Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration after peripheral nerve injury
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Abstract
After peripheral nerve injuries, successful axonal growth and functional recovery requires the reprogramming of Schwann cells into a reparative phenotype, a process dependent on the activation of the transcription factor c-Jun. Nevertheless, axonal regeneration is greatly impaired in aged organisms or after chronic denervation leading to important clinical problems. This regenerative failure has been associated to a diminished c-Jun expression by Schwann cells, but whether the inability of these cells to maintain a repair state is associated to the transition into a phenotype inhibitory for axonal growth, has not been evaluated so far. We find that repair Schwann cells transitions into a senescent phenotype, characterized by diminished c-Jun expression and secretion of factor inhibitory for axonal regeneration in both aging and chronic denervation. In both conditions, elimination of senescent Schwann cells by systemic senolytic drug treatment or genetic targeting improves nerve regeneration and functional recovery in aging and chronic denervation, associated with an upregulation of c-Jun expression and a decrease in nerve inflammation. This work provides the first characterization of senescent Schwann cells and their impact over axonal regeneration in aging and chronic denervation, opening new avenues for enhancing regeneration, and functional recovery after peripheral nerve injuries.
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Reply to the reviewers
Manuscript number: RC-2022-01784
Corresponding author(s): Felipe, Court
1. General Statements [optional]
We submit a revision plan for our manuscript *“Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration after peripheral nerve injury” *by Fuentes-Flores et al. from the groups of Felipe Court, Judith Campisi, Jose Gomez, and Ahmet Hoke.
One of the greatest challenges in the field of peripheral nerve regeneration is the decrease in the nerve regenerative capacity in aged patients or after delayed repair, a condition also known as chronic denervation. For the last two decades, several research groups have focused on …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Manuscript number: RC-2022-01784
Corresponding author(s): Felipe, Court
1. General Statements [optional]
We submit a revision plan for our manuscript *“Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration after peripheral nerve injury” *by Fuentes-Flores et al. from the groups of Felipe Court, Judith Campisi, Jose Gomez, and Ahmet Hoke.
One of the greatest challenges in the field of peripheral nerve regeneration is the decrease in the nerve regenerative capacity in aged patients or after delayed repair, a condition also known as chronic denervation. For the last two decades, several research groups have focused on understanding this phenomenon, but the main drivers of unsuccessful regeneration and poor functional recovery have been elusive, remaining an important clinical problem.
In the work described in this manuscript we found an unexpected property of Schwann cells in the denervated nerves. Aged and chronically denervated Schwann cells are not just passive participants in the impaired regeneration process, but they actively inhibit the regeneration of peripheral axons. Using a combination of morphological, behavioral and molecular techniques in a collaborative multi-lab approach we demonstrate for the first time that senescent Schwann cells accumulate in aged or chronically denervated peripheral nerves modifying the nerve environment, increasing proinflammatory and regeneration-inhibitory factors. Elimination of senescent Schwann cells using a systemic intervention with senolytic or genetically targeting p16-positive senescent cells, greatly improve axonal regeneration in both chronic denervation and aging conditions. Importantly, the enhanced axonal regeneration observed after senescent cell elimination is accompanied by improved functional recovery after chronic denervation. Chronic denervation and aging are the main clinical problems associated to peripheral nerve injuries. Our approach, using FDA approved drugs currently in clinicals trials for its application as senotherapeutics, effectively broadens the spectrum of its clinical use and effectiveness.
We foresee this work will be of interest to a wide audience, including experts in nerve regeneration, senescent cells, aging and those studying the effect of chronic insults in regenerative medicine.
We have now received the comments from two reviewers and we are prepared to experimentally approach the issues raised. We thank their criticism and suggestions, as well as their very enthusiastic comments. We are extremely pleased as both reviewers recognized the important implication of this work, from reviewer 1:
“The findings reported in this manuscript are very interesting and will move the field of nerve repair forward. This paper will be of interest for basic science audience in the fields of aging and neurobiology and has also potential interest to the broader clinical and translational fields. Indeed, this paper provides data that Schwann cells entering a senescent stage not only fail to support axon regeneration in aged animals, but actively inhibit axon regeneration…. Furthermore, the use of an FDA approved drug, currently in clinicals trials for its application as senotherapeutics, to increase axon regeneration in aged and chronic denervation conditions will provide new avenues for clinical applications”.
Which is backed up by reviewer 2:
“Overall, this is an interesting study that undertake fundamental question in the field of nerve physiopathology and also could open a good opportunity in developing therapeutic strategies for translational research”.
We understand the reviewers have raised issues associated with the manuscript format and we are prepared to profoundly edit the manuscript as suggested. In addition, after discussion the experimental issues raised by the reviewers, we are prepared to perform all the experiments and controls suggested (some of them are currently underway), including new animal experiments and in vitro work. This information is detailed in the point-by point revision plan below.
Thank you in advance for the consideration and we look forward to hearing from you in due course. Please do not hesitate to contact me if you want to discuss anything associated to the manuscript and the revision plan.
