Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/−) show numerous accelerated ageing features that limit their lifespan to 4-6 months1,2,3,4. 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)1,5. 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 syndrome6, 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.
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We thank P. de With, J. Rigters, E. Haasdijk, S. Gabriels, E. J. M. Stynenbosch, N. van Vliet, Y. van Loon, J. Baan and the animal caretakers for general assistance with mouse experiments. We thank A. H. J. Danser and J. P. van Leeuwen for support. We acknowledge financial support from the National Institute of Health (NIH)/National Institute of Ageing (NIA) (1PO1 AG-17242-02), the National Institute for Public Health and the Environment and the Ministry of Health, Welfare and Sport of The Netherlands (S/340005), European Research Council Advanced Grant DamAge and Proof of Concept Grant Dementia to J.H.J.H., the European commission FP7 Markage (FP7-Health-2008-200880), DNA Repair (LSHG-CT-2005-512113), EU ITN Address (GA-316390), the KWO Dutch Cancer Society (5030), the Dutch CAA Foundation and the Royal Academy of Arts and Sciences of the Netherlands (academia professorship to J.H.J.H.). The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement number HEALTH-F2-2010-259893. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Reviewer Information Nature thanks C. Lopez-Otin, M. Mattson and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Effect of dietary restriction on body weight and various healthspan parameters of Ercc1∆/− mice are primarily related to glucose metabolism and liver pathology.
a–d, Body weights curves of Ercc1∆/− (a, b) and Xpg−/− (c, d) male (a, c) and female (b, d) mice with ad libitum (blue) access to AIN93G diet or on 30% dietary restriction (red) shown as mean ± s.e. at weekly intervals; n = 4 animals per group, solitary housed at the EMC. Dietary restriction was initiated at 7 weeks of age at a restriction of 10% and increased weekly by 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 ad libitum and dietary restriction wild-type and Ercc1∆/− mice at 16 weeks. n ≥ 3 animals per group. h, Quantification of 16N nuclei in hepatocytes32 of 11-week-old male wild-type and Ercc1∆/− mice under ad libitum or dietary restriction regimens by FACS analyses; n = 5 animals per group. i, Total numbers of splenic CD4+ T cell from spleen of 16-week old Ercc1∆/− mice under dietary restriction or ad libitum and aged-matched wild-type controls. n ≥ 3 animals per group. j, IgA blood levels in male Ercc1∆/− mice at different ages under dietary restriction or ad libitum regimes. n = 5 animals per group. k, Average grip strength of the forelimbs and all limbs of 16-week old Ercc1∆/− and wild-type mice is similar under ad libitum and dietary restriction conditions; n = 4 animals per group. Mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Figure 2 Dietary restriction improves ageing-related histopathological phenotypes in different tissues of Ercc1∆/− mice.
a, Representative pictures of haematoxylin-eosin-stained slides from liver, kidney, and sciatic nerve. From left to right: ALErcc1, DRErcc1, ALWT and DRWT. Lesions were semiquantitatively assessed, with scores ranging from absent (0) to massive (5). The liver of a female ALErcc1 mouse shows moderate anisokaryosis (score = 3) and intranuclear inclusions (score = 3, arrowheads). The liver of a female DRErcc1mouse shows moderate hydropic degeneration with mild anisokaryosis (score = 1) and a few hepatocellular intranuclear inclusions (score = 1, arrowhead). Histologically normal liver tissue from ALWT and DRWT mice. The kidney of a female ALErcc1and DRErcc1mouse with severe tubular attenuation and degeneration (score = 5, arrows) with marked anisokaryosis (score = 4, arrowheads) next to histologically normal kidneys from female ALWT and DRWT mice. The sciatic nerve of a female ALErcc1 mouse with severe axonal swellings (score = 3, arrowheads). These axonal swellings probably 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 DRErcc1 mouse displays mild vacuole-like structures (score = 1, arrowhead) with pyknosis of Schwann cell nuclei (arrow), while the histologically normal sciatic nerves of female ALWT and DRWT mice display no axonal swellings. Scale bar in liver, 50 μm; in kidney, 100 μm; in sciatic nerve, 20 μm. b, Pathology assessment of anisokaryosis in livers from Ercc1∆/− mice at different ages under ad libitum (blue) or