Adenosine A_2A-dopamine D₂ receptor heteromers operate striatal function: impact on Parkinson's disease pharmacotherapeutics

The basal ganglia (BG) assemble a series of deep gray matter structures forming recurrent loops that include the cortex and thalamus, and that participate in the regulation of a plethora of brain functions, including elicitation and learning of reward-and aversive stimuli-associated behaviors, motor activity control and sensorimotor gating (Bromberg-Martin et al., 2010). The striatum is the main input BG structure, thus it receives cortical glutamatergic projections from widespread areas of cortex and projects into other BG nuclei, including globus pallidus pars externa and the BG outputsglobus pallidus pars interna and substantia nigra pars reticulata. On the other hand, the substantia nigra pars compacta-ventral tegmental area (SNpc-VTA) modulates cortical-BG-thalamic circuits by means of dopaminergic innervation of the striatum. Interestingly, the main population of striatal neurons, the medium spiny neurons (MSNs), provide the origin of two different striatal efferent pathways, the direct and indirect pathways (Schiffmann et al., 2007). Both project to the BG outputs, and the direct pathway also projects to brainstem, to the SNpc-VTA. The MSN originating these two pathways are characterized by the differential expression of several key genes. Thus, while MSNs from the direct pathway (direct MSNs) express dopamine D₁ receptors (D₁R) and contain the neuropeptides dynorphin and substance P, indirect MSNs express dopamine D₂ receptors (D₂R) and contain the neuropeptide enkephalin (Fuxe et al., 2007; Schiffmann et al., 2007). Striatal dopamine from SNpc-VTA projections potentiates direct and inhibits indirect pathway MSN, which leads to a net inhibition of thalamocortical areas.

Interestingly, D₁Rs and D₂Rs, respectively localized in the direct and indirect MSNs, are functionality tuned by direct receptor-receptor interactions (i.e., heteromerization) established with other endogenously expressed G protein-coupled receptors, for instance adenosine, metabotropic glutamate and cannabinoid receptors, among others. In this way, these heteromers may positively or negatively regulate D₁R and/or D₂R activation, a fact that may ultimately constitute a very complex scenario in which the control of BG functioning is regulated by a myriad of complex interactions between different neurotrasmistters (Fuxe et al., 2007). From these receptor-receptor complexes, one of the most studied is the one formed by D₂R and the adenosine A_2A receptor (A_2AR) in the indirect MSN. In such way, it has been shown that reciprocal antagonistic interactions occur within the A_2AR-D₂R heteromer (Fuxe et al., 2007). Thus, an allosteric interaction was initially described, by which A_2AR ligands decrease both the affinity and intrinsic efficacy of D₂R ligands (Ferré et al., 2016). And, more recently, an opposite interaction was described, by which D₂R agonists decrease the binding of A_2AR ligands (Fernández-Dueñas et al., 2013) (Figure 1). In addition, a strong antagonistic interaction (namely canonical) at the adenylyl cyclase (AC) level has been described, which depends on the ability of D₂R-mediated Gi activation to inhibit Gs activation elicited by A_2AR activation (Ferré et al., 2016) (Figure 1). Interestingly, the occurrence of both kinds of reciprocal antagonistic interactions (canonical and allosteric) led to conjecture the existence of different populations of receptors: forming and non-forming oligomers. Accordingly, in such a model A_2AR-D₂R heterodimers would preferentially allow the allosteric interaction, while the formation of D₂R-D₂R or A_2AR-A_2AR homodimers would be mainly responsible of the canonical interaction. Alternatively, the recent hypothesis of the formation of heterotetrameric structures could explain the simultaneous existence of both types of receptor-receptor interactions (Bonaventura et al., 2015; Ferré et al., 2016) (Figure 1). In fact, most existing experimental data on the biochemical and behavioral effects of A_2AR and D₂R ligands can be explained in the frame of one main population of striatal A₂R forming A_2AR-D₂R heteromers (Taura et al., 2017). However, it is important to note that although the existence of the direct A_2AR-D₂R interaction has been widely accepted for many years, the demonstration of its existence in native tissue was only made recently. First, co-immunoprecipitation experiments and immunoelectron microscopy studies showed the association and co-distribution of D₂R and A_2AR in rat striatum (Cabello et al., 2009). Next, it was possible to ascertain the close proximity of D₂R and A_2AR by means of the proximity ligation assay (PLA) in mice and sheep striatum (Trifilieff et al., 2011; Bonaventura et al., 2015), in which it was also observed the ability of a synthetic peptide to specifically disrupt the PLA in sheep striatum (Bonaventura et al., 2015). Finally, the clearest demonstration of A_2AR-D₂R heteromers in striatal tissue came from a complementary approach using immunoelectron microscopy, PLA and time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) with specific fluorescence ligands in rats (Fernández-Dueñas et al., 2015).

