The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice.

Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3-32 months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain.

Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay.

DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.

Male mice retain a metabolic memory of improved glucose tolerance induced during adult onset, short-term dietary restriction.

BACKGROUND: Chronic dietary restriction (DR) has been shown to have beneficial effects on glucose homeostasis and insulin sensitivity. These factors show rapid and robust improvements when rodents were crossed over from an ad libitum (AL) diet to DR in mid life. We aimed to determine whether the beneficial effects induced by short-term exposure to DR can be retained as a 'metabolic memory' when AL feeding is resumed (AL-DR-AL) and vice versa: whether the effects of long-term DR can be reversed by a period of AL feeding (DR-AL-DR). C57BL/6 male and female mice were used to examine sex differences (N = 10/sex/group). Mice were fed AL or DR from 3 until 15 months (baseline) and each dietary crossover lasted approximately 5 months. RESULTS: In females, body and fat mass were proportional to the changes in feeding regime and plasma insulin and glucose tolerance were unaffected by the crossovers. However, in male mice, glucose tolerance and plasma insulin levels were reversed within 6 to 12 weeks. When males returned to AL intake following 5 months DR (AL-DR-AL), body mass was maintained below baseline, proportional to changes in fat mass. Glucose tolerance was also significantly better compared to baseline. CONCLUSIONS: Male mice retained a metabolic memory of 5 months of DR feeding in terms of reduced body mass and improved glucose tolerance. This implies that some of the beneficial effects induced by a period of DR in adult life may be beneficial, even when free feeding is resumed at least in males. However, under continuous DR, lifespan extension was more prominent in females than in males.

Gross energy metabolism in mice under late onset, short term caloric restriction.

Late onset, short-term moderate caloric restriction (CR) may have beneficial health effects. A 26% CR regime induced at 14 months of age for 70 days in male C57Bl/6 (ICRFa) mice resulted in a reduction in body mass of 17%. A decrease in daily energy expenditure was associated with decreased body mass in CR mice. There was no difference in total levels of physical activity between the CR and ad libitum (AL) groups; however, activity patterns were different. We developed a Bayesian model to dissect the impact of food anticipation activity (FAA) and feeding on physical activity. FAA was stronger in CR mice and remaining basal activity was higher in AL mice, but CR mice displayed larger diurnal variations as well as a phase shift in their diurnal activity. CR mice displayed lower body temperature, especially late during the dark phase. This was due to lower basal (activity-independent) temperature at all times of the day, coupled to a phase shift in the diurnal rhythm. The correlation between body temperature and physical activity was independent of feeding regimen and light/dark cycles. Reduction of body mass and basal temperature were major compensatory mechanisms to reduced food availability during late-onset, short-term CR.

Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay.

During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many 'real-life' studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate 'tissues' such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 µM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.

Reduction of dietary energy density reduces body mass regain following energy restriction in female mice.

Restriction of energy intake induces a loss of body mass that is often regained when the restriction ends. We aimed to determine whether dietary energy density (independent of macronutrient composition) modulates postrestriction regain of body mass. Fifteen female mice consumed ad libitum a standard rodent diet (with 20% added cellulose). They were then subjected to a 20% energy restriction on this diet for 10 d. Following restriction, mice consumed ad libitum the same diet with either 0 or 40% added cellulose. The study utilized a crossover design so all mice consumed both diets. Body temperature, physical activity, and digestibility were all lower when consuming the 40% cellulose diet (P < 0.001). Mice regained less mass (9%) when consuming the 40% than the 0% cellulose diet, because net energy intake was reduced by 26% (P < 0.001), despite having a greater gross energy intake (P < 0.001) (29%). To test whether there might be a constraint on intake and digestibility of the 40% cellulose diet, 20 different female mice consumed this diet at room temperature and were then transferred to the cold (7°C) to determine whether they would increase intake of this diet in response to increased energy demands. It took up to 5 d after transfer for body mass, food intake, and digestibility to increase. This suggests a digestion constraint might have limited intake of the low-energy density diet immediately following restriction. Modulation of dietary energy density in the postrestriction phase may be a valuable strategy for maintaining mass loss achieved on energy-restricted diets.