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. 2013 May;123(5):2049-63.
doi: 10.1172/JCI65634. Epub 2013 Apr 8.

Atrx deficiency induces telomere dysfunction, endocrine defects, and reduced life span

L Ashley Watson  1 , Lauren A Solomon, Jennifer Ruizhe Li, Yan Jiang, Matthew Edwards, Kazuo Shin-ya, Frank Beier, Nathalie G Bérubé
Affiliations

Atrx deficiency induces telomere dysfunction, endocrine defects, and reduced life span

L Ashley Watson et al. J Clin Invest. 2013 May.

Abstract

Human ATRX mutations are associated with cognitive deficits, developmental abnormalities, and cancer. We show that the Atrx-null embryonic mouse brain accumulates replicative damage at telomeres and pericentromeric heterochromatin, which is exacerbated by loss of p53 and linked to ATM activation. ATRX-deficient neuroprogenitors exhibited higher incidence of telomere fusions and increased sensitivity to replication stress-inducing drugs VSports手机版. Treatment of Atrx-null neuroprogenitors with the G-quadruplex (G4) ligand telomestatin increased DNA damage, indicating that ATRX likely aids in the replication of telomeric G4-DNA structures. Unexpectedly, mutant mice displayed reduced growth, shortened life span, lordokyphosis, cataracts, heart enlargement, and hypoglycemia, as well as reduction of mineral bone density, trabecular bone content, and subcutaneous fat. We show that a subset of these defects can be attributed to loss of ATRX in the embryonic anterior pituitary that resulted in low circulating levels of thyroxine and IGF-1. Our findings suggest that loss of ATRX increases DNA damage locally in the forebrain and anterior pituitary and causes tissue attrition and other systemic defects similar to those seen in aging. .

