PROJECT SUMMARY/ABSTRACT Data from our mouse model of Botulinum Toxin A (BTxA) induced muscle paralysis has revealed that neuromuscular function, outside the axis of mechanical loading deficits, is a critical modulator of bone homeostasis. Consistent with this thesis, we have observed that transient muscle paralysis triggers acute inflammatory signaling within bone marrow that precedes the onset of focal RANKL-mediated osteoclastogenesis, which is responsible for the profound cortical and trabecular bone resorption observed in the model. However, the intercellular signaling that initiates acute bone marrow inflammation and subsequent bone resorption has not been elucidated and therefore presents a barrier to identifying translational strategies that would decouple neuromuscular dysfunction from bone loss. One potential initiator of this rapid response is neurogenic inflammation, which is triggered by neuropeptide release from sensory nerves and is amplified by mast cell mediated histamine release. We therefore pursued a series of preliminary studies to assess activation of this pathway following muscle paralysis and found that: 1) Substance P, a classic initiator of neurogenic inflammation, was upregulated in tibia bone marrow within 1 d of calf paralysis, 2) genes associated with connective tissue mast cell activation were acutely elevated following muscle paralysis, and 3) muscle paralysis induced bone resorption was significantly diminished in mast cell deficient KitW-sh/W-sh mice. We therefore hypothesize that: Bone resorption following muscle paralysis is initiated by neuropeptide signaling and is amplified by mast cell dependent histamine release. We will pursue this thesis via four complementary Specific Aims (SA), each with a corresponding sub-hypothesis. First, we anticipate that neuropeptides within bone marrow will be elevated by BTxA induced muscle paralysis prior to evidence of mast cell activation or bone resorption (SA#1). SA#2 will then demonstrate that successful antagonism of these neuropeptides will be required to inhibit mast cell activation and bone resorption induced by muscle paralysis. In SA#3, we will leverage a cKit independent, connective tissue mast cell deficient mouse to demonstrate that mast cell mediated histamine signaling is responsible for the profound osteoclastogenesis induced by muscle paralysis. SA#4 will then provide proof of concept that treatment with histamine receptor antagonists will significantly attenuate bone resorption caused by muscle paralysis. Each aspect of the proposed signaling pathway (neurogenic inflammation, neuropeptide signaling, mast cell activation, paralysis induced bone resorption) has been explored in other contexts but has not been integrated into a cellular signaling cascade that integrates muscle, nerve, and bone physiology. Importantly, if our thesis is supported, the broad clinical experience with histamine antagonists will enable repurposing of approved drugs toward the goal o...