The Caffeine-Binding Adenosine A2A Receptor Induces Age-Like HPA-axis Dysfunction by Targeting Glucocorticoid Receptor Function

Abstract

Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer's disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer's and age-related cognitive impairments may rely on its ability to modulate GR actions.

Figure 1. Tg(CaMKII-hA_2AR) rats overexpress hA_2AR in forebrain areas.

(a) Construct used to generate Tg(CaMKII-hA_2AR) rats. (b,c) Animals present an overexpression of total A_2AR in the forebrain confirmed by qPCR and Western blotting. The endogenous (right axis) rA_2AR mRNA levels were not modified in the hippocampus as assessed with specific rat A_2Aprimers. (d) A_2AR protein levels increase from 2 weeks old onwards in the hippocampus and (e) no changes were detected in adenosine A₁R mRNA levels for 12–14 weeks old animals. Results were analysed using unpaired Student’s t-test for each gene/brain area, *P < 0.05 compared to WT.

Figure 2. Neuronal overexpression of adenosine A_2A receptor (A_2AR) disrupts HPA-axis function.

(a) GR protein and (b) mRNA levels are decreased in the hippocampus (n = 5) of Tg(CaMKII-hA_2AR) compared to WT animals, and (c) CRH mRNA levels are increased in the hypothalamus (n = 5) of Tg(CaMKII-hA_2AR) compared to WT animals, calculated using a unpaired Student’s t-test. (d) Corticosterone levels evaluated at 8 AM and 8 PM are elevated in Tg(CaMKII-hA_2AR) and do not oscillate in a circadian manner (n = 6–9); Results were analysed with a two-way ANOVA followed by a Bonferroni post hoc. *P < 0.05 compared to WT, ^#P < 0.05 compared with WT at AM. High frequency stimulation (HFS: 100 Hz, 1s) was used to evaluate synaptic plasticity in hippocampal rat slices according to scheme depicted in lower panel (f). (e) At 32 °C, Tg(CaMKII-hA_2AR) animals present a lower post-tetanic potentiation (PTP) and no effect on long term potentiation (LTP). (f) At 30.5 °C, Tg(CaMKII-hA_2AR) animals present higher LTP magnitude which was rescued by one-month oral administration of an A_2AR selective antagonist, KW 6002 (5mg/Kg/day). LTP and PTP magnitudes were quantified in (g). *P < 0.05 unpaired Student’s t test, compared to WT; and are presented as mean ± SEM of (n = 6–11) experiments; ^#P < 0.05 using one-way ANOVA, followed by a Bonferroni post hoc and (h) Input/Output (I/O) curve corresponding to fEPSP slope evoked by different stimulation intensities (0.6 – 3 mA). There is a shift to the left in the curve from Tg(CaMKII-hA_2AR) animals. (i) Short-term reference memory, using the modified Y-maze test. Impairments in Tg(CaMKII-hA_2AR) animals were rescued by oral KW6002 administration (n = 10–16). Results were analysed with a one-way ANOVA followed by a Bonferroni post hoc and are presented as mean ± SEM of n experiments. *^,#P < 0.05 compared to Novel (N) arm.

Figure 3. Dexamethasone induced deficits in synaptic plasticity are prevented by adenosine A_2A receptor (A_2AR) blockade and exacerbated by A_2AR overexpression.

High frequency stimulation (HFS: 100 Hz, 1s) was used to evaluate synaptic plasticity in hippocampal slices. (a) Incubation of slices from WT rats with dexamethasone (100 nM) for 1 h decreases LTP magnitude an effect prevented by the A_2AR antagonist SCH58261 (50 nM). (b) Magnitude of the effects of dexamethasone, RU486 (100 nM) and SCH58261 (n = 3–8) in hippocampal slices from WT rats. Results are presented as mean ± SEM of n experiments analyzed using a one-way ANOVA followed by a Bonferroni post hoc test. *P < 0.05 compared to CTR; ^ϕP < 0.05 compared with dexamethasone 60 min. (c) Incubation of slices with dexamethasone (100 nM) for 20 minutes has no effect on LTP magnitude in WT animals (n = 3–8), whereas (e) in Tg(CaMKII-hA_2AR) animals is sufficient to induce a significant decrease in LTP magnitude (n = 6–9). (d) Bar plots of the effects of dexamethasone and (f) the prevention of these effects by the GR antagonist RU486 (n = 3–9). Results are presented as mean ± SEM of n experiments analyzed using an unpaired t-test for comparisons between WT and Tg(CaMKII-hA_2AR) and one-way ANOVA followed by a Bonferroni post hoc test for drug effects.*P < 0.05 compared to control, ^#P < 0.05 compared with dexamethasone 20 min, ^ϕP < 0.05 compared with dexamethasone 60 min.