2. Description of the planned revisions
Point-by point Reponses and revision plan in blue
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary This manuscript by Fuentes-Flores et al reports that elimination of senescent Schwann cells by systemic senolytic drug treatment or genetic targeting improves nerve regeneration and functional recovery in aging and chronic denervation. This improved regeneration is associated with an upregulation of c-Jun expression. Mechanistically the authors provide data to show that senescent Schwann cells secrete factors that are inhibitory to axon regeneration. These findings are very interesting and move the field forward beyond the notion that Schwann cells fail to support axon regeneration in aged animals and identify potential targets to enhance nerve repair. The use of a senolytic drug to increase regeneration in aged and chronic denervation conditions provides new avenues for clinical interventions. However, some of the claims are overstated since this is not the first characterization of senescent Schwann cells and the manipulations used in the study are not entirely specific for Schwann cells. The manuscript is also poorly written and difficult to follow, given the complex set of surgeries and terminology, and lack of explanation of the rationale for the surgery model used. Figures are poorly labeled and difficult to follow without figure legends, and figure legends do not match the figures.
We thank the reviewer for the positive comments, we also acknowledge the problems detected in the manuscript format, including the lack of a clear explanation of the complex procedures used. We are prepared to work carefully on the format, including clear explanations of the procedures and new schemes to complement the text. As detailed below we are also prepared to perform all the experimental work proposed by the reviewer, which will strengthen the conclusion of this manuscript.
Below are suggestions for improvements:
Major comments:
In the axon regeneration assays, how is the reconnection site defined in longitudinal images stained for SCG10? Of particular concern is that Figure 1B "adult chronic dmg" nerve section image appears to be identical to the image in Figure 5A "Vehicle adult (47dpi)". However, the reconnection site is located at different sites along the nerve. Also, the scale bar appears identical but the legend states different sizes. In Figure 1 chronic damage is 42dpi, and figure 5 is 47dpi, yet with what appears as the same image.
Revisions incorporated in the transferred manuscript, see section 3, below.
The authors need to provide a rationale for the choice of this complex injury model and what are the advantages over other models. Please clearly describe the timepoints for each experiment and why the time points were chosen for analysis. Provide the scheme of injury in the main Figures to ease comprehension. A scheme is provided in what appears to be Figure S2, but the legend of Figure S2 does not match. Please compare same time points between aged and adults. Days post injury is sometimes referred to 7 or 42, and it is difficult to follow if it is days post initial transection of the tibial nerve or days post reconnection of transected tibial to peroneal. Revise all Figure legends and supplementary Figure legends to match figures.
We thank the reviewer for this comment. In a revised manuscript we will provide a clear explanation for the injury model used, as well as references, including one from the group of Tessa Gordon that describe for the first time this model in rats (PMID: 30215557), and the one applying this model to the mouse from the groups of Rhona Mirsky and Kristjan Jessen (PMID: 33475496). Briefly, this model has two advantages: first, it allows to generate chronic denervation for months and then be able to connect the distal denervated stump with a proximal one without the need of a nerve bridge; And second, neurons in the different groups that have different denervation times (1 week versus 6 weeks) are all damaged at the same time, eliminating variability associated to chronic axonal damage. We will include this information in the results section of a revised manuscript along with the above references.
We will include schemes of the injuries performed in each experiment in each figure, also adding a timeline. This is an excellent suggestion to clearly understand the different procedures performed. We will also check all figures and legends, including correcting the problem detected by the reviewer (legends of Figures 2 and 5 were swapped). We understand the problem of referring to days post-injury, then we will introduce a new form of referring to the initial transection and the experiment, which include reconnection. Adding schemes per figure will also help to understand the different timelines used in different experiments, including the ones using the senolytics.
As detailed above, we will perform a very careful revision of the text and legends for consistency.
The authors' main conclusion is that Senescent Schwann cells inhibit axon regeneration. The authors need to tune down this statement and acknowledge that their manipulations are not entirely Schwann cells specific. While the data nicely shows a contribution of senescent Schwann cells, it does not sufficiently acknowledge the possibility that other senescent cells in the nerve contribute to this effect. First, the authors refer in the discussion that 60% of the senescent cells are SOX10 negative, and thus represent other cells beyond Schwann cells. This quantification needs to be shown in Figure 1. Second, the genetic and pharmacology manipulation eliminate all senescent cells, including Schwann cells. Third, while the culture experiment may be Schwann cells specific, the authors need to provide detailed information on how they purify these cells, how they induce repair Schwann cells (rSC) as claimed in Figure 3, and demonstrate whether these are pure Schwann cells. This is important because other cells in the nerve contribute to nerve repair, including mesenchymal cells. Finally, the claim that using Mpz-cre will lead to c-jun overexpression only in SC also needs to be demonstrated, since Mpz is also expressed in satellite glial cells in the DRG.
We thank the reviewer for these comments and suggestions. We will tone down the statement that senescence Schwann cells are the only cell candidates for modulating regeneration. We discussed this in the original manuscript, but we agree we need to review this statement, including new data detailed below.
We will include the data requested by the reviewer (60% SOX-10 negative senescent cells) in a new graph in Figure 1. Also, we are currently performing new experiments and quantifications using specific markers for macrophages, epithelial cells, and fibroblasts, to identify the cell identity of the 40% SOX-10 negative senescent cells in aging and chronic denervation.
