Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson’s Disease Post-Mortem Brain through a New AlphaScreen-Based Assay

Abstract

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.

Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor–Receptor Interaction in a Model of Movement Disorders

“…Importantly, the density of each individual D 2 R containing oligomer may be altered in disease conditions, which may constitute a putative pathological fingerprint. Precisely, we recently reported that D 2 R/A 2A R heteromers would be increased in the caudate from human postmortem PD patients (Fernandez-Duenas et al, 2019). This fact would negatively affect dopaminergic neurotransmission, thus providing the rationale for using A 2A R antagonists in PD (see above).…”

“…The AlphaLISA R immunoassay was performed as previously described (Fernandez-Duenas et al, 2019). Briefly, WT and D 2 R KO animals were euthanized by cervical dislocation, followed by dissection of striata on an ice-cold plate.…”

“…High-magnification images of the dorsal striatum from WT mice showed a high degree of D 2 R and M 1 R co-distribution (Figure 2A, arrows). Subsequently, to further demonstrate a close proximity (<200 nm) between both receptor types, we applied an AlphaLISA R immunoassay, as described previously (Fernandez-Duenas et al, 2019). Briefly, striatal membrane extracts were first incubated with specific primary antibodies against the receptor, which can be recognized by secondary antibodies tagged with beads able to engage in an energy transfer after the production of a singlet oxygen (Fernandez-Duenas et al, 2019).…”

“…Subsequently, to further demonstrate a close proximity (<200 nm) between both receptor types, we applied an AlphaLISA R immunoassay, as described previously (Fernandez-Duenas et al, 2019). Briefly, striatal membrane extracts were first incubated with specific primary antibodies against the receptor, which can be recognized by secondary antibodies tagged with beads able to engage in an energy transfer after the production of a singlet oxygen (Fernandez-Duenas et al, 2019). A significant higher energy transfer was observed in the WT compared to its corresponding negative control [WT vs. WT (one Prim Ab); p ≤ 0.05, Figure 2B].…”

Special Issue: G Protein-Coupled Adenosine Receptors: Molecular Aspects and Beyond

“…Borroto-Escuela et al present further evidence that A 2A R-D 2 R heteromers in the nucleus accumbens, through A 2A R mediated allosteric inhibition of the D 2 R, can increase anti-reward in the ventral striatopallidal GABA neurons and inhibit cocaine self-administration, whereas the A 2A R homodimer does not appear to be involved in this allosteric mechanism [19]. Subsequently, Fernández-Dueñas et al describe the development of a new AlphaScreen assay to detect GPCR oligomers in post-mortem human brains, thus confirming for the first time the existence of A 2A R/D 2 R heteromers in human caudate [20]. In brief, antibodies against A 2A R and D 2 R were selectively labelled with donor and acceptor beads to engage in a singlet oxygen energy transfer, dependent on the formation of A 2A R/D 2 R heteromers.…”