Fig 1: PP2A Expression in the SCN. Western blots and densitometric analysis of PP2A expression during the day (CT6) and night (CT18) in the SCN of hamsters (mean ± SEM of three independent experiments). Densitometric analysis of three independent experiments conducted at night shows no significant differences between day and night expression of PP2A (1.29 ± 0.017 vs 1.28 ± 0.012 arbitrary units, respectively, n = 4 per group; p = 0.6, Student’s unpaired t-test).
Fig 2: A Light Pulse During the Late Night Inhibits Enzymatic Activity of PP2A in the SCN. The PP2A activity was measured in SCN lysates after the administration of a sub-saturating light pulse to hamsters at ZT18, or controls kept in the dark (mean ± SEM of three independent experiments). Okadaic acid was administered in samples taken from dark-exposed hamsters, as an internal control. Okadaic acid has a similar effect on PP2A activity than light. A significant decrease of the PP2A activity for both the light pulse (LP) and the Okadaic acid administration was found compared to the Dark group, and there were no differences between LP and Okadaic (Dark: 6.23 ± 2.23 pmol/μg; LP: 0.62 ± 0.33 pmol/μg; Okadaic: 1.01 ± 0.20 pmol/μg; n = 3 per group; ANOVA: p < 0.01; multiple comparisons: Dark vs LP p < 0.01; Dark vs Okadaic, p < 0.01; LP vs Okadaic p = 0.89, Tukey’s adjusted p values).
Fig 3: PP2A Inhibition Increased the Light-Induced Phase Advance of the Circadian Rhythms. A–C: Actograms, plotted modulo tau, showing wheel-running activity rhythms of hamsters under constant darkness treated with an icv. administration of vehicle (A) or 100 nM Okadaic acid (B, one animal with the maximum response, and C, individual with a response no different to vehicle) 15 min before a subsaturating LP (100 lux, 15 min) at circadian time CT18, yellow circles mark the moment of the icv. administration. The top bar with ticks marks the circadian time (CT) for the pre-treatment period, numbers at the y-axis dentate the number of days at DD. D: The panel shows a scatter dot plot with the mean ± SEM for the phase advances. The treatment with Okadaic acid significantly increased the phase advance compared to the vehicle (LP: 158.5 ± 38.73 min; LP + Oka: 467.5 ± 124.4 min, n = 4 per group; p < 0.05, Student’s unpaired t-test).
Fig 4: Interaction Between p-GS and PP2A in the SCN With and Without a Light Pulse Stimulus. Western blot (WB) analysis for p-GS and PP2A, of samples immunoprecipitated (IP) with p-GS, PP2A antibodies or with non-immune serum (Control), and whole SCN extract, with or without light pulse at ZT18.
Fig 5: Model of the NO/cGMP/PKG/GS/PP2A Signal Transduction Pathway Responsible for Photic Entrainment by Advances of the Circadian Clock. Light stimulation at late night reaches the SCN retinorecipient neurons by glutamatergic neurotransmission, opening NMDAR postsynaptic receptor increasing intracellular calcium (Ca2+) and activates cascade of kinases and second messengers [Ca2+- calmodulin dependent kinase II (CamKII), nitric oxide synthase (NOS), NO, guanylate cyclase (GC), cyclic guanosine monophosphate (cGMP)], which increases cGMP dependent protein kinase (PKG) activity to phosphorylate GS. The phosphorylated GS peptide (p-GS) interacts with PP2A, inhibiting its activity. This will stabilize putative substrates of PP2A, such as phosphorylated cyclic adenosine monophosphate responding element binding protein (p-CREB), otherwise dephosphorylated by basal PP2A (shaded area without light pulse). p-CREB interacts with E-boxes increasing per1-2 genes transcription, a key endpoint needed for resetting the circadian clock. In the figure, the signal transduction pathway players already confirmed are shown in orange, darker colors mark the activated form of the proteins, while lighter colors indicate the inactive forms. The dotted arrow indicates the hypothetical p-CREB-PP2A interaction.
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