The ESCRT protein CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence

Reviewer #1 (Public review):

Summary:

The manuscript presents a significant and rigorous investigation into the role of CHMP5 in regulating bone formation and cellular senescence. The study provides compelling evidence that CHMP5 is essential for maintaining endolysosomal function and controlling mitochondrial ROS levels, thereby preventing the senescence of skeletal progenitor cells.

Strengths:

The authors demonstrate that the deletion of Chmp5 results in endolysosomal dysfunction, elevated mitochondrial ROS, and ultimately enhanced bone formation through both autonomous and paracrine mechanisms. The innovative use of senolytic drugs to ameliorate musculoskeletal abnormalities in Chmp5-deficient mice is a novel and critical finding, suggesting potential therapeutic strategies for musculoskeletal disorders linked to endolysosomal dysfunction.

Weaknesses:

The manuscript requires a deeper discussion or exploration of CHMP5's roles and a more refined analysis of senolytic drug specificity and effects. This would greatly enhance the comprehensiveness and clarity of the manuscript.

Reviewer #2 (Public review):

Summary:

The authors try to show the importance of CHMP5 for skeletal development.

Strengths:

The findings of this manuscript are interesting. The mouse phenotypes are well done and are of interest to a broader (bone) field.

Weaknesses:

The mechanistic insights are mediocre, and the cellular senescence aspect poor.

In total, it has not been shown that there are actual senescent cells that are reduced after D+Q-treatment. These statements need to be scaled back substantially.

Reviewer #3 (Public review):

Summary:

In this study, Zhang et al. reported that CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence. The effects of CHMP5 on osteoclastic bone resorption and bone turnover have been reported previously (PMID: 26195726), in which study the aberrant bone phenotype was observed in the CHMP5-ctsk-CKO mouse model, using the same mouse model, Zhang et al., report a novel role of CHMP5 on osteogenesis through affecting cell senescence. Overall, it is an interesting study and provides new insights in the field of cell senescence and bone.

Strengths:

Analyzed the bone phenotype OF CHMP5-periskeletal progenitor-CKO mouse model and found the novel role of senescent cells on osteogenesis and migration.

Weaknesses:

(1) There are a lot of papers that have reported that senescence impairs osteogenesis of skeletal stem cells. In this study, the author claimed that Chmp5 deficiency induces skeletal progennitor cell senescence and enhanced osteogenesis. Can the authors explain the controversial results?

(2) Co-culture of Chmp5-KO periskeletal progenitors with WT ones should be conducted to detect the migration and osteogenesis of WT cells in response to Chmp5-KO-induced senescent cells. In addition, the co-culture of WT periskeletal progenitors with senescent cells induced by H2O2, radiation, or from aged mice would provide more information.

(3) Many EVs were secreted from Chmp5-deleted periskeletal progenitors, compared to the rarely detected EVs around WT cells. Since EVs of BMSCs or osteoprogenitors show strong effects of promoting osteogenesis, did the EVs contribute to the enhanced osteogenesis induced by Chmp5-defeciency?

(4) EVs secreted from senescent cells propagate senescence and impair osteogenesis, why do EVs secreted from senescent cells induced by Chmp5-defeciency have opposite effects on osteogenesis?

(5) The Chmp5-ctsk mice show accelerated aging-related phenotypes, such as hair loss and joint stiffness. Did Ctsk also label cells in hair follicles or joint tissue?

(6) Fifteen proteins were found to increase and five proteins to decrease in the cell supernatant of Chmp5Ctsk periskeletal progenitors. How about SASP factors in the secretory profile?

(7) D+Q treatment mitigates musculoskeletal pathologies in Chmp5 conditional knockout mice. In the previously published paper (CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts), inhibition of osteoclastic bone resorption rescues the aberrant bone phenotype of the Chmp5 conditional knockout mice. Whether the effects of D+Q on bone overgrowth is because of the inhibition of bone resorption?

(8) The role of VPS4A in cell senescence should be measured to support the conclusion that CHMP5 regulates osteogenesis by affecting cell senescence.

(9) Cell senescence with markers, such as p21 and H2AX, co-stained with GFP should be performed in the mouse models to indicate the effects of Chmp5 on cell senescence in vivo.

