Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics

Abstract

Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day–night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the well-described role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3β), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3β and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock.

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Acknowledgments

This work was supported by the International Graduate School of Neuroscience of the Ruhr-University, Bochum, Germany. The authors acknowledge Soner Cinar and Beatrix Feldhaus (BSc and Diplom students from the Dept. of Molecular Neurobiochemistry, RUB, Germany) for their help with T-24 cell culture experiments and anti-Ras-scFv cloning, respectively. We thank the RUBION service center for providing facilities.

Conflict of Interest

The authors declare no competing financial interests.

Compliance with Ethical Standards

The authors declare that all animal procedures were performed in accordance to the German Law on Animal Welfare.

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Correspondence to Rolf Heumann.

Additional information

Tsvetan Serchov and Antje Jilg contributed equally to this work.

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Suppl. Fig. 1
figure8

Circadian regulation of MKP-1 protein expression in the SCN of WT and synRas mice. Representative Western blot and densitometric quantification of MKP-1 levels normalized to tubulin. Data were presented as a mean ±SEM (n=4; #p<0.05 in comparison to CT18 for both genotypes, two-way ANOVA with Bonferroni post hoc test). (GIF 44 kb)

Suppl. Fig. 2
figure9

Photic regulation of pERK1,2 and pCREB in the SCN of WT and synRas mice at early and late subjective night. a Representative Western blot and densitometric quantification of pERK1,2 normalized to the total ERK1,2. Representative immunohistochemical images of pCREB and densitometric quantification of pCREB induction in the SCN of WT and synRas animals at early subjective night (CT14) (b) and late subjective night (CT22) (c). Data are presented as mean ±SEM (n=4 per group, *p<0.05, **p<0.01 two-way ANOVA with Bonferroni post hoc test). (GIF 190 kb)

Suppl. Fig. 3
figure10

Representative Western blot demonstrating in vitro expression of anti-Ras-scFv antibody in T24 cells. (GIF 16 kb)

High resolution image (EPS 734 kb)

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High resolution image (EPS 593 kb)

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Serchov, T., Jilg, A., Wolf, C.T. et al. Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics. Mol Neurobiol 53, 1843–1855 (2016). https://doi.org/10.1007/s12035-015-9135-0

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Keywords

  • Ras
  • SCN
  • Photoentrainment
  • Tau
  • GSK3β
  • ERK1,2