Ablation of Chmp5 in mouse osteogenic lineage cells causes aberrant bone formation.

(A) Micro-CT and H&E staining showing periskeletal overgrowth in Chmp5Ctsk mice in comparison with Chmp5fl/fl mice at 7 weeks of age. Dot line representing the approximate bone border. n = 10 animals per group. (B) Micro-CT images displaying periskeletal overgrowth in the ankle and foot in Chmp5Ctsk versus Chmp5fl/fl mice at one year of age. n = 4 animals per group. (C) Alizarin red staining of skeletal preparations on postnatal day 10. n = 4 mice per group, arrows indicating periskeletal bone overgrowth. (D) Three-point test showing femur bone stiffness and ultimate stress (fracture stress) in Chmp5Ctsk and Chmp5fl/fl mice. n = 5 male animals per group. Similar changes observed in both sexes. (E) Confocal images mapping Ctsk+ (GFP+) progenitors in periskeletal tissues in Chmp5Ctsk/+;Rosa26mTmG/+ and Chmp5Ctsk;Rosa26mTmG/+ mice at 2 weeks of age. Asterisks indicating periskeletal overgrowths; dot-line representing the approximate bone border. n = 4 animals each group. (F) X-ray images demonstrating progressive cortical bone expansion (arrows) and decreased skeletal muscle mass (asterisks) in Chmp5Dmp1 in comparison with Chmp5fl/fl mice. n = 3, 3, 10, 8 animals per group for 3 weeks, 5 weeks, 10 weeks, and 1 year of age, respectively. (G) Micro-CT analyses displaying cortical bone expansion in Chmp5Dmp1 relative to Chmp5fl/fl mice at 10 weeks of age. n = 6 Chmp5fl/fl and 5 Chmp5Dmp1 male mice, similar changes found in both genders. (H) Skeletal muscle mass in Chmp5Dmp1 compared to Chmp5fl/fl mice at 10-12 weeks of age. n = 4 animals (male) per group, similar changes found in both genders. (I) Hindlimb abduction of Chmp5Dmp1 mice in comparison with Chmp5fl/fl littermate controls at 10 weeks of age. More than 20 animals per group observed. (J) Four-limb handing time (n = 10 per group, pooled data from both sexes), and forelimb pull strength (n = 6 per group, male mice) in Chmp5Dmp1 compared to Chmp5fl/fl mice at 10-12 weeks of age. Similar changes found in both genders. All data are mean ± s.d.; two-tailed unpaired Student’s t-test. Scale bars, 1 mm for micro-CT and skeletal preparation images, 200 μm for histological images, and 10 mm in panel (F).

CHMP5 restricts osteogenesis in skeletal progenitor cells.

(A) Flow cytometry showing the strategy for sorting CD45-;CD31-;GFP+ and CD45-;CD31-;GFP- periskeletal progenitors from periskeletal tissues of Chmp5Ctsk/+;Rosa26mTmG/+ and Chmp5Ctsk;Rosa26mTmG/+ mice at 2 weeks of age. n = 10 mice per group. (B) Quantitative PCR determining the expression of Chmp5 in Chmp5Ctsk relative to Chmp5Ctsk/+ periskeletal progenitors. n = 6 mice per group. (C) Alizarin red staining, von Kossa staining and alkaline phosphatase activity assay determining osteogenesis in Chmp5Ctsk compared to Chmp5Ctsk/+ periskeletal progenitors. n = 3 replicates each group, representative results of cells from 3 different mice per group. (D) Western blot confirming Chmp5 deletion in mouse MC3T3-E1 cells by lentiviral CRISPR/CAS9. (E) Alizarin red staining demonstrating osteogenesis in MC3T3-E1 cells with or without Chmp5 deletion by lentiviral CRISPR/CAS9. n = 4 replicates per group, experiments repeated twice for each time point. (F) Ingenuity pathway analysis of RNA-seq data showing increased activity of osteoblast differentiation in Chmp5Ctsk vs. Chmp5Ctsk/+ periskeletal progenitors. All data are mean ± s.d.; two-tailed unpaired Student’s t-test.

CHMP5 controls skeletal progenitor cell senescence.

