Mechanistic and Therapeutic Studies of Autosomal Dominant Osteopetrosis

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Abstract Autosomal dominant osteopetrosis type 2 (ADO2) is an osteosclerotic disorder resulting from missense mutations in the Chloride Channel 7 gene (CLCN7), which cause defects in osteoclastic bone resorption by a dominant negative mechanism. Disease severity varies widely, even within the same family, and one third of individuals with mutations are asymptomatic carriers. However, of the two thirds who are affected by the disease (ADO2 patients), all have high bone mineral density with at least one clinical manifestation including fracture, osteonecrosis (particularly of the jaw and/or maxilla), osteomyelitis, blindness, and/or bone marrow failure. To investigate the pathophysiology of the disease and to perform “proof of principle” experiments to treat the disease we created a novel ADO2 “knock-in” mouse with a disease causing G213R mutation in the Clcn7 gene. We found that phenotypic severity differs among genetic strains and the ADO2 mouse on the129 background (used in this proposal) is an excellent model of moderate disease [bone volume per total volume (BV/TV) 2-fold over WT at 12 weeks of age, due primarily to osteoclast dysfunction]. To begin to understand the mechanism, we examined intracellular trafficking in WT and ADO2 osteoclasts which revealed defects in early endosomal trafficking, which is necessary for sorting internalized cell surface proteins into late endosomes and lysosomes, or recycling them back to the plasma membrane. In addition, we found that the pharmacologic agents, chloroquine, which modulates early endosomal trafficking, and forskolin and roflumilast, which increase intracellular cAMP by different mechanisms, enhance the bone-degrading activity of ADO2 osteoclasts. Our hypotheses are: 1) The bone-resorbing activity of ADO2 osteoclasts can be functionally restored by modulating either endosomal trafficking or increasing intracellular cAMP levels; 2) Chloroquine treatment will rescue the phenotype of the ADO2 mouse in young (6 weeks) and adult (9 months) mice; 3) Discontinuation of chloroquine therapy in ADO2 mice will result in an increase in BV/TV, as measured by in vivo µCT, but not at an accelerated rate and not to a degree similar to that of age-matched vehicle treated mutant mice; and 4) Bone marrow transplantation (BMT) will reverse the skeletal defects in ADO2 mice when performed either early (6 weeks of age) or late (9 months of age) in the disease course. Successful completion of the proposed aims may provide proof of principle that low dose chloroquine and BMT will substantially improve or fully rescue the ADO2 phenotype in a mouse model that mimics the human disease. Young and adult mice will be used to more closely align our studies with questions that occur in the treatment of ADO2 patients. Our studies will also provide important insight into the underlying mechanism of how Clcn7 dysfunction in osteoclasts leads to the development of ADO2 in patients.