Our long-term goal is to understand the role and regulation of mitochondrial metabolism in bone physiology and pathology. Unlike other fields, such as cardiovascular and muscle physiology and neuroscience, to date very little effort has been directed towards mitochondrial research in the bone field. This presents an immense knowledge gap and a critical barrier to developing novel mitochondria-targeted strategies for bone pathologies, such as aging and trauma which are known to be associated with mitochondrial dysfunction. This also gives special significance to our mitochondria-centered proposal. Our objective here is to determine the mechanism controlling mitochondrial activity during osteoblastic (OB) differentiation of bone marrow stromal cells (BMSC, a.k.a. bone marrow mesenchymal stem cells) and test if strategies aimed at improving mitochondrial metabo- lism stimulate OB differentiation and bone formation. Our published and new unpublished data indicate that during OB differentiation, mitochondria fuse into a network, a phenomenon known to maximize the fidelity for oxidative phosphorylation (OxPhos). A dangerous byproduct of active OxPhos is oxidative stress which pro- motes opening of a large Mitochondrial Permeability Transition Pore (MPTP). MPTP opening impairs mito- chondrial integrity and function. Cyclophilin D (CypD) is a key positive regulator of MPTP. It is, thus beneficial for cells undergoing a shift towards oxidative metabolism, e.g. BMSCs differentiating into OBs, to inactivate CypD/MPTP. We indeed found that as mitochondria become fused and activated during OB differentiation, CypD is downregulated at the mRNA level ensuring protection against oxidative stress and supporting OxPhos and progression of OB program. Moreover, our data indicate that CypD genetic deletion in knockout (KO) mice or pharmacological inhibition is especially efficient in supporting OB oxidative and bone forming function under pathological stress, such as in aging and fracture. We recently reported that CypD KO mice are well protected against bone loss in aging. This is consistent with the literature showing that brain, heart, and kidney tissues of CypD KO mice are protected against degeneration in aging or ischemic injury. All this led us to hypothesize that mitochondrial fusion and CypD downregulation leading to activation of OxPhos and inhibition of MPTP dur- ing OB differentiation, are critical for OB differentiation. To test this hypothesis and fulfill our objective, we will: 1) characterize the mechanism by which mitochondria are activated during OB differentiation focusing on mito- chondrial fusion; 2) characterize the role and regulation of CypD/MPTP during OB differentiation; and 3) evalu- ate CypD as a therapeutic target to improve bone formation in fracture healing and aging. These studies will provide comprehensive understanding of regulation of mitochondrial metabolism during OB differentiation and a rationale for developing new mitochondria-...