Regarding in vitro experiments, we will provide detailed methods for Schwann cell purification, and induction of rSC phenotype. Related to the purity of these cultures, in past experiments we have obtained numbers ranging from 95-98% of Schwann cell purity; we will repeat these experiments and quantification for this manuscript and include this data in the method section of a revised text.
Regarding c-jun overexpression in the Mpz-cre, we agree with the reviewer that there is probably overexpression in satellite glial cells in the DRG. Satellite glial cells (SGCs) are a subset of cells in the Schwann cell (SC) lineage that express several early myelination markers, such as Mbp, Mag, and Plp, and the transcription factor Sox10. SGCs express early SC markers, such as CDH19, and are transcriptionally and morphologically similar to SCs, even in the absence of axonal contact. Regarding the possibility that SGCs are contributing to the enhanced regeneration presented by mice with c-jun overexpression, this issue was somehow approached previously by Wagstaff et al. (PMID: 33475496), as they showed that in this mouse strain, increased axonal regeneration was equally observed in sensory neurons, in contact with SGCs, and motor neurons, which are not associated to SGCs. This observation suggests that the effect is associated with c-jun overexpressing Schwann cells in the distal stump. In addition, in our work, the changes in senescent cells observed in the c-jun overexpressed mouse, were associated to the distal nerve stump, which was mechanically separated from the proximal region. We agree it is important to include this discussion, and we will do so in the revised manuscript.
In Figure 5, c-jun is shown after denervation (42 dpi). The results describe 28 days of denervation, 5 days of GCV and 7 days post reconnection, which makes 40 days. If that is not the case, results need to better explain timeline of this procedure. Also, what is the basal c-jun expression in p16-3MR mice? In addition to the number of c-jun positive cells shown in Figure 5G, the authors need to quantify the percent of c-jun puncta that co-localize with Sox10. The size of the c-jun puncta appears different in size in vehicle and GCV, is that an expected phenotype?
As expressed above, we will include schemes and timelines for all the surgical experiments in a revised manuscript, including detailed information for the experiments using the p16-3MR mice.
Regarding c-jun expression in the p16-3MR mice, we are currently performing the suggested control experiment which is important to draw conclusions of this research. We will use immunofluorescence, but also include western blots as an extra analytical method in this and other experiments. All this information will be included in a revised manuscript.
The observation of the apparent difference c-jun puncta is intriguing. Is not an expected phenotype, but it will important to check if there is a quantifiable change in the pattern of expression. We will quantify this in the different groups and include the results in a revised Figure 5.
Minor comments
Improve labeling of Figures or at the very least describe in the Figure legend. For example: Figure 5B-C, which of the graphs is from adult mice and which is from aged mice?
We are sorry about the lack of clear labeling in the figures; we will carefully review all figures in the manuscript and their corresponding legends, adding better labeling. Labelling of Figure 5B-C has been corrected.
The authors need to carefully describe where the high magnification images were taken in the injured nerve and keep the comparison at same site between groups. Please check the scale bar for each image. For example, the images in Figure 1D/F/I/K used same scale, but the cell size and cell morphology are different. The images for split individual channels need to match the merge channel images. For example, the individual channel and merge images are not properly aligned in Figure 4C, ABY-263 group.
As suggested by the reviewer, we will show the regions in which high magnification were taken. All quantifications were performed in comparable sites, but we will include information in a revised manuscript to clearly describe the methodology used. We will check all scale bars in a revised manuscript.
For the comment on alignment problems we have incorporated this in the transferred manuscript, see section 3 below.
The analysis method used to quantify axon regeneration should be consistent throughout. For example, in Figure 1C, number of axons/nerve width(um) was used for regeneration assay, but axon density (width corrected) was used in Figure 5B-C in regeneration assay.
Revisions incorporated in the transferred manuscript, see section 3, below.
**Referees cross-commenting**
I agree with all Referee #2's comments. Both sets of comments are important, complementary and point to the same major concerns that need to be addressed. Agree as well that both reviewer think this is an interesting and relevant study for the field of nerve repair, if revised appropriately.
Reviewer #1 (Significance (Required)):
__Significance____ __The findings reported in this manuscript are very interesting and will move the field of nerve repair forward. This paper will be of interest for basic science audience in the fields of aging and neurobiology and has also potential interest to the broader clinical and translational fields. Indeed, this paper provides data that Schwann cells entering a senescent stage not only fail to support axon regeneration in aged animals, but actively inhibit axon regeneration. While this reviewer raises questions on whether only senescent Schwann cells or other senescent cells in the nerve contribute to this effect, the identification of potential targets to enhance nerve repair is highly significant. Furthermore, the use of an FDA approved drug, currently in clinicals trials for its application as senotherapeutics, to increase axon regeneration in aged and chronic dennervation conditions will provide new avenues for clinical applications.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary The reported study by Fuentes-Flores et al. shows that Schwann cells (SCs) in peripheral nerves undergo senescence with aging or in chronic denervation. This senescent SC phenotype correlates with downregulation of c-Jun expression and axon regeneration capacity and consequently, affecting functional recovery. The study has been undertaken by using in vivo mice model of chronically denervated sciatic nerve and in vitro of rat-primary cell cultures (Schwann cell, DRG-explants, and their coculture). Schwann cell exosome manipulation was also included to exploit their released factor as media for cell cultures.
