Impaired genome maintenance suppresses the growth hormone--insulin-like growth factor 1 axis in mice with Cockayne syndrome

Abstract

Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb(m/m)/Xpa(-/-) mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb(m/m)/Xpa(-/-) mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb(m/m)/Xpa(-/-) and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.

Figure 1. Growth Retardation, Cachexia, and Premature Death in Csb^m/m/Xpa^−/− and Csb^m/m/Xpc^−/− Mice

(A) UV repair characteristics of wt, single-mutant, and double-mutant primary MEFs. UV-induced UDS (left panel) and recovery of RNA Synthesis (RRS, right panel) are indicative for GG-NER and TC-NER capacity, respectively. For a detailed explanation of the procedure, see Methods. Error bars indicate standard error of the mean (SEM). (B) Survival of primary MEFs exposed to increasing doses of UV-C light (254 nm), as determined using the with [³H]-thymidine incorporation assay. Error bars (in most cases smaller than symbols used) indicate SEM. (C) Photograph of a 14-d-old Csb^m/m/Xpa^−/− mouse with an Xpa^−/− littermate (hybrid 129Ola/C57BL/6J background). (D) Photograph of an 8-d-old Csb^m/m/Xpc^−/− mouse with a Csb^m/m littermate (hybrid 129Ola/C57BL/6J background). (E) Body weight curve of Csb^m/m/Xpa^−/− and Csb^m/m/Xpc^−/− mice (n = 7) compared to those defective in a single NER gene (n = 7) all in a hybrid 129Ola/C57BL/6J background. The arrow indicates birth. Error bars (in most cases smaller than symbols used) indicate SEM. (F) Photographs of day 18.5 Csb^m/m/Xpa^−/− and Xpa^−/− embryos (C57BL/6J).

Figure 2. Skeletal and Neurological Abnormalities in Csb^m/m/Xpa^−/− Mice

(A) Radiographs of wt and Csb^m/m/Xpa^−/− mice (age as indicated) and photograph of a 15-d-old Csb^m/m/Xpa^−/− mouse (C57BL/6J). (B) 2-D images of micro-CT scans of tibiae taken from Csb^m/m/Xpa^−/− animals and wt littermate controls (age indicated in the figure). Horizontal sections are shown of the upper and lower part of the tibiae. e, epiphysis; g, growth plate; and d, diaphysis. I indicates the section through the smaller part of the diaphysis, II indicates section through the broader part of the diaphysis. (C) Growth of tibiae, taken from Csb^m/m/Xpa^−/− animals (light squares) and wt littermate controls (dark squares). (D) Representative footprint patterns of 19-d-old wt and Csb^m/m/Xpa^−/− mice. Arrows indicate the trajectory of each mouse. Stride length and front base width measurements on 15-d-old wt, Xpa^−/−, Csb^m/m, and Csb^m/m/Xpa^−/− mice. The significantly (asterisk; p < 0.001) greater base width in the double mutant mouse indicates ataxia. (E) Representative pictures of a TUNEL staining in the retina of wt and Csb^m/m/Xpa^−/− mice and quantification of the number of TUNEL positive cells. Arrows indicate TUNEL positive cells in the ONL and INL. Note the significantly higher number of TUNEL-positive cells in both the ONL and the INL in the retina of Csb^m/m/Xpa^−/− compared to wt mice (asterisk; p < 0.05).

Figure 3. Histological Examination of Csb^m/m/Xpa^−/− Tissues

(A) BrdU staining of the jejunum of Csb^m/m/Xpa^−/− and littermate control mice, showing normal proliferative capacity of the intestine in the double mutant mouse. (B–E) Histological examination of liver sections of 15-d-old Csb^m/m/Xpa^−/− and littermate control mice, stained with HE (B), immunostained for the proliferation markers (incorporated) BrdU (C) and PCNA protein (D), or TUNEL-stained for the presence of apoptotic cells (E). Quantification of the number of proliferative or apoptotic cells did not reveal significant differences between Csb^m/m/Xpa^−/− and wt littermate mice. (F) HE staining of the pituitary of Csb^m/m/Xpa^−/− and littermate control mice.

Figure 4. Enhanced Sensitivity of Csb^m/m/Xpa^−/− Retinal Photoreceptor Cells to Genotoxic Insults

Representative pictures of TUNEL stained retinas of 19-d-old wt, Csb^{m/ m} , Xpa^−/−, and Csb^m/m/Xpa^−/− mice (right panel), 20 h after exposure of animals to 10 Gy of ionizing radiation, and quantification of the number of TUNEL positive cells in the ONL (left panel). Note the significantly higher number of TUNEL-positive cells in the retina of Csb^m/m/Xpa^−/− mice, as compared to wt and single-mutant littermate controls (p < 0.05). Single asterisks indicate statistically significant differences between unirradiated Csb^m/m/Xpa^−/− and littermate control mice (p < 0.05), double asterisks indicate statistically significant differences between unirradiated and irradiated Csb^m/m/Xpa^−/− mice (p < 0.05).

