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Lifelong caloric restriction increases expression of apoptosis repressor with a caspase recruitment domain (ARC) in the brain¹

University of Florida, Biochemistry of Aging Laboratory, College of Health and Human Performance, Gainesville, Florida, USA

²Correspondence: University of Florida, Biochemistry of Aging Laboratory, 25 FLG, Stadium Road, P.O. Box 118206, Gainesville, FL 32611, USA. E-mail: cleeuwen{at}ufl.edu

SPECIFIC AIMS

We investigated apoptosis and apoptotic regulatory proteins in the brain frontal cortex of 12-month-old, 26-month-old ad libitum fed, and 26-month-old caloric restricted (CR) male Fischer-344 rats.

PRINCIPAL FINDINGS

1. We found that specific DNA fragmentation indicative of apoptosis was increased with age (+124%) in the cortices of the brain and that CR attenuated this increase significantly (-36%).

2. We determined levels of ARC (apoptosis repressor with a caspase recruitment domain), which inhibits caspase-2 activity and attenuates cytochrome c release from the mitochondria, and found a significant age-associated decline in ARC level that was attenuated in the brains of the CR rats.

3. In accordance with the changes in ARC expression observed, CR attenuated the increases in cytosolic cytochrome c and caspase-2 activity with age and suppressed the age-associated rise in cleaved caspase-9 and cleaved caspase-3.

4. These data provide evidence for an increased incidence of apoptosis in rat brain with age and that CR has the ability to attenuate this. Our data suggest that CR may provide neuroprotection through ARC by suppressing cytochrome c release and caspases-2 activity.

CONCLUSIONS AND SIGNIFICANCE

Apoptosis plays an important role in neurological diseases, but its role during normal aging of the brain is unclear. With advancing age, the brain undergoes subtle morphological and functional alterations. Cognitive impairment is caused partly by the structural alterations of macromolecules resulting from direct oxidation or products formed from oxidation and glyco-oxidation mechanisms, possibly leading to neuron loss. Neuron loss and loss of cognitive function may be a major cause of death; there has been evidence suggesting that neuron loss by apoptosis plays a role within the brain during normal aging. Although the mechanism(s) of brain aging are complex, oxidant production and oxidative stress increase within the brain with age and this organ is thought to be highly susceptible to oxidative stress and apoptosis. In the normally aging brain, oxidative stress may activate mitochondrial-mediated apoptotic pathways, since mitochondria of aged neurons produce more oxidants, accumulate calcium, and exhibit increased oxidative damage, all known stimuli for apoptosis.

Mitochondrial-mediated pathway can release cytochrome c from the mitochondria into the cytosol, where it triggers the oligomerization of Apaf-1, which in turn recruits dATP, pro-caspase-9, and pro-caspase-3 into the apoptosome, the caspase activation multiprotein complex. Serum levels of TNF- have been shown to be significantly higher in old vs. young mice and could thereby activate receptor-mediated neuronal death, which subsequently activates procaspase-2 and procaspase-8. However, intricate mechanisms to prevent apoptosis exist and may operate to spare cell loss. The prodomain of proximal caspase-2 and -8 contain a caspase recruitment domain (CARD) for modulation of their activity. ARC (apoptosis repressor with a CARD) can also interact with caspase-2 and -8 and function as an inhibitor of receptor-mediated apoptosis. Recently, ARC has been shown to block mitochondrial cytochrome c release, thus maintaining mitochondrial function.

In the following experiment, we tested whether caloric restriction could prevent the accumulation of DNA fragmentation and activation of markers of apoptosis. Caloric restriction increases mean and maximum life span, improves calcium homeostasis, and reduces oxidant production; mice subjected to long-term caloric restriction showed reductions in TNF-. Improvement in cognitive function in humans and rodents has been attributed to caloric restriction. Apoptosis and caloric restriction in the brain has not been investigated. The present study attempted to determine the mechanisms of neuronal cell death by examining apoptosis, cytosolic cytochrome c content, caspases, and ARC expression with age and lifelong caloric restriction.