Major comments:
- __ __ Chronic denervated nerve model and Schwann cell phenotype
*Tibial nerve transection and chronic denervated nerve: As the referee have no information about this model (no reference is cited), a detailed description should be provided highlighting the interest of such model compared to standard sciatic nerve lesion model.
We thank the reviewer for this comment. We will provide a clear explanation for the injury model used, as well as references, including one from the group of Tessa Gordon that describe for the first time this model in rats (PMID: 30215557), and the one applying this model to the mouse from the groups of Rhona Mirsky and Kristjan Jessen (PMID: 33475496). Briefly, this model has two advantages: first, it allows to generate a chronic denervation for months and then be able to connect the distal denervated stump with a proximal one without the need of a nerve bridge; second, neurons in the different groups that have different denervation times (1 week versus 6 weeks) are all damaged at the same time, eliminating variability associated to chronic axonal damage. We will include this information in the result section of a revised manuscript along with the above references.
*Should be shown, histological analysis of denervated tibial branch prior reconnection with freshly cut proximal peroneal branch with specific immunostainings of rSCs v.s. sSC associated with dapi nuclear staining (as used along this study). Specific staining for other cells should be also provided (i.e., macrophage and endothelial cells). In simple words, "how chronically denervated nerve looks like and what is his cellular content? This is necessary to responds to the following main referee question: does the increase/decrease of rSCs or sSC under specific condition all through the study concerns the SCs that have migrated from freshly cut peroneal branch into denervated tibial distal branch, or resident SCs that have survived in chronically denervated tibial distal branch. In other words, whether rSCs that migrate (and accompanying regenerating axons) into chronically denervated branch nerve undergo phenotype change into sSC because of the environment of chronically denervated nerve. This is not clearly described or discussed, and remain confusing for reader.
We are sorry about the lack of clarity in the text and figures. The immunostaining analysis of denervated tibial branch prior reconnection is included in the original manuscript, specifically in Figure 1D-K, and Figure 4. In a revised manuscript we will include schemes in the Figures to shown the regions analyzed in each case.
We thank for the suggestion of including staining for other cell types. As suggested by the reviewer we are currently performing these experiments and analysis for macrophages, endothelial cells and fibroblasts, together with staining for cell senescence (p16), in both aged and chronically denervated conditions. We will include this data in a revised manuscript
About the specific question of the reviewer: “*does the increase/decrease of rSCs or sSC under specific condition all through the study concerns the SCs that have migrated from freshly cut peroneal branch into denervated tibial distal branch, or resident SCs that have survived in chronically denervated tibial distal branch”. *Our data demonstrate that senescence Schwann cells appear in the distal nerve stump in aged mice and after chronic denervation. The distal stump is physically disconnected from the proximal part of the nerve. Therefore, after reconnection the regenerating axons encounters a tissue which is already populated with senescent cells. To clearly explain this, we will add extra text in the results and discussion section to clarify these findings.
Support of up- and down-regulation of gene expression illustrated in fig.4
The conclusions and statements on up- or down-regulation of c-jun, yH2AX, beta-gal, P16, arise from quantitative and qualitative analysis from immunostaining of these specific markers by determining the number of positive cells rSCs vs. sSC. For these strong statements appropriate methods for quantification of protein levels such as western blots are required. For example, the statement of down regulation of c-jun expression, the quantitative graph shows strong increase in c-jun cell number under ABT263 treatment but the histological photo does not illustrate such decrease in number of the cell. It shows rather an increase in brightness of c-jun staining. Thus, only appropriate method for protein level quantification could be conclusive. This would also remove the doubt that some photos are under- or over- exposed as it appears in the figure. For example, in aged animal under vehicle condition, there is no variability in staining intensity. Accordingly, one question to the authors: for quantification of cell number, are weakly stained cells considered as positive cell?
We agree with the reviewer that a more quantitative method is required to complement the immunofluorescence data. As the reviewer correctly states, quantification of the immunofluorescence data corresponds to cell positive for the specific marker, expressed as % of cell positive for that maker. Therefore, we will perform western blot for c-jun, yH2AX, and p16 for the different models, including treatments with senolytics.
Regarding the method for quantification, we performed all these quantifications using Imaris software, in which we set up the same threshold for all conditions for a specific antibody marker. Then, in addition to the quantitative western blot analysis, we will include a graph representing the distribution of the labelling (intensity histogram) for all cell number quantification from immunofluorescence data, comparing control with the experimental condition. Finally, the methods used for quantification will be expanded in a revised manuscript.
The method section should be revised in general
Methods could be described in brief only when are supported by provided refs in which the reader could find details. Several refs are missing, i.e., 4.4 for thermal allodynia, 4.5 for ABT263 gavage administration; senescence induction, ...
Quantification methods should be more detailed, several information are missing and not found in result section or legends (i.e., number of nerve section per animal, neurite length, ...).
We completely agree with the reviewer that the method section was not developed adequately in the original manuscript. As described in previous responses, we will detail the methods used, especially those associated with quantifications performed. We will also include references for the different methods used, including those detailed by the reviewer.
