Aging modifies daily variation of antioxidant enzymes and oxidative status in the hippocampus
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.
Section snippets
Animal model
Male Holtzman rats bred in our animal facilities (LABIR, National University of San Luis, San Luis, Argentina), were weaned at 21-day old and immediately assigned randomly to each group: young adults (3-month-old, n = 24) or older rats (22-month-old, n = 24). Animals were maintained in a 21–23 °C controlled environment, with ad libitum access to food and water and under a 12 h-light:12 h-dark (LD) cycle (lights on at 07:00 a.m.). In order to analyze the endogenously-driven circadian rhythms, each group
Consequences of aging on circadian rhythms of antioxidant enzymes expression and activity in the rat hippocampus
First, we were aimed to test whether antioxidant enzymes expression and activity follow a circadian pattern in the hippocampus of young adults and older rat. We found CAT mRNA levels oscillate significantly in the absence of light in the young adults rat hippocampus (ANOVA: p < 0.01; Chronos-Fit: p = 0.00006, % rhythm = 73), peaking at the second half of the subjective day (rhythm's acrophase at CT 10:07 ± 00:23; Fig. 1A and Table 2). Consistently with that, CAT enzymatic activity follows its
Discussion
Aging is a complex biological process that leads to the decline of the functionality of many physiological systems, including the circadian system. Likewise, alteration of the circadian system has a profound effect on the longevity of different organisms (Lee, 2005, Kondratov et al., 2006). Even though, functional weakening of circadian rhythms and antioxidant function has been observed during aging (Siqueira et al., 2005, Kondratova and Kondratov, 2012, Manikonda and Jagota, 2012, Farajnia et
Conclusions
Taken together, these results strongly suggest an interplay between circadian system, aging and the cellular oxidative status. 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. This could cause the organisms were not able to be adequately prepared for daily changes in metabolism, thereby favoring the aging process and age-related
Acknowledgments
This work was supported by the National Agency for Scientific and Technology Promotion [grant PICT 2010-1139-ANPCyT, Argentina], the Florencio Fiorini Foundation [grant 2013, Argentina] and the National University of San Luis [grant PROICO 2-0314, UNSL, Argentina]. We acknowledge the Laboratory of Chronobiology (IMIBIO-SL, CONICET, UNSL). Maria G Lacoste and Ana C Anzulovich are member career of the National Council of Science and Technology (CONICET). We thank Dr Sofía Giménez and Dr Gabriela
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