Long Term Running Biphasically Improves Methylglyoxal-Related Metabolism, Redox Homeostasis and Neurotrophic Support within Adult Mouse Brain Cortex

Oxidative stress and neurotrophic support decline seem to be crucially involved in brain aging. Emerging evidences indicate the pro-oxidant methylglyoxal (MG) as a key player in the age-related dicarbonyl stress and molecular damage within the central nervous system. Although exercise promotes the overproduction of reactive oxygen species, habitual exercise may retard cellular aging and reduce the age-dependent cognitive decline through hormetic adaptations, yet molecular mechanisms underlying beneficial effects of exercise are still largely unclear. In particular, whereas adaptive responses induced by exercise initiated in youth have been broadly investigated, the effects of chronic and moderate exercise begun in adult age on biochemical hallmarks of very early senescence in mammal brains have not been extensively studied. This research investigated whether a long-term, forced and moderate running initiated in adult age may affect the interplay between the redox-related profile and the oxidative-/MG-dependent molecular damage patterns in CD1 female mice cortices; as well, we investigated possible exercise-induced effects on the activity of the brain derived neurotrophic factor (BDNF)-dependent pathway. Our findings suggested that after a transient imbalance in almost all parameters investigated, the lately-initiated exercise regimen strongly reduced molecular damage profiles in brains of adult mice, by enhancing activities of the main ROS- and MG-targeting scavenging systems, as well as by preserving the BDNF-dependent signaling through the transition from adult to middle age

Late-onset running biphasically improves redox balance, energy- and methylglyoxal-related status, as well as SIRT1 expression in mouse hippocampus.

Despite the active research in this field, molecular mechanisms underlying exercise-induced beneficial effects on brain physiology and functions are still matter of debate, especially with regard to biological processes activated by regular exercise affecting the onset and progression of hippocampal aging in individuals unfamiliar with habitual physical activity. Since such responses seem to be mediated by changes in antioxidative, antiglycative and metabolic status, a possible exercise-induced coordinated response involving redox, methylglyoxal- and sirtuin-related molecular networks may be hypothesized. In this study, hippocampi of CD1 mice undergoing the transition from mature to middle age were analyzed for redox-related profile, oxidative and methylglyoxal-dependent damage patterns, energy metabolism, sirtuin1 and glyoxalase1 expression after a 2- or 4-mo treadmill running program. Our findings suggested that the 4-mo regular running lowered the chance of dicarbonyl and oxidative stress, activated mitochondrial catabolism and preserved sirtuin1-related neuroprotection. Surprisingly, the same cellular pathways were negatively affected by the first 2 months of exercise, thus showing an interesting biphasic response. In conclusion, the duration of exercise caused a profound shift in the response to regular running within the rodent hippocampus in a time-dependent fashion. This research revealed important details of the interaction between exercise and mammal hippocampus during the transition from mature to middle age, and this might help to develop non-pharmacological approaches aimed at retarding brain senescence, even in individuals unfamiliar with habitual exercise

MG-related protein damage and expression of MG-targeting detoxification system in hippocampi of mice undergoing 2- or 4-mo moderate treadmill running.

<p>Immunoreactivity levels against arg-pyrimidine and protein expression of glyoxalase 1 (GLO1) in hippocampi of mature CD-1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 12 per group). As shown in panel a, an age-dependent increase in the levels of MG-damaged protein was detected (S4 vs S2), and an increasing trend in MG-related protein damage was observed after 2 months of running (E2 vs S2). The immunoreactivity against arg-pyrimidine decreased after 4 months of regular exercise (E4 vs S4). Dot-blots signals were normalized against the Comassie Brilliant Blue-based total protein staining. In the panel a, right section, representative dot immunoblots of three independent experiments were reported. As reported in panel b, an age-dependent increase in the GLO1 protein expression levels was detected (S4 vs S2). 2-mo exercise increased GLO1 protein levels (E2 vs S2), while the expression levels of GLO1 decreased after four months of regular running (E4 vs S4). Immunosignals were normalized against the housekeeping β-actin. Representative western immunoblots of three independent experiments were reported in panel b. Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: *** P<0.001; ** P<0.01. Experiments were performed in triplicate.</p

NAD redox balance and expression of SIRT1 in hippocampi of mice undergoing 2- or 4-mo moderate treadmill running.

