Loss of circadian rhythmicity in aging mPer1-/-mCry2-/- mutant mice

Abstract

The mPer1, mPer2, mCry1, and mCry2 genes play a central role in the molecular mechanism driving the central pacemaker of the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus. In vitro studies suggest a close interaction of all mPER and mCRY proteins. We investigated mPER and mCRY interactions in vivo by generating different combinations of mPer/mCry double-mutant mice. We previously showed that mCry2 acts as a nonallelic suppressor of mPer2 in the core clock mechanism. Here, we focus on the circadian phenotypes of mPer1/mCry double-mutant animals and find a decay of the clock with age in mPer1-/- mCry2-/- mice at the behavioral and the molecular levels. Our findings indicate that complexes consisting of different combinations of mPER and mCRY proteins are not redundant in vivo and have different potentials in transcriptional regulation in the system of autoregulatory feedback loops driving the circadian clock.

Figure 1.

Generation of mPer1mCry double-mutant mice and representative locomotor activity records. (A) Southern blot analysis of wild-type, mPer1^-/- mCry1^-/-, and mPer1^-/- mCry2^-/- tail DNA. The mPer1 probe hybridizes to a 20-kb wild-type and a 11.8-kb mutant fragment of EcoRI-digested genomic DNA. The mCry1 probe detects a 9-kb wild-type and a 4-kb NcoI-digested fragment of the targeted locus. In mCry2 mutants, the wild-type allele is detected by hybridization of the probe to a 7-kb EcoRI fragment, whereas the mutant allele yields a 3.5-kb fragment. (B–D,F) Representative locomotor activity records of wild-type (B), mPer1^-/- mCry1^-/- (C), young mPer1^-/- mCry2^-/- (D), and old mPer1^-/- mCry2^-/- (F) animals kept in a 12-h light/12-h dark (LD) cycle and in constant darkness (DD; transition indicated by the horizontal line). Activity is represented by black bars and is double-plotted with the activity of the following light/dark cycle plotted to the right and below the previous light/dark cycle. The top bar indicates light and dark phases in LD. For the first 5 d in DD, wheel rotations per day were 20,000 ± 2500 (n = 17) for wild-type animals, 21,500 ± 7300 (n = 15) for mPer1^-/- mCry1^-/- mutants, 25,100 ± 6200 (n = 14) for young mPer1^-/- mCry2^-/- mutants, and 17,200 + 7900 (n = 9) for old mPer1^-/- mCry2^-/- mutants. (E,G,I) Periodogram analysis of young mPer1^-/- mCry2^-/- animals in DD (E corresponds to activity plot in D), and old mPer1^-/- mCry2^-/- animals in LD (G corresponds to activity plot in F) and DD (I corresponds to activity plot in F). Analysis was performed on 10 consecutive days in LD or DD after animals were allowed to adapt 5 d to the new light regimen. The ascending straight line in the periodograms represents a statistical significance of p < 0.001 as determined by the ClockLab program. (H) Age dependence of rhythmicity in wild-type (dark gray bar), mPer1^-/- (white bar), mCry2^-/- (black bar), and mPer1^-/- mCry2^-/- (light gray bar) mice. The animals tested were divided into three groups according to their age (2–6 mo, 6–12mo, and >12 mo old). Rhythmicity in DD was determined by periodogram analysis. The values on top of each bar indicate the total numbers of animals tested per group and genotype.