(A) Cell number counting and AlamarBlue assay determining cell proliferation in Chmp5Ctsk and Chmp5Ctsk/+ periskeletal progenitors from 2-week-old animals. n = 3 replicates per group per time point; experiments repeated 3 times using cells from 3 different mice. (B) Volcano plot of RNA-seq data showing differentially expressed genes in Chmp5Ctsk vs. Chmp5Ctsk/+ periskeletal progenitors. n = 3 per group with cells from 3 different animals. (C) ConsensusPathDB gene set enrichment analysis of RNA-seq data showing positive enrichment of genes in multiple molecular pathways related to cell senescence in Chmp5Ctsk relative to Chmp5Ctsk/+ periskeletal progenitors. (D) Western blot determining the expression of p16 and p21 proteins in Chmp5Ctsk compared to wild-type (control) periskeletal progenitors. The numbers above lanes indicating the intensity of the p16 or p21 band relative to that of Control cells after normalization by β-Actin. Results repeated 3 times with cells from 3 different animals. (E) Gross image demonstrating aging-related phenotypes in Chmp5Ctsk compared to Chmp5Ctsk/+ mice at 1 year of age. Images are representative of 5 animals per group. (F) Western blot confirming deletion of Chmp5 in ATDC5 cells by lentiviral CRISPR/CAS9. (G) Cell cycle analysis in ATDC5 cells with or without Chmp5 deletion. n = 3 replicates per group, results repeated twice. All data are mean ± s.d.; 2-way ANOVA followed by multiple comparisons.

Secretory phenotype of Chmp5-deficient skeletal progenitors.

(A) GSEA of RNA-seq data showing positive enrichment of genes associated with the molecular pathway of senescence-associated secretory phenotype (Reactome database) in Chmp5Ctsk relative to Chmp5Ctsk/+ periskeletal progenitors. (B) Confocal images showing and quantifying extracellular vesicles (arrowheads) around Chmp5Ctsk periskeletal progenitors. Images are representative of 30 cells from 3 animals per group. Scale bars, 10 μm. (C and D) Nanoparticle tracking analysis of extracellular vesicles in culture medium of Chmp5Ctsk and wild-type periskeletal progenitors. Data pooled from 3 replicates per group, 5 reads for each; repeated twice using cells from 2 mice per group. (E) Cell number counting determining cell proliferation in ATDC5 cells treated with culture supernatant from Chmp5-deficient or Chmp5-sufficient cells. n = 3 per group per time point, repeated twice. (F and G) Cell number counting and alizarin red staining examining cell proliferation (F) and osteogenesis (G) in neighboring CD45-;CD31-;GFP- progenitors sorted from periskeletal tissues of Chmp5Ctsk;Rosa26mTmG/+ or Ctsk-Cre;Rosa26mTmG/+ mice. n = 3 replicates per group per time point, repeated 3 times using cells from 3 different animals. (H) Volcano plot showing differentially secreted protein in supernatants of Chmp5Ctsk vs. wild-type periskeletal progenitors analyzed by Nano LC-MS/MS. n = 3 with cells from 3 different animals per group. (I) Western blot verifying the increase in COL1A1 and TAGLN proteins in supernatants of Chmp5Ctsk relative to wild-type periskeletal progenitors. Commas blue stain as the loading control. Results repeated 3 times using cells from 3 different mice each group. All data are mean ± s.d.; two-tailed unpaired Student’s t-test for comparison of two groups or 2-way ANOVA followed by multiple comparisons.

Senolytic treatment mitigates musculoskeletal pathologies in Chmp5 conditional knockout mice.

(A) Incucyte live-cell apoptosis analysis of Chmp5Ctsk and wild-type periskeletal progenitors treated with 50 μM Quercetin and 500 nM dasatinib (Q + D) and labeled by Annexin V. n = 6 for each group, experiment repeated twice using cells from different animals. (B) Gross images, radiography, and histology (H&E) respectively demonstrating hindlimb abduction and periskeletal bone overgrowth (arrows or dot-line) in Chmp5Ctsk in comparison with Chmp5fl/fl mice after treatment with Q + D or vehicle PEG400 weekly for 7 weeks. n = 8-10 mice per group. Continuous lines in H&E images indicating an approximate edge of the cortical bone and dot lines indicating the edge of periskeletal overgrowing bones. Scale bars, 100 μm. (C) Four-limb hanging time and forelimb pull strength in Chmp5Dmp1 and Chmp5fl/fl mice after treatment with Q + D or the vehicle PEG400. n = 12 animals per group for hanging time test pooled from both genders; n = 7 male mice per group for the forelimb pull strength test, similar changes found in both genders. (D) Gross images showing hindlimb abduction in Chmp5Dmp1 compared to Chmp5fl/fl mice after treatment with Q + D or the vehicle PEG400. n = 12 animals per group. (E) Micro-CT analysis demonstrating cortical bone thickness in Chmp5Dmp1 mice after treatment with Q + D or the vehicle PEG400. n = 5 male mice per group, similar changes found in both genders. All data are mean ± s.d.; 2-way ANOVA followed by multiple comparisons or two-tailed unpaired Student’s t-test for comparison of two groups.

