An intracerebroventricular injection of amyloid-beta peptide (1-42) aggregates modifies daily temporal organization of clock factors expression, protein carbonyls and antioxidant enzymes in the rat hippocampus.

Alzheimer disease (AD) is the most frequent form of dementia in the elderly. It is characterized by the deterioration of memory and learning. The histopathological hallmarks of AD include the presence of extracellular deposits of amyloid beta peptide, intracellular neurofibrillary tangles, neuron and synapse loss, in the brain, including the hippocampus. Accumulation of Aß peptide causes an increase in intracellular reactive oxygen species (ROS) and free radicals associated to a deficient antioxidant defense system. Besides oxidative stress and cognitive deficit, AD patients show alterations in their circadian rhythms. The objective of this work was to investigate the effects of an intracerebroventricular injection of amyloid beta peptide Aß(1-42) aggregates on temporal patterns of protein oxidation, antioxidant enzymes and clock factors in the rat hippocampus. Four-month-old male Holtzman rats divided into the groups control (CO) and Aß-injected (Aß), were maintained under 12 h-light12h-dark conditions and received water and food ad-libitum. Hippocampus samples were isolated every 6 h during a 24 h period. Our results showed daily patterns of protein carbonyls, catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as Rorα and Rev-erbß mRNA, in the rat hippocampus. Interestingly, an intracerebroventricular injection of Aß aggregates modified daily oscillation of protein carbonyls levels, phase-shifted daily rhythms of clock genes and had a differential effect on the daily expression and activity of CAT and GPx. Thus, Aß aggregates might affect clock-mediated transcriptional regulation of antioxidant enzymes, by affecting the formation of BMAL1:CLOCK heterodimer, probably, as a consequence of the alteration of the redox state observed in rats injected with Aß.

Aging modifies daily variation of antioxidant enzymes and oxidative status in the hippocampus.

BACKGROUND: Aging is a complex and multifactorial biological process that leads to the progressive deterioration of physiological systems, including the circadian system. In addition, oxidative stress has been associated with the aging of the normal brain and the development of late-onset neurodegenerative diseases. Even though, functional weakening of circadian rhythms and antioxidant function has been observed during aging, the mechanisms by which the circadian system signaling and oxidative stress are interrelated have not yet been elucidated. The objectives of this study were to evaluate the consequences of aging on the temporal organization of the antioxidant defense system and oxidative status as well as to analyze the endogenous clock activity, in the hippocampus of aged rats. METHODS: Young adults (3-month-old) or older (22-month-old) male Holtzman rats were maintained under constant darkness conditions, during 15days before the sacrifice. Levels of catalase (CAT) and glutathione peroxidase (GPx) mRNA and activity, reduced glutathione (GSH), lipoperoxidation (LPO) and BMAL1 protein were analyzed in hippocampus samples isolated every 4h during a 24-h period. Locomotor activity was recorded during 20days before the experiment. RESULTS: Our results show that aging modifies temporal patterns of CAT and GPx expression and activity in the hippocampus in a different way. On the one hand, it abolishes the oscillating CAT expression and specific enzymatic activity while, on the other, it increases the mesor of circadian GPx activity rhythm (p<0.01). Additionally, we observed increased GSH (p<0.05) and reduced LPO (p<0.01) levels in the hippocampus of aged rats. Moreover, the nocturnal locomotor activity was reduced in the older animals in comparison to the young adult rats (p<0.01). Interestingly, the 22month-old animals became arrhythmic and showed a marked fragmentation as well as a significant decline in daily locomotor activity when they were maintained under constant darkness conditions (p<0.05). Aging also abolished circadian rhythms of the core clock BMAL1 protein. CONCLUSION: The loss of temporal organization of the antioxidant enzymes activity, the oxidative status and the cellular clock machinery could result in a temporally altered antioxidant defense system in the aging brain. Learning about how aging affects the circadian system and the expression of genes involved in the antioxidant defense system could contribute to the design of new strategies to improve the quality of life of older people and also to promote a healthy aging.

Rhythmic Bdnf and TrkB expression patterns in the prefrontal cortex are lost in aged rats.

Aging brain undergoes several changes leading to a decline in cognitive functions. Memory and learning-related genes such as Creb, Bdnf and its receptor TrkB, are expressed in different brain regions including prefrontal cortex. Those genes' proteins regulate a wide range of functions such as synaptic plasticity and long-term potentiation. In this work, our objectives were: 1) to investigate whether Creb1, Bdnf and TrkB genes display endogenous circadian expression rhythms, in the prefrontal cortex of rats maintained under constant darkness conditions; 2) to study the synchronization of those temporal patterns to the local cellular clock and 3) to evaluate the aging consequences on both cognition-related genes and activating clock transcription factor, BMAL1, rhythms. A bioinformatics analysis revealed clock-responsive (E-box) sites in regulatory regions of Creb1, Bdnf and TrkB genes. Additionally, cAMP response elements (CRE) were found in Bdnf and TrkB promoters. We observed those key cognition-related factors expression oscillates in the rat prefrontal cortex. Creb1 and TrkB mRNAs display a circadian rhythm with their highest levels occurring at the second half of the 24h period. Interestingly, the cosinor analysis revealed a 12-h rhythm of Bdnf transcript levels, with peaks occurring at the second half of the subjective day and night, respectively. As expected, the BMAL1 rhythm's acrophase precedes Creb1 and first Bdnf expression peaks. Noteworthy, Creb1, Bdnf and TrkB expression rhythms are lost in the prefrontal cortex of aged rats, probably, as consequence of the loss of BMAL1 protein circadian rhythm and altered function of the local cellular clock.