dietary restriction (red) regimen and young ad libitum (black) and dietary restriction (purple) wild-type controls. Scores range from absent (0) to massive (5); n ≥ 10 animals per group; bars indicate group medians. c, d, Pathology assessment of anisokaryosis (c) and tubulonephrosis (d) in the kidneys of Ercc1∆/− and wild-type mice at different ages under ad libitum and dietary restriction regimes. Scores range from absent (0) to massive (5); n ≥ 10 animals per group. e, Pathology assessment of axonal swellings in sciatic nerves of Ercc1∆/− mice at different ages under ad libitum or dietary restriction regimens. Scores range from absent (0) through massive (5); n ≥ 10 animals per group. f, Representative pictures to the testicular lesions observed in Ercc1∆/− males. The ALErcc1 testes (upper panel) exhibited moderate testicular degeneration and atrophy (arrows). Also, the Leydig cells (yellow asterisk) appeared more prominent, probably owing to the tubular loss and attenuation, or possibly owing to true Leydig cell hyperplasia (a common ageing lesion in rodent testes). These phenotypes were slightly rescued in the testes of DRErcc1 mice (lower panel). g, h, Pathology assessment of seminiferous tubular degeneration and atrophy (g) and Leydig cell hyperplasia (h) in the testes of Ercc1∆/− mice at 16 weeks of age under ad libitum (blue) or dietary restriction (red) regimen. Scores were given as absent (0), subtle (1), mild (2), moderate (3), severe (4), and massive (5) for each criteria, with a 0.5 interval; n = 10 animals per group; bars indicate group medians. Note that testicular development is mostly completed at the start of dietary restriction. *P < 0.05, **P < 0.01, ***P < 0.001. The values for the wild-type mice do not change significantly in the timeframes used here (see ref. 40). Pathological scores, including those of other liver and kidney ageing-related histopathological phenotypes, are given in Supplementary Table 1.
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 wild-type male mice (b) as well as Ercc1∆/− and wild-type female mice (c) expressed as percentage measured using micro-CT. d, e, Femur length of Ercc1∆/− and wild-type male (d) and female (e) mice. f, g, Trabecular thickness in the femur VOI of Ercc1∆/− and wild-type male (f) and female (g) mice. Ad libitum- and dietary restriction-treated animals were measured at different ages with n ≥ 3 animals per group. Values of Ercc1∆/− mice are depicted in blue (ad libitum) and red (dietary restriction). Young wild-type controls are depicted in black (ad libitum) and purple (dietary restriction). Error bars denote mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001.
a–c, Onset of neurological abnormalities as tremors (a), imbalance (b), and paresis of the hind limbs (c) with age in Xpg−/− mice under ad libitum and dietary restriction regimens. n = 8 animals per group. The onset of continuous dietary restriction is indicated by the red arrows. Average age at the onset of tremors is delayed from 9 to 24 weeks, imbalance from 15 to 20 weeks, and paresis from 18 to 26 weeks. Temporary dietary restriction was given between 6 and 12 weeks of age and is indicated in green. This short period of dietary restriction yielded a median delay in onset of tremors of 7 weeks while the median age of onset of both imbalance and paresis was delayed by 3 weeks. P values were calculated against Xpg−/−-ad libitum mice using the log-rank test.
Extended Data Figure 5 Dietary restriction improves microgliosis and astrocytosis in brain and spinal cord of Ercc1∆/− 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 per 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). Mac2-immunoreactivity, which outlines a subset of phagocytosing microglia cells, is also reduced in the neocortex of 16-week-old DRErcc1 mice (n = 4) when compared to ad-libitum mice (n = 3). e–g, Representative pictures of spinal cord sections of 16-week-old ALErcc1 and DRErcc1 mice immunoperoxidase-stained for Mac2 (e) and GFAP (f) reflecting reduced microgliosis and astrocytosis, respectively, in the nervous system of diet-restricted mice. 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 the nervous system of diet-restricted mice. 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-week-old ALErcc1 and DRErcc1 mice immunoperoxidase-stained for GFAP, showing reduced GFAP staining in the nervous system of diet-restricted mice. Six 40 μm slices are shown per animal, with 360 μm cerebrum thickness between each slice. Mean ± s.e. ***P < 0.001.