The use of rat for in vitro DRG and SC culture while in vivo study is undertaken on mice The switch of species from in vivo to in vitro (mice vs. rat), is not justified as mice DRG and SC culture are also commonly used. In addition, the use of transgenic mice (used here only for in vivo) could also be exploited to address specific and reinforce the data.
We agree with the reviewer that using mouse SC in in vitro experiments will be a better approximation to support our findings. We have been using rat SC in this and other publications as they were the standard model used in in vitro experiments. Nevertheless, as the author states, now there are suitable methods for culturing mouse SC, that we have incorporated in our lab. Therefore, we will perform key in vitro experiments using mouse SC together with mouse DRGs.
Regarding the use of the transgenic mice (3MR), we thank the reviewer for this suggestion; we will perform new experiments using SC derived from 3MR mice in order to demonstrate induction of senescence (by expression of the red fluorescent protein in this transgenic line) and senolysis in vitro.
Use of conditioned exosome/media
Should be explained why the use of exosomes directly in cell culture was not tested. This would be close to physiological condition, regarding the concentration of released factors.
This is an important point that was not explored. As we have plenty of experience using SC-derived exosomes, we will perform the suggested experiments comparing the effect of exosomes from conditioned media from senescent-induced SC and include the results from these experiments in a revised manuscript.
The statement on the effect of rSC vs. sSC cell on growth cone dynamic
The provided data illustrated in fig. 3 are not in support that sSC affect growth cone dynamics. Only what would be "suggested" is that the decrease in neurite length could be associated to changes of growth cone morphology, on fixed tissue, that appeared to be affected. If such statement has to be maintained, time-laps is required. The image does not reflect a retracting neurite nor collapsed growth cone. In addition, other mechanisms could be at the basis of observed decrease in neurite length, which are not evaluated here. This is an important point to address as the authors state that sSC release inhibitory factors.
We completely agree with the reviewer: we are not exploring growth cone dynamics. We will change the manner these results are presented as we are not demonstrating a dynamic process in our results. We prefer to modify the text associated with these experiments rather than perform a time-lapse analysis at this moment. This is part of a future exploration we want to achieve, that will take some time to develop, and we consider at this moment lies outside the scope of the present work. Included in section 4, below.
other comments
- The surgical description is complicated, also annotation to be added in supp fig #2A; provide
We will work in the description of the model, including references to other papers using this nerve anastomosis model for assessing regenerative potential. As stated above we will also include schemes in all figures to help the reader with the surgical procedure and different timelines used.
- M&M 4.2: lign #8, error referring to Fig 2A, correct by. Supp fig 2A
Revisions incorporated in the transferred manuscript, see section 3, below.
- Review refs list, ref#17, full info needed
Revisions incorporated in the transferred manuscript, see section 3, below.
- Fig 3H would be interesting only if contain a column of the 21 proteins exclusively expressed in senescent-induced SCs
Revisions incorporated in the transferred manuscript, see section 3, below.
- The immunostaining of Lamin B1 positive nuclear invaginations in fig S4 should better described in results for non-familiar reader
We will describe better this staining pattern in the result section of the revised manuscript.
- Title of Fig 3, the expression "neuronal growth" is not appropriate here (neurite outgrowth)
Revisions incorporated in the transferred manuscript, see section 3 below.
**Referees cross-commenting**
I agree with referee#1's comments. He/she has raised complementary and important points that should be taken into account by the authors as well as we share the same major concerns. Furthermore, we both expressed the interest of such study if revised appropriately.
Reviewer #2 (Significance (Required)):
Significance
Overall, this is an interesting study that undertake fundamental question in the field of nerve physiopathology and also could open a good opportunity in developing therapeutic strategies for translational research. However, additional investigations are needed to support the main conclusions.
3. Description of the revisions that have already been incorporated in the transferred manuscript
Reviewer 1
Major comments
In the axon regeneration assays, how is the reconnection site defined in longitudinal images stained for SCG10? Of particular concern is that Figure 1B "adult chronic dmg" nerve section image appears to be identical to the image in Figure 5A "Vehicle adult (47dpi)". However, the reconnection site is located at different sites along the nerve. Also, the scale bar appears identical but the legend states different sizes. In Figure 1 chronic damage is 42dpi, and figure 5 is 47dpi, yet with what appears as the same image.
We thank the reviewer for detecting this issue. The problem arises as the data shown in Figure 1a corresponds to the controls (vehicle) of Figure 5a. The data in Figure 1 is a known phenomenon in the field of peripheral regeneration (i.e., decreased regeneration in aged animals as well as in chronically denervated nerves); nevertheless, we decided to add this data at the start of the manuscript to clearly shown the reader (thinking in a broader scientific audience) the baseline of the evident decrease in axonal regeneration in these two conditions. To make this very clear, we have included the same image Figure 1 and 5 for the controls and detailed this in the legends of both Figure 1 and 5 in the uploaded manuscript.
We have checked the scales and modify the scale in Figure 5 as it was not correct. We have also corrected the nomenclature for days post injury in this image as well as in the corresponding legend.
In addition, for transparency, we have uploaded in a public repository (EBI BioStudies database, https://www.ebi.ac.uk/biostudies/) all the microscopy images used in this work, which is detailed in the uploaded manuscript in a new section named data availability.
