cAMP-dependent signaling as a core component of the mammalian circadian pacemaker

Abstract

The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3',5'-monophosphate (cAMP) signaling constitutes an additional, bona fide component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase, and period. This role is general and is evident in peripheral mammalian tissues and cell lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.

Fig. 1. Dampened and desynchronised cellular circadian gene expression in SCN after suppression of cAMP and CRE rhythms

(A) Circadian oscillation of cAMP concentration (blue) and PER2::LUC bioluminescence (red), and cAMP concentration in SCN slices treated with MDL-12,330A (MDL) or with forskolin plus IBMX. **p<0.01 vs. other samples, ANOVA and post-hoc Duncan’s. (B) Circadian oscillation of CRE activity in two representative SCN slices reported by CRE:luciferase adenovirus. (C) Reversible, dose-dependent suppression of SCN PER2::LUC expression by MDL. Arrows indicate medium changes. (D) Effect of MDL on PER2::LUC expression in individual SCN neurons (n= 20), bioluminescence expressed as relative grey scale (0-255 pixel intensity). Videomicrographs illustrate distribution of PER2::LUC expression before (left) and during (right) treatment with MDL, V= 3rd ventricle, bar= 500μm. (E) Desynchronisation of cellular pacemakers of SCN revealed by (above) raster plots and (below) Rayleigh plots of PER2::LUC oscillations of 20 representative SCN neurons before and after addition of MDL. Data representative of 3 or more slices.

Fig. 2. Influence of effectors of cAMP signalling on SCN circadian pacemaking

(A) Effect of HCN channel blocker ZD7288 (arrowhead) on SCN mPER2:LUC circadian gene expression. (B) Damping of peak bioluminescence in SCN slices treated with vehicle or ZD7288 (Pre= pre-treatment, Mean±SEM, n>4). (C) Brefeldin A suppresses circadian gene expression in PER2::LUC SCN. (D) Transient re-activation of MDL-suppressed circadian PER2::LUC expression in SCN slices by Sp-8-CPT-2′-O-Me-cAMPS. (E) Acute activation of cellular circadian gene expression (expressed as relative grey scale units) by Epac agonist in presence of MDL, illustrated by raster (upper panel) and graphical plots (lower panel) of 20 representative cells. (F) Phase shifts of SCN circadian PER2::LUC bioluminescence rhythm by Epac agonist (Sp-8-CPT-2′-O-Me-cAMP, red) but not vehicle (black, mean±SEM, n>3 per time point, ** p<0.01 vs. vehicle, ANOVA and Bonferroni).

Fig. 3. Alterations in the phase of the SCN oscillator after acute transitions in cAMP concentrations

(A) PER2::LUC bioluminescence rhythms from SCN treated with vehicle or forskolin and IBMX (green arrowhead), followed by washout (blue arrowheads). Dotted lines highlight synchrony of For-IBMX-treated slices, but not control slices, after washout. (B) Phases of PER2::LUC rhythms in individual SCN immediately before (pre-) and four days after (post-) washout of vehicle or For/IBMX. Removal of For-IBMX caused resynchronisation, driving slices to a common phase regardless of phase before washout.

Fig. 4. Prolonged SCN circadian period in vitro and in vivo after inhibition of AC by THFA

(A) Effect of THFA on circadian period of representative SCN slices, reported by mPer1::luciferase (B) Sigmoidal curve fit to one-site inhibition model with 95% confidence limits. Red data point indicates period after washout. (C) Circadian period in SCN from wild-type and Clock mutant mice, before or during treatment with THFA (**p<0.01 vs. pre-treatment, ANOVA and Bonferroni test). All data plotted as mean±SEM, n≥3. (D) Representative double-plotted, wheel-running records of mice treated with (left) vehicle- and (right) THFA (delivered to SCN via osmotic mini-pump). Mice entrained to 12 hours light (shaded) and 12 hours dim red light were released into continuous dim-red. Asterisk indicates day of surgery and commencement of infusion.