Loss of the Ubiquitin-Associated Domain of sqstm1/p62 in Zebrafish Causes a Phenotype Resembling Paget's Disease of Bone

Abstract

The ubiquitin-binding protein p62, encoded by Sequestosome 1 (SQSTM1), is an essential molecular adaptor for selective autophagy. Heterozygous mutations deleting or disrupting the ubiquitin-associated (UBA) domain of p62 have been reported as the major genetic cause for Paget’s disease of bone (PDB), the second most common skeletal disease, characterized by hyperactive osteoclasts and focal increases of bone turnover. In this study, we aimed to determine the impact of a similar sqstm1/p62 mutation on the skeleton of zebrafish. We successfully established a sqstm1tmΔUBA zebrafish line with premature truncation of the UBA domain and performed skeletal phenotyping of heterozygous and homozygous mutant zebrafish. Homozygous sqstm1tmΔUBA zebrafish suffered from early lethality after 6 mpf, possibly related to a dysregulated autophagy process. Nevertheless, we detected skeletal abnormalities that were generally more severe in older animals and in homozygous versus heterozygous sqstm1tmΔUBA zebrafish. MicroCT analysis and histologic staining showed alterations in the vertebral bodies and/or bone density in heterozygous sqstm1tmΔUBA zebrafish. We also detected signs of osteocytic osteolysis in carriers of a mutant sqstm1tmΔUBA allele, shown by a higher percentage of enlarged osteocyte lacunae at 12mpf (36% in heterozygote mutants, 20% in wild types). By performing scale histomorphometry, we also detected a higher degree of scale resorption in homozygous sqstm1tmΔUBA zebrafish at 6 mpf. In conclusion, we have generated a Sqstm1 mutant zebrafish model with features of PDB, characterized by focal bone defects and increased osteoclast activity. This model may be useful to further define PDB disease mechanisms and other p62-related (patho)physiological processes.