Age-related changes in brain mitochondrial DNA deletion and oxidative stress are differentially modulated by dietary fat type and coenzyme Q₁₀

Mitochondria-related oxidative damage is a primary event in aging and age-related neurodegenerative disorders. Some dietary treatments, such as antioxidant supplementation or the enrichment of mitochondrial membranes with less oxidizable fatty acids, reduce lipid peroxidation and lengthen life span in rodents. This study compares life-long feeding on monounsaturated fatty acids (MUFAs), such as virgin olive oil, and n-6 polyunsaturated fatty acids, such as sunflower oil, with or without coenzyme Q₁₀ supplementation, with respect to age-related molecular changes in rat brain mitochondria. The MUFA diet led to diminished age-related phenotypic changes, with lipoxidation-derived protein markers being higher among the older animals, whereas protein carbonyl compounds were lower. It is noteworthy that the MUFA diet prevented the age-related increase in levels of mitochondrial DNA deletions in the brain mitochondria from aged animals. The findings of this study suggest that age-related oxidative stress is related, at the mitochondrial level, to other age-related features such as mitochondrial electron transport and mtDNA alterations, and it can be modulated by selecting an appropriate dietary fat type and/or by suitable supplementation with low levels of the antioxidant/electron carrier molecule coenzyme Q.     

Long-term potentiation in aged rats is restored when the age-related decrease in polyunsaturated fatty acid concentration is reversed

Several age-related changes have been identified in rat hippocampus; among these are deficits in glutamate release and long-term potentiation in dentate gyrus. These deficits correlate with a decrease in the concentration of arachidonic acid in hippocampus. In this study, the effects of dietary supplementation for 8 weeks with omega -6 or omega -3 fatty acids were assessed in groups of aged and young rats. The data presented indicate that dietary supplementation in aged rats restored the concentrations of arachidonic acid and docosahexanoic acid in hippocampal preparations to those observed in tissue prepared from young rats. In parallel, aged rats which received the experimental diets sustained long-term potentiation in a manner indistinguishable from young rats. The evidence presented supports the view that an age-related increase in reactive oxygen species production is linked with the decrease in polyunsaturated fatty acids and that a diet enriched in eicosapentanoic acid has antioxidant properties which may play a key role in reversal of the observed age-related deficits.     

Reversal of age-related oxidative stress prevents hippocampal synaptic plasticity deficits by protecting D-serine-dependent NMDA receptor activation

Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl-D-aspartate receptor (NMDA-R) by its co-agonist D-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous D-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent D-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in D-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC. Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the D-serine-dependent pathway.     

Phenotypic and gene expression modification with normal brain aging in GFAP-positive astrocytes and neural stem cells

Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in the expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked whether a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in the gene expression of GFAP, VEGF, and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits NSC and progenitor cell maintenance and contributes to decreased neurogenesis.     

Tissue-specific changes of mitochondrial functions in aged rats: effect of a long-term dietary treatment with N-acetylcysteine

The understanding of the involvement of mitochondrial oxidative phosphorylation (OXPHOS) in the aging process has often been biased by the different methodological approaches as well as the choice of the biological material utilized by the various groups. In the present paper, we have carried out a detailed analysis of several bioenergetic parameters and oxidative markers in brain and heart mitochondria from young (2 months) and old (28 months) rats. This analysis has revealed an age-related decrease in respiratory fluxes in brain but not in heart mitochondria. The age-related decrease in respiratory rate (-43%) by NAD-dependent substrates was associated with a consistent decline (-40%) of complex I activity in brain mitochondria. On the other hand, heart mitochondria showed an age-related decline of complex II activity. Both tissues showed, however, an age-associated accumulation of oxidative damage. We have then performed the same analysis on old (28 months) rats subjected to a long-term (16 months) diet containing the antioxidant N-acetylcysteine (NAC). The treated old rats showed a slight brain-specific improvement of mitochondrial energy production efficiency, mostly with NAD-dependent substrates, together with a decrease in carbonyl protein content and an increase in the amount of protein thiols of brain cytosolic fraction. A full recovery of complex II activity was detected in heart mitochondria from NAC-treated old rats. The present work documents the marked tissue specificity of the decline of bioenergetic functions in isolated mitochondria from aged rats and provides the first data on the effects of a long-term treatment with N-acetylcysteine.     