CHMP5 is essential for endolysosomal functions and maintaining VPS4A protein in skeletal progenitors.

(A) Representative confocal images demonstrating abnormally enlarged vesicles in cultured Chmp5Ctsk relative to wild-type periskeletal progenitors. Abnormal cells identified by containing enlarged GFP+ vesicles. n = 200 cells from 3 mice per group. (B) Schematic showing molecular markers utilized to analyze the endocytic pathway. (C) Representative confocal images showing LAMP1 immunostaining in Chmp5Ctsk and wild-type periskeletal progenitors. n = 20 cells per group. (D) Quantification of LAMP1+ vesicles in ATDC5 cells with or without Chmp5 depletion. n = 93, 66 cells, respectively. (E) Representative confocal images showing RAB7 immunostaining in Chmp5Ctsk and wild-type periskeletal progenitors. n = 15 cells per genotype. (F) Quantification of RAB7+ vesicles in ATDC5 cells with or without Chmp5 depletion. n = 94, 92 cells, respectively. (G) Transmission electron microscopy showing the accumulation of multivesicular body-like structures (arrows) and lysosome-like structures (arrowheads) in Chmp5Ctsk relative to wild-type periskeletal progenitors. n = 30 cells per group. (H) Confocal live cell images demonstrating delayed degradation of the EGF conjugate in Chmp5Ctsk vs. wild-type periskeletal progenitors. n = 10 cells each group per time point. (I) Volcano plot of proteomic analysis showing differentially expressed proteins in Chmp5Ctsk vs. wild-type periskeletal progenitors. n = 3 with cells from 3 different animals. (J) Western blot verifying the decrease in VPS4A protein in Chmp5Ctsk vs. wild-type periskeletal progenitors. The numbers above lanes indicating the intensity of the VPS4A band relative to that of wild-type cells after normalization by β-Actin. Results repeated 3 times with cells from 3 different mice each group. All data are mean ± s.d.; Mann-Whitney test for the comparison of the vesicle number and two-tailed unpaired Student’s t-test for the comparison of the vesicle size in (D) and (F). Scale bars, 10 μm except (G) as indicated.

Mitochondrial dysfunction is responsible for cell senescence in Chmp5-deleted skeletal progenitors.

(A) GSEA of RNA--seq data reporting positive enrichment of genes associated with the molecular pathway of oxidative stress-induced senescence in Chmp5Ctsk relative to Chmp5Ctsk/+ periskeletal progenitors. (B) Confocal fluorescence images showing intracellular ROS (CellROX Deep Red) and mitochondria (TMRE) in Chmp5Ctsk vs. wild-type periskeletal progenitors. Images are representative of 30 cells per group. Scale bars, 15 μm. The graph on the right shows the quantification of the fluorescence intensity of CellROX Deep Red. n = 10 each group, results repeated twice. (C) Quantification of TMRE fluorescence intensity in Chmp5-sufficient and Chmp5-deficient ATDC5 cells. n = 12 for each group, repeated 3 times. (D) Western blotting determining the expression of mitochondrial OXPHOS proteins NDUF88, SDHB, MT-CO1, UQCRC2, and ATF5A in Chmp5-deficient compared to Chmp5-sufficient ATDC5 cells. Experiment repeated twice. (E) Cell number counting to determine cell proliferation in galactose medium. n = 6 replicates per group, repeated three times. (F) Seahorse mitochondrial stress test showing mitochondrial respiratory capacity and extracellular acidification rate in Chmp5Ctsk relative to wild-type periskeletal progenitors. Data pooled from cells of 3 mice, 8 replicates for each sample. (G) Cell number counting and AlamarBlue assay determining cell proliferation in ATDC5 cells with or without Chmp5 depletion after treatment with the antioxidant N-Acetyl cysteine. n = 3 or 12 each group for cell number counting or AlamarBlue assay, respectively; results repeated 3 times. (H) Schematic showing the function of CHMP5 in maintaining VPS4A protein and endolysosomal homeostasis and restricting cell senescence and osteogenesis in skeletal progenitors. All data are mean ± s.d.; two-tailed unpaired Student’s t-test, except the Mann-Whitney test in panel (E).

