OBJECTIVE: ASTRONAUTS LOSE 1-1.5% OF THEIR BONE MASS PER MONTH DURING LOW EARTH ORBIT MISSIONS. SPACEFLIGHT PROFILES OF 2 YEARS OR LONGER AS PROJECTED FOR MARS MISSIONS WILL SIGNIFICANTLY INCREASE BONE FRAGILITY POTENTIALLY COMPROMISING THE ABILITY OF ASTRONAUTS TO PERFORM THEIR MISSION TASKS IN TRANSIT AND ON MARS. TO FACILITATE HUMAN EXPLORATION OF MARS IT IS THEREFORE ESSENTIAL TO UNDERSTAND THE UNDERLYING CAUSES OF MICROGRAVITY-INDUCED BONE LOSS AND DEVELOP EFFECTIVE COUNTERMEASURES TO SUSTAIN BONE STRUCTURE AND MECHANICAL INTEGRITY UNDER MICROGRAVITY CONDITIONS. OUR LONG-TERM GOAL IS TO PROTECT THE SKELETAL HEALTH OF ASTRONAUTS IN SPACE ENVIRONMENTS SO THEY CAN PERFORM THEIR MISSION OPERATIONS. THE OBJECTIVE OF THE CURRENT PROPOSAL IS TO DETERMINE THE EFFECTS OF MICROGRAVITY ON OSTEOBLAST-OSTEOCLAST COUPLING AND COMMUNICATION IN ORDER TO IDENTITY A COUNTERMEASURE FOR MICROGRAVITY-INDUCED DECOUPLING. OUR CENTRAL HYPOTHESIS IS THAT SIMULATED MICROGRAVITY WILL IMPAIR OSTEOBLAST-OSTEOCLAST COUPLING AND THAT BISPHOSPHONATE (ALENDRONATE) AND/OR ANTI-RANKL ANTIBODY (DENOSUMAB) TREATMENT WILL MITIGATE MICROGRAVITY-INDUCED OSTEOBLAST-OSTEOCLAST DECOUPLING. THE RATIONAL FOR THIS PROPOSAL IS THE FACT THAT MICROGRAVITY PROMOTES OSTEOCLASTIC BONE RESORPTION AND IMPAIRS OSTEOBLASTIC BONE FORMATION. IT IS THEREFORE EXPECTED THAT PHYSIOLOGICAL OSTEOBLASTOSTEOCLAST COUPLING IS IMPAIRED AND THAT DENOSUMAB A RANKL INHIBITOR WILL RESTORE OSTEOBLAST-OSTEOCLAST COUPLING AND MITIGATE MICROGRAVITY-INDUCED BONE LOSS TO A GREATER DEGREE THAN ALENDRONATE AN ANTI-CATABOLIC AGENT. SPECIFIC AIMS AND METHODS: THE CENTRAL HYPOTHESIS WILL BE TESTED BY COMPLETING FOLLOWING TWO SPECIFIC AIMS. 1. DETERMINE THE EFFECTS OF SIMULATED MICROGRAVITY ON OSTEOBLAST-OSTEOCLAST COUPLING AND COMMUNICATION UTILIZING IN VITRO CO-CULTURES. OUR WORKING HYPOTHESIS IS THAT MICROGRAVITY WILL ALTER OSTEOBLAST-OSTEOCLAST COMMUNICATION PROCESSES. PATHWAY ANALYSES WILL BE CONDUCTED BY ASSESSING THE DIFFERENTIAL EXPRESSION OF MRNA (E.G. RNASEQ QPCR) AND PROTEIN (I.E. ELISA WESTERN BLOTS) AS WELL AS HISTOLOGY AND HISTOMORPHOMETRY. COMBINED CONFOCAL RAMAN SCANNING ELECTRON MICROSCOPY (CRSEM) WILL BE USED TO DETERMINE FUNCTIONAL IMPAIRMENT OF BONE RESORPTION AND FORMATION. 2. ASSESS THE EFFICACY OF DENOSUMAB RELATIVE TO ALENDRONATE AS A COUNTERMEASURE TO MICROGRAVITY-INDUCED OSTEOBLAST-OSTEOCLAST DECOUPLING IN VITRO. OUR WORKING HYPOTHESIS IS THAT DENOSUMAB WILL BE MORE EFFICACIOUS IN RESTORING MICROGRAVITY-INDUCED IMPAIRMENT OF OSTEOBLASTOSTEOCLAST COUPLING AS IT FUNCTIONS BY BLOCKING EXCESS RANKL WHILE ALENDRONATE ONLY AFFECTS RESORPTION. OSTEOBLAST-OSTEOCLAST COMMUNICATION WILL BE ASSESSED USING PROTEOMIC AND GENETIC ANALYSIS AND FUNCTION RESTORATION WILL BE ASSESSED USING HISTOLOGY HISTOMORPHOMETRY AND CRSEM. SIGNIFICANCE AND OUTCOMES: BONE REMODELING IS A COMPLEX PROCESS THAT IS TIGHTLY REGULATED BY OSTEOBLAST-OSTEOCLAST COUPLING AND COMMUNICATION. MICROGRAVITY IMPAIRS THIS PROCESS BY INCREASING OSTEOCLAST ASSOCIATED RESORPTION AND DECREASING OSTEOBLASTIC BONE FORMATION. IN ORDER TO CONTINUE TO EXPAND OUR MANNED SPACEFLIGHT PROGRAM IT IS ESSENTIAL TO DETERMINE THE EFFECTS OF MICROGRAVITY ON OSTEOBLAST-OSTEOCLAST COUPLING AND DEVELOP TARGETED COUNTERMEASURES TO SUSTAIN BONE HEALTH IN MICROGRAVITY. AT THE COMPLETION OF THE STUDY WE EXPECT TO CHARACTERIZE THE EFFECTS OF MICROGRAVITY ON OSTEOBLAST-OSTEOCLAST COUPLING IDENTIFY COMMUNICATION PATHWAYS ASSOCIATED WITH MICROGRAVITY AS WELL AS ESTABLISH THE EFFICACY OF ALENDRONATE AND/OR DENOSUMAB AS POTENTIAL THERAPEUTICS TO RESTORE OSTEOBLAST-OSTEOCLAST COMMUNICATION AND FUNCTION. THE OUTCOMES OF THIS STUDY WILL FILL NASAS RECOGNIZED KNOWLEDGE GAP AND GUIDING QUESTION IN MICROGRAVITY EFFECTS ON OSTEOBLAST-OSTEOCLAST COMMUNICATION AND EVALUATE POTENTIAL THERAPEUTICS TO ABERRANT THE MICROGRAVITY-INDUCED BONE LOSS IN ASTRONAUTS (AH2 3 CMB1 2 6).