Melatonin ameliorates degenerative alterations caused by age in the rat prostate and mitigates high-fat diet damages

Imbalance of sexual steroids milieu and oxidative stress are often observed during aging and correlated to prostate disorders. Likewise, high-fat intake has been related to prostate damage and tumor development. Melatonin (MLT) is an antioxidant whose secretion decreases in elderly and is also suggested to protect the gland. This study evaluated the impact of a long-term high-fat diet during aging on prostate morphology and antioxidant system of rats and tested the effects of MLT supplementation under these conditions. Male rats were assigned into four groups: control, treated with MLT, high-fat diet and high-fat diet treated with MLT. The high-fat diet was provided from the 24th week of age, MLT from the 48th (100 μg/kg/day) and rats were euthanized at the 62nd week. The high-fat diet increased body weight, retroperitoneal fatness, glycaemia, and circulating estrogen levels. It aggravated the aging effects, leading to epithelial atrophy (∼32% reduction of epithelial height) and collagen fibers increase (83%). MLT alone did not alter biometric and physiological parameters, except for the prostate weight decrease, whereas it alleviated biometric as well as ameliorated acinar atrophy induced by high-lipid intake. Systemic oxidative stress increased, and prostatic glutathione peroxidase activity decreased fivefold with the high-fat diet despite the indole. Regardless of the diet, MLT triggered epithelial desquamation, reduced androgen receptor-positive cells, increased smooth muscle layer thickness (12%), decreased at least 50% corpora amylacea formation, and stimulated prostatic gluthatione-S-transferase activity. In conclusion, MLT partially recovered prostate damage induced by aging and the long-term high-fat diet and ameliorated degenerative prostate alterations.     

Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve

BackgroundAging is associated with structural, functional and biochemical alterations in the nervous system. Calorie restriction (CR) was found to retard most physiological indices of aging.ObjectivesThis work aimed to investigate the effect of CR on age-related changes in sciatic nerves.Materials and methodsThirty male albino rats aged 1 month were equally divided into three groups; Group I [control adult-ad libitum AL]: fed a regular diet and sacrificed at the age of 6 months, group II (aged-AL group): fed a regular diet AL and sacrificed at the age of 18 months, and group III (aged CR) fed a 40% calorie restricted diet and sacrificed at the age of 18 months. Rats were anesthetized and sciatic nerves were processed for light, electron microscope and morphometric studies. Oxidative stress in sciatic nerves was investigated by estimation of lipid perioxidation by product malondialdehyde (MDA) tissue level and antioxidant enzyme; superoxide dismutase activity (SOD).ResultsThe aged (AL) sciatic nerves appeared disorganized, with thick perineurium and increased collagen fibers associated with decreased g-ratio. Abnormal myelin forms were seen as outfolded myelin loops, thin denuded myelin, splitting of myelin into myelin figures and interlamellar vacuoles. Schwann cells revealed vacuolated cytoplasm. There was also significant increase in MDA level and a significant decrease in SOD activity in comparison to control adult (AL). Apparent structural and histomorphological improvement were noticed after CR in aged rats.ConclusionAging caused structural and biochemical alterations in sciatic nerves with alleviating effect of calorie restriction on such effects.     