Regarding the localization of the reconnection site, this is identified using the whole z-stack of the nerve and not a single section, the region in the z-stack can be recognized using two parameters: the difference in diameter between the proximal and distal stump and by identifying the filament used to suture both stumps. We have included a description of this procedure in the method section of the revised manuscript. As this is not always a perpendicular line, in the revised Figures we have now used an arrowhead to denote the reconnection site.
We are sorry for the confusion generated by the labeling of some images. We will review the text and figures and fix errors. In a revised manuscript we will also add schemes for several figures in order to explain better the experimental procedure and timelines.
Minor comments
Improve labeling of Figures or at the very least describe in the Figure legend. For example: Figure 5B-C, which of the graphs is from adult mice and which is from aged mice?
We have included the suggested labelling for Figure 5B-C.
The images for split individual channels need to match the merge channel images. For example, the individual channel and merge images are not properly aligned in Figure 4C, ABY-263 group.
We thank the reviewer for spotting the error in the split channels, we have now fixed this in Fig 4C, but also corrected other alignment problems detected in Fig 4A and 5F.
The analysis method used to quantify axon regeneration should be consistent throughout. For example, in Figure 1C, number of axons/nerve width(um) was used for regeneration assay, but axon density (width corrected) was used in Figure 5B-C in regeneration assay.
We have included the procedure used to quantify axonal regeneration in the method section of the uploaded manuscript, which is the same throughout the manuscript. We are sorry for the different texts in the axes of graphs included in Figure 1 and Figure 5. In the first version of the figures, we were using the term “axon density (width corrected)”, but then we decided to change it to “number of axons/nerve width (mm)”, which was more precise. Unfortunately, the text of the graph axis in Figure 5 was not changed by mistake. We have now fix this in the revised version.
Reviewer 2
- M&M 4.2: lign #8, error referring to Fig 2A, correct by. Supp fig 2A
We have corrected this error in the uploaded manuscript.
- Review refs list, ref#17, full info needed
We have fixed this reference in the uploaded manuscript.
- Fig 3H would be interesting only if contain a column of the 21 proteins exclusively expressed in senescent-induced SCs
We are sorry about this omission in Figure 3H. We included a list of all the identified proteins of repair and senescent-induced SCs in Supplementary Table 4 (Table S4) of the original manuscript, including the identity of the 21 proteins exclusively expressed in senescent-induced SCs. In the revised version, we have incorporated the information of the 21 proteins in Figure 3H as suggested by the reviewer.
- Title of Fig 3, the expression "neuronal growth" is not appropriate here (neurite outgrowth)
We thank the reviewer for detecting this error, we have changed the expression as suggested in the uploaded manuscript.
Other changes included in the revised manuscript and Figures
- Numeric data for graphs We have included a new supplementary excel file: Supplementary Table 8, including the data for each replicate associated to the graphs of text and supplementary figures.
Revision Figure 5G.
During our review on all the individual replicates of the manuscript data to upload into BioStudies, the first author noticed that the data in graph of Figure 5G corresponded to a pilot experiment performed to set up the protocol. The final experiment was not included, then we uploaded the correct images and included the final quantification. Data is comparable, and statistical differences remains.
Revision Figure 3A.
We have modified the pseudocolors of the S100 antibody channel from magenta to green for ease of visualization. The image and quantification remain exactly the same.
4. Description of analyses that authors prefer not to carry out
Reviewer 2
The statement on the effect of rSC vs. sSC cell on growth cone dynamic
The provided data illustrated in fig. 3 are not in support that sSC affect growth cone dynamics. Only what would be "suggested" is that the decrease in neurite length could be associated to changes of growth cone morphology, on fixed tissue, that appeared to be affected. If such statement has to be maintained, time-laps is required. The image does not reflect a retracting neurite nor collapsed growth cone. In addition, other mechanisms could be at the basis of observed decrease in neurite length, which are not evaluated here. This is an important point to address as the authors state that sSC release inhibitory factors.
We completely agree with the reviewer: we are not exploring growth cone dynamics. We will change the manner these results are presented as we are not demonstrating a dynamic process in our results. We prefer to modify the text associated with these experiments rather than perform a time-lapse analysis at this moment. This is part of a future exploration we want to achieve, that will take some time to develop, and we consider at this moment lies outside the scope of the present work.
-
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Referee #2
Evidence, reproducibility and clarity
The reported study by Fuentes-Flores et al. shows that Schwann cells (SCs) in peripheral nerves undergo senescence with aging or in chronic denervation. This senescent SC phenotype correlates with downregulation of c-Jun expression and axon regeneration capacity and consequently, affecting functional recovery. The study has been undertaken by using in vivo mice model of chronically denervated sciatic nerve and in vitro of rat-primary cell cultures (Schwann cell, DRG-explants, and their coculture). Schwann cell exosome manipulation was also included to exploit their released factor as media for cell cultures.
Major comments:
- Chronic …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
The reported study by Fuentes-Flores et al. shows that Schwann cells (SCs) in peripheral nerves undergo senescence with aging or in chronic denervation. This senescent SC phenotype correlates with downregulation of c-Jun expression and axon regeneration capacity and consequently, affecting functional recovery. The study has been undertaken by using in vivo mice model of chronically denervated sciatic nerve and in vitro of rat-primary cell cultures (Schwann cell, DRG-explants, and their coculture). Schwann cell exosome manipulation was also included to exploit their released factor as media for cell cultures.
