RasGrf1 deficiency delays aging in mice

Abstract

RasGRF1 is a Ras-guanine nucleotide exchange factor implicated in a variety of physiological processes including learning and memory and glucose homeostasis. To determine the role of RASGRF1 in aging, lifespan and metabolic parameters were analyzed in aged RasGrf1(-/-) mice. We observed that mice deficient for RasGrf1(-/-) display an increase in average and most importantly, in maximal lifespan (20% higher than controls). This was not due to the role of Ras in cancer because tumor-free survival was also enhanced in these animals. Aged RasGrf1(-/-) displayed better motor coordination than control mice. Protection against oxidative stress was similarly preserved in old RasGrf1(-/-). IGF-I levels were lower in RasGrf1(-/-) than in controls. Furthermore, SIRT1 expression was increased in RasGrf1(-/-) animals. Consistent with this, the blood metabolomic profiles of RasGrf1-deficient mice resembled those observed in calorie-restricted animals. In addition, cardiac glucose consumption as determined PET was not altered by aging in the mutant model, indicating that RasGrf1-deficient mice display delayed aging. Our observations link Ras signaling to lifespan and suggest that RasGrf1 is an evolutionary conserved gene which could be targeted for the development of therapies to delay age-related processes.

Figure 1.. Delayed Ageing in RasGrf1^−/−.

(A) Survival curve of RasGrf1^−/− male mice and of WT of the same genetic background. WT n = 47; RasGrf1^−/− n = 45. Report the Kaplan - Meyer representation of the two groups (p<0.05). (B) Percentage of mice alive after a hundred and thirty weeks (*p<0.02) and a hundred and forty-seven weeks (**p<0.005) of the two cohorts. Maximal lifespan in wild type was a hundred and forty-seven weeks. Thus 20% of all the RasGrf1^−/− cohort survived longer than the maximal lifespan of the wild types.

Figure 2.. Increased Neuromuscular Coordination Coincided With Biomarkers of Aging in RasGrf1^−/− Mice

(A) Neuromuscular coordination was quantified as the percentage of male mice that successfully passed the tightrope test. Numbers within the bars indicate animals that passed the test divided by the total number of animals which were subjected to the test. Young animals were 4-6 months old, and old animals were 20-22 months old. Significance is shown as *p<0.05; **p<0.01 vs. WT. (B) Liver extracts from male mice were used to assess 16S rRNA expression in 4-6 months old animals. Expression of 16S rRNA was significantly higher in RasGrf1^−/− mice. WT n = 4; RasGrf1^−/− = 3; *p=0.03.

Figure 3.. Decreased Oxidative Stress in RasGrf1^−/− Mice

(A) Oxidative stress was measured in brain from 4-6 months old animals. Glutathione redox ratio was significantly higher in WT (n=7) than in RasGrf1^−/− male mice (n=8). Malondialdehyde (MDA) levels were significantly lower in brain of RasGrf1^−/− mice (n=8) than in that of WT (n=7). MDA was measured by the formation of the aduct malondialdehyde-thiobarbituric acid (MDA-TBA) (*p<0.05). (B) Oxidised glutathione and malondialdehyde levels were significantly lower in liver of RasGrf1^−/− male mice. n for WT = 7 and for RasGrf1^−/− = 8 (*p<0.05) (4-6 months old). (C) Oxidised proteins in young (4-6 months old) and old (20-22 months old) WT mice were significantly higher than in RasGrf1^−/− male mice (*p<0.05). These were measured by western blot with antibodies directed against aldehydes in proteins. (D) Cytochrome c oxidase expression is significantly higher in RasGrf1^−/− than in WT male mice of 4-6 months old (*p=0.037). Total RNA from selected tissues was isolated and used to quantify cytochrome c oxidase by real time RT-PCR.

Figure 4.. Metabolic Analysis of RasGrf1^−/− Mice

(A) Liver of RasGrf1^−/− male animals (4-6 months old) contains a significant amount of glycogen after 24 hours of fasting contrasting with the complete absence in WT animals (4-6 months old). PAS staining was quantified using color deconvolution with ImageJ software (Broken Symmetry Software). Values represent relative staining of three different sections (*p=0.01 determined by a paired T-test). (B) Sirtuin mRNA expression in liver and heart of RasGrf1^−/− male mice is significantly higher than in WT mice (*p=0.033; #p=0.035). Wild-type n = 4, RasGrf1^−/− n = 3 (4-6 months old). (C) IGF-I plasma levels was measured in 3-5 months old male mice by RIA revealing a 30% reduction in RasGrf1^−/−. Wild-type n = 10, RasGrf1^−/− n = 9, *p<0.001.

Figure 5.. Analysis Of Metabolic Parameters in RasGrf1 Deficient and Control Mice

(A) Metabolomic analysis reveals a metabolic shift of RasGrf1^−/− fed male mice towards caloric restricted WT (4-6 months old). Multivariate analysis (PLS - DA) of NMR spectra of blood plasma showing global metabolic profiles of WT, RasGrf1^−/−, and calorie restricted WT mice. The graph represents the scores plot of the PLS-DA model for discrimination between WT and RasGrf1^−/−. The graph also shows the metabolic profile of WT animals on caloric restriction projected over the PLS-DA latent space. Each symbol represents an animal. (B) Glucose up-take in vivo by heart of WT and RasGrf1^−/− male mice. Positron emission tomography (PET) analysis was used to estimate in vivo glucose uptake in young (4-6 months old) and old (20-22 months old) animals. The image is the result of a representative experiment. Histograms represent the means of glucose up-take measured in four animals in each group. Wild type n = 7, RasGrf1^−/− n = 8; #p<0.01.

Figure 6.. Schematic Representation of the Molecular and Physiological Characteristics of RasGrf1^−/− Animals

RasGrf1−/− results in an increased longevity which is mediated by a lower insulin /IGF signaling (IIS) which eventually leads to metabolic benefits and in lower ROS production and subsequent oxidative protection. These two independent changes converge in a notable increase in longevity and fitness.