Alternate-day fasting protects the rat heart against age-induced inflammation and fibrosis by inhibiting oxidative damage and NF-kB activation

The free radical theory of aging is currently one of the most popular. In parallel, many studies have demonstrated the association of fibrosis and increased oxidative stress in the pathogenesis of some chronic human diseases, and fibrosis is often characteristic of aging tissues. One of the few interventions that effectively slow aging is calorie restriction and the protection against the age-associated increase of oxidative stress remains one of the foremost hypotheses to explain this action. As an alternative to traditional calorie restriction, another dietary regimen, termed alternate-day fasting, has also been tested, whose antiaging mechanisms have not been explored so much extensively. We thus studied the effects of alternate-day fasting, started at 2 months of age, on oxidative stress and fibrosis in the heart during aging. In the left ventricle of the heart of elderly (aged 24 months) versus young (aged 6 months) male rats we found a significant increase in oxidative stress paralleled by increased fibrosis. In parallel there was a significant increase in inflammatory cytokine levels and in NF-kB DNA binding activity with advancing age. Alternate-day fasting protected against all these age-related phenomena. These data support the hypothesis that this kind of dietary restriction protects against age-related fibrosis, at least in part by reducing inflammation and oxidative damage, and this protection can thus be considered a factor in the prevention of age-related diseases with sclerotic evolution.     

Molecular and cellular characterization of the age-related neuroinflammatory processes occurring in normal rat hippocampus: potential relation with the loss of somatostatin GABAergic neurons

Increased neuroinflammatory reaction is frequently observed during normal brain aging. However, a direct link between neuroinflammation and neurodegeneration during aging has not yet been clearly shown. Here, we have characterized the age-related hippocampal inflammatory processes and the potential relation with hippocampal neurodegeneration. The mRNA expression of the pro-inflammatory cytokines IL-1beta and tumor necrosis factor-alpha (TNF-alpha), and the iNOs enzyme was significantly increased in aged hippocampus. Accordingly, numerous activated microglial cells were observed in aged rats. These cells were differentially distributed along the hippocampus, being more frequently located in the hilus and the CA3 area. The mRNA expression of somatostatin, a neuropeptide expressed by some GABAergic interneurons, and the number of somatostatin-immunopositive cells decreased in aged rats. However, the number of hippocampal parvalbumin-containing GABAergic interneurons was preserved. Interestingly, in aged rats, the mRNA expression of somatostatin and IL-1beta was inversely correlated and, the decrease in the number of somatostatin-immunopositive cells was higher in the hilus of dentate gyrus than in the CA1 region. Finally, intraperitoneal chronic lipopolysaccharide (LPS) injection in young animals mimicked the age-related hippocampal inflammation as well as the decrease of somatostatin mRNA expression. Present results strongly support the neuroinflammation as a potential factor involved in the age-related degeneration of somatostatin GABAergic cells.     

Changes induced by aging and drug treatment on cerebral enzymatic antioxidant system

The age-related modifications of the participants to the cerebral enzymatic antioxidant system (superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase) were evaluated in four brain regions from male Wistar rats aged 5, 10, 15, 20, 25, 30, and 35 months. Both the specific enzyme activity and the profile of any enzyme tested markedly differ with age according to the region examined: parieto-temporal cortex, caudate-putamen, substantia nigra and thalamus. This inhomogeneous age-related profile of enzyme activities could explain both the controversial data of literature and the different regional vulnerability of the brain tissue to damage with aging. In rats aged 10, 20, or 30 months, the chronic i.p. treatment for two months with papaverine or ergot alkaloids (dihydroergocristine, dihydroergocornine, dehydroergocriptine) suggests that the antioxidant enzyme activities may be influenced according to the agent utilized, the brain region tested, and the age of the animal. In any case, small differences in the drug structure support marked differences in the type and extent of the intervention on the antioxidant enzymatic system.     

Molecular architecture of myelinated peripheral nerves is supported by calorie restriction with aging

Peripheral nerves from aged animals exhibit features of degeneration, including marked fiber loss, morphological irregularities in myelinated axons and notable reduction in the expression of myelin proteins. To investigate how protein homeostatic mechanisms change with age within the peripheral nervous system, we isolated Schwann cells from the sciatic nerves of young and old rats. The responsiveness of cells from aged nerves to stress stimuli is weakened, which in part may account for the observed age-associated alterations in glial and axonal proteins in vivo . Although calorie restriction is known to slow the aging process in the central nervous system, its influence on peripheral nerves has not been investigated in detail. To determine if dietary restriction is beneficial for peripheral nerve health and glial function, we studied sciatic nerves from rats of four distinct ages (8, 18, 29 and 38 months) kept on an ad libitum (AL) or a 40% calorie restricted diet. Age-associated reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated Schwann cell phenotype. The improvements in nerve architecture with diet restriction, in part, are underlined by sustained expression of protein chaperones and markers of the autophagy–lysosomal pathway. Together, the in vitro and in vivo results suggest that there might be an age-limit by which dietary intervention needs to be initiated to elicit a beneficial response on peripheral nerve health.     

