordingly, here we aimed to demonstrate the A2AR dependence of D2R/ß-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between ß-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 ß-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 ß-arrestin recruitment, and furthermore, reveal a pivotal role for A2AR in the antipsychotic-like activity of the ß-arrestin-biased D2R ligand, UNC9994.

The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A2A receptor controlling its cell surface expression and function in vivo.

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 A2A receptor (A2AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and A2AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and A2AR 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 A2AR cell surface expression that correlated well with an increased A2AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced A2AR agonist-induced catalepsy and an increased response to A2AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling A2AR biology in the striatum, which may be relevant for PD management.

Cognitive impairments associated with alterations in synaptic proteins induced by the genetic loss of adenosine A2A receptors in mice.

The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A2A adenosine receptor subtype (A2AR) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A2AR in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A2AR constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A2AR knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders.

Angiotensin II type 1/adenosine A 2A receptor oligomers: a novel target for tardive dyskinesia.

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between AT1R and A2AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of AT1R/A2AR 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 AT1R/A2AR 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 AT1R/A2AR oligomers with potential usefulness for the therapeutic management of TD.

The Exonuclease Trex2 Shapes Psoriatic Phenotype.

Trex2 is a keratinocyte-specific 3'-deoxyribonuclease that participates in the maintenance of skin homeostasis after DNA damage. Here, we show that this exonuclease is strongly upregulated in human psoriasis, a hyperproliferative and inflammatory skin disease. Similarly, the imiquimod (IMQ)- and Il23-induced mouse psoriasis was associated with a substantial upregulation of Trex2, which was recruited into fragmented chromatin in keratinocytes that were undergoing impaired proliferation, differentiation, and cell death, indicating an important role in DNA processing. Using Trex2 knockout mice, we have found that Trex2 deficiency attenuated IMQ-induced psoriasis-like skin inflammation and enhanced IMQ-induced parakeratosis. Also, Il23-induced ear swelling was diminished in Trex2 knockout mice in comparison with wild-type (wt) mice. Transcriptome analysis identified multiple genes that were deregulated by Trex2 loss after treatment with IMQ. Specifically, immune response genes and pathways normally associated with inflammation were downregulated, whereas those related to skin differentiation and chromatin biology showed increased expression. Interestingly, Trex2 deficiency led to decreased IMQ-induced keratinocyte death via both cell autonomous and noncell autonomous mechanisms. Hence, our data indicate that Trex2 acts as a critical factor in the pathogenesis of psoriasis by promoting keratinocyte apoptosis and enucleation and thereby influencing skin immune responses.

Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats.

Parkinson's disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction - i.e. of dopamine D2 receptor (D2R) with adenosine A2A receptor (A2AR) (forming D2R-A2AR oligomers) - finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A2AR assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A2AR oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A2AR oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments.

Uncovering caffeine's adenosine A2A receptor inverse agonism in experimental parkinsonism.

Caffeine, the most consumed psychoactive substance worldwide, may have beneficial effects on Parkinson's disease (PD) therapy. The mechanism by which caffeine contributes to its antiparkinsonian effects by acting as either an adenosine A2A receptor (A2AR) neutral antagonist or an inverse agonist is unresolved. Here we show that caffeine is an A2AR inverse agonist in cell-based functional studies and in experimental parkinsonism. Thus, we observed that caffeine triggers a distinct mode, opposite to A2AR agonist, of the receptor's activation switch leading to suppression of its spontaneous activity. These inverse agonist-related effects were also determined in the striatum of a mouse model of PD, correlating well with increased caffeine-mediated motor effects. Overall, caffeine A2AR inverse agonism may be behind some of the well-known physiological effects of this substance both in health and disease. This information might have a critical mechanistic impact for PD pharmacotherapeutic design.