Extended Data Figure 6 Dietary restriction dramatically preserves neurofunctioning of Ercc1∆/− mice.
a, Quantification of TUNEL-positive cells in the outer nuclear layer of retinal sections of 16-week-old ad libitum (blue) or diet-restricted (red) Ercc1∆/− mice; n = 4 animals per group. b, Analysis of the total number of motor neurons with abnormal Golgi apparatus (indicative of impaired cells, 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 diet-restricted and ad libitum Ercc1∆/− mice. n = 4 animals per group. TUNEL-positive cells (a) and neurons with abnormal Golgi morphology (b) were absent in both ad libitum17 and diet-restricted young wild-type 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-week-old ad libitum and diet-restricted Ercc1∆/− mice. n ≥ 3 animals per group. Mean ± s.e. ***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-week old ad libitum- (n = 3) and dietary restriction-(n = 4) treated Ercc1∆/− mice. Quantification of the number of p53-positive neurons is shown in Fig. 3e. 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 γH2AX (green, double-stranded DNA breaks) and DAPI (blue, for staining DNA) in 16-week-old ad libitum (n = 3) and diet-restricted (n = 4) Ercc1∆/− mice. The Purkinje (PkJ) neurons are present in a single layer (PL, the purkinje layer) in between the molecular layer (ML) and granular layer (GL)41. Quantification of the number of γH2AX-positive PkJ-neurons is shown in Fig. 3h. The analysis was performed using a Zeiss LSM700 laser scanning microscope.
Extended Data Figure 7 Effect of dietary restriction on mTorc1, mTorc2 and Ins/PDK1 signalling using immunoblot analysis of wild-type and Ercc1∆/− liver extracts.
a, b, Quantified relative S6 and Akt phosphorylation by dietary restriction in wild-type (a) and Ercc1∆/− (b) liver extracts. Six animals per group were used at 11 weeks of age. c–h, Representative images used to quantify the ratio of S6 and Akt phosphorylation versus total S6 and Akt respectively. Phosphorylation of Akt at position S473 seems to be increased by dietary restriction in liver homogenates of 11-week-old wild-type (e) and Ercc1∆/− (f) mice, but is suppressed at position T308 (g, h). Phosphorylation of S6 at S240 and S244 is unaffected by dietary restriction (c, d). For immunoblots, data for 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, β-actin is presented as a loading control.
