PROJECT SUMMARY/ABSTRACT – PROJECT 1 Osteoblasts are bone-forming cells that produce large amounts of collagen and must maintain protein homeostasis to prevent cell dysfunction and death. Altered protein homeostasis in osteoblasts may contribute to the pathology of skeletal diseases such as Osteogenesis Imperfecta (OI). Autophagy recycles cellular components by delivering them to lysosomes for degradation. In macroautophagy, cargo, such as aggregated protein, is sequestered in double-membrane vesicles and delivered to lysosomes, while in chaperone-mediated autophagy (CMA), individual proteins are delivered by chaperones. We have shown that deletion of Atg7, a gene essential for macroautophagy, from osteoblasts causes low bone mass and fractures. This was associated with low osteoblast number and disruption of the osteocyte network. Osteocytes, which differentiate from osteoblasts, are buried in the bone matrix and sense changes in biomechanical load to orchestrate bone formation and resorption. The mechanisms by which loss of macroautophagy in osteoblasts and osteocytes cause low bone mass remain unclear. To examine the role of CMA in osteoblasts and osteocytes, we created mice lacking Lamp2a, which is essential for CMA, and this also caused low bone mass, but to a milder degree than loss of macroautophagy. Studies in other cell types suggest that CMA may be important mainly under conditions of cellular stress, such as endoplasmic reticulum stress. In line with this, we found that osteoblasts lacking CMA are more vulnerable to apoptosis induced by endoplasmic reticulum stress and produce more RANKL, a cytokine that stimulates formation of bone-resorbing osteoclasts. Based on this, we hypothesize that macroautophagy and CMA promote the differentiation, survival, and function of osteoblast lineage cells by maintaining proteostasis and we propose the following aims. Aim 1 will identify mechanisms by which deletion of Atg7 reduces osteoblast number by measuring proliferation and apoptosis, and by using single cell RNA sequencing to examine differentiation. We will also determine if loss of Atg7 alters the response of the tibia to mechanical loading. Aim 2 will determine if loss of CMA worsens the skeletal phenotype of a murine model of OI to reveal if CMA protects osteoblasts and osteocytes from endoplasmic reticulum stress. Aim 3 will determine whether genetic stimulation of macroautophagy improves osteoblast function and reduces endoplasmic reticulum stress in a murine model of OI. Successful completion of these studies will clarify the role of macroautophagy and CMA in osteoblast lineage cells may identify new targets for therapeutic intervention in conditions such as OI.