Daily patterns of clock and cognition-related factors are modified in the hippocampus of vitamin A-deficient rats

Abstract

The circadian expression of clock and clock-controlled cognition-related genes in the hippocampus would be essential to achieve an optimal daily cognitive performance. There is some evidence that retinoid nuclear receptors (RARs and RXRs) can regulate circadian gene expression in different tissues. In this study, Holtzman male rats from control and vitamin A-deficient groups were sacrificed throughout a 24-h period and hippocampus samples were isolated every 4 or 5 h. RARα and RXRβ expression level was quantified and daily expression patterns of clock BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins, as well as temporal expression of cognition-related RC3 and BDNF genes were determined in the hippocampus of the two groups of rats. Our results show significant daily variations of BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins and, consequently, daily oscillating expression of RC3 and BDNF genes in the rat hippocampus. Vitamin A deficiency reduced RXRβ mRNA level as well as the amplitude of PER1, REVERB gene, and REVERB protein rhythms, and phase-shifted the daily peaks of BMAL1 and RORα mRNA, RORα protein, and RC3 and BDNF mRNA levels. Thus, nutritional factors, such as vitamin A and its derivatives the retinoids, might modulate daily patterns of BDNF and RC3 expression in the hippocampus, and they could be essential to maintain an optimal daily performance at molecular level in this learning-and-memory-related brain area.

Figure 1

Transcript levels of RARα and RXRβ in the hippocampus of control and vitamin A-deficient rats. Basal mRNA levels were determined by Real-Time PCR and normalized to β-actin. Each bar represents the mean ± SE of 4 samples in triplicates with ***P<0.001 in comparison to controls.

Figure 2

Schematic representation of RARE, RXRE, E-box and RORE sites on the 5’ regulatory region of BMAL1, PER1, RORα and REVERB genes. The Gene ID # for the sequences taken from the NCBI Gene database are: rBMAL1 (NCBI Gene ID # 29657), rPER1 (NCBI Gene ID # 287422), rRORa (NCBI Gene ID # 300807) and rREVERB (NCBI Gene ID # 252917). Arrows indicate the first translation codon, gray boxes represent exons, dashed circles are RXRE sites, black circles are RAREs, black ovals represent perfect E-boxes, white ovals Ebox-like elements and grey ovals are RORE sites. Negative (−) numbers indicate regulatory sites positions relative to the start of translation (+1).

Figure 3

Daily rhythms of clock genes expression in the hippocampus of control and vitamin A-deficient rats. (A–D) Cosine fitting curves for normalized BMAL1 (A), PER1 (B), RORα (C) and REVERB (D) mRNA levels throughout a day. Horizontal bars represent the distribution of light (open) and dark (closed) phases of a 24-h (ZT0-ZT24) photoperiod. Each point on the curves represents the mean ± SE of three pools of two hippocampus samples each at a given ZT (with ZT=0 when light is on). Significant daily variation was evaluated 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. (E) Representative patterns of PCR products at different ZTs throughout a day-night cycle.

Figure 4

Daily rhythms of RORα and REVERB protein levels in the rat hippocampus. (A–B) Cosine fitting curves for normalized RORα (A) and REVERB (B) protein levels throughout a day, obtained from the densitometric quantitation of the Immunoblots. Horizontal bars represent the distribution of light (open) and dark (closed) phases of a 24-h (ZT0-ZT24) photoperiod. Each value on the curves represents the mean ± SE of three pools of two hippocampus samples each at a given ZT (with ZT=0 when light is on). Significant daily variation was evaluated using one-way ANOVA followed by Tukey test with *P<0.05 and **P<0.01 when compared indicated means with the corresponding maximal value in each group. (C) Representative Immunoblot analysis of protein extracted from CO and DE rat hippocampi isolated at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22.

Figure 5

Schematic representation of E-box and RARE sites on the 5′ regulatory region of the BDNF and RC3 genes. Genes ID # are: 24225 and 64356 for the rBDNF and rRC3 sequences taken from the NCBI database, respectively. Arrow indicates the first translation codon, gray box represents first exon, black ovals are perfect E-boxes, the white oval is an E-box-like and white circles represent RARE elements. Negative (−) numbers indicate clock-and retinoic acid-responsive sites positions relative to the start of translation (+1).

Figure 6

Daily RC3 and BDNF expression in the hippocampus of control and vitamin A-deficient rats. (A–B) Cosine fitting curves for normalized RC3 (A) and BDNF (B) mRNA levels throughout a day. Horizontal bars represent the distribution of light (open) and dark (closed) phases of a 24-h (ZT0-ZT24) photoperiod. Each point on the curves represents the mean ± SE of three pools of two hippocampus samples each at a given ZT (with ZT=0 when light is on). Significant daily variation was evaluated 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. (C) Representative patterns of PCR products at different ZTs throughout a day-night cycle.