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Figures

Figure 1
Figure 1. Increased DNA damage leads to ATM activation and p53-dependent apoptosis in the Atrx-null embryonic brain.
(A) Immunostaining for γH2AX in E13.5 control (Ctrl), cKO, and cKO;p53–/– compound mutant cortical cryosections. Scale bar: 100 μm. DAPI staining of E13.5 forebrain highlights in green the hippocampal hem (H), cortex (Ctx), and basal ganglia (BG) regions where γH2AX foci per unit area were scored. Control, cKO, and cKO;p53–/– (n = 3); p53–/– (n = 2). (B) γH2AX staining in P0.5 control, cKO, and cKO;p53–/– cortical cryosections. Scale bar: 200 μm. DAPI staining of P0.5 forebrain highlights in green the hippocampus (H) and cortex (Ctx) regions where γH2AX foci per unit area were scored. Control and cKO (n = 3); cKO;p53–/– and p53–/– (n = 2). (C) Co-immunofluorescence detection of γH2AX (red) and activated caspase-3 (AC3; green) in E13.5 cortical cryosections. Scale bar: 30 μm. AC3+ cells were scored for the presence (AC3 + γH2AX) or absence (AC3 – γH2AX) of DNA damage (n = 3). (D) Western blot analysis of nuclear protein extracts obtained from E13.5 telencephalon (n = 3). While levels of ATR and phospho-ATR were not increased (left panels), phospho-ATM was noticeably increased in the cKO extracts compared with controls (indicated by an asterisk). Original magnification, ×100 (A and B); ×200 (C). *P < 0.05.
Figure 2
Figure 2. Increased DNA damage and telomere defects in cKO NPCs.
(A) Confocal immuno-FISH images of ATRX (red) and telomeres (Tel-FISH; green) in NPCs demonstrates colocalization of the ATRX protein with a subset of telomeres. Scale bar: 5 μm. (B) Confocal immuno-FISH images of γH2AX (red) and telomeres (Tel-FISH; green) shows increased incidence of TIF (γH2AX/Tel-FISH colocalization) in cKO compared with control NPCs (300 nuclei counted, n = 3 control/cKO littermate-matched pairs). Scale bar: 10 μm. (C) DAPI staining of control and cKO metaphase spreads shows representative chromosome fusion in cKO NPC metaphase (arrowhead). Frequency of fusions per metaphase was increased in cKO metaphases compared with control (control: 88 metaphases, cKO: 108 metaphases counted, n = 3). (D) Tel-FISH (green) demonstrates increased telomeric fusions in cKO metaphase chromosomes compared with control (1,475 chromosomes counted; n = 3). (E) Telomere defects (deletion, merge, bridge, and duplication) were scored in control and cKO metaphase chromosomes. Representative images of defects appear to the right of quantification. In all cases, cKO chromosomes showed an increase in telomeric defects compared with control (1,475 chromosomes counted; n = 3). Original magnification, ×1,000 (A and B); ×630 (C–E). *P < 0.05.
Figure 3
Figure 3. ATRX-deficient cells are hypersensitive to replication stress–inducing agents and the G4-DNA ligand TMS.
(A) AtrxloxP/Y MEFs were untransduced or transduced with adenovirus expressing Cre recombinase fused to GFP (Ad-CreGFP) or Ad-GFP and subsequently treated with HU for 24 hours or γ-irradiated at the indicated doses. Cell viability was measured at 24 hours after HU treatment (n = 4) and at 6 hours after irradiation (n = 3) via trypan blue dye exclusion. (B) Control and cKO NPCs were treated with HU or MMC for 24 hours or γ-irradiated at the indicated doses. Cell viability was measured at 24 hours after HU and MMC treatment and at 6 hours after irradiation (n = 3). (C) Co-immunofluorescence detection of PCNA, a marker of replication foci, and γH2AX in control and cKO E13.5 cortical cryosections. Results were quantified by measuring the ratio of γH2AX staining that localized to late-replicating PCNA foci to total γH2AX staining per cell, to account for overall lower levels of γH2AX signal in control cells (300 nuclei counted, n = 3). Scale bar: 12 μm. (D) Control and cKO NPCs were treated with 20 μm TMS for 2 hours, and γH2AX signal was imaged 6 hours after treatment. Scale bar: 70 μm. (E) Control and cKO NPCs were treated with TMS for 24 hours, and cell viability was measured 24 hours after treatment (n = 3). Original magnification, ×600 (C); ×100 (D). *P < 0.05.
Figure 4
Figure 4. Reduced growth and life span in mice lacking ATRX in the forebrain.
(A) Kaplan-Meier survival curve of Cre+ control (n = 12) and cKO mice raised with (+sibs, n = 13) or without (–sibs, n = 11) siblings. Survival of cKO mice was significantly decreased compared with that of control mice (P = 0.0001). The survival of cKO mice was not significantly different whether they were raised with or without siblings (P = 0.4974). (B) Representative pictures of P17 control and cKO littermates, illustrating size difference of the mice. (C) Body weight (g) and length (cm) measurements of control (Cre–, n = 25; Cre+, n = 8) and cKO (n = 24) mice. No significant difference was observed between Cre– and Cre+ control mice. (D) Skeletal elements of control and cKO mice were stained with alizarin red and alcian blue. (E) Length measurements of P17 control and cKO skeletal elements. (n = 5). *P < 0.05.
Figure 5
Figure 5. Postnatal phenotypes in Atrx-cKO mice.
(A) Whole skeletal isosurface images of P17 control and cKO mice were generated using microCT. Arrow points to kyphosis. Scale bar: 10 mm. (B) Horizontal view of the cKO skull illustrates decreased bone mineralization. Scale bar: 5 mm. (C) Tibial cross section shows decreased cortical thickness. Scale bar: 1 mm. (D) Decreased BMD (*P = 0.002), trabecular number (**P = 0.002), and cortical thickness (†P = 0.0001) in the cKO mice compared with controls. Data were obtained from hind legs using MicroView 3D software (n = 3). (E) Picrosirius red staining of P17 control and cKO tibia (representative image), femur, and humerus reveals a drastic loss of trabecular bone area in cKO compared with control mice (n = 4; *P < 0.05). Scale bar: 200 mm. (F) H&E staining of P20 skin cryosections shows loss of subcutaneous fat in cKO compared with control mice (n = 3; *P = 0.0002). Dermal thickness was not significantly different (n = 3; *P = 0.3545). Scale bar: 300 mm. sf, subcutaneous fat; d, dermis. (G) Dark field image of P20 control and cKO ocular lenses demonstrates appearance of cataracts (loss of lens transparency) in cKO compared with control. (H) Dark field image of P20 control and cKO spleen demonstrates the disproportionally smaller size of cKO spleen. Original magnification, ×50 (E and F). See also Supplemental Figure 5.
Figure 6
Figure 6. Endocrine defects and hypoglycemia in Atrx-cKO mice.
(A) Longitudinal growth of control and cKO tibia was measured after 7 days (d7) of ex vivo culture. Results are expressed as the ratio of length at d7 to that at d0. No difference in growth was detected between control and cKO mice (n = 3). Scale bar: 100 μm. (B) Serum and liver IGF-1 levels are decreased in cKO mice (n = 3). (C) Expression of several IGF-1 pathway genes is altered in cKO liver compared with controls (n = 3). Real-time data were normalized to Gapdh expression. (D) Circulating T4 levels are significantly decreased in P20 cKO mice compared with controls, while GH levels are only mildly affected (n = 3). (E) Thyroid hormone target genes exhibit decreased expression in the liver of P20 cKO mice compared with controls (n = 3). Real-time data were normalized to Gapdh expression. (F) Glucose levels are reduced in P20 cKO serum compared with controls (n = 5). Original magnification, ×50 (A). *P < 0.05.
Figure 7
Figure 7. Loss of ATRX in the developing anterior pituitary causes DNA damage, reduced Tsh expression, and altered thyroid function.
(A) Quantitative RT-PCR analysis of P23 control and cKO shows loss of Atrx expression in the pituitary (5.9-fold decrease) and thyroid (1.25-fold decrease). Tsha and Tshb subunits showed decreased expression in the pituitary, and a number of downstream targets of TSH showed decreased expression in the thyroid of cKO mice compared with controls (n = 3). (B) H&E staining of P23 control and cKO pituitary. Scale bar: 500 μm. (C) Immunofluorescence detection of ATRX expression in P23 control and cKO pituitary demonstrates specific loss of ATRX in the anterior and intermediate pituitary. Scale bars: 500 μm (left panels) and 50 μm (right panels). A, anterior; I, intermediate; P, posterior pituitary. (D) Immunofluorescence detection of ATRX in E13.5 control and cKO sagittal embryonic pituitary cryosections shows loss of ATRX expression. Scale bar: 100 μm. Pit, pituitary. (E) Immunofluorescence detection of γH2AX in E13.5 sagittal embryonic pituitary cryosections shows increased DNA damage in cKO embryonic pituitary compared with control. Scale bar: 100 μm. Original magnification, ×25 (C, left panels); ×100 (C, right panels, and E); ×50 (D). *P < 0.05.
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