Increased expression of heat shock proteins in rat brain during aging: relationship with mitochondrial function and glutathione redox state

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Abstract

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Oxidative damage plays a crucial role in the brain aging process and induction of Hsps is critically utilized by brain cells in the repair process following various pathogenic insults. In the present study, we investigated, in rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant redox status. In the brain expression of Hsp72 and Hsc70 increased with age up to 28 months; at this age the maximum induction was observed in the hippocampus and substantia nigra followed by cerebellum, cortex, septum and striatum. Hsps induction was associated with significant changes in glutathione (GSH) redox state and HNE levels. Interestingly, a significant positive correlation between decrease in GSH and increase in Hsp72 was observed in all brain regions examined during aging. Analysis of mitochondrial complexes showed a progressive decrease of Complex I activity and mRNA expression in the hippocampus and a significant decrease of Complex I and IV activities in the substantia nigra and septum. Our results sustain a role for GSH redox state in Hsp expression. Increase of Hsp expression promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage. Conceivably, heat shock signal pathway by increasing cellular stress resistance may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders.

Introduction

Although the term ‘aging’ is generally understood in broad terms, the aging process is extremely complex and multifaced. Increasing evidence supports the notion that reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are fundamental causes in the aging processes and neurodegenerative diseases (Halliwell, 2001; Golden et al., 2002). Experimental evidence indicates that increased rate of free radical generation and decreased efficiency of the reparative/degradative mechanisms, such as antioxidant defense and proteolysis, both are factors which primarily contribute to age-related elevation in the level of oxidative stress and brain damage. Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment and the tripeptide is a co-factor for many cytoplasmic enzymes and may also act as an important post-translational modification in a number of cellular proteins. The cysteine thiol acts, in fact, as a nucleophile in reactions with both exogenous and endogenous electrophilic species. As a consequence, reactive oxygen species (ROS) are frequently targeted by GSH in both spontaneous and catalytic reactions (Butterfield et al., 2002a, Butterfield et al., 2002b; Drake et al., 2002, Drake et al., 2003, Poon et al., 2004). Since ROS have defined roles in cell signaling events as well as in human disease pathologies, an imbalance in expression of GSH and associated enzymes has been implicated in a variety of pathological conditions (Calabrese et al., 2003, Butterfield et al., 2002c). Cause and effect links between GSH metabolism and diseases such as cancer, neurodegenerative diseases and aging have been shown (Droge, 2002). Moreover, an increase in protein oxidative damage, as indicated by the loss of protein sulfhydryl groups and by a decline in the activity of important metabolic enzymes, has been documentated to occur in brain during aging (Droge, 2002, Stadtman, 2001). The levels of oxidized proteins, exhibiting carbonyl groups, increase progressively with age in brain extracts of rats of different ages, and in old rats can represent 30–50% of the total cellular protein (Carney et al., 1991). Protein carbonyls can be generated by direct oxidative damage to proteins, by the binding to proteins of cytotoxic aldehyde such as 4-hydroxy-trans-nonenal (HNE), and by glycosidation of proteins (Hyun et al., 2003). HNE, which is highly neurotoxic, avidly binds to proteins, and HNE-protein adducts are demonstrable in senile plaques and tangles in AD, tissues from ALS patients, and lewy bodies in PD.

