Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice

Abstract

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1^∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg^-/- (also known as Ercc5^-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1^∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1^∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1^∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Extended Data Figure 1. Effect of dietary restriction on body weight and various healthspan parameters of Ercc1^Δ/− mice primarily related to glucose metabolism and liver pathology

a–d, Body weights curves of Ercc1^Δ/− (a–b) and Xpg^−/− (c–d) male (a, c) and female (b, d) mice with ad libitum (AL; blue) access to AIN93G diet or on 30% dietary restriction (DR; red) shown as mean±SE at weekly intervals; n=4 animals/group solitary housed at the ErasmusMC. DR was initiated at 7 weeks of age with 10%, when the mice almost reached the maximum body weight avoiding disruption of development of young animals, and increased weekly with 10%, until 30% was reached from 9 weeks of age onwards. e–g, Blood glucose after feeding (e), plasma fasting insulin (f), and plasma albumin levels (g), indicative of liver functioning, in AL and DR wt and Ercc1^Δ/− mice at 16 weeks. n≥3 animals/group. h, Quantification of 16N nuclei in hepatocytes of 11 week old male wt and Ercc1^Δ/− mice under AL or DR regimens by FACS analyses; n=5 animals/group. i, Total numbers of splenic CD4+ T cell levels from spleen of 16 weeks old Ercc1^Δ/− mice under DR or AL and aged-matched wt controls. n≥3 animals/group. j, IgA blood levels in male Ercc1^Δ/− mice at different ages under DR or AL regimes. n=5 animals/group. k, Average grip strength of the forelimbs and all limbs of 16-week old Ercc1^Δ/− and wt mice is similar under AL and DR conditions; n= 4 animals/group. Error bars denote mean ± SE. * p<0.05, ** p<0.01, ***p<0.001.

Extended Data Figure 2. Dietary restriction improves aging-related histopathological phenotypes in different tissues of Ercc1^Δ/− mice

a, Representative pictures of Hematoxylin-Eosin stained slides from liver, kidney, and sciatic nerve. Left panel represents Ercc1^Δ/− ad libitum (Ercc1^Δ/− AL), left middle panel represents DR-treated Ercc1^Δ/− group (Ercc1^Δ/− DR), right middle panel is from wt-AL group, and right panel represents DR-treated wt. Lesions were semi-quantitatively assessed in scores ranging from absent (0) through massive (5). Liver of female Ercc1^Δ/− AL mouse with moderate anisokaryosis (score=3) and intranuclear inclusions (score =3, arrowheads), liver of female Ercc1^Δ/− DR mouse with moderate hydropic degeneration with mild anisokaryosis (score=1) and presence of few hepatocellular intranuclear inclusions (score=1, arrowhead), and histologically normal liver from wt-AL and wt-DR. Kidney of female Ercc1^Δ/− AL and Ercc1^Δ/− DR mouse with severe tubular attenuation and degeneration (score=5, arrows) with marked anisokaryosis (score=4, arrowheads) next to histologically normal kidneys from female wt-AL and wt-DR mice. Sciatic nerve of a female Ercc1^Δ/− AL mouse with severe axonal swellings (score=3, arrowheads). These axonal swellings most likely represent vacuoles containing myelin debris and/or fragmented axons. The Schwann cell nuclei around vacuolated areas are pyknotic (arrow). The sciatic nerve of a female Ercc1^Δ/− DR mouse displays mild vacuole-like structures (score=1, arrowhead) with pyknosis of Schwann cell nuclei (arrow), while the histologically normal sciatic nerves of female wt-AL and wt-DR mice display no axonal swellings. Scale bar in liver = 50 μm, kidney = 100 μm, and in sciatic nerve = 20 μm. b, Pathology assessment of anisokaryosis in liver of Ercc1^Δ/− mice at different ages under AL (blue) or DR (red) regimen and young AL (black) and DR (purple) wt controls. Scores range from absent (0) through massive (5); n≥10 animals/group; bars indicate group medians. c–d, Pathology assessment of anisokaryosis (c) and tubulonephrosis (d) in kidney of Ercc1^Δ/− and wt mice at different ages under AL and DR regimes. Scores range from absent (0) through massive (5); n≥10 animals/group. e, Pathology assessment of axonal swellings in sciatic nerves of Ercc1^Δ/− mice at different ages under AL or DR regimen. Scores range from absent (0) through massive (5); n≥10 animals/group. f, Representative pictures to the testicular lesions observed in Ercc1^Δ/− males. The AL testes (upper panel) exhibited moderate testicular degeneration and atrophy (arrows). Also, the Leydig cells (yellow asterisk) appeared more prominent likely secondary to the tubular loss/attenuation or may be due to true Leydig cell hyperplasia (a common aging lesion in rodent testes). These phenotypes were slightly rescued in the DR testes (lower panel). g–h, Pathology assessment of seminiferous tubular degeneration and atrophy (g) and Leydig cell hyperplasia (h) in testis of Ercc1^Δ/− mice at 16 weeks of age under AL (blue) or DR (red) regimen. Scores were given as absent (0), subtle (1), mild (2), moderate (3), severe (4), and massive (5) for each criteria with 0.5 interval; n=10 animals/group; bars indicate group medians. Note that testicular development is mostly completed at the start of DR. * p<0.05, ** p<0.01, ***p<0.001. As for wt controls the values do not change significantly in the timeframes used here (see). Pathological scores, including those of other liver and kidney aging-related histopathological phenotypes are given in Supplementary Table 1.