<p>Assessment of nicotinamide adenine dinucleotide redox balance (panel a) and Western immunoblot against SIRT1 (panel b) in hippocampi of mature CD-1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 12 per group). Both the NAD redox ratio and the SIRT1 protein level were decreased in an age-dependent fashion (S4 vs S2) (panels a and b, respectively). 2-mo exercise reduced both the NAD⁺/NADH ratio and the expression level of SIRT1 (E2 vs S2) (panels a and b, respectively), whereas both the NAD redox balance and the protein level of SIRT1 increased after 4-mo exercise (E4 vs S4) (panels a and b, respectively). Western blot signals were normalized against the housekeeping β-actin. Representative western immunoblots of three independent experiments were reported in panel b. Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: *** P<0.001; ** P<0.01. Experiments were performed in triplicate.</p

Hippocampal peroxidative damage in mice undergoing 2- or 4-mo moderate treadmill running.

<p>Assessment of hippocampal levels of thiobarbituric acid-reactive substances (TBARS) levels in mature CD-1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 12 per group). TBARS levels were found to be increased by the sedentary aging (S4 vs S2). 2-mo exercise increased TBARS hippocampal concentrations (E2 vs S2), while 4-mo running decreased TBARS levels (E4 vs S4). Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: *** P<0.001; Experiments were performed in triplicate.</p

Ponderal profiles of mice undergoing a long-term moderate treadmill running.

<p>No significant age-dependent main effect was detected on body weight, neither did treadmill running induce significant alterations in ponderal state. Values were given as means ± std. dev.</p

Neurotrophic support in brains of mice undergoing a long-term moderate treadmill running.

<p>Western immunoblot against the brain derived neurotrophic factor (BDNF) (panel a) and the phosphorilated cAMP response element binding (p-CREB) transcriptional activator (panel b) in brain cortices of adult CD1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 6 per group). A significant age-dependent variation in BDNF protein expression was detected (S4 vs S2). BDNF levels were also significantly reduced in two-month exercised mice (E2 vs S2), yet strongly enhanced in four-month exercised mice (E4 vs S4), with respect to age-matched counterparts. Significant age-dependent reductions in the protein levels of p-CREB (S4 vs S2). Two months of exercise caused a conspicuous decrease in p-CREB protein levels (E2 vs S2), whereas p-CREB protein amounts increased after four months of regular running (E4 vs S4), with respect to age-matched unexercised mice. Immunosignals were normalized against the housekeeping β-actin. Representative PVDF-western immunoblots of three independent experiments are reported. Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: * P<0.05; ** P<0.01; *** P<0.001. Experiments were performed in triplicate.</p

Methylglyoxal-related enzymatic removal and molecular damage in brains of mice undergoing a long-term moderate treadmill running.

<p>Immunoreactivity levels against arg-pyrimidine (arg-pyr, panel a) and specific activities of glyoxalase 1 (GLO1, panel b) and glyoxalase 2 (GLO2, panel c) in brain cortices of adult CD1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 6 per group). Dot blot assays revelead an age-dependent increase in the levels of MG-damaged proteins (S4 vs S2). No significant variation of MG-damaged polypeptides was detected after two months of exercise (E2 vs S2), while immunoreactivity against MG-modified arginine residues decreased markedly after four-month physical activity, with respect to age-matched unexercised mice (E4 vs S4). Immunosignals were normalized against the Coomassie Brilliant Blue (CBB)-based total protein staining. In the panel a, right section, representative PVDF-dot immunoblots of three independent experiments are reported, together with analyses for total protein loading performed by the ImageJ software. Both GLO1 and GLO2 specific activities increased in an age-dependent manner (S4 vs S2). The exercise program elevated GLO2 activity exclusively after two months of regular exercise (E2 vs S2), whereas GLO1 specific activity was increased only after four months of physical activity (E4 vs S4). Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: * P<0.05; *** P<0.001. Experiments were performed in triplicate.</p

Glutathione redox status in hippocampi of mice undergoing 2- or 4-mo moderate treadmill running.

<p>Evaluation of the glutathione redox balance in hippocampi of mature CD1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 12 per group). A significant age-dependent decrease in the total vs oxidized glutathione ratio was found within mouse hippocampal formations (S4 vs S2). 2-mo running decreased the total vs disulfide glutathione ratio (E2 vs S2), whereas no significant change was induced by 4-mo exercise (E4 vs S4). Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: *** P<0.001; ** P<0.01. Experiments were performed in triplicate.</p