Osteoclast Methods in Protein Phosphatase Research.

Osteoclasts are specialized cells that degrade bone and are essential for bone formation and maintaining bone homeostasis. Excess or deficient activity of these cells can significantly alter bone mass, structure, and physical strength, leading to significant morbidity, as in osteoporosis or osteopetrosis, among many other diseases. Protein phosphorylation in osteoclasts plays critical roles in the signaling pathways that govern the production of osteoclasts and regulate their bone-resorbing activity. In this chapter, we describe the isolation of mouse splenocytes and their differentiation into mature osteoclasts on resorptive (e.g., bone) and non-resorptive (e.g., plastic or glass) surfaces, examining matrix resorption by osteoclasts, immunofluorescence staining of these cells, and knocking out genes by CRISPR in the mouse osteoclastogenic cell line RAW264.7.

The origins and formation of bone-resorbing osteoclasts.

Osteoclasts (OCLs) are hematopoietic cells whose physiological function is to degrade bone. OCLs are key players in the processes that determine and maintain the mass, shape, and physical properties of bone. OCLs adhere to bone tightly and degrade its matrix by secreting protons and proteases onto the underlying surface. The combination of low pH and proteases degrades the mineral and protein components of the matrix and forms a resorption pit; the degraded material is internalized by the cell and then secreted into the circulation. Insufficient or excessive activity of OCLs can lead to significant changes in bone and either cause or exacerbate symptoms of diseases, as in osteoporosis, osteopetrosis, and cancer-induced bone lysis. OCLs are derived from monocyte-macrophage precursor cells whose origins are in two distinct embryonic cell lineages - erythromyeloid progenitor cells of the yolk sac, and hematopoietic stem cells. OCLs are formed in a multi-stage process that is induced by the cytokines M-CSF and RANKL, during which the cells differentiate, fuse to form multi-nucleated cells, and then differentiate further to become mature, bone-resorbing OCLs. Recent studies indicate that OCLs can undergo fission in vivo to generate smaller cells, called "osteomorphs", that can be "re-cycled" by fusing with other cells to form new OCLs. In this review we describe OCLs and discuss their cellular origins and the cellular and molecular events that drive osteoclastogenesis.

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

Bone homeostasis is a complex, multi-step process, which is based primarily on a tightly orchestrated interplay between bone formation and bone resorption that is executed by osteoblasts and osteoclasts (OCLs), respectively. The essential physiological balance between these cells is maintained and controlled at multiple levels, ranging from regulated gene expression to endocrine signals, yet the underlying cellular and molecular mechanisms are still poorly understood. One approach for deciphering the mechanisms that regulate bone homeostasis is the characterization of relevant pathological states in which this balance is disturbed. In this article we describe one such "error of nature," namely the development of acute recessive osteopetrosis (ARO) in humans that is caused by mutations in sorting nexin 10 (SNX10) that affect OCL functioning. We hypothesize here that, by virtue of its specific roles in vesicular trafficking, SNX10 serves as a key selective regulator of the composition of diverse membrane compartments in OCLs, thereby affecting critical processes in the sequence of events that link the plasma membrane with formation of the ruffled border and with extracellular acidification. As a result, SNX10 determines multiple features of these cells either directly or, as in regulation of cell-cell fusion, indirectly. This hypothesis is further supported by the similarities between the cellular defects observed in OCLs form various models of ARO, induced by mutations in SNX10 and in other genes, which suggest that mutations in the known ARO-associated genes act by disrupting the same plasma membrane-to-ruffled border axis, albeit to different degrees. In this article, we describe the population genetics and spread of the original arginine-to-glutamine mutation at position 51 (R51Q) in SNX10 in the Palestinian community. We further review recent studies, conducted in animal and cellular model systems, that highlight the essential roles of SNX10 in critical membrane functions in OCLs, and discuss possible future research directions that are needed for challenging or substantiating our hypothesis.

An SNX10-dependent mechanism downregulates fusion between mature osteoclasts.

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 the 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- to 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 downregulates 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. This article has an associated First Person interview with the joint first authors of the paper.

Massive osteopetrosis caused by non-functional osteoclasts in R51Q SNX10 mutant mice.

The R51Q mutation in sorting nexin 10 (SNX10) was shown to cause a lethal genetic disease in humans, namely autosomal recessive osteopetrosis (ARO). We describe here the first R51Q SNX10 knock-in mouse model and show that mice homozygous for this mutation exhibit massive, early-onset, and widespread osteopetrosis. The mutant mice exhibit multiple additional characteristics of the corresponding human disease, including stunted growth, failure to thrive, missing or impacted teeth, occasional osteomyelitis, and a significantly-reduced lifespan. Osteopetrosis in this model is the result of osteoclast inactivity that, in turn, is caused by absence of ruffled borders in the mutant osteoclasts and by their inability to secrete protons. These results confirm that the R51Q mutation in SNX10 is a causative factor in ARO and provide a model system for studying this rare disease.

The effect of the waiting room's environment on level of anxiety experienced by children prior to dental treatment: a case control study.

BACKGROUND: In addition to visit purpose, one of the environmental factors that can cause anxiety prior to dental treatment includes the waiting room experience, specifically the amount of time spent awaiting treatment and the waiting room environment. The purpose of this study was to compare the effect of the waiting room's environment on the level of anxiety experienced by children in multisensory and traditional waiting rooms. METHODS: Case control study. Test group waited for treatment in a multisensory waiting room, which consisted of a lighting column that children could touch and climb; as well as, rhythmic music played on loudspeakers. Control group waited for treatment in a traditional waiting room. Study participants were asked to answer the "Venham Picture Test", a dental anxiety scale, while in the waiting room prior to entering the treatment room. Chi-squared, Fisher's Exact tests, and linear regression were utilized. A p-value less than 0.05 was considered statistically significant. RESULTS: No significant difference in dental anxiety scores was found between the test and control groups according to waiting room type (p > .05). Dental anxiety was significantly higher in patients who had longer waiting time prior to treatment (p = 0.019). In addition, dental anxiety was significantly associated with visit purpose (p < .001): children waiting for dental examination or those scheduled for dental treatment with conscious sedation were less anxious than children waiting for emergency treatment. CONCLUSIONS: A sensory adapted waiting room environment may be less important in reducing children's anxiety prior to dental treatment. Children's dental anxiety can be reduced by preventing emergency treatments, scheduling routine dental visits and decreasing waiting time. TRIAL REGISTRATION: TRN NCT03197129, date of registration June 20, 2017.