A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging

Abstract

Although DNA damage is considered a driving force for aging, the nature of the damage that arises endogenously remains unclear. Replicative stress, a source of endogenous DNA damage, is prevented primarily by the ATR kinase. We have developed a mouse model of Seckel syndrome characterized by a severe deficiency in ATR. Seckel mice show high levels of replicative stress during embryogenesis, when proliferation is widespread, but this is reduced to marginal amounts in postnatal life. In spite of this decrease, adult Seckel mice show accelerated aging, which is further aggravated in the absence of p53. Together, these results support a model whereby replicative stress, particularly in utero, contributes to the onset of aging in postnatal life, and this is balanced by the replicative stress-limiting role of the checkpoint proteins ATR and p53.

Figure 1. Generation of a humanized allele of the Seckel Syndrome

a, Schematic representation of the strategy to generate the mutant allele. The rearranged allele contains the genomic region encompassing human exons 8-10 (pink) inserted into the equivalent region of the murine Atr gene (red exons). The SS mutation is indicated in E9. b, Visualization of the location and sequence homology of the humanized ATR protein in the region encoded in E8-E10. The full-length quimeric protein presents a 99.1% homology with murine ATR. c, PCR genotyping, d, RT-PCR of ATR with primers at E8 and E10 and e, ATR western blot, of littermate MEF lines (c-e). f, ATR western blot in human fibroblast lines 1BR3 (control) and F02-98 (Seckel), together with ATR^+/+ and ATR^S/S MEF.

Figure 2. ATR^S/S mice recapitulate the human SS

a, Representative pictures and weights of ATR^+/+ and ATR^S/S mice at 3 months of age a,b or at birth c,d. e, Picture of the heads of ATR^+/+ and ATR^S/S littermates. An outline is drawn to illustrate the protruding nose appearance (red arrow) due to the receding forehead. f, Computerized tomography (CT) of the heads of ATR^+/+ and ATR^S/S littermates illustrating the receding forehead (1) and micrognathia (2). g, MRI scans illustrating the AgCC (1) and the presence of cysts (2) on Seckel brains. h, Schema of the control allele (ATR^HS) which is identical to ATR^S but lacks the SS mutation. i, Representative picture of a couple of 3 month-old ATR^+/+ and ATR^HS/HS littermates.

Figure 3. Premature ageing of ATR^S/S mice

a, Kaplan-Meyer representation of the lifespan of ATR^+/+ (n=20) and ATR^S/S (n=27) mice. b, CT of a pair of 2 month-old Seckel and control littermates, as well as an old (25 months) ATR^+/+ mouse. The yellow arrow indicates the presence of kyphosis. c, H/E staining from the femoral head of a pair of 2 month-old Seckel and control male littermates, as well as an old (25 months) ATR^+/+ male mouse to illustrate the accumulation of fat in the BM (black arrows). Scale bar indicates 200 μm. d, Distribution of the femoral bone density in ATR^+/+, ATR^S/S and old (25-26 months) ATR^+/+ mice as a measure of the osteoporosis (n=7) (H.u.: Hounsfield unit). No significant gender differences were observed. e, Counts of platelets, red (RBC) and white (WBC) blood cells obtained from 3 month old ATR^+/+ and ATR^S/S mice.

Figure 4. Accumulation of RS in ATR^S/S MEF

a, Growth curves of ATR^+/+ and ATR^S/S MEF. b, Percentages of cells at the G2 stage of the cell cycle derived from the analysis of DNA content by flow cytometry. c, Distribution of γH2AX and 53BP1 on ATR^+/+ and ATR^S/S MEF. Scale bar indicates 5 μm. d, Average number of chromosome breaks per cell on control and Seckel metaphases. f, Percentage of the Fra8E1 alleles that were found to be at a break by FISH analyses. e, Representative images of the type of genomic aberrations found on ATR^S/S MEF. Metaphases were stained with probes recognizing the telomeres and the Fra8E1 fragile site. 50 metaphases were analyzed per condition in 3 replicates.

Figure 5. Response of ATR^S/S MEF to PIKK inhibitors

a, High-Throughput microscopy data illustrating the distribution of γH2AX signal in wt and ATR^S/S MEF treated with ATM and DNAPKcs inhibitors. b, Representative image from the analysis shown in a in the presence of both drugs. The distribution of 53BP1 is also shown below. c, Effect of the inhibitors on the G2 arrest observed on ATR^S/S MEF. d, Percentage of dead cells found on ATR^+/+ and ATR^S/S MEF cultures after 24 hrs of treatment with the inhibitors.

Figure 6. Accumulation of RS on ATR^S/S embryos

a, Immunohistochemistry (IHC) of γH2AX on the liver of 13.5 dpc wt and Seckel littermate embryos. b, Examples of two representative areas of ATR^S/S embryos, which were chosen from organs that appear compromised on adult mice -such as the forebrain or the face-, that illustrate the generalized accumulation of p53 or apoptotic cells (as measured with activated caspase 3) on the mutant embryos. Scale bar (white) indicates 150 μm. c, Quantification of the signals from b. d, Quantification of the frequency of cells with pan-nuclear γH2AX on different organs of 13.5dpc embryo and 2 month old mice.

Figure 7. Effect of p53 depletion on ATR^S/S cells and mice

a, Relative number of cells alive on ATR^+/+ and ATR^S/S cultures after p53 depletion (see Methods). The number was normalized to cells infected with a control shRNA expressing lentivirus. b, Representative images of the type of abnormal nuclei found on p53 depleted Seckel MEF. Scale bar indicates 5 μm. Effect of p53 depletion on the G2 arrest (c) and γH2AX accumulation (d) observed on ATR^S/S MEF. Scale bar indicates 100 μm. e, γH2AX IHC on a sagital section of a 13.5 dpc ATR^S/S/p53^−/−embryo. f, Magnification of a region close to the jaw on embryos from the different genotypes processed as in e. Scale bar indicates 50 μm.