Snx10 and PIKfyve are Required for Lysosome Formation in Osteoclasts

MORSE L; SUMMERS M; WEIMIN L; BATTAGLINO R; PICOTTO G

Montreal

Congreso; Annual Meeting of the American Society for Bone and Mineral Research (ASBMR); 2018

ASBMR

Snx10 and PIKfyve are Required for Lysosome Formation in Osteoclasts ID: A18018811Osteoclasts rely on intracellular trafficking and organelle homeostasis for normal function. Snx10 is a PI3P-binding protein which localizes to osteoclastic early endosomes. Snx10-deficent osteoclasts show impaired endocytosis, extracellular acidification, ruffled border formation and bone resorption, suggesting that Snx10 regulates vesicular trafficking in endosome/lysosome formation. In order to elucidate the cellular mechanisms by which Snx10 regulates vesicular transport to control osteoclastic resorption we studied PIKfyve, another PI3P-binding kinase, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is required for the biogenesis of lysosomes. Genetic or pharmacological inhibition of PIKfyve (and diminished PI(3,5)P2 synthesis) causes endosome enlargement and cytoplasmic vacuolation. Overexpression of SNX10 also induces accumulation of early endosomes. These changes in the number and size of endosomes and lysosomes suggests that both Snx10 and PIKfyve are involved in membrane traffic between these organelles. Apilimod, a small molecule with cytotoxic activity used for the treatment of inflammatory diseases, inhibits PIKfyve with nanomolar specificity. Intriguingly, expression of CLCN7, OSTM1, and SNX10 is essential for sensitivity to apilimod which suggests that endosome/lysosome dysfunction mediates apilimod?s cytotoxic effect. Snx10 and PIKfyve co-localize to early endosomes in osteoclasts, as determined by subcellular fractionation studies and immunofluorescent staining. Treatment with 10nM Apilimod results in accumulation of early endosomes, inhibition of RANKL-induced osteoclast formation, inhibition of lysosome formation and secretion of TRAP from differentiated osteoclasts. Apilimod, on the other hand, does not inhibit lysosome biogenesis in Snx10-deficient osteoclasts. Taken together, these results suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2. These findings may suggest novel therapeutic approaches targeting the secretory pathway in osteoclasts.