Abbreviations used : Aβ, amyloid β-peptide ; DHR, dihydrorhodamine ; DR, dietary restriction ; GRP, glucose-regulated protein ; HSP, heat-shock protein ; 3-NP, 3-nitropropionic acid.
Beneficial Effects of Dietary Restriction on Cerebral Cortical Synaptic Terminals
Preservation of Glucose and Glutamate Transport and Mitochondrial Function After Exposure to Amyloid β-Peptide, Iron, and 3-Nitropropionic Acid
Article first published online: 25 DEC 2001
Journal of Neurochemistry
Volume 75, Issue 1, pages 314–320, July 2000
How to Cite
Guo, Z., Ersoz, A., Butterfield, D. A. and Mattson, M. P. (2000), Beneficial Effects of Dietary Restriction on Cerebral Cortical Synaptic Terminals. Journal of Neurochemistry, 75: 314–320. doi: 10.1046/j.1471-4159.2000.0750314.x
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- Alzheimer's disease;
- Cerebral cortex;
- Glucose-regulated protein;
- Heat-shock protein;
- Huntington's disease;
Abstract : Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid β-peptide and Fe2+) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a “preconditioning” effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.
The lifespans of laboratory rodents can be increased by reducing their calorie intake (Sohal and Weindruch, 1996). Such dietary restriction (DR) with maintenance of micronutrient intake can be accomplished either by intermittent feeding, e.g., an alternate-day feeding regimen (Goodrick et al., 1983 ; Talan and Ingram, 1985), or by “pair-feeding” using food pellets that contain 60-70% of the calories of the food pellets of the ad libitum-fed control animals (Sohal et al., 1994). DR reduces the development of age-related cancers, immune and neuroendocrine alterations, and motor dysfunction (Kritchevsky and Klurfeld, 1986 ; Ingram et al., 1987 ; Spaulding et al., 1987 ; Wachsman, 1996). Recent findings suggest that, like in other organ systems, DR has beneficial effects in the brain. Rats maintained on DR perform better on learning and memory tasks than do adlibitum-fed, age-matched rats (Idrobo et al., 1987 ; Stewart et al., 1989 ; Pitsikas and Algeri, 1992). In addition, age-related increases in levels of glial fibrillary acidic protein in the brain are suppressed in DR rats (Morgan et al., 1997). Studies of animal models of neurodegenerative disorders suggest that DR can increase resistance of neurons to age-related and disease-specific stresses. For example, rats maintained on an alternate-day DR regimen exhibit increased resistance of hippocampal neurons to excitotoxic injury and reduced deficits in learning and memory in models of Alzheimer's disease (Bruce-Keller et al., 1999 ; Zhu et al., 1999). Damage to substantia nigra dopaminergic neurons and associated motor dysfunction are markedly reduced in a mouse model of Parkinson's disease (Duan and Mattson, 1999). In a stroke model, brain damage is reduced, and behavioral outcome is improved in rats that had been maintained on a DR regimen (Yu et al., 1999).
Two mechanisms have been proposed to explain the beneficial effects of DR on aging and disease. One mechanism is based on the fact that most oxyradicals in cells are produced in mitochondria during the process of oxidative phosphorylation. Because less glucose is available to mitochondria in cells of DR animals, fewer oxyradicals are produced over time, and so there is less free radical-mediated damage to proteins, DNA, and membrane lipids (Sohal et al., 1994). A second possible mechanism for the beneficial effects of DR is that the mild metabolic stress associated with DR induces cells to express “stress proteins” that increase their resistance to disease processes. Evidence supporting the latter mechanism comes from studies showing that levels of heatshock proteins (HSPs) are increased in several tissues of rats and mice maintained on a DR regimen (Aly et al., 1994 ; Ehrenfried et al., 1996 ; Heydari et al., 1996 ; Lee et al., 1999). In addition, when rats and mice are administered 2-deoxy-D-glucose, a metabolic stress that mimics the effects of DR, neurons in their brains exhibit increased expression of stress proteins and increased resistance to oxidative and ischemic injury (Duan and Mattson, 1999 ; Yu et al., 1999).
