Co‐activation of adenosine and glutamate receptors modulates signaling in primary brain cell cultures

Abstract

G protein‐coupled receptors (GPCRs) represent the largest class of targets for current therapeutics. Two distinct modulatory GPCRs within the CNS, the adenosine A1 receptor (A1AR) and the metabotropic glutamate receptor subtype 5 (mGlu5), have been implicated in Alzheimer's disease (AD) pathology and as potential targets to treat cognitive impairments and disease progression. Both GPCRs are present on the same CNS‐resident cell types, e.g. neurons and astrocytes, and are expressed in brain regions implicated in AD pathology. GPCR discovery programs generally only consider GPCR activity in isolation, without factoring in the influence of other GPCRs or stimuli present. Of note, both glutamate and adenosine are often present within culture medium and/or released from cultured cells. GPCR cross‐talk and/or heteromerization can introduce pharmacological heterogeneity and offer new avenues for targeted drug development. We sought to test the hypothesis that coincident activation of co‐located GPCRs modulates signaling in primary cultures of CNS resident cells. Primary cultures were derived from striatum and cortices of E16 mice and cultured for 6–8 days. High‐throughput signaling assays (iCa2+ mobilization, inositol phosphate (IP1) and cAMP accumulation) and radioligand binding studies were performed to confirm expression and function of mGlu5 and A1AR in the cultures by assessing the ability of: 1) A1AR selective (MeCCPA) and non‐selective (NECA) agonists to inhibit forskolin stimulation of cAMP accumulation; and 2) mGlu5 selective allosteric agonist (VU0424465) and group I mGlu selective orthosteric agonist (DHPG) to stimulate iCa2+ and IP1 accumulation. Experiments performed in the presence and absence of adenosine deaminase revealed that ambient adenosine was not influencing mGlu5 signalling. Coincident activation of A1AR enhanced mGlu5‐mediated iCa2+ mobilization in response to both orthosteric and allosteric agonists in primary striatal and cortical neuronal cultures. However, A1AR activation had no effect on mGlu5‐mediated IP1 accumulation in cortical neurons. Conversely, coincident activation of mGlu5 had little influence on A1AR‐mediated inhibition of cAMP accumulation, but inhibited AR‐stimulation of ERK1/2 phosphorylation. Collectively, our data demonstrate that coincident activation of mGlu5 and A1AR differentially modulates intracellular signaling pathways. Future work exploring the underlying mechanisms may reveal new strategies for targeting these GPCRs to treat AD and cognitive disorders.Support or Funding InformationThis work was supported by National Health and Medical Research Council of Australia (APP1123722 to KJG and LTM) and Australian Research Council Future Fellowships to KJG and KL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.