Autophagy in Osx1-Cre-targeted cells is essential for development, growth, and maintenance of bone.

Autophagy is a cellular recycling pathway in which cytoplasmic components are delivered to lysosomes for degradation. Changes in autophagy levels have been implicated in various skeletal pathologies including osteoporosis. Elimination of autophagy at different stages of the osteoblast lineage reduces bone formation and bone mass. However, whether autophagy plays a role specifically during development, growth, or maintenance of bone remains unclear. To start addressing this question, we eliminated autophagy in the entire osteoblast lineage prenatally or at weaning and subjected the mice to skeletal phenotyping at 4.5 and 10 months of age. We found that regardless of when autophagy was eliminated from the osteoblast lineage, autophagy deficiency reduced bone mineral density (BMD), cortical thickness, and cancellous bone volume in the femur and spine. Serial BMD analysis revealed that autophagy-deficient mice had consistently lower BMDs from 3 to 9 months of age, and the BMD difference between genotypes became progressively greater in the spine. The reduction in vertebral cancellous bone volume of autophagy-deficient mice was associated with reduced bone formation. To assess autophagy-induced changes at the cellular and molecular level, we performed single-cell RNA-sequencing (scRNA-seq) analysis of periosteal mesenchymal cells and compared autophagy deficiency-induced changes in periosteal and endosteal cell preparations. This analysis revealed that autophagy deficiency disrupts proteostasis, causes mitochondrial dysfunction, induces senescence, and increases stress response pathways like TNF and TGF-β. Overall, we conclude that autophagy is important for skeletal growth and maintenance of bone, and we identify potential cellular populations and cellular processes via which autophagy support bone formation.