CHMP5 restricts bone formation in osteogenic lineage cells.

(A) Alizarin red staining of skeletal preparations on postnatal day 2. n = 4 mice per group. Scale bar, 1 mm. (B) Confocal images of Rosa26mTmG/+ (Cre-) control mice. Related to Fig. 1E. Scale bar, 200 μm. (C) Tartrate-resistant acid phosphatase (TRAP) staining demonstrating the absence of osteoclasts in peri-skeletal overgrowth (asterisk) of Chmp5Ctsk mice at 2 weeks of age. TRAP+ osteoclasts in the bone marrow as positive control; dot-line representing approximate border between peri-skeletal overgrowth and the bone at femoral condyle of the knee. Images are representative of 3 animals per group. Scale bar, 200 μm. (D) Gross image and measurement of femur thickness showing bone expansion in Chmp5Dmp1 versus Chmp5fl/fl mice at 10 weeks of age. n = 13 Chmp5fl/fl and 12 Chmp5Dmp1 mice. Scale bar, 1 mm. (E) H&E histology showing bone expansion at the endosteum (arrows) in Chmp5Dmp1 mice. The left images showing the midshaft and the right images showing the metaphysis of the femur bone. Images are representative of 3 mice per group. Scale bars, 0.5 mm. Data are mean ± s.d.; two-tailed unpaired Student’s t-test.

Sorting of CD45-;CD31-;CTSK+ periskeletal progenitors.

(A) Unstained cells from Rosa26-mTmGfl/+ mice as a negative control for sorting CD45-;CD31-;CTSK+ skeletal progenitors. Related to Fig. 2A. (B) Quantification of CD45-;CD31-;GFP+ and CD45-;CD31-;GFP- cell population in samples of Chmp5Ctsk;Rosa26mTmG/+ mice. n = 10 mice per group.

Cell apoptosis is not the main cause of the decreased proliferative rate in Chmp5-deficient skeletal progenitors.

(A) Annexin V stain examining cellular apoptosis in Chmp5Ctsk and Chmp5Ctsk/+ periskeletal progenitors. n = 3 replicates each group; repeated twice using cells from 2 mice per group. (B) TUNEL staining and quantification of TUNEL+ cells (%) in the periskeletal tissues of Chmp5Ctsk and Chmp5Ctsk/+ mice. Arrows indicating positive cells in periskeletal overgrowth in Chmp5Ctsk mice, dot-line representing approximate border between periskeletal overgrowth and bone in the femoral condyle of the knee; n = 3 animals per group; scale bars, 100 μm. (C) Cell number counting determining cell proliferation in ATDC5 cells after deleting Chmp5 by lentiviral CRISPR/CAS9. n = 4 replicates per group per time point, experiment repeated three times. (D) Annexin V-PI stain analyzing cellular apoptosis in ATDC5 cells with or without Chmp5 deletion. n = 3 replicates for each group, repeated 3 times. (E) Cell number counting determining cell proliferation in ATDC5 cells with or without Chmp5 deletion after treatment with the pan-caspase inhibitor Q-VD-OPh. n = 3 replicates of each group each dose, repeated twice. All data are mean ± s.d.; two-tailed Student’s t-test for comparison of two groups or 2-way ANOVA followed by multiple comparisons.

Secretory phenotype of Chmp5-deficient skeletal progenitors.