A unique feature of the nervous system that may underlie the selective vulnerability of neurons in neurodegenerative disorders is the presence of synapses. Synapses are sites of intense ion (Na+ and Ca2+) fluxes and metabolic activity associated with excitatory synaptic transmission. Accordingly, ion-motive ATPases, glucose and glutamate transporters, and mitochondria are present at very high levels in synaptic terminals (Haber et al., 1993 ; Mark et al., 1995 ; Gloor, 1997 ; Keller et al., 1997). Whereas synaptic activity normally mediates adaptive processes such as learning and memory, excessive calcium influx and oxyradical production in synaptic terminals may initiate a neurodegenerative process, and such synaptic degeneration has been implicated in several different neurodegenerative disorders, including Alzheimer's and Huntington's diseases and stroke (for review, see Mattson and Duan, 1999). In light of the recently documented neuroprotective effects of DR (Bruce-Keller et al., 1999 ; Duan and Mattson, 1999 ; Yu et al., 1999) and its ability to retard age-related deficits in learning and memory (Idrobo et al., 1987 ; Ingram et al., 1987 ; Stewart et al., 1989 ; Pitsikas and Algeri, 1992), we tested the hypothesis that DR induces beneficial biochemical changes at the level of the synapse.
MATERIALS AND METHODS
Animals and diets
Adult (3-month-old) male Sprague-Dawley rats (Harlan Sprague Dawley) were maintained under temperature- and light-controlled conditions (20-23°C, 12-h light/12-h dark cycle). Rats were divided into two groups : an ad libitum group that had continual access to food (n = 24) and a DR group that was provided food on alternate days (n = 24). Previous studies have shown that rats and mice maintained on such an alternate-day feeding schedule will consume ~30% less food over time compared with animals fed ad libitum (Weindruch and Walford, 1988). Rats were maintained on the DR regimen for 3 months and were then killed for synaptosome preparation. As expected, the body weights of rats maintained on DR were significantly decreased by ~15% compared with rats fed ad libitum (ad libitum, 492 ± 9 g ; DR group, 423 ± 7 g ; p < 0.0001). All procedures complied with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee.
Synaptosome preparation and experimental treatments
Rats from DR and ad libitum groups were killed alternately in pairs, and cerebral hemispheres from individual rats were homogenized in a solution containing 0.32 M sucrose, 4 μg/ml pepstatin, 5 μg/ml aprotinin, 20 μg/ml trypsin inhibitor, 4 μg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM EGTA, and 20 mM HEPES. Synaptosomes were prepared using a discontinuous sucrose density gradient protocol described previously (Keller et al., 1997 ; Begley et al., 1999). Protein concentrations in synaptosomal preparations were determined (Pierce BCA kit), and the synaptosomes were diluted in Locke's buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, and 5 mM HEPES, pH 7.2) to a final concentration of 200 μg of protein/ml. Synaptosomes were then aliquoted into 1.5-ml Eppendorf tubes for experimental treatment. Synaptosomes were either left untreated or exposed to 10 μM FeSO4, 50 μM amyloid β-peptide (Aβ) 25-35 (Bachem, Torrance, CA, U.S.A.), or 5 mM 3-nitropropionic acid (3-NP). Following an experimental treatment period of 4 h, synaptosomes were pelleted and subjected to glucose and glutamate transport assays or to analyses of mitochondrial reactive oxygen species and transmembrane potential as described below. In preliminary studies we oberved no differences in brain weight or yield of synaptosomes (in mg of synaptosome protein per brain) between the DR and ad libitum groups.
Measurements of glucose and glutamate uptake
The methods for quantifying glutamate and glucose uptake in synaptosomes have been described previously (Keller et al., 1997). For the glucose uptake assay, synaptosomes (200 μg per tube) were subjected to experimental treatments and then washed three times in glucose-free Locke's buffer, and the assay was started by addition of 1.5 μCi of 2-deoxy[3H]glucose. Seven minutes later the assay was stopped by pelleting the synaptosomes, washing twice with glucose-free Locke's solution, and lysing the synaptosomes in 200 μl of a 1% sodium dodecyl sulfate in phosphate-buffered saline solution. For the glutamate uptake assay, synaptosomes (200 μg per tube) were incubated for 7 min with [3H]glutamate (0.1 μCi/ml). The synaptosomes were then pelleted, washed three times in Locke's buffer, and lysed in 200 μl of 1% sodium dodecyl sulfate in phosphate-buffered saline solution. The lysates were placed in scintillation vials containing Scintiverse, and radioactivity was counted in a Packard model 2500TR liquid scintillation counter. Results are expressed as cpm per milligram of protein. The rates of glucose and glutamate uptake were linear during the 7-min assay period and remained constant under basal conditions throughout the time course of experiments (up to 4 h).
