Retinoic acid receptors move in time with the clock in the hippocampus. Effect of a vitamin-A-deficient diet

Abstract

An endogenous time-keeping mechanism controls circadian biological rhythms in mammals. Previously, we showed that vitamin A deficiency modifies clock BMAL1 and PER1 as well as BDNF and neurogranin daily rhythmicity in the rat hippocampus when animals are maintained under 12-h-light:12-h-dark conditions. Retinoic acid nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been detected in the same brain area. Our objectives were (a) to analyze whether RARα, RARβ and RXRβ exhibit a circadian variation in the rat hippocampus and (b) to investigate the effect of a vitamin-A-deficient diet on the circadian expression of BMAL1, PER1 and retinoic acid receptors (RARs and RXRβ) genes. Holtzman male rats from control and vitamin-A-deficient groups were maintained under 12-h-light:12-h-dark or 12-h-dark:12-h-dark conditions during the last week of treatment. RARα, RARβ, RXRβ, BMAL1 and PER1 transcript and protein levels were determined in hippocampus samples isolated every 4 h in a 24-h period. Regulatory regions of RARs and RXRβ genes were scanned for clock-responsive sites, while BMAL1 and PER1 promoters were analyzed for retinoic acid responsive elements and retinoid X responsive elements. E-box and retinoid-related orphan receptor responsive element sites were found on regulatory regions of retinoid receptors genes, which display an endogenously controlled circadian expression in the rat hippocampus. Those temporal profiles were modified when animals were fed with a vitamin-A-deficient diet. Similarly, the nutritional vitamin A deficiency phase shifted BMAL1 and abolished PER1 circadian expression at both mRNA and protein levels. Our data suggest that vitamin A deficiency may affect the circadian expression in the hippocampus by modifying the rhythmic profiles of retinoic acid receptors.

Figure 1

Daily rhythms of RARα (A), RARβ (B) and RXRβ (C) mRNA expression in the rat hippocampus. Horizontal bars represent the distribution of light (open) and dark (closed) phases of a 24 h (ZT0-ZT24) photoperiod. Cosine fitting curves represent normalized mRNA levels versus ZT. Each value on the curve represents the mean ± SE of three pools of two hippocampus samples each at a given ZT (with ZT= 0 when light is on). Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0.05, **P< 0.01 and ***P< 0.001 when indicated means were compared to the corresponding maximal value in each group. Top of the figures, p ≤ 0.05 indicates detection of a rhythm, p = 0 indicates p < 0.0005 from analysis by the cosinor method. D) Representative patterns of PCR products at different ZTs throughout a day-night cycle.

Figure 2

Day-night cycles of RARα (A), RARβ (B), and RXRβ (C) protein levels in the rat hippocampus. Cosine fitting curves for rhythmic normalized RARα, RARβ, and RXRβ protein levels obtained from the densitometric quantification of the Immunoblots representative data. Each point represents the mean ± SE of two pools of two hippocampus samples each at a given ZT (with ZT= 0 when light is on). Horizontal bars represent the distribution of light (open) and dark (closed) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0.05 when indicated means were compared to the corresponding maximal value in each group. Top of the figures, p ≤ 0.05 indicates detection of a rhythm, p = 0 indicates p < 0.0005 from analysis by the cosinor method. D) Representative Immunoblots at different ZTs throughout a day-night cycle.

Figure 3

Circadian rhythms of RARα, RARβ, and RXRβ mRNA levels in the hippocampus of CO and VAD rats. Cosine fitting curves for normalized RARα (A), RARβ (B), and RXRβ (C) transcripts levels throughout a day. Horizontal bars represent the distribution of dark-dark (DD) phases of a 24 h photoperiod (CT0-CT24). Each point on the curve represents the mean ± SE of three pools of two hippocampus samples each at a given CT. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0.05, **P< 0.01 and ***P< 0.001 when indicated means were compared to the corresponding maximal value in each group. Top of the figures, p ≤ 0.05 indicates detection of a rhythm, p = 0 indicates p < 0.0005 from analysis by the cosinor method. D) Representative patterns of PCR products at different CTs in a 24 h cycle.

Figure 4

Circadian profiles of RARα (A), RARβ (B), and RXRβ (C) protein levels in the hippocampus of CO and VAD rats. Cosine fitting curves for rhythmic normalized RARα, RARβ, and RXRβ protein levels were obtained from the densitometric quantification of the Immunoblots representative data. Each point in the curve represents the mean ± SE of two pools of two hippocampus samples each at a given CT. Horizontal bars represent the distribution of dark-dark (DD) phases of a 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0.05, **P< 0.01 and ***P< 0.001 when indicated means were compared to the corresponding maximal value in each group. Top of the figures, p ≤ 0.05 indicates detection of a rhythm, p = 0 indicates p < 0.0005 from analysis by the cosinor method. D) Representative Immunoblots at different CTs in a 24 h cycle.

Figure 5

Schematic representation of RARE, RXRE, and E-box sites on the 5’ regulatory region of RARα, RARβ and RXRβ genes. Arrows indicate the first translation codon, gray boxes represent exons, black circles are RARE sites, dashed circles RXREs, white circles represents RORE sites and gray ovals are E-box-like (CATATG, CATGTG, or CACTTG) sites. Negative (-) numbers indicate regulatory sites positions relative to the start of translation (+1).

Figure 6

Circadian patterns of BMAL1 and PER1 mRNA levels in the hippocampus of CO and VAD rats. Cosine fitting curves for normalized BMAL1 (A) and PER1 (B) mRNA levels throughout a day. Each point in the curve represents the mean ± SE of two pools of two hippocampus samples each at a given CT. Horizontal bars represent the distribution of dark-dark (DD) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0.05 and **P< 0.01 when indicated means were compared to the corresponding maximal value in each group. Top of the figures, p ≤ 0.05 indicates detection of a rhythm, p = 0 indicates p < 0.0005 from analysis by the cosinor method. C) Representative patterns of PCR products at different CTs in a 24 h cycle.

Figure 7

Circadian profiles of BMAL1(A) and PER1(B) protein levels in the hippocampus of CO and VAD rats. Cosine fitting curves for rhythmic normalized BMAL1 and PER1 protein levels were obtained from the densitometric quantification of the Immunoblots representative data. Each point in the curve represents the mean ± SE of two pools of two hippocampus samples each at a given CT. Horizontal bars represent the distribution of dark-dark (DD) phases of the 24-h photoperiod. Statistical analysis was performed using one-way ANOVA followed by Tukey test with *P<0.05, **P< 0.01 and ***P< 0.001 when indicated means were compared to the corresponding maximal value in each group. Top of the figures, p ≤ 0.05 indicates detection of a rhythm, p = 0 indicates p < 0.0005 from analysis by the cosinor method. C) Representative Immunoblots at different CTs in a 24 h cycle.

Figure 8

Schematic representation of RARE and RXRE sites on the 5’ regulatory region of BMAL1 and PER1 genes. Arrows indicate the first translation codon, gray boxes represent exons, dashed circles are RXRE sites and black circles are RAREs. Negative (-) numbers indicate regulatory sites positions relative to the start of translation (+1).