Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Abstract

Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P‐binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod‐specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10‐deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing

“…Apilimod has been found to be well tolerated in humans, exhibiting a desirable safety profile at doses of up to 125 mg twice daily 2,48 and a peak serum concentration of 0.265 ± 0.183 μM, indicating that therapeutic dosing may be achieved in patients at concentrations likely to promote antiviral activity. Apilimod has been evaluated in phase II clinical trials for the treatment of active Crohn's disease, rheumatoid arthritis and common variable immunodeficiency 2,3 , and in phase I studies for the treatment of follicular lymphoma 49 . Apilimod also efficiently inhibits Ebola virus, Lassa virus and Marburg virus in human cell lines, underscoring its potential broad-spectrum antiviral activity 21,22 .…”

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals

“…Apilimod has been evaluated in phase II clinical trials for the treatment of active Crohn's disease and rheumatoid arthritis (RA) 86 , and in additional phase II trials for the oral treatment of common variable immunodeficiency (CVID), but did not show efficacy for these indications 85,87 . In 2019, orphan drug designation was granted to apilimod in the U.S. for the treatment of follicular lymphoma 88 , with phase I clinical trials ongoing. Notably, it has been reported that apilimod efficiently inhibits EBOV, Lassa virus (LASV), and Marburg virus (MARV) in human cell lines, underscoring its potential broad-spectrum antiviral activity 44,89 .…”

Ostm1 from Mouse to Human: Insights into Osteoclast Maturation

“…Impaired release of these proteases confirms that Ostm1 plays a role in osteoclast secretory lysosome trafficking and possibly in exocytosis ( Figure 3 ). Additional support for a major role of Ostm1 in endosome/lysosome trafficking was described in the context of B cell lymphoma drug sensitivity [ 89 ] as well as in lysosome formation in osteoclasts [ 90 ]. These results demonstrate an osteoclast cell-intrinsic role for Ostm1 as a positive regulator of secretory lysosome dispersion, independent of other bone cells such as osteoblasts.…”