Measurements of mitochondrial reactive oxygen species and transmembrane potential
The dye dihydrorhodamine (DHR), which enters mitochondria and fluoresces when oxidized by reactive oxygen species (particularly peroxynitrite and hydroxyl radical) to the positively charged rhodamine-123 derivative, was used to measure relative levels of mitochondrial oxyradicals. Previous studies have characterized the use of DHR in similar studies of synaptosomes and cultured neurons (Keller et al., 1997 ; Mattson et al., 1997). Following experimental treatment, synaptosomes were incubated for 30 min in the presence of 10 μM DHR and then were washed twice in Locke's buffer. Following washing, the synaptosomes were seeded into 35-mm-diameter, glassbottom, polyethylenimine-coated culture dishes and allowed to settle on the glass surface during a 10-15-min incubation. DHR fluorescence in synaptosomes was imaged using a confocal laser scanning microscope with excitation at 488 nm and emission at 510 nm, and the average pixel intensity in userdefined areas corresponding to synaptosomal aggregates was determined using Imagespace software (Molecular Dynamics). All images were coded and analyzed without knowledge of experimental treatment history of the synaptosomes.
Mitochondrial transmembrane potential was assessed using the dye rhodamine-123 by methods similar to those described previously (Guo and Mattson, 2000). In brief, synaptosomes were incubated for 30 min in the presence of 5 μM dye, washed tiwce with Locke's buffer, and then analyzed as described for DHR fluorescence.
These methods were similar to those described previously (Begley et al., 1999). In brief, 50 μg of solubilized synaptosomal proteins was separated by electrophoresis in a polyacrylamide gel, transferred to a nitrocellulose sheet, and immunoreacted with primary antibody overnight at 4°C. The nitrocellulose sheet was further processed using horseradish peroxidase-conjugated anti-mouse secondary antibody and a chemiluminescence detection method (Amersham). The primary antibodies included a mouse monoclonal antibody against the inducible form of HSP-70 (Sigma ; 1:5,000 dilution), a rabbit polyclonal antibody against glucose-regulated protein (GRP)-78 (StressGen ; 1:2,000 dilution), and a mouse monoclonal antibody against HSP-60 (Sigma ; 1:500 dilution).
Preservation of glucose and glutamate uptake following exposure to oxidative and metabolic insults in cortical synaptosomes from rats maintained on DR
Previous studies have shown that cortical synaptosomes exhibit relatively high level of basal glucose uptake and that glucose uptake is reduced following exposure to oxidative insults, including Fe2+ and Aβ (Keller et al., 1997). Basal levels of glucose uptake were similar in synaptosomes from DR rats and rats fed ad libitum (Fig. 1). As expected, exposure of synaptosomes from ad libitum-fed control rats to Fe2+ for 4 h resulted in a marked impairment of glucose uptake to a value <30% of the basal level. In contrast, exposure of synaptosomes from DR rats to the same concentration of Fe2+ resulted in only a 25-30% decrease in glucose uptake (Fig. 1). Aβ, a neurotoxic protein believed to promote neuronal degeneration in Alzheimer's disease (for review, see Mattson, 1997), caused a 25% decrease in glucose uptake in synaptosomes from ad libitum-fed rats but did not impair glucose uptake in synaptosomes from DR rats. Synaptosomes from DR rats were also resistant to impairment of glucose uptake following exposure to the mitochondrial toxin 3-NP, compared with synaptosomes from rats fed ad libitum (Fig. 1).
Glutamate transport plays an important role in removing glutamate from the extracellular space at synapses and thereby protecting neurons against excitotoxicity (for review, see Sims and Robinson, 1999). Exposure of synaptosomes to Fe2+ resulted in a decreased level of glutamate uptake, the magnitude of which was significantly less in synaptosomes from DR rats than in synaptosomes from rats fed ad libitum (Fig. 2). Glutamate uptake was also impaired following exposure of synaptosomes to Aβ and 3-NP, and synaptosomes from DR rats were more resistant to impairment of glutamate uptake by these insults compared with synaptosomes from rats fed ad libitum.
