Our group recently identified the tryptophan-kynurenine (Kyn) catabolic pathway as a novel causal mechanism in age-associated musculoskeletal complications (stem cell dysfunction, muscle, and bone loss). Our published studies reported that the Kyn pathway is activated with age and decreases bone marrow-derived mesenchymal stem cells (BMSCs) differentiation, decreased autophagy flux, induces premature senescence, and promotes bone loss. Furthermore, our pilot study demonstrated that inhibiting AhR prevented bone deterioration and reduced systemic stress markers in aged mice. Based on our published and preliminary data, our central hypothesis is that elevated levels of Kyn metabolites and AhR activation with age in bone and BMSCs lead to altered autophagy/mitophagy flux, which underlies mitochondrial dysfunction and BMSC’s vulnerability to premature senescence, leading to decreased osteogenic differentiation, and consequently increased bone loss. This hypothesis will be tested with two independent but related aims. Specific Aim 1: To test the hypothesis that the accumulation of Kyn and its metabolites alters mitophagy and induces premature senescence with age in BMSCs thereby affecting osteogenic pathways (in vitro and in vivo). We will define the roles of several Kyn metabolites (e.g., Kyn, KynA) in the inhibition of mitophagy and senescence in vitro and in vivo. We will assess autophagy and senescent biomarkers, senescence-associated secretory phenotype (SASP) gene expression, and Kyn metabolite profiles to identify potential translational approaches. Specific Aim 2: Test the hypothesis that inhibiting AhR signaling in response to Kyn pathway metabolites restores mitophagy and inhibits premature senescence, rescuing osteogenesis, thereby reducing or preventing bone loss with age. For this specific aim, we will use pharmacological inhibitors (e.g., CH- 223191) and genetic manipulation (stem cell and bone-specific AhR KO mice using, e.g., Prrx1-Cre, Osx1- Cre) to examine the ability of AhR inhibition to restore autophagy and inhibit senescence in BMSCs. The knowledge gained from our proposed studies will establish a conceptual framework to advance the therapeutic potential of AhR inhibition in age-related musculoskeletal fragility.

In the current application, we will test our hypothesis that inhibition of KYN-AhR signaling in bone and bone marrow stromal cells can effectively prevent or reduce age-dependent bone loss. Furthermore, we will determine the molecular mechanisms by which KYN-AhR signaling modulates mitophagy/autophagy and premature senescence in bone marrow stromal cells and bone cells.