Regional differences in PACAP transport across the blood-brain barrier in mice: a possible influence of strain,amyloid beta protein,and age

The blood–brain barrier (BBB) controls the exchange of peptides and regulatory proteins between the central nervous system (CNS) and the blood. Transport across the BBB of such regulatory substances is altered in animal models of Alzheimer’s disease. These alterations could lead to cognitive impairments or diminish their therapeutic potential. Here, we measured the transport rate of radioactively labeled pituitary adenylate cyclase-activating polypeptide (PACAP) from blood into whole brain and into 11 brain regions in three groups of mice: young (2 months old) ICR, young (2 months old) SAMP8, and aged (12 months old) SAMP8 mice. The SAMP8 is a strain which develops impaired learning and memory with aging that correlates with an age-related increase in brain levels of amyloid β protein (AβP). PACAP is a powerful neurotrophin that may have a therapeutic role in neurodegenerative diseases. We found that I-PACAP crossed the BBB fastest at the hypothalamus and the hippocampus in all three groups. Slower transport rates into the whole brain, the olfactory bulb, the hypothalamus, and the hippocampus for aged SAMP8 mice was likely related to differences both from strain and expression of AβP with aging.     

Protective effect of dietary squalene supplementation on mitochondrial function in liver of aged rats

Mitochondria are an important intracellular source and target of reactive oxygen species. The life span of a species is thought to be determined, in part, by the rate of mitochondrial damage inflicted by oxygen free radicals during the course of normal cellular metabolism. In the present study, we have investigated the protective effect of squalene supplementation for 15 days and 30 days on energy status and antioxidant defense system in liver mitochondria of 18 young and 18 aged rats. The dietary supplementation of 2% squalene significantly minimized aging associated alterations in mitochondrial energy status by maintaining the activities of TCA cycle enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) at higher level in the liver mitochondria of aged rats compared with unsupplemented controls. It exerted an antioxidant effect by inhibiting mitochondrial lipid peroxidation (malondialdehyde) in liver of young and aged rats. Supplementation with squalene also maintained the mitochondrial antioxidant defense system at higher rate by increasing the level of reduced glutathione and the activities of glutathione-dependent antioxidant enzymes (glutathione peroxidase and glutathione-S-transferase) and antiperoxidative enzymes (superoxide dismutase and catalase) in the liver of young and aged rats. The results of this study provide evidence that dietary supplementation with squalene can improve liver mitochondrial function during aging and minimize the age-associated disorders in which reactive oxygen species are a major cause.     

Dietary and behavioral interventions protect against age related activation of caspase cascades in the canine brain