Preservation of mitochondrial function after oxidative and metabolic insults in synaptosomes from rats maintained on DR
Mitochondria are present in synaptic terminals, wherein they play important roles in supplying ATP and maintaining calcium homeostasis. Dysfunction of mitochondria in various neurodegenerative conditions is associated with increased levels of mitochondrial reactive oxygen species and a decrease in the mitochondrial transmembrane potential (Dugan et al., 1995 ; Keller et al., 1997). We previously reported that exposure of cortical synaptosomes to Fe2+ and Aβ results in increased levels of mitochondrial reactive oxygen species and mitochondrial membrane depolarization (Keller et al., 1997) ; Mark et al., 1997a). When cortical synaptosomes from ad libitum-fed rats were exposed to Fe2+, levels of DHR fluorescence, a measure of mitochondrial reactive oxygen species, were increased approximately sixfold within 4 h (Fig. 3). In contrast, levels of DHR fluorescence were increased only threefold in synaptosomes from DR rats 4 h following exposure to Fe2+. The magnitude of the increase in DHR fluorescence following exposures to Aβ and 3-NP was also significantly less in synaptosomes from DR rats compared with synaptosomes from rats fed ad libitum (Fig. 3).
Previous studies have shown that a decrease in mitochondrial transmembrane potential occurs in neurons exposed to Aβ and iron and that this change in potential can contribute to neuronal apoptosis in experimental models of neurodegenerative disorders (Keller et al., 1998 ; Guo et al., 1999 ; Jellinger, 1999). When synaptosomes from ad libitum-fed rats were exposed to Fe2+, levels of rhodamine-123 fluorescence (a measure of mitochondrial membrane potential) were decreased by ~50% within 4 h (Fig. 4). Levels of rhodamine-123 fluorescence were significantly greater in synaptosomes from DR rats 4 h following exposure to Fe2+. The magnitude of the decrease in rhodamine-123 fluorescence following exposures to 3-NP was also significantly less in synaptosomes from DR rats compared with synaptosomes from rats fed ad libitum (Fig. 4). The difference in the level of rhodamine-123 fluorescence following exposure to Aβ in synaptosomes from DR- and ad libitum-fed rats did not reach statistical significance.
Increased levels of stress proteins in synaptosomes from rats maintained on DR
Emerging data suggest that the mechanism whereby DR increases neuronal resistance in experimental models of neurodegenerative disorders is by inducing the expression of “stress proteins” (Duan and Mattson, 1999 ; Yu and Mattson, 1999). We therefore determined if such stress proteins are present in cortical synaptosomes and whether DR affected levels of the stress proteins. Immunoblot analyses were performed on proteins in synaptosomal homogenates using antibodies against HSP-70, HSP-60, and GRP-78. All three stress proteins were present at readily detectable levels in cortical synaptosomes from ad libitum-fed control rats (Fig. 5). Levels of HSP-70 and GRP-78 were significantly increased in synaptosomes from DR-fed rats compared with levels in synaptosomes from rats fed ad libitum (Fig. 5). Levels of HSP-60 were similar in synaptosomes from DR-fed rats and synaptosomes from rats fed ad libitum.
The present findings provide the first direct evidence that DR can affect synaptic homeostasis in a manner consistent with an “antiaging” effect. We found that cortical synaptosomes from DR rats were more resistant to dysfunction caused by oxidative and metabolic insults than were synaptosomes from rats fed ad libitum. The extent of impairment of glucose uptake following exposure of synaptosomes to Fe2+, Aβ, and 3-NP was significantly less in synaptosomes from rats maintained on an alternate-day DR regimen. Basal levels of glucose and glutamate uptake were not different in synaptosomes from DR and ad libitum-fed rats, indicating that the enhanced levels of glucose and glutamate uptake following oxidative insults were not the result of synaptosomes from DR rats having a greater capacity for glucose and/or glutamate uptake. The mechanism whereby the three different insults impair glucose uptake involves oxidative stress and membrane lipid peroxidation (Keller et al., 1997, 1998 ; Mark et al., 1997b), suggesting that DR lessens the impact of such oxidative insults on synaptic function. Mitochondrial homeostasis, as indicated by levels of reactive oxygen species and membrane potential, were relatively preserved following the oxidative and metabolic insults in synaptosomes from DR rats, suggesting that DR exerts its protective effect, at least in part, by suppressing mitochondrial oxyradical production and preserving mitochondrial function. The measurements of mitochondrial reactive oxygen species and potential used in the present study likely represent signal coming from intraterminal mitochondria because previous analyses of similar synaptosome preparations have shown that most mitochondria are located within the nerve terminal endings, although some free mitochondria may also be present (Deutsch et al., 1981).