(A) Nanoparticle tracking analysis showing the size distribution of extracellular vesicles in Chmp5Ctsk and wild-type skeletal progenitors. Data pooled from 3 replicates, 5 reads for each; repeated twice using cells from 2 mice; D10, D50, and D90 indicating percent undersize, for example D50 = 229 nm representing that 50% of vesicles are 229 nm or smaller. (B) AlarmaBlue assay examining cell proliferation in neighboring CD45-;CD31-;GFP- progenitors sorted from periskeletal tissues of Chmp5Ctsk;Rosa26mTmG/+ or Ctsk-Cre;Rosa26mTmG/+ mice. n = 12 replicates per group per time-point, repeated 3 times using cells from 3 animals. (C) Alizarin red staining determining osteogenesis in neighboring CD45-;CD31-;GFP- progenitors sorted from periskeletal tissues of Chmp5Ctsk;Rosa26mTmG/+ or Ctsk-Cre;Rosa26mTmG/+ mice after induction in osteogenic medium for 3 weeks. n = 3 replicates per group per time point, repeated 3 times using cells from 3 animals. (D) Immunostaining and quantification of cell proliferation marker Ki-67 in the periskeletal tissues around the knee of Chmp5Ctsk and Chmp5Ctsk/+ mice. n = 3 animals per group; dot-line representing the approximate border between the periskeletal overgrowth and the bone in the femoral condyle; scale bars, 100 μm. (E) Heatmap of LC-MS/MS results showing differentially secreted proteins in supernatants of Chmp5Ctsk relative to wild-type skeletal progenitors. n = 3 with cells from 3 different animals per group for Nano LC-MS/MS analysis. All data are mean ± s.d., 2-way ANOVA followed by multiple comparisons or two-tailed unpaired Student’s t-test for 2-group comparison.

Additional x-ray images of Chmp5Ctsk mice after treatment with Q + D or the vehicle PEG400 for 7 weeks.

Arrows indicating periskeletal bone overgrowths.

CHMP5 is essential for endolysosomal functions and maintaining VPS4A protein in skeletal progenitors.

(A) Representative confocal fluorescence images and quantification of the fluorescence intensity of LysoTracker Red DND-99 in Chmp5Ctsk compared to wild-type periskeletal progenitors. n = 11 replicates per group for quantitative analysis; repeated 3 times using cells from 3 mice. Scale bars, 50 μm. (B) Isotype controls for immunofluorescence staining. Scale bars, 20 μm. (C) Co-localization of LAMP1 and RAB7 in Chmp5-sufficient or Chmp5-deficient ATDC5 cells. Scale bars, 10 μm. (D) Colocalization analysis of LAMP1 and RAB7 performed by the ImageJ Coloc2 programme and Pearson’s R value (above threshold) shown, n = 20 cells per group. (E) Representative confocal images of immunostaining for early endosome marker EEA1, cycling endosome marker RAB11, and trans-Golgi network marker TGN38 in Chmp5Ctsk vs. wild-type periskeletal progenitors. n = 30 cells per group. Scale bars, 10 μm (EEA1), 20 μm (RAB11), 25 μm (TGN38). (F) RNA-seq data showing Vps4a mRNA expression in Chmp5Ctsk vs. Chmp5Ctsk/+ periskeletal progenitors. n = 3 with cells from 3 different animals for each group. (G) Western blot showing that CHMP5 and VPS4A protein expression after transfection of CHMP5 or empty vector in HEK-293T cells. Experiments repeated 3 times with independent cells and transfections at different times. (H) Quantitative PCR showing the expression of CHMP5 and VPS4A mRNA after transfecting CHMP5 or empty vector in HEK-293T cells. n = 3 replicates for each group, experiments repeated 3 times with independent cells and transfections at different times. All data shown as mean ± s.d.; two-tailed unpaired Student’s t-test.

Mitochondrial dysfunction and activation of DNA damage-induced senescence in Chmp5-deficient skeletal progenitors.

(A) Representative TMRE confocal images showing accumulation of mitochondria in Chmp5Ctsk periskeletal progenitors. n = 30 cells per group, scale bars, 25 μm. (B) Confocal fluorescence imaging mapping intracellular ROS (CellROX Deep Red) and mitochondria (MitoTracker Green) in ATDC5 cells with or without Chmp5 depletion. n = 20 cells for each group; scale bars, 10 μm. The right histogram showing the quantification of CellROX Deep Red in ATDC5 cells by flow cytometry. n = 3 per group, experiment repeated twice. (C) GSEA of RNA-seq data showing positive enrichment of genes associated with the molecular pathway of DNA damage-induced senescence in Chmp5Ctsk relative to Chmp5Ctsk/+ periskeletal progenitors. (D) Quantitative PCR determining expression of H2afx and Trp53 genes in Chmp5Ctsk relative to Chmp5Ctsk/+ periskeletal progenitors. n = 6 with cells from different animals per group. Data shown as mean ± s.d.; two-tailed unpaired Student’s t-test.