Lifestyle interventions such as diet, exercise, and cognitive training represent a quietly emerging revolution in the modern approach to counteracting age-related declines in brain health. Previous studies in our laboratory have shown that long-term dietary supplementation with antioxidants and mitochondrial cofactors (AOX) or behavioral enrichment with social, cognitive, and exercise components (ENR), can effectively improve cognitive performance and reduce brain pathology of aged canines, including oxidative damage and Aβ accumulation. In this study, we build on and extend our previous findings by investigating if the interventions reduce caspase activation and ceramide accumulation in the aged frontal cortex, since caspase activation and ceramide accumulation are common convergence points for oxidative damage and Aβ, among other factors associated with the aged and AD brain. Aged beagles were placed into one of four treatment groups: CON--control environment/control diet, AOX--control environment/antioxidant diet, ENR--enriched environment/control diet, AOX/ENR--enriched environment/antioxidant diet for 2.8 years. Following behavioral testing, brains were removed and frontal cortices were analyzed to monitor levels of active caspase 3, active caspase 9 and their respective cleavage products such as tau and semaphorin7a, and ceramides. Our results show that levels of activated caspase-3 were reduced by ENR and AOX interventions with the largest reduction occurring with combined AOX/ENR group. Further, reductions in caspase-3 correlated with reduced errors in a reversal learning task, which depends on frontal cortex function. In addition, animals treated with an AOX arm showed reduced numbers of cells expressing active caspase 9 or its cleavage product semaphorin 7A, while ENR (but not AOX) reduced ceramide levels. Overall, these data demonstrate that lifestyle interventions curtail activation of pro-degenerative pathways to improve cellular health and are the first to show that lifestyle interventions can regulate caspase pathways in a higher animal model of aging.     

Metabonomic alterations in hippocampus, temporal and prefrontal cortex with age in rats

The metabolic changes in hippocampus, temporal cortex and prefrontal cortex in SD rats along with aging were explored using a metabonomic approach, which based on high resolution “magic angle spinning” ¹H NMR spectroscopy. The metabolite profiles were analyzed by partial least squares-discriminant analysis, and the results showed that the metabolites of the above three brain regions in old rats were dramatically different from that in the adult and young rats. The old rats showed increased myo-inositol and lactate in all of the three brain regions, and decreased N-acetylaspartate in temporal and frontal cortex, Glutamate–GABA level became imbalance in temporal cortex of old rats. In addition, compared with the adult female rats, male rats had higher levels of N-acetylaspartate, taurine, and creatine in temporal or frontal cortex. The age-related metabolic changes may indicate the early functional alterations of neural cells in these brain regions, especially the temporal cortex. The gender-related metabolic changes suggest the significance of the hormonal regulation in brain metabolism. Our work highlights the potential of metabolic profiling to enhance our understanding of biological mechanisms of brain aging.     

Antioxidant enzymes in erythrocytes from old and diet restricted old rats

Diet restriction, prolonging the lifespan of rodents, represents an interesting model for gerontological studies. We analyzed the activity of antioxidant enzymes, Superoxide Dismutase, Catalase and Glutathione Peroxidase in erythrocytes from young, old and old food restricted Wistar rats. Diet restriction was applied feeding the animals on every-other-day schedule starting from the age of 3.5 months. The age-dependent decrease of Catalase and Glutathione Peroxidase activities was prevented by food restriction, whereas Superoxide Dismutase activity was not influenced either by aging and dietary intervention. Present results support the hypothesis that diet restriction increases the protection of cell structure against the peroxidative damage, preserving the activity of antioxidant enzymes.     

Oxidative-stress-dependent up-regulation of Bcl-2 expression in the central nervous system of aged Fisher-344 rats

Oxidative stress has been shown to play a role in aging and in neurodegenerative disorders. Some of the consequences of oxidative stress are DNA base modifications, lipid peroxidation, and protein modifications such as formation of carbonyls and nitrotyrosine. These events may play a role in apoptosis, another factor in aging and neurodegeneration, in response to uncompensated oxidative stress. Bcl-2 is a mitochondrial protein that protects neurons from apoptotic stimuli including oxidative stress. Using immunohistochemistry and western blot analysis, here we show that Bcl-2 is up-regulated in the hippocampus and cerebellum of aged (24 months) Fisher 344 rats. Treatment with the free radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN) effectively reverses this age-dependent Bcl-2 up-regulation indicating that this response is redox sensitive. This conclusion was further supported by inducing the same regional Bcl-2 up-regulation in young (3 months) Fisher 344 rats exposed to 100% normobaric O(2) for 48 h. Our results indicate that Bcl-2 expression is increased in the aged brain, possibly as a consequence of oxidative stress challenges. These results also illustrate the effectiveness of antioxidants in reversing age-related changes in the CNS and support further research to investigate their use in aging and in age-related neurodegenerative disorders.