This report suggests that apoptosis is increased with normal aging in the frontal cortex of the brain and that lifelong caloric restriction reduces the DNA fragmentation that occurs with age (Fig. 1 ). We show that cytosolic levels of cytochrome c and caspase-2, -3, and -9 activities (data not shown) are increased with age in the frontal cortex and that CR is able to attenuate these changes through ARC (Fig. 2 ). The decreased availability of ARC with age may increase the susceptibility for apoptosis by increasing cytochrome c release and the activity of caspase-2. In striking contrast, caloric restriction may increase the resistance to apoptosis by increasing the expression of ARC, thereby attenuating both mitochondrial and receptor-mediated susceptibility for apoptosis (Fig. 3 ). The potential role of apoptosis in the brain during aging and its possible prevention by caloric restriction could partly explain the increase in longevity observed in caloric restricted mammals.

View larger version (25K): [in this window] [in a new window]   Figure 1. Ad libitum fed and caloric restricted male Fischer 344 rats were obtained from the National Institute of Aging colony (Harlan Sprague Dawley, Indianapolis, IN). We used 12-month ad libitum fed (12AD, n=6), 26-month ad libitum fed (26AD, n=6), and 26-month caloric restricted animals (26CR, n=6). Caloric restriction (10% restriction) was started at 3.5 months of age, increased to 25% restriction at 3.75 months, and maintained at 40% restriction from 4 months onward and throughout the individual animal’s life. The frontal brain cortices were removed and cytosolic and mitochondrial protein was separated by differential centrifugation. Apoptosis determined by the quantification of mono- and oligonucleosomes (apoptotic index) in the frontal brain cortices of 12-month adult (12AD), 26-month-old (26AD), and 26 month-old caloric restricted (26CR) male Fischer 344 rats using a quantitative ELISA (Roche Molecular Biochemicals, Germany). Increased cell death was observed in 26AD group when compared to the 12AD group (*P<0.0001). Caloric restriction attenuated the age associated increase of the levels of mono- and oligonucleosomes compared to the age-matched ad libitum fed animals (**P<0.0001). Results (mean±SD; n=6 in each group) are reported as arbitrary OD units/mg protein.

View larger version (34K): [in this window] [in a new window]   Figure 2. ARC levels in the brain of adult (12AD), old (26AD), and old caloric restricted (26CR) Fischer 344 male rats determined by Western blot analysis (1:1000 dilution; Oncogene Research, Products, San Diego, CA). ARC protein content was significantly decreased in the 26AD group when compared to 12AD group (*P<0.0001). The caloric restricted animals showed a significant increased level of ARC compared with the age-matched ad libitum fed controls (**P<0.0001). Results (mean±SD; n=6 per group) are reported as % of 12AD group.

View larger version (49K): [in this window] [in a new window]   Figure 3. Brief overview of proposed protective roles of ARC (apoptosis repressor with a caspase recruitment domain) in the frontal brain cortex due to lifelong caloric restriction. With age, receptor-mediated pathways may be initiated by ligand binding of TNF- to receptors resulting in caspase activation, such as caspase-2 and -8 and further activation of procaspase-3. An increase in ARC may be able to attenuate this receptor-mediated pathway. The mitochondrial-mediated pathway may be stimulated by oxidants or increases in calcium levels resulting in loss of mitochondrial membrane potential and mitochondrial dysfunction, a phenomenon often observed in neurons of aging animals. Anti-apoptotic regulatory proteins of the bcl-2 family and the newly found protein ARC (increased with CR in cytosol) regulate the release of cytochrome c from the mitochondria and therefore caspase-3 and -9 activation. Other caspase-independent pathways that originate from the mitochondria can release proapoptotic regulatory proteins, such as apoptosis-inducing factor (AIF). Inhibitors of apoptosis (IAPs) may also play a significant role in determining the fate of a neuron. Hence, a diminished activation of mitochondrial-mediated and death receptor-mediated pathways with lifelong caloric restriction could have a profound affect on apoptosis in the brain and therefore cognition.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0803fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0803fje

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