(10) ADTC5 cell as osteochondromas cells line, is not a good cell model of periskeletal progenitors. Maybe primary periskeletal progenitor cell is a better choice.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript presents a significant and rigorous investigation into the role of CHMP5 in regulating bone formation and cellular senescence. The study provides compelling evidence that CHMP5 is essential for maintaining endolysosomal function and controlling mitochondrial ROS levels, thereby preventing the senescence of skeletal progenitor cells.

Strengths:

The authors demonstrate that the deletion of Chmp5 results in endolysosomal dysfunction, elevated mitochondrial ROS, and ultimately enhanced bone formation through both autonomous and paracrine mechanisms. The innovative use of senolytic drugs to ameliorate musculoskeletal abnormalities in Chmp5-deficient mice is a novel and critical finding, suggesting potential therapeutic strategies for musculoskeletal disorders linked to endolysosomal dysfunction.

Weaknesses:

The manuscript requires a deeper discussion or exploration of CHMP5's roles and a more refined analysis of senolytic drug specificity and effects. This would greatly enhance the comprehensiveness and clarity of the manuscript.

We thank the reviewer for these insightful comments. The tissue-specific roles of CHMP5 and the specificity of quercetin and dasatinib treatments in Chmp5-deficient mice will be further discussed and clarified in the revised manuscript.

Reviewer #2 (Public review):

Summary:

The authors try to show the importance of CHMP5 for skeletal development.

Strengths:

The findings of this manuscript are interesting. The mouse phenotypes are well done and are of interest to a broader (bone) field.

Weaknesses:

The mechanistic insights are mediocre, and the cellular senescence aspect poor.

In total, it has not been shown that there are actual senescent cells that are reduced after D+Q-treatment. These statements need to be scaled back substantially.

We thank the reviewer for these suggestive comments. Although multiple hallmarks of cell senescence were shown in CHMP5-deficient skeletal progenitors, we will detect and add additional markers of cell senescence in the revised manuscript.

In addition, the effects and specificity of the Q+D treatment will be further discussed and clarified with the revision.

Reviewer #3 (Public review):

Summary:

In this study, Zhang et al. reported that CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence. The effects of CHMP5 on osteoclastic bone resorption and bone turnover have been reported previously (PMID: 26195726), in which study the aberrant bone phenotype was observed in the CHMP5ctsk-CKO mouse model, using the same mouse model, Zhang et al., report a novel role of CHMP5 on osteogenesis through affecting cell senescence. Overall, it is an interesting study and provides new insights in the field of cell senescence and bone.

Strengths:

Analyzed the bone phenotype OF CHMP5-periskeletal progenitor-CKO mouse model and found the novel role of senescent cells on osteogenesis and migration.

Weaknesses:

(1) There are a lot of papers that have reported that senescence impairs osteogenesis of skeletal stem cells. In this study, the author claimed that Chmp5 deficiency induces skeletal progennitor cell senescence and enhanced osteogenesis. Can the authors explain the controversial results?

Different skeletal stem cell populations in time and space have been identified and reported. This study shows that Chmp5 deficiency in periskeletal and endosteal skeletal progenitors causes cell senescence and aberrant bone formation. Although cell senescence during aging can impair osteogenesis of certain skeletal stem cells, which contributes to diseases with low bone mass such as osteoporosis, aging can also increase heterotopic mineralization/calcification in musculoskeletal soft tissues such as ligaments and tendons, which is consistent with our results in this study. These reflect out-of-order musculoskeletal mineralization during aging. We will expand the discussion and clarify the results of CHMP5-regulated cell senescence in osteogenesis in the revised manuscript.

(2) Co-culture of Chmp5-KO periskeletal progenitors with WT ones should be conducted to detect the migration and osteogenesis of WT cells in response to Chmp5-KO-induced senescent cells. In addition, the co-culture of WT periskeletal progenitors with senescent cells induced by H2O2, radiation, or from aged mice would provide more information.

Increased osteogenesis of WT skeletal progenitors in the periskeletal lesion was shown to be a paracrine mechanism of abnormal bone formation in Chmp5Ctsk mice. The coculture experiment will help confirm the effect of Chmp5-deficient skeletal progenitors on the osteogenesis of neighboring WT skeletal progenitors.