It is well known that brain cells are continually challenged by conditions which may cause acute or chronic stress. To adapt to these environmental changes and survive different types of injuries, a network of different responses have evolved which detect and control diverse forms of cellular stress. One of these responses, known as the heat shock response, has attracted a great deal of attention as a fundamental mechanism necessary for cell survival under a variety of unfavorable conditions (Calabrese et al., 2000a). In the central nervous system, heat shock protein (Hsp) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment, exposure to heavy metals, amino acid analogs or cytotoxic drugs (Calabrese et al., 2002a, Calabrese et al., 2000c). While prolonged exposure to conditions of extreme stress is harmful and can lead to cell death, induction of Hsp synthesis can result in stress tolerance and cytoprotection against stress-induced molecular damage (Calabrese et al., 2000a). Hence, the heat shock response contributes to establishing a cytoprotective state in a variety of metabolic disturbances and injuries, including hypoxia, stroke, epilepsy, cell and tissue trauma, neurodegenerative disease, and aging (Mayer, 2003, Motterlini et al., 2000, Calabrese et al., 2002b). This has opened new perspectives in medicine and pharmacology, as molecules activating this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In aged animals, various denatured proteins such as enzymes with lowered activity, unfolded proteins, and proteins modified by oxidation and glycation have been detected. These abnormal proteins may lead to protein aggregation, cell damage, and decreased function of organs. Stress proteins (molecular chaperones) are thought to have a role in protecting cells from damages through defense against denaturation, and restoration or resolution of denatured proteins. Therefore, alterations of the expression and function of stress proteins are supposed to be linked to the protein denaturation with aging. Recently, it was suggested that the basal Hsp70 increased by accumulation of modified proteins in aged rat kidney (Unno et al., 2000). Accumulation of denatured proteins in long-lived cells like neurons might be related to the decreased function of the brain with aging. In addition, mitochondrial respiratory chain is considered a powerful source of reactive oxygen species (ROS), and increasing body of evidence suggests that the dysfunction of cell energy metabolism is an important factor in the pathogenesis of most important neurodegenerative disorders (Calabrese et al., 2001). The implication of mitochondria both as producers and as targets of ROS has been, in fact, the basis for the mitochondrial theory of aging. Recent studies have confirmed that Complex I is a major source of superoxide production in several types of mitochondria and localized the oxygen-reducing site between the ferricyanide and the quinone reduction sites (Lenaz et al., 2002). In view of the recent findings suggesting that mitochondria are selective target of Hsp protection against oxidative insults (Tsuchiya et al., 2003), we investigated, in different brain regions of rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant status. We found that heat shock protein expresion in some brain regions, such as cortex, substantia nigra (S. nigra), striatum, septum, and hippocampus, were higher in aged than young animals and this increase was associated with mitochondrial dysfunction and with disruption of thiol homeostasis, as indicated by a decrease in the GSH and increase in GSSG and HNE levels. The possible implications of redox-dependent mechanisms and heat shock response in neurodegenerative disorders are discussed.

Section snippets

Animals

All animal protocols were approved by the University of Catania Laboratory Animal Care Advisory Committee. Male Wistar rats purchased from Harlan (Udine, Italy) were maintained in a temperature and humidity-controlled room with a 12 h light:dark cycle. Rats aged 6, 12, and 28 months, (n=8 per age group) were fed ad libitum a certified diet prepared according to the recommendations of the AIN, and the percentage energy composition is given in Table 1. After sacrifice, brains were quickly removed

Glutathione redox state analysis

When different brain regions were examined for GSH levels as a function of aging, all brain regions showed a diminution in GSH at 28 months, and all but cerebellum and septum showed the decline in GSH levels at 12 months (Fig. 1A and B). Conversely, the level of oxidized GSH, i.e., GSSG, increased with the same pattern (Fig. 1A and B). To test the hypothesis that this loss of redox status in the brain as a consequence of aging would induce an Hsp response, we measured the expression of Hsp72 in

Discussion

Aging is characterized by a general decline in physiological functions that affects many tissues and increases the risk of death. Mitochondria are important participants in the regulation of the reduction–oxidative status of the cell. As generators of ROS, mitochondria have antioxidant defense systems to counteract oxidative stress. Lacking many endogenous antioxidant mechanisms, mitochondria depend on glutathione (GSH) as an endogenous combatant against H2O2 (Coll et al., 2003, Fernandez-Checa

Acknowledgements

This work was supported by grants of Italian Cofin 2000, FIRB RBNE01ZK8F, and by NIH grants to D.A.B. [AG-05119; AG-10836].

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