Role of Mitochondrial Deacetylase Sirt3 in Skeletal Homeostasis

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Mitochondria are the major source of cellular ATP generated through the process of oxidative phosphorylation. Recent insights have revealed that mitochondria are also involved in other tasks, such as cell death, reactive oxygen species (ROS) generation, immunity, and calcium homeostasis. Mitochondrial protein lysine acetylation plays a key role in these metabolic processes. Although still not completely understood, this post-translational modification of mitochondrial protein networks seem to be important for maintenance of healthy, properly functioning mitochondria. Unsurprisingly, in human diseases as well as mouse models, mitochondrial dysfunction has been linked to numerous aspects of aging, including neurodegeneration, cardiopathologies, insulin resistance, and bone loss. However, it is largely unknown whether the reduced bone mass with advancing age is due to a change in mitochondrial function(s) in osteoclasts or in other cell types. The primary goal of the work proposed in this application is to establish the role of mitochondrial function in bone cell physiology and disease. In order to investigate mitochondrial function, we will manipulate Sirtuin 3 (Sirt3), a molecule with critical roles for both mitochondrial function and energy homeostasis that has not clearly been studied in osteoclasts and osteoblasts. Sirt3 is an NAD+- dependent deacetylase localized primarily to the mitochondria, with a unique role in the activation of metabolic enzymes and Complex I subunits. Sirt3 deacetylates and activates Ndufa9, an important Complex I subunit, leading to enhancement of cellular ATP levels and oxidative phosphorylation. Sirt3 can also interact with the acetyl-CoA synthetase, affect G1 arrest induced by loss of Bcl-2 and, thereby, regulate cell death. Loss of all of these roles can contribute to mitochondrial dysfunction and aberrantly enhanced ROS levels, most likely by down-regulating Complex I activity. Our preliminary data shows that the anti-osteoclastogenic actions of estrogens are associated with decreased Complex I activity and ATP production in early osteoclast precursors, and that Sirt3 expression is upregulated during osteoclastogenesis. Further, global deletion of Sirt3 in mice prevents age-related bone loss, accompanied by a decrease in bone resorption. Removal of Sirt3 from osteoblast progenitors in vitro also reduces osteoblast differentiation and mineralization. These observations form the foundation for the hypothesis that Sirt3 plays an essential role in skeletal homeostasis by regulating mitochondrial function(s) in osteoclasts and osteoblasts. To test these hypotheses, we will examine whether deletion of Sirt3 in osteoclasts (Aim 1) and osteoblasts (Aim 2) prevents the loss of bone mass caused by advancing age or estrogen deficiency. Further, we will perform in vitro studies to identify Sirt3 target proteins in osteoclasts and osteoblasts that are responsible for their effects on differentiation and function using quantitative analysis of global proteome and lysine acetylome in primary bone marrow-derived macrophages with or without Sirt3 genes (Aim 3). Successful completion of this work will shed light on novel mechanisms that contribute to ost oporosis and will advance knowledge of how the skeleton responds to changes in mitochondrial function.