An SNX10-dependent mechanism downregulates fusion between mature osteoclasts

Abstract

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10-100 fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous, SNX10-dependent mechanism that down-regulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo.

Sorting Nexin 10 as a Key Regulator of Membrane Trafficking in Bone-Resorbing Osteoclasts: Lessons Learned From Osteopetrosis

“…prominent in late endosomes (Haucke, 2005;Wallroth and Haucke, 2018;Barnea-Zohar et al, 2021). In agreement with these findings, studies in RAW264.7 cells indicated that SNX10 binds the lipid kinase PIKfyve, which phosphorylates PI3P into PI(3,5)2P, and both PIKfyve and SNX10 co-fractionate in these cells together with EEA1, a marker of early endosomes (Battaglino et al, 2019;Sultana et al, 2020).…”

“…OCLs from these mice lacked RBs and could not acidify the cell-bone interface, in agreement with their complete lack of activity in in vitro pit resorption assays. R51Q SNX10 OCLs also stained poorly for TRAP, appeared to be less adherent, and were noticeably more fragile and shorter-lived than their wild-type or heterozygous counterparts (Barnea-Zohar et al, 2021). Levels of CTX in circulation were elevated in these mice despite clear OCL inactivity (Stein et al, 2020), similar to the SNX10 KD mice (Ye et al, 2015).…”

“…One prominent example concerns OCL fusion, a process that initiates at the plasma membrane. When grown in vitro on mineralized or non-mineralized surfaces, R51Q SNX10 OCLs fuse repeatedly and continuously to form gigantic OCLs that can become 10-to 100-fold larger than controls (Barnea-Zohar et al, 2021). Examination of the fusion of mutant OCLs in culture by live-cell imaging revealed widespread fusion between mature OCLs, a fusion modality that was rarely observed among WT OCLs (Figure 5).…”

“…In particular, the PX domain of SNX10, like those of most other SNX proteins (Teasdale and Collins, 2012), predominantly binds PI3P, which is found in early endosomes; SNX10 also binds phosphatidylinositol-3,5-diphosphate (PI(3,5)P 2 ) that is FIGURE 6 | Overview of the known functions of SNX10 along the plasma membrane-to-ruffled border axis. Functions or abilities indicated include support of endocytosis (references Ye et al, 2015 andBarnea-Zohar et al, 2021 noted in the figure), binding to PI3P and PI(3,5)P 2 (Barnea-Zohar et al, 2021), association with PIKfyve, EEA1, and early endosomal markers (Qin et al, 2006;Battaglino et al, 2019;Sultana et al, 2020), inhibition of chaperone mediated autophagy (You et al, 2018;Zhang et al, 2018Zhang et al, , 2020, association with LAMP1 and ATG5 and promotion of lysosome-autophagosome fusion (Zhang et al, 2018(Zhang et al, , 2020, and ruffled border formation (Ye et al, 2015;Stein et al, 2020;Barnea-Zohar et al, 2021). See the section "Cell-Based Studies," for additional details.…”

“…Fine spatial and temporal tuning of membrane composition, compartmentalization, and dynamics play essential roles throughout the formation and development of OCLs, starting with the early stimulation of monocytes by M-CSF and RANKL and culminating in full OCL maturation and activation. This is evident in the extensive cell-cell fusion that occurs throughout osteoclastogenesis, until OCLs gain full resorption competence and stop fusing with other mature OCLs (Barnea-Zohar et al, 2021). It is also evident in the ability of OCLs to sense the properties of the underlying bone surface and to translate this information into cues that drive SZ formation at specific locations of the ventral cell membrane.…”