Loss of ATRX does not confer susceptibility to osteoarthritis

Abstract

The chromatin remodelling protein ATRX is associated with the rare genetic disorder ATR-X syndrome. This syndrome includes developmental delay, cognitive impairment, and a variety of skeletal deformities. ATRX plays a role in several basic chromatin-mediated cellular events including DNA replication, telomere stability, gene transcription, and chromosome congression and cohesion during cell division. We have used a loss-of-function approach to directly investigate the role of Atrx in the adult skeleton in three different models of selective Atrx loss. We specifically targeted deletion of Atrx to the forelimb mesenchyme, to cartilage and to bone-forming osteoblasts. We previously demonstrated that loss of ATRX in forelimb mesenchyme causes brachydactyly while deletion in chondrocytes had minimal effects during development. We now show that targeted deletion of Atrx in osteoblasts causes minor dwarfism but does not recapitulate most of the skeletal phenotypes seen in ATR-X syndrome patients. In adult mice from all three models, we find that joints lacking Atrx are not more susceptible to osteoarthritis, as determined by OARSI scoring and immunohistochemistry. These results indicate that while ATRX plays limited roles during early stages of skeletal development, deficiency of the protein in adult tissues does not confer susceptibility to osteoarthritis.

Figure 1. Characterisation of joint morphology in two year-old Atrx^Col2 mice.

A) Minor fibrillation and fissuring of the tibial and femoral articular surfaces was observed in both genotypes, demonstrating mild osteoarthritis in both genotypes. OARSI scoring confirmed the absence of significant differences between genotypes. Scale bar = 96 µm. B) Immunohistological staining of articular cartilage, demonstrating that ATRX protein is present in normal aged articular cartilage, but absent from the nuclei of mutant chondrocytes. Scale bar = 48 µm.

Figure 2. Characterisation of joint osteoarthritis in Atrx ^Prx1 mice.

A) Mild fibrillation and fissuring of the articular surfaces was observed in elbows of control or mutant mice, and no difference was seen between genotypes. Scale bar = 96 µm. B) Integrity of the knee was similar in control and mutant mice. C) Immunohistological staining of articular cartilage from the elbows of mice, demonstrating that ATRX protein is present in normal aged articular cartilage, but absent from the nuclei of chondrocytes derived from ATRX deficient forelimb mesenchyme. Scale bar = 48 µm.

Figure 3. Atrx ^Col1 mice exhibit reduced growth without growth plate abnormalities.

A) Mice lacking Atrx in osteoblasts are born at normal Mendelian ratios. B) Overall skeletal morphology of Atrx ^Col1 examined by alican blue/alizarin red stain reveals reduced overall size and shortened limbs. C) Longitudinal measurements of the bones of the axial skeleton demonstrate that all bones in the mutant are shortened, and forelimb bones show significant shortening compared to controls N=5 (* p<0.05). D) Mineralised trabecular bone area below the growth plate at weaning is not different between genotypes upon visualization of sections stained with picrosirius red staining. Scale bar = 192 µm. E) Atrx ^Col1 mice are slightly smaller by body weight compared to littermates at birth, weaning and adulthood. F) IHC staining for Atrx protein demonstrates reduced Atrx in Atrx^Col1 cortical and subchondral bone. Scale bar = 48 µm.

Figure 4. Atrx ^Col1 mice have normal mineralisation.

A) Representative tibias from weaned Atrx ^Col1 showing that there was no gross morphological differences in the long bones between genotypes. B) No difference in bone mineral density (p = 0.90) was observed between Atrx ^Col1 mice and controls. Error Bars - SD, N=3 C) Trabecular number was unchanged between Atrx ^Col1 mice compared with controls (p = 0.46). Micro-CT data were obtained from hind legs using MicroView 3D software Error Bars - SD, N =3.

Figure 5. Ossification is not impeded or delayed by Atrx depletion in osteoblasts.

A) Ossification of the skull is normal in Atrx ^Col1 mice, and no changes are seen in suture formation. B) Skull length and width are not significantly affected in Atrx ^Col1 mice compared to controls. N=5, Error bars - SD C) Mineralisation of high-density osteoblast cultures. Control and Atrx-null osteoblasts show mineralised nodules (black) after two weeks in osteogenic media, and mineralise at similar rates at three and four weeks. Representative images from five trials.

Figure 6. Characterisation of joint osteoarthritis in Atrx ^Col1 mice.

A) Fibrillation or fissuring of the articular surfaces was not observed in elbows of control or mutant mice, demonstrating very mild OA in both genotypes. Scale bar = 96 µm. B) Joint integrity of the medial surface of the knee was similar in control and mutant mice. N = 4, Error bars = SD.