Nuclear inclusions and dysregulated autophagy in osteoclasts in a mouse model of Paget's disease of bone caused by P394L SQSTM1 mutation

Introduction: Paget's disease of bone (PDB) is characterized by focally increased bone turnover, driven by abnormal and hyperactive osteoclasts. We have recently reported upon the generation of a PDB mouse model with a knock-in sqstm1 P394L mutation, equivalent to the SQSTM1 P392L mutation, most commonly linked to PDB in humans. Dysregulation in autophagy has been reported in myoblasts in a related condition of inclusion body myopathy, Paget's disease and fronto-temporal dementia (IBMPFD) however the role of autophagy in osteoclasts is relatively unknown. SQSTM1, the autophagy related gene 5 (ATG5) and the microtubule associated protein 1 light chain 3 gene (LC3) play important roles in autophagy. Here we report that mice with the P394L sqstm1 mutation develop nuclear inclusions and dysregulated autophagy in osteoclasts. Methods: M-CSF dependent macrophages, osteoclast precursors and osteoclasts were generated from bone marrow cultures of the P394L+/+, P394L+/− and WT mice. Transcription of SQSTM1, ATG5 and LC3 was assessed by quantitative real-time PCR. Protein levels of LC3-II were analysed by Western Blot. Twenty osteoclasts from 2 bone lesions of P394L+/+ mice were assessed for inclusions by transmission electron microscopy. Results: No significant difference in SQSTM1, LC3 or ATG5 expression was observed in P394L+/+ as compared to the WT M-CSF dependent macrophages. However, after 48 h stimulation with RANKL, transcription of SQSTM1, LC3 and ATG5 increased by 70 ± 13% (p < 0.05), 75 ± 7% (p < 0.01) and 63 ± 10% (p < 0.01) respectively in P394L+/+ compared to WT osteoclast precursors. There was no significant difference in LC3-II protein level between P394L+/− or P394L+/+ and WT osteoclasts. However, after inhibiting autophagosome–lysosome fusion with Bafilomycin A, we observed increased accumulation of LC3-II in cells carrying the P394L+/− and P394L+/+ mutations by 182 ± 80% (p < 0.05) and 80 ± 6% (p < 0.001) respectively as compared to WT. A nuclear inclusion containing micro cylindrical structures similar to human inclusions seen in PDB was identified in 1/20 (5%) osteoclasts in bone lesions. Conclusions: These results are consistent with increased autophagy gene expression, autophagosome formation and relatively increased autophagosome flux in the sqstm1 P394L mutant osteoclast lineage cells. This may reflect their hyperactivity at a functional level and/or suboptimal cellular protein catabolism, given the finding of nuclear inclusions in mutant osteoclasts.