Increased mammalian lifespan and a segmental and tissue-specific slowing of aging after genetic reduction of mTOR expression

Abstract

We analyzed aging parameters using a mechanistic target of rapamycin (mTOR) hypomorphic mouse model. Mice with two hypomorphic (mTOR(Δ/Δ)) alleles are viable but express mTOR at approximately 25% of wild-type levels. These animals demonstrate reduced mTORC1 and mTORC2 activity and exhibit an approximately 20% increase in median survival. While mTOR(Δ/Δ) mice are smaller than wild-type mice, these animals do not demonstrate any alterations in normalized food intake, glucose homeostasis, or metabolic rate. Consistent with their increased lifespan, mTOR(Δ/Δ) mice exhibited a reduction in a number of aging tissue biomarkers. Functional assessment suggested that, as mTOR(Δ/Δ) mice age, they exhibit a marked functional preservation in many, but not all, organ systems. Thus, in a mammalian model, while reducing mTOR expression markedly increases overall lifespan, it affects the age-dependent decline in tissue and organ function in a segmental fashion.

Figure 1

A mouse model of reduced mTOR expression extends lifespan. A) Genomic organization of the wild type allele (+) and the hypomorphic mTOR allele (Δ). B) Representative mTOR protein expression in the liver of two wild type (mTOR^+/+) and two mTOR^Δ/Δ mice. GAPDH is used as a loading control and the normalized expression (WT=1) of mTOR to GAPDH is shown for each animal. C) Leucine-stimulated S6 Kinase phosphorylation (pS6K) in primary mouse embryonic fibroblasts isolated from wild type or mTOR^Δ/Δ mice. D) Insulin-stimulated mTOR activity in pairs of wild type or mTOR^Δ/Δ mice. E) Survival of a cohort of male WT and mTOR^Δ/Δ mice. F) Survival of female members of the cohort. G) Survival of the overall cohort. H) Incidence of malignant tumors found at necropsy denoted by shaded portion of each bar. While the overall incidence of cancer was different between the two genotypes, the spectrum of tumors observed was similar. **p<0.001 Fisher’s Exact test.

Figure 2

The mTOR^Δ/Δ mice are smaller but have no significant alterations in glucose homeostasis and metabolism. All measurements were performed using male mice. A) Representative size of a wild type and mTOR^Δ/Δ adult mouse. B) Body weight of wild type and mTOR^Δ/Δ mice (n=7 WT and n=7 mTOR^Δ/Δ, curves are statistically different using a one-way Anova followed by two tailed t-test, p<0.01). C) Daily food intake is indistinguishable between wild type (shaded bar) and mTOR^Δ/Δ mice (open bar) (n=7 WT and n=6 mTOR^Δ/Δ, food intake is normalized to body weight). D) Glucose tolerance of 8–12 week old wild type and mTOR^Δ/Δ mice (n=7 WT and n=6 mTOR^Δ/Δ). E) Insulin tolerance test of 8–12 week old wild type and mTOR^Δ/Δ mice (n=12 WT and n=7 mTOR^Δ/Δ). F) Respiratory exchange ratio (RER) of WT (n=7) and mTOR^Δ/Δ (n=5). G) Measurement of rates of total body fatty acid oxidation normalized to body weight in WT (n=7) and mTOR^Δ/Δ mice (n=5). H) Total oxygen consumption normalized to body weight (n=7 WT and n=5 mTOR^Δ/Δ). I) Total daily energy expenditure is not altered in mTOR^Δ/Δ mice (n=7 WT and n=5 mTOR^Δ/Δ). For all panels, shaded bars represent the wild type mice and the open bars represent the mTOR^Δ/Δ mice. Where indicated, metabolic parameters are adjusted to body weight raised to the 0.75 power as indicated by the symbol (BW). All pooled data is presented as mean ± SEM.

Figure 3

Molecular and biochemical biomarkers of aging are reduced in old mTOR^Δ/Δ mice. A) Assessment of the age-dependent increase in kidney mRNA levels for the cell cycle inhibitor p16^INK4a normalized to GAPDH expression (n=3 mice per genotype and age with each animal performed in triplicate). B) A similar assessment in old and young liver samples (n=6 young WT, n=5 young mTOR^Δ/Δ, n=4 old WT and n=4 old mTOR^Δ/Δ with each sample performed in triplicate). C) Representative brain sections stained for nitrotyrosine (red, upper panels) obtained from young WT mice, old WT mice and old mTOR^Δ/Δ mice. Cell nuclei with stained concurrently with DAPI (blue, lower panels). D) Intensity of nitrotyrosine staining in the brains of old WT (n=3 animals with three to five determinations per animal) and mTOR^Δ/Δ (n=4 animals with three to five determinations per animal) mice. E) Staining for polyubquitinated proteins in brain tissue sections obtained from young WT mice, old WT mice or old mTOR^Δ/Δ mice. Upper panels (red) are stained with an antibody that recognizes proteins that are polyubiquitinated, lower panels are analyzed by nuclear DAPI staining. F) Quantification of polyubiquitinated protein levels in brain sections of WT (n=3 animals with three to five determinations per animal) and mTOR^Δ/Δ (n=4 animals with three to five determinations per animal) mice. All pooled data is presented as mean ± SEM. *p<0.05, **p<0.01.

Figure 4

The effects of reduced mTOR expression on a range of tissue specific age-related parameters. A) Escape latency times on day 3 of training for the Barnes maze test for both young female (n=6 mice per genotype) and old female (n=9 WT and n=13 mTOR^Δ/Δ) mice. Wild type mice are shown in the shaded bars, while the open bars are mTOR^Δ/Δ mice. *p <0.05. B) Learning strategy of old mice in the acquisition phase for training in the Barnes maze. Arrows indicate transition point between random to directed searching, an indicator of the speed in which new spatial learning is obtained (n=9 WT female mice and n=13 mTOR^Δ/Δ female mice). C) Duration on the Rotarod, a measure of coordination and balance (n=6 male mice per genotype for young animals and n=4 old male WT, n=7 old male mTOR^Δ/Δ mice, *p <0.05). D) Stride width variance in young and old mice (n= 6 young female mice per genotype and n=6 old WT male and female mice and n=13 old male and female mTOR^Δ/Δ mice, *p <0.05). E) Grip strength, normalized to gram of body weight, in young female mice (n=6 per genotype) and old female mice (n=4 WT and n=11 for mTOR^Δ/Δ mice, *p <0.05). F) Assessment of the age-dependent decline in bone volume (BV) to tissue volume (TV) (n= 4 young mice per genotype and n=6 old mice per genotype, *p <0.05). G) Age-dependent incidence of visibly apparent superficial infections of the skin, eyes or mouth of the total cohort of wild type and mTOR^Δ/Δ mice (n=34 WT and n=43 mTOR^Δ/Δ mice, statistical analysis by Fisher’s Exact test, *p<0.05, **p<0.01). All bar graph data is presented as mean ± SEM.