Figure 2.

mPer2 mRNA and mPER2 protein expression profiles of young and old mPer1^-/- mCry2^-/- mice. (A) Diurnal expression of mPer2 in the SCN of wild-type (solid line), young mPer1^-/- mCry2^-/- (dotted line), and old mPer1^-/- mCry2^-/- (dashed line) mice in LD. In old double mutants, mPer2 cycling is significantly dampened (p < 0.05). The black and white bars on the X-axis indicate the dark and light phases, respectively. All data presented are mean ± S.D. for three different experiments. (Right panels) Representative micrographs of SCN probed with an mPer2 antisense probe at time points of minimal(ZT0) and maximal(ZT12) expression. Tissue was visualized by Hoechst dye nuclear staining (blue); silver grains are artificially colored (red) for clarification. Bar, 200 μm. (B) Circadian expression of mPer2 in the SCN of wild-type (solid line) and young mPer1^-/- mCry2^-/- (dotted line) mice on the fourth day in DD. Gray and black bars on the X-axis indicate subjective day and night, respectively. (C) Diurnal variation of mPER2 immunoreactivity in the SCN of wild-type (solid line), young mPer1^-/- mCry2^-/- (dotted line), and old mPer1^-/- mCry2^-/- (dashed line) mice in LD. Quantification was performed by counting immunoreactive nuclei in the area of the SCN. In old double mutants, oscillation of mPER2 immunoreactivity is significantly dampened (p < 0.05) with medium numbers of immunoreactive nuclei. (Right panels) Representative micrographs of immunostained SCN at time points of minimal(ZT0) and maximal(ZT12) immunoreactivity. Bar, 100 μm. (D) Quantified Northern analysis of diurnal expression of mPer2 in the kidney of wild-type (solid line), young mPer1^-/- mCry2^-/- (dotted line), and old mPer1^-/- mCry2^-/- (dashed line) mice in LD. (E) Representative Northern blot from kidney tissue from wild-type (left), young mPer1^-/- mCry2^-/- (middle), and old mPer1^-/- mCry2^-/- (right) mice sequentially hybridized with mPer2 (top row) and Gapdh (bottom row) antisense probes. The black and white bars below the blots indicate dark and light phase, respectively.

Figure 3.

mCry1 and Bmal1 mRNA, and mCRY1 protein expression profiles of wild-type (solid line), young mPer1^-/- mCry2^-/- (dotted line), and old mPer1^-/- mCry2^-/- (dashed line) mice. (A) Diurnal expression of mCry1 in the SCN in LD. The black and white bars on the X-axis indicate dark and light phases, respectively. (Right panels) Representative micrographs of SCN probed with the mCry1 antisense probe at time points of minimal(ZT0) and maximal(ZT12) expression. Bar, 200 μm. (B) Circadian expression of mCry1 in the SCN on the fourth day in DD. The gray and black bars on the X-axis indicate subjective day and night, respectively. (C) Diurnal variation of mCRY1 immunoreactivity in the SCN in LD. Quantification was performed by counting immunoreactive nuclei in the area of the SCN. In old double mutants, oscillation of mCRY1 immunoreactivity is significantly dampened (p < 0.05), with constantly high numbers of immunoreactive nuclei throughout the LD cycle. (Right panels) Representative micrographs of immunostained SCN at time points of minimal(ZT0) and maximal(ZT12) immunoreactivity. Bar, 100 μm. (D) Diurnal expression of mCRY1 protein in the SCN of wild-type (solid line), mPer1^-/- (dotted line), and mCry2^-/- (hatched line) mice. (E) Diurnal variation of Bmal1 mRNA expression in the SCN in LD. In old double mutants, Bmal1 cycling is significantly dampened (p < 0.05). (F) Circadian expression of Bmal1 mRNA in the SCN on the fourth day in DD. (G) Diurnal time course of Avp mRNA expression in the SCN in LD. (H) Diurnal time course of Dbp mRNA expression in the SCN in LD. Tissue was visualized by Hoechst dye nuclear staining (blue); silver grains are artificially colored (red) for clarification. All data presented are mean ± S.D. for three different experiments.

Figure 4.