Extended Data Figure 3. Dietary restriction preserves skeletal structure in Ercc1^Δ/− mice

a, Illustration depicting the femural volume of interest (VOI) for microCT analyses. b–c, Trabecular bone volume fraction (BV/TV) representing the amount of trabecular bone in the femur VOI of (b) wt male mice as well as (c) Ercc1^Δ/− and wt female mice expressed as percentage measured using micro-CT. d–e, Femur length of Ercc1^Δ/− and wt (d) male and (e) female mice. f–g, Trabecular thickness in the femur VOI of Ercc1^Δ/− and wt (f) male and (g) female mice. AL- and DR-treated animals were measured at different ages with n≥3 animals/group. Values of Ercc1^Δ/− mice are depicted in blue (AL) and red (DR). Young wt controls are depicted in black (AL) and purple (DR). Error bars denote mean ± SE. * p<0.05, ** p<0.01, ***p<0.001.

Extended Data Figure 4. Dietary restriction preserves neurofunctional behavior of Xpg^−/− mice

a–c, Onset of neurological abnormalities as tremors (a), imbalance (b), and paresis of the hind limbs (c) with age in Xpg^−/− mice under AL and DR regimens. n=8 animals/group. The onset of continuous DR is indicated by the red arrows. Average age in the onset of tremors is delayed from 9 to 24 weeks of age, imbalance from 15 to 20, and paresis from 18 to 26 weeks of age. Temporary DR was given between 6 and 12 weeks of age and is indicated in green. This short period of DR yielded in a median delay in onset of tremors of 7 weeks while the median age of onset of both imbalance and paresis was 3 weeks delayed. p values were calculated against Xpg^−/−-AL using the log-rank test.

Extended Data Figure 5. Dietary restriction improves microgliosis and astrocytosis in brain and spinal cord of Ercc1^Δ/− mice

a–c, Quantification of the relative intensity of consecutive transverse brain and spinal cord sections immunoperoxidase-stained for Mac2 in spinal cord (a), and GFAP in spinal cord (b) and cerebrum (c). n>3 animals/group; bars indicate group medians. d, Iba1, Mac2, and GFAP immunofluorescent confocal images showing that reduced astrocytosis (GFAP) in cortex is paralleled by reduced staining for microglia (Iba1). Also Mac2-immunoreactivity, which outlines a subset of phagocytosing microglia cells, is reduced in 16 week old DR (n=4) as compared to AL (n=3) cortex in Ercc1^Δ/− mice. e–g, Representative pictures of spinal cord sections of 16 weeks old AL and DR Ercc1^Δ/− mice immunoperoxidase-stained for Mac2 (e) GFAP (f) reflecting reduced microgliosis and astrocytosis respectively, in the DR nervous system. Immunoperoxidase-stained spinal cord sections for ATF3 (g) showed that activation of the stress-inducible transcription factor ATF3 (which is induced following genotoxic stress via p53-dependent and -independent pathways) is less pronounced in DR nervous system. Per condition, sections from two different animals are presented next to each other. Black arrows indicate cells with high nuclear ATF3 staining. h, Representative pictures of consecutive transverse brain sections of 16 weeks old AL and DR Ercc1^Δ/− mice immunoperoxidase-stained for GFAP, showing reduced GFAP staining, in the DR nervous system. Per animal, represented in one row, six 40μm slices are shown with 360μm cerebrum thickness in between each slice. Error bars denote mean ± SE. ***p<0.001.

