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 A_2AR-deficient mice (A_2AR^−/−) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A_2AR antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A_2AR. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A₁R and A_2AR 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 A_2AR^−/− mice. Notably, while guanosine was not able to modify MRS7396 binding to A_2AR-expressing cells, a partial blockade was observed in cells co-expressing A₁R and A_2AR. The relevance of the A₁R and A_2AR interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A_2AR agonist-mediated effects in doubly expressing A₁R and A_2AR cells. Interestingly, while guanosine did not affect A₁R/A_2AR heteromer formation, it reduced A_2AR agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A₁R and A_2AR co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.

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

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 (L-DOPA) in unilaterally 6-hydroxidopamine-lesioned rats. In addition, at 5 and 7.5 mg/kg, GUO inhibited L-DOPA-induced dyskinesia (LID) 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.