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

Highlights

• CAT and GPx expression and activity are temporally altered in the aging hippocampus.

• Aging increases GSH and reduces LPO levels in the in the same brain area.

• Aging abolishes circadian rhythms of the core clock BMAL1 protein in the hippocampus.

• Temporal patterns of locomotor activity are disrupted in the aged rats.

Abstract

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 15 days 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 4 h during a 24-h period. Locomotor activity was recorded during 20 days 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 22 month-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.

Introduction

Aging is a complex and multifactorial biological process that leads to the progressive deterioration of organisms. Normal aging is often associated with cognitive decline, and the hippocampus is particularly vulnerable to it (Shankar, 2010).

Several studies over the years have associated the oxidative stress with the aging of the normal brain and the development of late-onset neurodegenerative diseases, such as Alzheimer and Parkinson's diseases (Finkel and Holbrook, 2000, Balaban et al., 2005, Wang et al., 2010, Dias et al., 2013, Zhao and Zhao, 2013). Oxidative stress is generated by a combination of increased production of free radicals and oxidant agents with decreased antioxidant levels and dysregulation of the antioxidant defense system (Wang et al., 2010, Gilca et al., 2011, Dias et al., 2013, Zhao and Zhao, 2013). The main free radical is the superoxide anion, which is converted to hydrogen peroxide by the superoxide dismutase (SOD) enzyme. The hydrogen peroxide is then decomposed into oxygen and water by catalase (CAT) and glutathione peroxidase (GPx) enzymes, which are part of the cellular antioxidant defense system (Balaban et al., 2005). Furthermore, GPx also reduces lipid peroxides; in both cases GPx activity depends on the levels of reduced glutathione (GSH), since the reduction of hydrogen and lipid peroxides is coupled to the oxidation of this compound. GSH is the most abundant endogenous antioxidant in cells and besides its participation as substrate in enzymatic reactions, it exerts a powerful antioxidant effect by itself in the elimination of free radicals; therefore, this metabolite is particularly important in the regulation of the cellular redox state and protection against oxidative stress (Wu et al., 2004, Lu, 2009). Several studies have demonstrated that the activity of antioxidant enzymes decreases with the age in rat (Tsay et al., 2000, Cao et al., 2004, Rodrigues Siqueira et al., 2005, Wang et al., 2010). Additionally, the intracellular GSH concentration was also found to decrease with the age in the rat brain (Suh et al., 2004, Suh et al., 2005). Given the brain is rich in polyunsaturated fatty acids, it is highly susceptible to lipid peroxidation (LPO). It has been reported the levels of LPO increase with aging in several organs, including the brain (Radak et al., 2011). Particularly, and as mentioned before, GSH and GPx are especially important protecting lipids against oxidative stress (Radak et al., 2011). Investigations revealed an age-associated decrease in the activity of CAT, SOD and GPx as well as the GSH level, along with an increase in the level of lipid peroxidation during aging and Alzheimer's disease in the brain (Haddadi et al., 2014, Casado et al., 2008).

Previously, we and others have reported a well-orchestrated temporal expression and activity of the antioxidant defense system in different tissues, such as liver and brain (Pablos et al., 1998, Baydas et al., 2002, Fonzo et al., 2009, Ponce et al., 2012, Navigatore-Fonzo et al., 2014). It has been demonstrated that most living organisms have a circadian system that synchronizes internal events to the environmental time. In mammals, the circadian system has a hierarchic architecture. It is constituted by a master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus, which synchronizes several peripheral and subordinated clocks in the rest of the body, through a wide variety of mechanisms (Mendoza and Challet, 2009, Albrecht, 2012, Bollinger and Schibler, 2014). Thus, the SCN regulates the circadian rhythmicity of peripheral clocks by a direct way, through neural and humoral signals, as well as through an indirect way, by controlling the daily activity/rest patterns and, consequently, the body temperature and food cycle signalling (Dibner et al., 2010, Buhr and Takahashi, 2013). The persistence of rhythms when an individual is isolated from environmental cues, for example light for mammals, and kept under constant darkness conditions, is indicative of the endogenous clock control (Wollnik, 1989, Golombek and Rosenstein, 2010). The molecular clock machinery comprises: 1- transcriptional-translational feedback loops that encompass interconnected positive and negative mechanisms, 2- oscillating posttranslational modifications, and 3 -epigenetic changes. In the positive loop of the mammalian cellular clock, the transcriptional activator protein, BMAL1 (from Brain and Muscle ARNT Like protein 1) dimerizes with CLOCK (Circadian Locomoter Output Cycles Kaput protein) and binds to E-box (CANNTG) promoter sequences to activate the transcription of other clock and clock-controlled genes (Reppert and Weaver, 2002, Buhr and Takahashi, 2013). Thus, the BMAL1:CLOCK heterodimer drives the transcription of three clock Period (Per1, Per2, and Per3), two Cryptochromes (Cry1 and Cry2) and other clock and clock-controlled genes. As Per and Cry mRNAs are translated and proteins accumulate in the cytoplasm, they form PER-CRY heterodimers, which, once phosphorylated, translocate into the nucleus to negatively interfere with BMAL1:CLOCK-dependent transcription (Mendoza and Challet, 2009, Reppert and Weaver, 2002, Buhr and Takahashi, 2013). It has been shown in in vitro models, that the reduced forms of the redox NADH and NADPH cofactors, strongly stimulates the binding of BMAL1:CLOCK heterodimers to the E-box sites in the promoter of the target genes, whereas the oxidized forms thereof (NAD⁺ and NADP⁺) inhibit it (Rutter et al., 2001). These observations suggest the possibility that oxidative stress and cellular redox imbalance observed in the senescence may have some effects on the activity of the circadian clock, and its target gene expression, probably, by modulating the binding activity of BMAL1:CLOCK to the DNA. This might constitute the biochemical and molecular basis and probably explain the altered temporal organization of behavioral and physiological parameters in older individuals.

To date, at least in our knowledge, there is no report on the aging consequences on circadian patterns of the antioxidant system in a peripheral clock such as the hippocampus. Taking into account above background information, the objectives of this study were: 1) to evaluate the consequences of aging on the temporal organization of the antioxidant defense system and the oxidative status in the aged hippocampus and 2) to analyze the endogenous clock activity in the same brain area of aged rats.