Coincident activation of adenosine A1 receptors and metabotropic glutamate receptor 5 differentially influences signalling in primary brain cell cultures

[Speaker] Karen J Gregory:1
[Co-author] Shane D Hellyer:1, Sabine Albold:1, Phuc T Nguyen:1, Katie Leach:1, Lauren T May:1
1:Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Australia

G protein-coupled receptors (GPCRs) are highly 'druggable' proteins and represent the largest class of current therapeutic targets. Two distinct neuromodulatory GPCRs, 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 co-located in the same brain regions and CNS-resident cell types, e.g. neurons and astrocytes, and 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 signalling in primary neuron and astrocyte cultures. Primary neuronal cultures were derived from striatum and cortices of E16 mice and astrocyte cultures derived from cortices of rat pups (p1-4). High-throughput signaling assays were performed (iCa2+ mobilization, inositol phosphate (IP1) and cAMP accumulation) to confirm functional mGlu5 and A1AR in the cultures by assessing the ability of: 1) A1AR selective agonist (MeCCPA) to inhibit forskolin stimulation of cAMP accumulation; and 2) mGlu5 selective allosteric agonist (VU0424465) to stimulate iCa2+ and IP1 accumulation. Coincident activation of A1AR enhanced mGlu5-mediated iCa2+ mobilization in response to both orthosteric and allosteric agonists in striatal and cortical neurons. 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. Collectively, our data demonstrate that coincident activation of mGlu5 and A1AR differentially modulates intracellular signalling pathways in primary neurons. Future work exploring the underlying mechanisms may reveal new strategies for targeting these GPCRs to treat AD and cognitive disorders.
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