Major comments:
- Chronic denervated nerve model and Schwann cell phenotype
- Tibial nerve transection and chronic denervated nerve: As the referee have no information about this model (no reference is cited), a detailed description should be provided highlighting the interest of such model compared to standard sciatic nerve lesion model.
- Should be shown, histological analysis of denervated tibial branch prior reconnection with freshly cut proximal peroneal branch with specific immunostainings of rSCs v.s. sSC associated with dapi nuclear staining (as used along this study). Specific staining for other cells should be also provided (i.e., macrophage and endothelial cells). In simple words, "how chronically denervated nerve looks like and what is his cellular content? This is necessary to responds to the following main referee question: does the increase/decrease of rSCs or sSC under specific condition all through the study concerns the SCs that have migrated from freshly cut peroneal branch into denervated tibial distal branch, or resident SCs that have survived in chronically denervated tibial distal branch. In other words, whether rSCs that migrate (and accompanying regenerating axons) into chronically denervated branch nerve undergo phenotype change into sSC because of the environment of chronically denervated nerve. This is not clearly described or discussed, and remain confusing for reader.
- Support of up- and down-regulation of gene expression illustrated in fig.4 The conclusions and statements on up- or down-regulation of c-jun, yH2AX, beta-gal, P16, arise from quantitative and qualitative analysis from immunostaining of these specific markers by determining the number of positive cells rSCs vs. sSC. For these strong statements appropriate methods for quantification of protein levels such as western blots are requiered. For example, the statement of down regulation of c-jun expression, the quantitative graph shows strong increase in c-jun cell number under ABT263 treatment bu the histological photo does not illustrate such decrease in number of the cell. It shows rather an increase in brightness of c-jun staining. Thus, only appropriate method for protein level quantification could be conclusive. This would also remove the doubt that some photos are under- or over- exposed as it appears in the figure. For example, in aged animal under vehicle condition, there is no variability in staining intensity. Accordingly, one question to the authors: for quantification of cell number, are weakly stained cells considered as positive cell?
- The method section should be revised in general Methods could be described in brief only when are supported by provided refs in which the reader could find details. Several refs are missing, i.e., 4.4 for thermal allodynia, 4.5 for ABT263 gavage administration; senescence induction, ... Quantification methods should be more detailed, several information are missing and not found in result section or legends (i.e., number of nerve section per animal, neurite length, ...).
- The use of rat for in vitro DRG and SC culture while in vivo study is undertaken on mice The switche of species from in vivo to in vitro (mice vs. rat), is not justified as mice DRG and SC culture are also commonly used. In addition, the use of transgenic mice (used here only for in vivo) could also be exploited to address specific and reinforce the data.
- Use of conditioned exosome/media Should be explained why the use of exosomes directly in cell culture was not tested. This would be close to physiological condition, regarding the concentration of released factors.
- The statement on the effect of rSC vs. sSC cell on growth cone dynamic The provided data illustrated in fig. 3 are not in support that sSC affect growth cone dynamics. Only what would be "suggested" is that the decrease in neurite length could be associated to changes of growth cone morphology, on fixed tissue, that appeared to be affected. If such statement has to be maintained, time-laps is required. The image does not reflect a retracting neurite nor collapsed growth cone. In addition, other mechanisms could be at the basis of observed decrease in neurite length, which are not evaluated here. This is an important point to address as the authors state that sSC relase inhibitory factors.
Other comments
- The surgical description is complicated, also annotation to be added in supp fig #2A; provide
- M&M 4.2: lign #8, error referring to Fig 2A, correct by. Supp fig 2A
- Review refs list, ref#17, full info needed
- Fig 3H would be interesting only if contain a column of the 21 proteins exclusively expressed in senescent-induced SCs
- The immunostaining of Lamin B1 positive nuclear invaginations in fig S4 should better described in results for non-familiar reader
- Title of Fig 3, the expression "neuronal growth" is not appropriate here (neurite outgrowth)
Referees cross-commenting
I agree with referee#1's comments. He/she has raised complementary and important points that should be taken into account by the authors as well as we share the same major concerns. Furthermore, we both expressed the interest of such study if revised appropriately.
Significance
Overall, this is an interesting study that undertake fundamental question in the field of nerve physiopathology and also could open a good opportunity in developing therapeutic strategies for translational research. However, additional investigations are needed to support the main conclusions.