Figure 1.

Scheme showing the proposed striatal adenosine A_2A receptor (A_2AR)-dopamine D₂ receptors (D₂R) heteromer-dependent modulation of basal ganglia behavioral outputs.

The heterotetrameric structure of the A_2AR-D₂R heteromer allows a multimodal functional interplay between adenosine (ADO) and dopamine (DA). Thus, ADO decreases D₂R agonist affinity through an allosteric interaction (1) and it reduces D₂R signaling through G protein-dependent (3) and G protein-independent (5) pathways. Conversely, DA activation of D₂R decreases A_2AR agonist affinity through an allosteric interaction (2) and it inhibits A_2AR agonist-mediated activation of adenylyl cyclase (AC), by means of the antagonistic Gs-Gi canonical interaction at the AC level (4). When uninterrupted by the canonical interaction, A_2AR signals through activation of AC and protein kinase A (PKA), resulting in an increasein the excitability of indirect medium spiny neurons (MSNs), which facilitates prepulse inhibition (PPI) and promotes catalepsy. When uninterrupted by the allostericinteraction, D₂R signals through phospholipase C (PLC), which leads to activation of protein phosphatase 2B (PP2B) and a decrease in the excitability of indirect MSNs, which facilitates locomotion. Interestingly, A_2AR and D₂R activation can also modify gene expression through different mechanisms, including G protein-dependent and independent mitogen activated protein kinase (MAPK) activation, respectively.

At this point, we can clearly state that striatal D₂ Rs form homo- and hetero-complexes with other receptors, for instance the A_2AR, and that the above-described receptor-receptor interactions may lead to a fine-tuning regulation of BG function. However, apart from the obvious interest of revealing the functional role of A_2AR-D₂R heteromers in these brain areas, a high effort is being dedicated to elucidating their role in the pathophysiology and pharmacotherapeutics of Parkinson's disease (PD). PD is a neurodegenerative disease that affects approximately 1% over the age of 60, which turns to 5% in subjects up to 85 years.The etiology of this pathology is not well determined, and both genetic and environmental factors may be involved. Conversely, it is well-accepted that the main symptoms of PD (bradykinesia, rigidity, resting tremor and posture instability) appear when a large proportion of dopaminergic fibers from the SNpc projecting into the striatum are lost. Therefore, the main PD treatment consists of trying to restore dopamine levels by administrating dopamine-based drugs, from which the precursor L-DOPA has been the most extensively used since the seventies. However, chronic treatment with L-DOPA invariably leads to the occurrence of severe side-effects, such as dyskinesia, thus a lot of efforts are being directed to find out novel non-dopaminergic-based drugs with a lower incidence of these side effects. One of these new approaches consists of the development of selective A_2AR antagonists (for review, see Vallano et al., 2011), whose mode of action may consist off acilitating D₂R functionor by an independent effect on A_2AR not interacting with D₂R. Thus, as recently addressed in (Taura et al., 2017), there is evidence for an upregulation of A_2AR upon dopamine denervation, which should lead to the presence of a significant population of A_2AR not forming heteromers with D₂R in PD. Accordingly, either by the direct blockade of A_2AR function, which may be over-activatedin PD, or by precluding the negative allosteric interaction within the A_2AR-D₂R heteromer, A_2AR antagonists have been demonstrated to exert beneficial effects in PD animal models and also in PD patients (Vallano et al., 2011).