Notably, the cause and outcome of cell senescence are highly heterogeneous, and different causes of cell senescence can cause significantly different outcomes. Although the coculture of WT periskeletal progenitors with senescent cells induced by H2O2, radiation, or from aged mice would be very interesting, these are beyond the scope of the current study.

(3) Many EVs were secreted from Chmp5-deleted periskeletal progenitors, compared to the rarely detected EVs around WT cells. Since EVs of BMSCs or osteoprogenitors show strong effects of promoting osteogenesis, did the EVs contribute to the enhanced osteogenesis induced by Chmp5-defeciency?

The WT skeletal progenitor cells from Chmp5Ctsk mice have an increased capacity of osteogenesis compared to the corresponding cells from control animals, suggesting that the EVs of the Chmp5-deleted periskeletal progenitors could promote osteogenesis of the WT skeletal progenitors, which represents a paracrine mechanism of abnormal bone formation in Chmp5 deficient animals. We will discuss and clarify these results in the revised manuscript.

(4) EVs secreted from senescent cells propagate senescence and impair osteogenesis, why do EVs secreted from senescent cells induced by Chmp5-defeciency have opposite effects on osteogenesis?

The question is similar to comment #1. The functional heterogeneity of cellular senescence will be discussed in further detail and clarified in the revised manuscript.

(5) The Chmp5-ctsk mice show accelerated aging-related phenotypes, such as hair loss and joint stiffness. Did Ctsk also label cells in hair follicles or joint tissue?

This is an interesting question. Although we did not check the expression of CHMP5 in hair follicles, which is outside the scope of the present study, the result in Fig. 1E showed the expression of CHMP5 in joint ligaments. Notably, abnormal periskeletal bone formation occurs predominantly at the joint ligament insertion site in Chmp5Ctsk mice, which will be elucidated and discussed in the revised manuscript.

(6) Fifteen proteins were found to increase and five proteins to decrease in the cell supernatant of Chmp5Ctsk periskeletal progenitors. How about SASP factors in the secretory profile?

As mentioned above, the SASP phenotype and related factors of senescent cells could be highly heterogeneous depending on inducers, cell types, and timing of senescence. Most of the proteins we identified in the secretome analysis have previously been reported in the secretory profile of osteoblasts. Although we were also interested in the change of some common SASP factors, such as inflammatory cytokines, the experiment did not detect these factors because of their small molecular weights and the technical limitations of mass spec analysis.

(7) D+Q treatment mitigates musculoskeletal pathologies in Chmp5 conditional knockout mice. In the previously published paper (CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts), inhibition of osteoclastic bone resorption rescues the aberrant bone phenotype of the Chmp5 conditional knockout mice. Whether the effects of D+Q on bone overgrowth is because of the inhibition of bone resorption?

Although in Chmp5Ctsk mice we cannot exclude the effect of D+Q on osteoclasts, the effect of D+Q on osteoblast lineage cells, which is the focus of the current study, was verified in Chmp5Dmp1 mice. We will expand the discussion and make these results clearer with the revision.

(8) The role of VPS4A in cell senescence should be measured to support the conclusion that CHMP5 regulates osteogenesis by affecting cell senescence.

We agree that additional experiments examining the role of VPS4A in cell senescence will provide more mechanistic insights. The focus of the current study is to report that CHMP5 restricts abnormal bone formation by preventing endolysosome-mitochondrion-mediated cell senescence. The roles of VPS4A in cell senescence and skeletal biology will be explored in separate studies.

(9) Cell senescence with markers, such as p21 and H2AX, co-stained with GFP should be performed in the mouse models to indicate the effects of Chmp5 on cell senescence in vivo.

We will examine additional markers of cell senescence, as the reviewers suggest, in the revised manuscript.

(10) ADTC5 cell as osteochondromas cells line, is not a good cell model of periskeletal progenitors. Maybe primary periskeletal progenitor cell is a better choice.

We were aware that ATDC5 cells are typically used as a chondrocyte progenitor cell line. However, our previous study showed that ATDC5 cells could also be used as a reasonable cell model for periskeletal progenitors. Furthermore, the corresponding results from primary periskeletal progenitors were shown. We will further clarify this in the revision.

In general, the comments of these reviewers will help clarify our results and further strengthen our conclusion. We will address these comments and questions point to point in more detail in the revised manuscript.