Extended Data Figure 6. Dietary restriction dramatically preserves neurofunctioning of Ercc1^Δ/− mice

a, Quantification of TUNEL-positive cells in the outer nuclear layer of retinal sections of 16 week old AL (blue) or DR (red) Ercc1^Δ/− mice; n=4 animals/group. b, Analysis of the total number of motor neurons with an abnormal Golgi apparatus (indicative of cells with ill health; see thick arrows in representative image; neuron with normal Golgi is indicated by a thin arrow) in C6 cervical spinal cord sections from 16 week old DR and AL Ercc1^Δ/− mice. n=4 animals/group. TUNEL-positive cells (a) and neurons with abnormal Golgi morphology (b) were absent in both AL- and DR-treated young wt mice. c, Quantitative stereological analysis of the total number of non-neuronal cells (Dapi+/NeuN-; p=0.2744) in the neocortex of transverse brain sections of 16 weeks old AL and DR Ercc1^Δ/− mice. n≥3 animals/group. Error bars indicate mean ± SE. ***p<0.001. d, Representative images of neocortex stained for NeuN (neurons), P53 and Dapi (for staining DNA) used for quantitative stereological analysis of the total number of neurons (NeuN+) and non-neuronal cells (Dapi+/NeuN−) in 16 weeks old AL- (n=3) and DR-(n=4) treated Ercc1^Δ/− mice. Quantification of the number of P53-positive neurons is shown in Figure 3f. The analysis was performed using the optical dissector probe from StereoInvestigator on a Zeiss LSM700 laser scanning microscope. e, Representative image of cerebellum stained for yH2AX (green; double stranded DNA breaks) and Dapi (blue; for staining DNA) in 16 weeks old AL (n=3) and DR (n=4) treated Ercc1^Δ/− mice. The Purkinje (PkJ) neurons are present in a single layer (PL; purkinje layer) in between the molecular layer (ML) and granular layer (GL). Quantification of the number of yH2AX-positive PkJ-neurons is shown in Figure 3i. The analysis was performed using a Zeiss LSM700 laser scanning microscope.

Extended Data Figure 7. Effect of DR on mTorc1, mTorc2 and Ins/PDK1 signaling using immunoblot analysis of wt and Ercc1^Δ/− liver extracts

a–b, Quantified relative S6 and AKT phosphorylation by DR in both wt (a) and Ercc1^Δ/− (b) liver extracts. 6 Animals per group were used; 11 weeks-of-age. c–h, Representative images used for the quantification of the ratio of S6 and AKT phosphorylation versus total S6 and AKT respectively. Phosphorylation of AKT at position S473 seems increased by DR in liver homogenates of 11 week old wt (e) and Ercc1^Δ/− (f) mice but is suppressed at position T308 (g, h). Phosphorylation of S6 at S240/244 is unaffected by DR (c, d). For immunoblots, three animals per group are shown. For graphs and statistics, six animals per group were used. The blue arrow indicates signals used for quantification. Below each blot b-actin is presented as a loading control.

Extended Data Figure 8. Molecular analysis of expression changes by diet, DNA damage, or aging