Light responsiveness in the SCN of young and old mPer1^-/- mCry2^-/- mice. (A) In situ hybridization analysis of mPer2 light inducibility in the SCN of wild-type (wt, top row), young mPer1^-/- mCry2^-/- (middle row), and old mPer1^-/- mCry2^-/- mice (bottom row). Shown are representative micrographs of SCN probed with an mPer2 antisense probe with (light) or without light administration (control) at ZT14 (15-min light pulse, 400 lx; animals were sacrificed 1 h later). Tissue was visualized by Hoechst dye nuclear staining (blue); silver grains are artificially colored (red) for clarification. Bar, 200 μm. (B) Quantification of mPer2 induction after a light pulse at Z14. (Left panel) Control animals without light exposure. (Right panel) Relative mPer2 mRNA induction after light exposure (wild-type control was set as 1). Data presented are mean ± S.D. of three different animals each. Statistical significance is indicated by asterisks (*, p < 0.05; ***, p < 0.001). (C) Immunohistochemistry analysis of CREB Ser 133 phosphorylation by light in the SCN of wild-type (wt, top row), young mPer1^-/- mCry2^-/- (middle row), and old mPer1^-/- mCry2^-/- mice (bottom row). Shown are representative micrographs of SCN sections immunostained for ^Ser133P-CREB with (light) or without light administration (control) at ZT14. As a control, SCN sections for all genotypes were stained for CREB (unphosphorylated) at the same time points. (D) Quantification of CREB phosphorylation after a light pulse at ZT14. Panels show numbers of ^Ser133P-CREB immunoreactive nuclei in the SCN with or without light exposure (***, p < 0.001). (E) Light-induced phase shifts in mPer1^-/- mCry1^-/- mice using the Aschoff Type II protocol to assess phase shifts. Animals were kept for at least 10 d in LD and released into DD after a light pulse at ZT14 or ZT22. Negative values indicate phase delays; positive values indicate phase advances. The data presented are mean ± S.D. of 10–14 animals (***, p < 0.001). (F) Light-induced phase shifts in young mPer1^-/- mCry2^-/- mice using the Aschoff Type I protocolto assess phase shifts. Animals were kept for at least 10 d in DD before a light pulse at CT14 or CT22. Data presented are mean ± S.D. of 10–13 animals.

Figure 5.

Histology and light responsiveness in the retina of wild-type, young, and old mPer1^-/- mCry2^-/- mice. Gomori trichrome (A) and lipofuscin (B) staining of retinal sections of wild-type (first row), young mPer1^-/- mCry2^-/- (second row), old mPer1^-/- mCry2^-/- (third row), mPer1^-/- (fourth row), and mCry2^-/- mice (fifth row). Retinal layers are indicated on the left. PRL, potoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (C) Immunohistochemistry analysis of light-induced CREB Ser 133 phosphorylation in the retina. (Left panels) Immunostained retinal sections of control animals without light exposure. (Right panels) Animals 1 h after light exposure (400 lx, 15 min) at ZT14. (D) Immunohistochemistry analysis for (unphosphorylated) CREB in the retina at ZT14. Bar, 10 μm.

Figure 6.

Working model for PER/CRY-driven inhibition of CLOCK/BMAL1. (A) mPer and mCry transcripts show diurnal/circadian cycling in the SCN. Whereas mPer1 expression peaks between ZT/CT 4 and 8, mCry1 and mPer2 mRNA rhythms have their maxima at ZT/CT10 to ZT/CT12, respectively. Note that mCry2 is also expressed in the SCN but without a clearly defined rhythm. Protein peaks are delayed by ∼4–6 h with regard to mRNA. (B) mPER and mCRY proteins form heteromeric complexes that form with certain preferences according to protein–protein affinity and temporal abundance. The complexes are color coded, with mPER1/mCRY1 and mPER2/mCRY2 representing the most abundant ones. The green horizontal line indicates a threshold above which a PER/CRY complex is abundant enough to influence CLOCK/BMAL1 transcription. (C) Time course of the overall inhibitory potential of the mPER/mCRY heteromers on CLOCK/BMAL1 activity. The strong inhibition of CLOCK/BMAL1 during (subjective) night corresponds to the low transcriptional activity of (CLOCK/BMAL1-induced) mPer and mCry genes.