Role of Autophagy in skeletal diseases

/Abstract Macroautophagy, here referred to as autophagy, is a recycling process that facilitates lysosomal degradation of cytoplasmic content, including protein aggregates and damaged organelles. In previous work, we showed that autophagy deficiency reduces osteoblast number and bone formation. In work leading to this proposal, we created a novel mouse model in which we stimulate autophagy by increasing expression of endogenous transcription factor EB (Tfeb) using CRISPR activation. This maneuver increased bone formation in cancellous and cortical compartments, leading to high bone mass and increased bone strength. These studies establish that autophagy in osteoblast lineage cells promotes bone formation. However, the mechanisms underpinning this effect are unknown. To fill this gap in knowledge, we used single-cell RNA sequencing (scRNA-seq) of bone cells from autophagy-deficient mice. This analysis revealed that autophagy deficiency increases senescence in osteoblast lineage cells. Moreover, we provide evidence that the autophagy insufficiency in two skeletal diseases, namely aging and Osteogenesis imperfecta (OI), is associated with reduced bone formation and elevated senescence. Specifically, we used scRNA-seq to compare skeletal aging and autophagy deficiency and show that many biological processes altered by aging are similarly changed by autophagy deficiency, including senescence. OI is another condition where insufficient autophagy may lead to osteoblast senescence and reduced bone formation. Misfolded procollagen aggregates are cleared by autophagy, and mutations in collagen or collagen processing enzymes exaggerate collagen misfolding to levels beyond osteoblasts' clearance capacity, leading to intercellular collagen aggregation. We found that insufficiency of autophagy in clearing procollagen aggregates in OI is associated with increased senescence markers in mouse models of OI. Importantly, we found that stimulation of autophagy in a model of OI increased bone mineral density. Together, our results reveal an association between autophagy insufficiency, increased senescence, and reduced bone formation in aging and OI. Therefore, we hypothesize that autophagy promotes bone formation by inhibiting cellular senescence in osteoblasts, and insufficiency of autophagy in aging and OI contributes to the loss of bone mass in these conditions. To test this hypothesis, we propose the following aims. Aim 1 will examine if autophagy promotes bone formation by preventing cellular senescence. Aims 2 and 3 will determine if there is a cause- effect relationship between autophagy insufficiency, senescence, and bone formation in aging and OI. Project Number: 1R01AG094715-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Melda Onal | Institution: UNIV OF ARKANSAS FOR MED SCIS, LITTLE ROCK, AR | Award Amount: $487,847 | Activity Code: R01 | Study Section: Skeletal Biology Development and Disease Study Section[SBDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11363966