a–b, Ghr and Igf1r gene expression changes measured by quantitative real-time PCR in liver samples of 11 week old wt and Ercc1^Δ/− mice by DR (n=5). Gene-specific real-time PCR primers as described in Methods. c, MicroRNA expression profile comparison of wt and Ercc1^Δ/− liver under AL and DR conditions. 188 significant regulated miRNAs (FRD ≤ 5%) between groups are shown. Five of the most significantly changed microRNAs are zoomed in. miR-34a, a downstream target of p53 and involved in cell cycle and apoptosis induced by DNA-damage^,, showed a differential effect between liver homogenates of 11 week old wt and Ercc1^Δ/− mice. It was downregulated by DR in liver of wt mice (1.62 fold, p=0.02), but strongly upregulated in liver of AL-fed Ercc1^Δ/− mice compared to AL-fed wt mice (4.7 fold, p=0.0001) and seems suppressed in DR-treated Ercc1^Δ/− profiles. These chances were confirmed by qPCR (data not shown). d, Heatmap of key antioxidant defense genes in liver and brain of wt and Ercc1^Δ/− mice. Fold changes were calculated for wt-DR, Ercc1-AL, and Ercc1-DR against wt-AL using microarray expression profiles of liver at 11 weeks of age (n=5) or quantitative real-time PCR for cerebellum at 16 weeks of age (n=4). DR induced an antioxidant response in liver, which is less pronounced in brain specimens, consistent with earlier findings of Swindell, likely due to the already high antioxidant defense levels in the nervous system. The difference in antioxidant response between liver and brain by genotype is conform previous results. Interestingly, the Purkinje-neuron marker Calbindin is clearly reduced in cerebella of Ercc1-AL mice and is less reduced in Ercc1-DR mice, confirming the drastic reduction in DNA damage induced neuronal (Purkinje cell) loss by DR. Blue color represents decreased expression, red represents increased expression. Hierarchical clustering on liver and cerebellum genes was performed using Pearson correlation. e, DR reduces the p16-RB branch of senescence and the senescence-associated secretary phenotype as assessed by next generation sequencing expression analysis of liver RNA of Ercc1^Δ/− mice. To assess the p16-RB branch of the senescence phenotype, we followed a next generation sequencing approach as described in using 16 week old livers from AL- and DR-fed wt and AL- and DR-fed Ercc1^Δ/− mice. By sequencing >150M sequence reads per sample, we detected the p16-ink4a (Cdkn2a) transcript at sufficient levels. P16-ink4 (Cdkn2a) is considered a key marker for cellular senescence, but difficult to quantitatively analyze using other methods due to the high ratio normal cells compared to senescent cells. Datasets were normalized by calculating reads per kilobase million (RPKM). Subsequently, z-scores were calculated and plotted in a heatmap: red refers to increased expression, blue to decreased expression. In AL-fed Ercc1^Δ/− liver RNA p16-ink4a (Cdkn2a) is upregulated compared to wt AL animals, but down-regulated after DR, indicating that Ercc1^Δ/− mice have increased cellular senescence that is reduced upon DR. Secondly, we monitored the transcriptionally-induced senescence-associated secretory phenotype (SASP) as described in. Many, if not all, SASP factors are not exclusively specific for cellular senescence. To reduce the probability that observed SASP factor expression changes are contributed by other cells, we only selected those SASP factors that have an absolute expression (RPKM) in the same range as p16-ink4a in across these datasets, since these are most likely the result of cellular senescence. The figure shows that most SASP factors are down-regulated after DR such as IL-6, the most prominent SASP cytokine supporting the idea that cellular senescence and associated SASP is increased in AL-fed Ercc1^Δ/− liver and is reduced by DR. Hierarchical clustering was performed using Pearson correlation. f, Suppression of long genes in normal aging of rat liver. Relative frequency plot of gene length of DEGs in livers of 24 month old rat versus 6 month young rat. Upregulated genes are depicted in red and downregulated genes in green. The DEGs from rat liver were selected using a fold change cut-off of 1.5 and FDR < 0.05. The dataset is publicly available in the NCBI gene expression omnibus under number GSE66715.

Figure 1. Dietary restriction extends life- and healthspan of Ercc1^Δ/− and Xpg^−/− mouse mutants

a–d, Survival of mice with ad libitum (AL) access to AIN93G diet or on 30% dietary restriction (DR; red, AL; blue throughout) at two separate test sites. Male (a) and female (b) Ercc1^Δ/− mice, group housed at the RIVM (n=20–25 animals/group, separate experiments), and Ercc1^Δ/− (c) and Xpg^−/− (d) mice, solitary housed at the ErasmusMC (n=8 animals/group, 4 of each gender), under AL or DR regimens. DR was initiated at 7 weeks of age with 10%, (when the mice almost reached maximum body weight and development was completed), and increased weekly with 10%, until 30% was reached from 9 weeks onward. Remaining median and maximum lifespan are indicated (week 8 was considered the start of effective DR). Simultaneously, a cohort of Ercc1^Δ/− (c) and Xpg^−/− (d) mice received a temporary DR regime for a period of 6 weeks (green; n=8 animals/group, 4 of each gender). At the age of 6 weeks they received 30% DR and were switched back to AL from 12 weeks onward. p values were calculated by the log-rank test. e, Quantification of 16N nuclei in hepatocytes of AL or DR male Ercc1^Δ/− mice by FACS analyses; n=5 animals/group. f, Trabecular bone volume fraction (Bone Volume/Tissue Volume of interest, BV/TV, in %) in femurs of Ercc1^Δ/− male mice, measured using micro-CT. AL- and DR-treated animals were analyzed at different ages with n≥6 animals/group. g, Age-dependent decline of vasodilatation in Ercc1^Δ/− aorta segments, ex vivo. DR-Ercc1^Δ/− aorta segments show significantly more relaxation at age of 16 weeks than AL-Ercc1^Δ/− aorta. h, Frequency of CD4+CD25+Foxp3+ T regulatory cells among all CD4+ T cells from spleen of 16 weeks old Ercc1^Δ/− mice under DR or AL and aged-matched wt controls. n≥3 animals/group. Error bars denote mean ± SE. * p<0.05, ** p<0.01, ***p<0.001.

