Leucine Repeat Rich Kinase 1 Controls Osteoclast Activity by Managing Lysosomal Trafficking and Secretion.

We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 ( Lrrk1 ) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis.