Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis.

Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses.

rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG.

Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments.

Osteocytes are terminally differentiated long-lived cells and account for greater than 95% of the bone cell population. It has been established that osteocytes are connected through their highly developed dendritic network, which is necessary for the maintenance of optimal bone homeostasis. However, little is known on how osteocytes use the network to coordinate their cellular function and communication that requires energy and protein turnover. Here using super-resolution confocal imaging on both live and fixed osteocytes, we demonstrated conclusively that mitochondria are widely distributed and dynamically shared between osteocytes. Using confocal live cell imaging analysis we showed that inhibiting the contact between mitochondria and endoplasmic reticulum (ER) by the knockdown of MFN2 in osteocytes impedes the transfer of mitochondria suggesting the involvement of ER contact with mitochondria in the transfer process. Moreover, we showed that transport of mitochondria between osteocytes within the network enables rescue of osteocytes with dysfunction of mitochondria. Using the 3D tetraculture system with confocal imaging, we identify the transfer of mitochondria from healthy osteocytes enables recovery of mitochondria activities in osteocytes that devoid of mitochondrial DNA by ethidium bromide. The results indicated that when osteocytes are depleted of functional mitochondria, normal parental osteocytes can transfer mitochondria to these stressed osteocytes to provide them with energy. Collectively we show for the first time that the utilisation of mitochondrial transfer enables osteocytes to function with a network and coordinate their cellular activities in response to different energy demands.