Figure 2. Dietary restriction dramatically preserves neurofunctioning of Ercc1^Δ/− mice

a–c, Onset of neurological abnormalities: tremors (a), imbalance (b), paresis of the hind limbs (c) with age in AL and DR Ercc1^Δ/− mice. n=8 animals/group. The onset of continuous DR is indicated by red arrows and the 6-week DR interval as green horizontal line. d–e, Average time spent on an accelerating rotarod of wt and Ercc1^Δ/− mice on different diets at 16 weeks of age (d; n=8 animals/group) or weekly monitored beyond the lifespan of Ercc1-AL mice (e; Ercc1^Δ/− n=4, wt n=3). A daily training period was given at 25 weeks of age. f–g, Quantitative stereological analysis of the total number of neurons (f; NeuN+; p=0.0008) in the neocortex of transverse brain sections and motor neurons (g; ChAT+; p=0.0176) in C6 cervical spinal cord sections of 16-weeks old AL and DR Ercc1^Δ/− mice. Note that the selective effect on neurons is consistent with earlier observations that neurons are the primary target of Ercc1 deficiency. n≥3 animals/group. Error bars indicate mean ± SE. * p<0.05, ** p<0.01, ***p<0.001.

Figure 3. Dietary restriction in Ercc1^Δ/− mice resembles wtDR and preserves genome function

a, Principal component analysis (PCA) of full genome liver RNA expression profiles of 11-week old Ercc1^Δ/− mice under AL (blue squares) and DR (red triangles) and wt controls (AL: black circles; DR: purple triangles). This analysis takes into account all the genes in the microarray platform. The two main principal components PC1 and PC2 explain 52% of the variability in the original dataset: PC1 (x-axis, 33%) differentiates on the basis of expression changes induced by DR, independent of genotype; PC2 (y-axis, 19%) reflects differences associated with genotype. b–c, Relative frequency plot of gene length (log scale) of DEGs in: wt-DR vs. wt-AL (purple interrupted lines), Ercc1^Δ/− AL vs. wt-AL (blue lines) and Ercc1^Δ/− DR vs. wt-AL (red lines). Plots are shown for only up-regulated genes (b), and only down-regulated genes (c). Black arrows indicate the extra peak of up-regulated short genes (b) and peak of down-regulated long genes (c) in Ercc1-AL. d, Relative frequency plot of gene length of DEGs in hippocampus of approximately 80 year old humans versus approximately 20 year young humans. Upregulated genes are depicted in red and downregulated genes in green. The DEGs from human hippocampus were selected using a log2-fold change cut-off of 0.5 and FDR <0.05. The dataset used corresponds to NCBI gene expression omnibus, number GSE11882. e–f, P53-positive cells were counted in neocortex of 3 consecutive transverse brain sections (e) at the level of the Bregma (Mouse brain atlas, Paxinos) and 3 consecutive C6 cervical spinal cord sections (f). Sections from 16-week old DR Ercc1^Δ/− mice (n=4) show significantly reduced levels of P53-positive cells (p<0.0001 for neocortex and p=0.0002 for spinal cord) as compared to sections from AL mice (n=4). g, Relative expression changes in the P53 target gene p21 in 11-week old wt and Ercc1^Δ/− mice by DR (n=5) h, yH2AX-positive Purkinje (PkJ) neurons were counted in cerebellum of 5 consecutive transverse brain sections in AL- and DR-treated Ercc1^Δ/− mice (p=0.014). Error bars indicate mean ± SE. * p<0.05, ** p<0.01, ***p<0.001. i. Mechanistic model for the anti-aging effect of DR. With aging DNA damage from exogenous and endogenous (metabolic) sources accumulates, which is accelerated in repair-deficient Ercc1^Δ/− mice. Stochastic DNA lesions reduce transcriptional output in a gene-size dependent manner leading to cell dysfunction and death, stem cell exhaustion, organ/tissue atrophy and functional decline, causing aging-related diseases. Accumulating DNA damage and DR trigger an anti-aging response, which involves suppression of growth, up-regulation of anti-oxidant defense and presumably metabolic redesign which reduces steady state levels of reactive metabolites, thereby preserving genome integrity and delaying aging-related functional decline.