Autosomal dominant osteopetrosis type II (ADO2) is a disease characterized by abnormally dense bones that are prone to fractures. ADO2 is caused by mutations in the CLCN7 gene which leads to dysfunctional osteoclasts, a type of bone cell responsible for bone being broken down (bone resorption) while maintaining a constant equilibrium with bone being built (bone formation) by osteoblasts. When osteoclasts don’t work properly, bone is continuously built but bone resorption is impaired, causing dense bone structure with poor bone quality. The mechanism for osteoclast dysfunction in ADO2 is not fully understood. One of the hallmarks for active osteoclasts resorbing bone is the formation of the ruffled border, a specialized cell membrane structure resembling fingerlike processes. Within cells, an organelle called a lysosome takes part in the formation of the ruffled border, and release of calcium from lysosomes is crucial for this process. By triggering calcium release from lysosomes, formation of ruffle border can be enhanced and osteoclasts’ ability to resorb bone might be improved. In this study, a drug that can stimulate lysosomal calcium release was tested to identify its effect on gene expression changes by real-time PCR, osteoclasts number by TRAP-staining and osteoclasts’ resorption capability by pit formation assay on bone discs using both wild-type and ADO2 osteoclasts.

There is an ongoing struggle in my lab to further understand the mechanism by which the mutation in the CLCN7 gene causes osteopetrosis. The work that I've done this semester in my research project has attempted to identify a piece of that overall pathway. If I receive positive results in my experiments that the addition of calcium from lysosomes increase the function of osteoclasts, then that proves that calcium from lysosomes plays a part in the function of osteoclasts. This would be an important discovery because it would open the door for us to begin testing multiple types of drugs or therapies that induce calcium release. These would all be tested in mice and if any prove effective they could even move on to a clinical trial in humans. If I receive negative results in my experiment, it will still be useful because it shows that lysosomal calcium release does not play a major role in the function of osteoclasts and that this route of research should no longer be pursued.