a, b, Ghr and Igf1r gene expression changes measured by quantitative real-time PCR (qRT–PCR) in liver samples of 11-week old wild-type and Ercc1∆/− mice with restricted diets (n = 5). Gene-specific real-time PCR primers are described in Methods. c, MicroRNA expression profile comparison of wild-type and Ercc1∆/− mouse liver tissue under ad libitum and diet-restricted conditions. Shown are 188 significantly regulated miRNAs (FRD ≤ 5%) between groups. Five of the most significantly changed microRNAs are zoomed in. miR-34a, a downstream target of p53 that is involved in cell cycle regulation and apoptosis, is induced by DNA damage42,43. It showed differential expression between liver homogenates of 11-week-old wild-type and Ercc1∆/− mice. It was downregulated by dietary restriction in the liver of wild-type mice (1.62 fold, P = 0.02), but strongly upregulated in the liver of ALErcc1 mice compared to ALWT mice (4.7-fold, P = 0.0001) and seems suppressed in DRErcc1 expression profiles. These changes were confirmed by qPCR (data not shown). d, Heat map of key antioxidant defence genes in liver and brain of wild-type and Ercc1∆/− mice. Fold changes were calculated for DRWT, ALErcc1, and DRErcc1mice against ALWT mice, using microarray expression profiles of liver tissue at 11 weeks of age (n = 5) or qRT–PCR for cerebellum tissue at 16 weeks of age (n = 4). Dietary restriction induced an antioxidant response in liver, which is less pronounced in brain specimens, consistent with earlier findings44. This is likely to be due to the high endogenous antioxidant defence levels in the nervous system. The difference in antioxidant response between liver and brain by genotype conforms to previous results6. Interestingly, the Purkinje neuron marker calbindin is clearly reduced in cerebella of ALErcc1 mice but is less reduced in DRErcc1 mice, confirming the strong reduction in DNA-damage-induced Purkinje cell loss induced by dietary restriction. Blue, decreased expression; red, increased expression. Hierarchical clustering on liver and cerebellum genes was performed using a Pearson correlation. e, Dietary restriction reduces the p16-RB branch of senescence and the senescence-associated secretary phenotype (SASP) as assessed by next-generation sequencing expression analysis of the liver RNA of Ercc1∆/− mice. To assess the p16-RB branch of the senescence phenotype45, we followed a next-generation sequencing approach as previously described36 using 16-week-old liver tissue from ALWT, DRWT, ALErcc1 and DRErcc1 mice (n = 1). 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 is difficult to quantitatively analyse using other methods owing to the high ratio of normal cells to senescent cells. Data sets were normalized by calculating reads per kilobase million (RPKM). Subsequently, z-scores were calculated and plotted in a heat map. Red, increased expression; blue, decreased expression. In ALErcc1- liver RNA, p16-ink4a (Cdkn2a) is upregulated compared to levels in ALWT animals, but downregulated after dietary restriction. This indicates that Ercc1∆/− mice have increased cellular senescence that is reduced upon dietary restriction. Second, we monitored the transcriptionally induced SASP as described previously46. 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 to by other cells, we selected only those SASP factors that have an absolute expression (RPKM) in the same range as p16-ink4a in across these data sets, since these are most likely to be the result of cellular senescence. The figure shows that most SASP factors such as IL-6, the most prominent SASP cytokine are downregulated after dietary restriction. This supports the idea that cellular senescence and associated SASP are increased in ALErcc1 liver and are reduced by dietary restriction. Hierarchical clustering was performed using a Pearson correlation. f, Suppression of long genes in normal ageing of rat liver. A relative-frequency plot of the gene length of DEGs in liver tissue from 24-month old rat versus that seen in 6-month old rat. Upregulated genes, red; downregulated genes, green. The DEGs from rat liver were selected using a fold-change cut-off of 1.5 and an FDR <0.05. The data set is publicly available in the NCBI Gene Expression Omnibus under accession number GSE66715.
Pathology scores of aging-related histopathological phenotypes in liver, kidney and sciatic nerve of AL and DR wt and Ercc1δ/− mice (XLSX 15 kb)
Gene ontology pathway and upstream regulator transcription factor analysis of DEG response by DR in wt and Ercc1δ/− (XLSX 20 kb)
AL-fed Ercc1δ/− mouse (shown on the right), at 16 weeks of age, shows abnormal gait, trembling and balance problems. These age-related neurological symptoms are absent in the littermate DR Ercc1δ/− mouse (left) at 16 weeks but eventually may develop after 35 weeks of age (see Figure 2a-c). Video contains audio comments. (reference to website with access to the video: http://cluster15.erasmusmc.nl/drvideos/index.html). (MP4 27918 kb)
Wildtype AL and DR and Ercc1δ/− AL and DR mice, form right to left respectively, were tested for locomotor performance on an accelerating rotarod. The average time of four animals per group is shown in Figure 2d. Video contains audio comments. (reference to website with access to the video: http://cluster15.erasmusmc.nl/drvideos/index.html). (MP4 24342 kb)
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Vermeij, W., Dollé, M., Reiling, E. et al. Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice. Nature 537, 427–431 (2016). https://doi.org/10.1038/nature19329
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