Circadian rhythms of locomotor activity and hippocampal clock genes expression are dampened in vitamin A-deficient rats.

The main external time giver is the day-night cycle; however, signals from feeding and the activity/rest cycles can entrain peripheral clocks, such as the hippocampus, in the absence of light. Knowing that vitamin A and its derivatives, the retinoids, may act as regulators of the endogenous clock activity, we hypothesized that the nutritional deficiency of vitamin A may influence the locomotor activity rhythm as well as the endogenous circadian patterns of clock genes in the rat hippocampus. Locomotor activity was recorded during the last week of the treatment period. Circadian rhythms of clock genes expression were analyzed by reverse transcription-polymerase chain reaction in hippocampus samples that were isolated every 4 hours during a 24-hour period. Reduced glutathione (GSH) levels were also determined by a kinetic assay. Regulatory regions of clock PER2, CRY1, and CRY2 genes were scanned for RXRE, RARE, and RORE sites. As expected, the locomotor activity pattern of rats shifted rightward under constant dark conditions. Clock genes expression and GSH levels displayed robust circadian oscillations in the rat hippocampus. We found RXRE and RORE sites on regulatory regions of clock genes. Vitamin A deficiency dampened rhythms of locomotor activity as well as modified endogenous rhythms of clock genes expression and GSH levels. Thus, vitamin A may have a role in endogenous clock functioning and participate in the circadian regulation of the cellular redox state in the hippocampus, a peripheral clock with relevant function in memory and learning.

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

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, RARß and RXRß 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 RXRß) 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, RARß, RXRß, 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 RXRß 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.

Daily oscillation of glutathione redox cycle is dampened in the nutritional vitamin A deficiency.

Examples of hormonal phase-shifting of circadian gene expression began to emerge a few years ago. Vitamin A fulfills a hormonal function by binding of retinoic acid to its nuclear receptors, RARs and RXRs. We found retinoid- as well as clock-responsive sites on regulatory regions of Glutathione reductase (GR) and Glutathione peroxidase (GPx) genes. Interestingly, we observed retinoid receptors, as well as GSH, GR and GPx, display daily oscillating patterns in the rat liver. We also found that feeding animals with a vitamin A-free diet, dampened daily rhythms of RARα and RXRß mRNA, GR expression and activity, GSH, BMAL1 protein levels and locomotor activity. Differently, day-night oscillations of RXRα, GPx mRNA levels and activity and PER1 protein levels, were phase-shifted in the liver of vitamin A-deficient rats. These observations would emphasize the importance of micronutrient vitamin A in the modulation of biological rhythms of GSH and cellular redox state in liver.

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

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 RXRß 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 RXRß 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.

Temporal patterns of lipoperoxidation and antioxidant enzymes are modified in the hippocampus of vitamin A-deficient rats.

Animals can adapt their behavior to predictable temporal fluctuations in the environment through both, memory-and-learning processes and an endogenous time-keeping mechanism. Hippocampus plays a key role in memory and learning and is especially susceptible to oxidative stress. In compensation, antioxidant enzymes activity, such as Catalase (CAT) and Glutathione peroxidase (GPx), has been detected in this brain region. Daily rhythms of antioxidant enzymes activity, as well as of glutathione and lipid peroxides levels, have been described in brain. Here, we investigate day/night variations in lipoperoxidation, CAT, and GPx expression and activity, as well as the temporal fluctuations of two key components of the endogenous clock, BMAL1 and PER1, in the rat hippocampus and evaluate to which extent vitamin A deficiency may affect their amplitude or phase. Holtzman male rats from control, vitamin A-deficient, and vitamin A-refed groups were sacrificed throughout a 24-h period. Daily levels of clock proteins, lipoperoxidation, CAT and GPx mRNA, protein, and activity, were determined in the rat hippocampus obtained every 4 or 5 h. Gene expression of RARalpha and RXRbeta was also quantified in the hippocampus of the three groups of rats. Our results show significant daily variations of BMAL1 and PER1 protein expression. Rhythmic lipoperoxidation, CAT, and GPx, expression and activity, were also observed in the rat hippocampus. Vitamin A deficiency reduced RXRbeta mRNA level, as well as the amplitude of BMAL1 and PER1 daily oscillation, phase-shifted the daily peak of lipoperoxidation, and had a differential effect on the oscillating CAT and GPx mRNA, protein, and activity. Learning how vitamin A deficiency affects the circadian gene expression in the hippocampus may have an impact on the neurobiology, nutritional and chronobiology fields, emphasizing for the first time the importance of nutritional factors, such as dietary micronutrients, in the regulation of circadian parameters in this brain memory-and-learning-related region.