Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices.

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson's disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 µM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A1 (A1R) and/or A2A receptors (A2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A1R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A1R agonist, did not alter GUO effects. Regarding A2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A1R antagonist DPCPX, and the A2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A1R-A2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

A2A-D2 Heteromers on Striatal Astrocytes: Biochemical and Biophysical Evidence.

Our previous findings indicate that A2A and D2 receptors are co-expressed on adult rat striatal astrocytes and on the astrocyte processes, and that A2A-D2 receptor⁻receptor interaction can control the release of glutamate from the processes. Functional evidence suggests that the receptor⁻receptor interaction was based on heteromerization of native A2A and D2 receptors at the plasma membrane of striatal astrocyte processes. We here provide biochemical and biophysical evidence confirming that receptor⁻receptor interaction between A2A and D2 receptors at the astrocyte plasma membrane is based on A2A-D2 heteromerization. To our knowledge, this is the first direct demonstration of the ability of native A2A and D2 receptors to heteromerize on glial cells. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, the findings that adenosine A2A and dopamine D2 receptors can form A2A-D2 heteromers on the astrocytes in the striatum (and that these heteromers can play roles in the control of the striatal glutamatergic transmission) may shed light on the molecular mechanisms involved in the pathogenesis of the disease.

Homocysteine and A2A-D2 Receptor-Receptor Interaction at Striatal Astrocyte Processes.

The interaction between adenosine A2A and dopamine D2 receptors in striatal neurons is a well-established phenomenon and has opened up new perspectives on the molecular mechanisms involved in Parkinson's disease. However, it has barely been investigated in astrocytes. Here, we show by immunofluorescence that both A2A and D2 receptors are expressed in adult rat striatal astrocytes in situ, and investigate on presence, function, and interactions of the receptors in the astrocyte processes-acutely prepared from the adult rat striatum-and on the effects of homocysteine on the A2A-D2 receptor-receptor interaction. We found that A2A and D2 receptors were co-expressed on vesicular glutamate transporter-1-positive astrocyte processes, and confirmed that A2A-D2 receptor-receptor interaction controlled glutamate release-assessed by measuring the [3H]D-aspartate release-from the processes. The complexity of A2A-D2 receptor-receptor interaction is suggested by the effect of intracellular homocysteine, which reduced D2-mediated inhibition of glutamate release (homocysteine allosteric action on D2), without interfering with the A2A-mediated antagonism of the D2 effect (maintained A2A-D2 interaction). Our findings indicate the crucial integrative role of A2A-D2 molecular circuits at the plasma membrane of striatal astrocyte processes. The fact that homocysteine reduced D2-mediated inhibition of glutamate release could provide new insights into striatal astrocyte-neuron intercellular communications. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, these findings may shed light on the pathogenic mechanisms of the disease and contribute to the development of new drugs for its treatment.