Figure 4. Overexpression of adenosine A_2A receptor (A_2AR) induces age-like modifications in adenosine neuromodulation.

(a,b) The A_2AR selective agonist CGS21680, 30 nM, has an effect on basal fEPSP slope in Tg(CaMKII-hA_2AR) but not in WT animals ; Results were analyzed using a unpaired Student’s t-test (*P < 0.05 comparing to WT); (c) A_2AR tonically increase excitatory transmission in Tg(CaMKII-hA_2AR) animals, an effect revealed by the inhibitory effect of the A_2AR selective antagonist SCH58261 (50 nM) on basal synaptic transmission, that was not observed in WT animals. (d) The effect of CGS21680, 30 nM is blocked by H89 (1 μM), a PKA antagonist, but not GF (1 μM) a PKC antagonist. Results were analyzed using One-way ANOVA followed by a Bonferroni’s multiple comparison post hoc test (^#P < 0.05 comparing with Tg(CaMKII-hA_2AR with CGS21680 alone). (e) The effect of A_2AR activation does not change in the presence of the selective adenosine A₁R antagonist DPCPX (100 nM;n = 3); P > 0.05 using a paired Student’s t-test analysis). (f) The effect of CGS58261 upon CPA, 30 nM, on fEPSP is lost in Tg(CaMKII-hA_2AR) animals. Results were analyzed using a paired Student’s t-test comparing to CPA alone (n = 4/7, *P < 0.01 in WT animals comparing to CPA alone). (g) Time course of a representative experiment testing the A₁/A_2A crosstalk in WT versus Tg(CaMKII-hA_2AR).

Figure 5. Adenosine A_2A receptors (A_2AR) modulate glucocorticoid response element (GRE) regulated luciferase expression in N1E115 cells and promote dexamethasone induced Glucocorticoid Receptor (GR) translocation to the nucleus.

(a) Dexamethasone induced an increase in luciferase activity (n =  8–17) (b) which is decreased upon A_2AR blockade by two antagonists, SCH 58261 (10–100 nM) and KW 6002 (50 nM) (n = 5–11) and (c) increased upon direct A_2AR activation with CGS 21680 (10–50 nM) (n = 3–9), as depicted in the upper schemes. Activation of A_2AR alone is sufficient to modulate endogenous GR transcriptional activity (d) A_2AR antagonist decreases luciferase activity (n = 6–14) while (e) A_2AR agonist increases it (n = 3–11). (f) A_2AR effects are prevented by the glucocorticoid receptor (GR) antagonist, RU 486 (100 nM, n = 5–10). Results are presented as mean ± SEM of n experiments. In (d–f) results were normalized to CTR, i.e., to the condition without dexamethasone, while in b) and c) results are normalized to DEX-induced activity. *P < 0.05 compared to control, ^ΦP < 0.05 compared with dexamethasone induced luciferase activity calculated using a one-way ANOVA followed by a Bonferroni post hoc test. (g) Left panel illustrates the gradual enrichment of GR in the nuclear fraction of neuronal cultures over time of exposure to dexamethasone (n = 2–4). This increase is completely prevented by blocking A_2AR with SCH 58261- 50 nM (in right panel). Results are presented as mean ± SEM of n experiments. *P < 0.05 compared to control, ^#P < 0.05 compared with dexamethasone calculated using two-way ANOVA followed by a Bonferroni post hoc. (h) Expression levels of GR and A_2AR in N1E115, compared to hippocampal (Hip) and cortical (Ctx) tissue.

Figure 6. Adenosine A_2A receptors (A_2AR) regulates transcription of GR target genes.

mRNA levels of GILZ (a), PER1 (b) and Bcl2 (c) increase in primary neurons after incubation with dexamethasone (100 nM) for 1 h an effect prevented by the presence of the selective A_2AR antagonist SCH 51280 (50 nM), n = 3–5. *P < 0.05 compared to control, calculated using one-way ANOVA followed by a Bonferroni post hoc test (d) mRNA levels of GILZ, PER1 and Bcl2 in the hippocampus of Tg(CaMKII-hA_2AR) compared to WT animals. Results are presented as mean ± SEM of n experiments. *P < 0.05 compared to control, calculated using two-way ANOVA followed by a Bonferroni post hoc. The upper right scheme depicts the GR-mediated GRE activation to induce transcription of GILZ, PER1 and Bcl2.