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Referee #1
Evidence, reproducibility and clarity
Summary
This manuscript by Fuentes-Flores et al reports that elimination of senescent Schwann cells by systemic senolytic drug treatment or genetic targeting improves nerve regeneration and functional recovery in aging and chronic denervation. This improved regeneration is associated with an upregulation of c-Jun expression. Mechanistically the authors provide data to show that senescent Schwann cells secrete factors that are inhibitory to axon regeneration. These findings are very interesting and move the field forward beyond the notion that Schwann cells fail to support axon regeneration in aged animals and identify potential …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
This manuscript by Fuentes-Flores et al reports that elimination of senescent Schwann cells by systemic senolytic drug treatment or genetic targeting improves nerve regeneration and functional recovery in aging and chronic denervation. This improved regeneration is associated with an upregulation of c-Jun expression. Mechanistically the authors provide data to show that senescent Schwann cells secrete factors that are inhibitory to axon regeneration. These findings are very interesting and move the field forward beyond the notion that Schwann cells fail to support axon regeneration in aged animals and identify potential targets to enhance nerve repair. The use of a senolytic drug to increase regeneration in aged and chronic denervation conditions provides new avenues for clinical interventions. However, some of the claims are overstated since this is not the first characterization of senescent Schwann cells and the manipulations used in the study are not entirely specific for Schwann cells. The manuscript is also poorly written and difficult to follow, given the complex set of surgeries and terminology, and lack of explanation of the rationale for the surgery model used. Figures are poorly labeled and difficult to follow without figure legends, and figure legends do not match the figures. Below are suggestions for improvements:
Major comments:
- In the axon regeneration assays, how is the reconnection site defined in longitudinal images stained for SCG10? Of particular concern is that Figure 1B "adult chronic dmg" nerve section image appears to be identical to the image in Figure 5A "Vehicle adult (47dpi)". However, the reconnection site is located at different sites along the nerve. Also, the scale bar appears identical but the legend states different sizes. In Figure 1 chronic damage is 42dpi, and figure 5 is 47dpi, yet with what appears as the same image.
- The authors need to provide a rationale for the choice of this complex injury model and what are the advantages over other models. Please clearly describe the timepoints for each experiment and why the time points were chosen for analysis. Provide the scheme of injury in the main figures to ease comprehension. A scheme is provided in what appears to be Figure S2, but the legend of Figure S2 does not match. Please compare same time points between aged and adults. Days post injury is sometimes referred to 7 or 42, and it is difficult to follow if it is days post initial transection of the tibial nerve or days post reconnection of transected tibial to peroneal. Revise all figure legends and supplementary figure legends to match figures.
- The authors' main conclusion is that Senescent Schwann cells inhibit axon regeneration. The authors need to tune down this statement and acknowledge that their manipulations are not entirely Schwann cells specific. While the data nicely shows a contribution of senescent Schwann cells, it does not sufficiently acknowledge the possibility that other senescent cells in the nerve contribute to this effect. First, the authors refer in the discussion that 60% of the senescent cells are SOX10 negative, and thus represent other cells beyond Schwann cells. This quantification needs to be shown in Figure 1. Second, the genetic and pharmacology manipulation eliminate all senescent cells, including Schwann cells. Third, while the culture experiment may be Schwann cells specific, the authors need to provide detailed information on how they purify these cells, how they induce repair Schwann cells (rSC) as claimed in Figure 3, and demonstrate whether these are pure Schwann cells. This is important because other cells in the nerve contribute to nerve repair, including mesenchymal cells. Finally, the claim that using Mpz-cre will lead to c-jun overexpression only in SC also needs to be demonstrated, since Mpz is also expressed in satellite glial cells in the DRG.
- In Figure 5, c-jun is shown after denervation (42 dpi). The results describe 28 days of denervation, 5 days of GCV and 7 days post reconnection, which makes 40 days. If that is not the case, results need to better explain timeline of this procedure. Also, what is the basal c-jun expression in p16-3MR mice? In addition to the number of c-jun positive cells shown in Figure 5G, the authors need to quantify the percent of c-jun puncta that co-localize with Sox10. The size of the c-jun puncta appears different in size in vehicle and GCV, is that an expected phenotype?
Minor comments
- Improve labeling of Figures or at the very least describe in the Figure legend. For example: Figure 5B-C, which of the graphs is from adult mice and which is from aged mice?
- The authors need to carefully describe where the high magnification images were taken in the injured nerve and keep the comparison at same site between groups. Please check the scale bar for each image. For example, the images in Figure 1D/F/I/K used same scale, but the cell size and cell morphology are different. The images for split individual channels need to match the merge channel images. For example, the individual channel and merge images are not properly aligned in Figure 4C, ABY-263 group.
- The analysis method used to quantify axon regeneration should be consistent throughout. For example, in Figure 1C, number of axons/nerve width(um) was used for regeneration assay, but axon density (width corrected) was used in Figure 5B-C in regeneration assay.
Referees cross-commenting
I agree with all Referee #2's comments. Both sets of comments are important, complementary and point to the same major concerns that need to be addressed. Agree as well that both reviewer think this is an interesting and relevant study for the field of nerve repair, if revised appropriately.
Significance
The findings reported in this manuscript are very interesting and will move the field of nerve repair forward. This paper will be of interest for basic science audience in the fields of aging and neurobiology and has also potential interest to the broader clinical and translational fields. Indeed, this paper provides data that Schwann cells entering a senescent stage not only fail to support axon regeneration in aged animals, but actively inhibit axon regeneration. While this reviewer raises questions on whether only senescent Schwann cells or other senescent cells in the nerve contribute to this effect, the identification of potential targets to enhance nerve repair is highly significant. Furthermore, the use of an FDA approved drug, currently in clinicals trials for its application as senotherapeutics, to increase axon regeneration in aged and chronic dennervation conditions will provide new avenues for clinical applications.
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