The insults used and end points measured in the present study were selected because of their relevance to several different age-related neurodegenerative disorders. Fe2+ and Aβ induce membrane lipid peroxidation in hippocampal neurons (Goodman and Mattson, 1994 ; Mark et al., 1997b) and cortical synaptosomes (Keller et al., 1997). Increased levels of membrane lipid peroxidation have been documented in association with the neurodegenerative process in postmortem tissues from patients with Alzheimer's disease (Lovell et al., 1997 ; Sayre et al., 1997), Parkinson's disease (Jellinger, 1999), amyotrophic lateral sclerosis (Pedersen et al., 1998), and stroke (Chan, 1994). 3-NP is an irreversible inhibitor of mitochondrial succinate dehydrogenase that induces selective damage to striatal neurons and is therefore used in rodent models of Huntington's disease (Beal, 1994). Reduced glucose transport has been documented in Alzheimer's patients and following exposure of neurons and synaptosomes to Aβ (for review, see Mattson et al., 1999), and impaired glutamate transport has been linked to Alzheimer's disease (Masliash et al., 1996) and amyotrophic lateral sclerosis (Rothstein, 1995). Impairment of these two membrane transporters is expected to increase neuronal vulnerability to excitotoxicity, a mechanism of neuronal degeneration increasingly implicated in many different neurodegenerative conditions. Preservation of synaptic glucose and glutamate transport may therefore contribute to the neuroprotection and improved behavioral outcome previously documented in animal models of Alzheimer's disease (Bruce-Keller et al., 1999 ; Zhu et al., 1999), Parkinson's disease (Duan and Mattson, 1999), Huntington's disease (Bruce-Keller et al., 1999), and stroke (Yu and Mattson, 1999).
It is well established that exposure of neurons in cell culture or in vivo to moderate levels of stress, such as an elevated temperature or mild ischemia, can increase resistance of the neurons to subsequent more severe insults (Lowenstein et al., 1991 ; Matsushima and Hakim, 1995 ; Barone et al., 1998). Associated with such “preconditioning” mechanisms is an increase in production of “stress proteins,” including HSP-70 (States et al., 1996 ; Lee et al., 1999) and GRP-78 (Yu et al., 1999). We have proposed that the neuroprotective effect of DR is mediated by a similar preconditioning mechanism (Lee et al., 1999 ; Yu and Mattson, 1999). The present findings suggest that this beneficial stress response results in increased levels of stress proteins in synaptic terminals that is correlated with increased resistance of the synaptic terminals to oxidative and metabolic insults. Previous immunohistochemical and biochemical analyses have documented the presence of both the inducible (Karunanithi et al., 1999) and constitutive (Suzuki et al., 1999) forms of HSP-70 in synaptic terminals. In addition to the present findings, support for a synaptoprotective action of stress proteins comes from a recent study showing that synaptic transmission at Drosophila neuromuscular synapses is protected by prior heat shock and that the protection is tightly correlated with increased production of HSP-70 (Karunanithi et al., 1999).
Synaptosomes from DR rats were resistant to a wide range of toxic insults in a wide range of ways. The beneficial effects of DR may therefore result from a change at a point(s) in the neurotoxic cascade that is pivotal to each of the neurotoxic insults. Previous studies of nonneuronal cells have shown that heat shock can prevent cell death induced by oxidative insults (Polla et al., 1996) and mitochondrial toxins (Jaattela et al., 1998). In addition, HSPs can protect mitochondria against various insults (Mizzen et al., 1991 ; Polla et al., 1996), consistent with our data showing preservation of mitochondrial function after oxidative and metabolic insults in synaptosomes from rats maintained on a DR regimen. Thus, stimuli that increase levels of stress proteins in synapses may preserve both the structure and function of the synapses under various adverse conditions.
Our data indicate that such beneficial stress responses can be realized by a simple dietary manipulation—reducing food intake. If similar mechanisms are operative in humans, then DR may be a useful approach for reducing risk for, and severity of, the many neurodegenerative conditions that involve synaptic dysfunction and degeneration. In support of this possibility, recent data from prospective epidemiological studies indicate that individuals with low daily calorie intakes have a reduced risk for Parkinson's disease (Logroscino et al., 1996) and Alzheimer's disease (Mayeux et al., 1999).
This work was supported by the National Institute on Aging.
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