Noteworthy, to date, just one A_2AR antagonist has been approved for human use and introduced into clinics (Vallano et al., 2011); while others have not survived clinical trials (for several reasons) or are still under investigation. In view of that, and within the A_2AR-D₂R heteromerization context, it would seem likely to revisit the pharmacological strategy used to select A_2AR-based drugs for PD therapeutics. Accordingly, we propose that it would be important to characterize the in vivo efficacy of the putative effects of A_2AR ligands, considered to be implemented in PD therapeutics, on the striatal A_2AR forming and not forming heteromers with D₂R. An example of this kind of approach is the recent study we set to readdress the in vivo pharmacological properties of the A_2AR antagonist SCH442416 (Taura et al., 2017). Of note, we took advantage of genetic manipulation, using wild-type and D₂R or A_2AR deficient mice (D₂R^–/– and A_2AR^–/–, respectively), in order to dissect the role of A_2AR-D₂R heteromerization on the behavioral effects of this ligand. First, a significant but partial reduction of activity was observed when evaluating spontaneous locomotor activity in D₂R^–/– or A_2AR^–/– mice, thus pointing to the existence of neuroadaptations that counteract the respective loss of D₂R- and A_2AR- mediated tonic stimulation and inhibition of psychomotor activity. When examining the effects of SCH442416-mediated activation of locomotion, we observed a substantial diminished effect in D₂R^–/– mice, which would be consistent with a dependence on the allosteric interaction within the A_2AR-D₂R heteromer (Taura et al., 2017). On the other hand, our data also provided further support to the importance of A_2AR mediation in the behavioral effects secondary to the activation or interruption of D₂R signaling, such as inhibition of sensorimotor gating or catalepsy, respectively, consistent with a dependence on the canonical interaction within the A_2AR-D₂R heteromer. Thus, SCH442416 counteracted the inhibitory effect the D₂R agonist sumanirole on prepulse inhibition (PPI) and the cataleptic effect of the D₂R antagonist haloperidol. On the other hand, the A_2AR agonist CGS21680-induced catalepsy was completely counteracted by the genetic blockade of A_2AR and only a partial counteraction was observed in D₂R^–/– mice (Taura et al., 2017). Nevertheless, SCH442416 can counteract CGS21680-mediated catalepsy in D₂R^–/– mice (unpublished data), indicating that this can be used as a method to evaluate the potency and efficacy of A_2AR antagonists on A_2AR not forming heteromers with D₂R.

We have recently proposed a heuristic model of the operation of the A_2AR and D₂R in the indirect MSN that integrates the information available in the literature together with our more recent behavioral results (see Taura et al., 2017 and Figure 1). The model proposes that D₂R mainly signals by activating phospholipase C (PLC) through α β γ subunit-dependent mechanism, thus activating the Ca²⁺/calmodulin-dependent protein phosphate calmodulin (PP2B), which leads to enhancing locomotor activity (Figure 1). Similarly, A_2AR mainly signals through activation of AC and protein kinase A (PKA), which facilitates PPI and catalepsy (Figure 1). In addition, D₂R and A_2AR activation also modify gene expression by G protein-independent or dependent mitogen-activated protein kinase (MAPK) activation (Figure 1). In the frame of the A_2AR-D₂R heteromer, it seems clear that these signaling cascades are determined by the receptor that is predominantly activated and the consequent predominant interaction, allosteric or canonical (Taura et al., 2017) (Figure 1). Obviously, these interactions should be absent in the frame of the A_2AR not forming heteromers, which should be independent from D₂R signaling.

Overall, the accumulation of data since the early 1990's regarding the interactions between D₂R and A_2AR has prompted to a more comprehensive representation of the functioning of the A_2AR-D₂R heteromer in the BG context. However, although the formation of A_2AR-D₂R heteromers in native tissue and their significant functional and pharmacological significance is becoming generally accepted, we need to determine their role in the pathophysiology and treatment of PD and other neuropsychiatric disorders, particularly in view of the possible changes in the stoichiometry of A_2AR and D₂R, when attempting to develop novel pharmacological tools such as new A_2AR antagonists¹¹.

This work was supported by MINECO/ISC III (SAF2014-55700-P and PIE14/00034), IWT (SBO-140028) and Fundació la Marató de TV3 (Grant 20152031) to FC. Also, FC and VFD belong to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya,2014 SGR 1251), and by the intramural funds of the National Institute on Drug Abuse to SF.