Figure 5. Expression Levels of Genes Associated with the Somatotroph Axis, Antioxidant Defense, and Metabolism in Mutant and wt Mouse Liver and Other Organs at Various Ages

(A) Q-PCR evaluation of mRNA levels of genes associated with antioxidant defense (dark gray bars), oxidative metabolism (light gray bars), and the GH/IGF1 axis (black bars) in the liver, kidney, heart, and spleen of 15-d-old Csb^m/m/Xpa^−/−, Csb^m/m, and Xpa^−/− pups. For each gene, expression levels in the mutant tissue are plotted relative to that of age-matched wt control tissues (dotted line). Error bars indicate SEM between replicates (n ≥ 3). (B) Relative mRNA expression levels (fold changes, relative to embryonic day 18) of genes involved in the GH/IGF1 growth axis, antioxidant defense, and oxidative metabolism in the liver of wt and Csb^m/m/Xpa^−/− pups, plotted as a function of time. Error bars indicate SEM between replicates (n ≥ 3).

Figure 6. Transcriptome Similarities between Csb^m/m/Xpa^−/− and Naturally Aged Mice

(A) Spearman's r correlation of 16-, 96- and 130-wk-old mice with 15-d-old Csb^m/m/Xpa^−/− mice, where −1.0 is a perfect negative (inverse) correlation, 0.0 is no correlation, and +1.0 is a perfect positive correlation. (B) Similarities between significantly overrepresented biological processes. Note that in both Csb^m/m/Xpa^−/− and naturally aged mice, transcriptional changes were mostly associated with metabolic processes. (C) Correlation in significant expression changes of genes associated with the GH/IGF1 axis and oxidative metabolism in the livers of Csb^m/m/Xpa^−/− and naturally aged (96- and 130-wk-old) mice. An extensive overview is listed in Tables S3 and S4.

Figure 7. Carbohydrate/Fat Metabolism and IGF1 Serum Levels

IGF1 (A) and glucose (B) in the serum of 7-, 10-, 15-, and 17-d-old wt, Xpa^−/−, Csb^m/m, and Csb^m/m/Xpa^−/− mice (n = 6). The levels of IGF1 (ng/ml) and glucose (mmol/l) in the serum of Csb^m/m/Xpa^−/− mice are significantly lower than that of control littermates (p < 0.0004 and p < 0.04, respectively). (C) PAS staining for glycogen and Oil Red O staining for triglycerides in livers of 15-d-old wt and Csb^m/m/Xpa^−/− mice and 96-wk-old wt mice. Pictures were taken at 100× magnification. Note the large polyploid nuclei in the 96-wk-old wt mouse liver and the reduced glycogen levels in the Csb^m/m/Xpa^−/− liver after overnight fasting.

Figure 8. Expression Levels of Genes Associated with the GH/IGF1 Axis, Oxidative Metabolism, and Antioxidant Defense in DEHP-Treated wt Mice

Relative mRNA levels of genes involved in the GH/IGF1 growth axis, oxidative metabolism, and antioxidant defense in 13-wk-old wt mice treated with a low dose of the pro-oxidant DEHP. For each gene, expression levels in the treated wt mouse livers are plotted relative to that of age-matched untreated wt littermate controls (dotted line). Error bars indicate SEM. Asterisks indicate statistically significant differences (one-tailed p ≤ 0.05, see also Text S1).

Figure 9. The Proposed Link between DNA Damage and the Decline of the GH/IGF1 Somatotroph Axis

ROS are natural byproducts of metabolism and can injure several macromolecules including DNA, thus contributing to a slow but steady accumulation of damage, transcriptional stress, impaired replication, and eventually the progressive loss of tissue homeostasis and gradual organismal deterioration. Cells and tissues will respond by (1) up-regulating their antioxidant defense responses that would moderate the harmful effects of ROS, (2) using a battery of genome maintenance pathways that would repair or remove damaged macromolecules and help to resist the oxidative stress, and (3) suppressing their GH/IGF1 somatotroph axis along with the oxidative metabolism, thus substantially moderating their metabolic activity that would otherwise lead to high oxygen consumption and increased generation of oxidants. To this end, the physiologic reduction of the somatotroph axis and oxidative metabolism is envisaged to be beneficial in terms of net lifespan.