Author Response
The following is the authors’ response to the original reviews.
eLife assessment
This manuscript describes valuable information on how the extraocular muscles (EOM) are preserved in a mouse model of familial Amyotrophic lateral sclerosis (ALS) that carries a G93A mutation in the Sod1 gene. The authors provide convincing evidence of how the integrity of neuromuscular junction is preserved in EOM but not in limb and diaphragm muscles of G93A mice. Overall, this interesting work provides new evidence regarding the etiopathogenesis of ALS and insights for the development of therapeutic targets to slow the loss of neuromuscular function in ALS.
Public Reviews:
Reviewer#1 (Public Review):
Summary:
The study explores the mechanisms that preserve satellite cell function in extraocular muscles (EOMs) in a mouse model of familial Amyotrophic lateral sclerosis (ALS) that carries the G93A mutation in the Sod1 gene. ALS is a fatal neuromuscular disorder driven by motor neuron degeneration, leading to progressive wasting of most skeletal muscles but not EOM. The study first established that integrity of neuromuscular junction (NMJ) is preserved in EOM but not in limb and diaphragm muscles of G93A mice, and sodium butyrate (NaBu) treatment partially improves NMJ integrity in limb and diaphragm muscles of G93A mice. They also found a loss of synaptic satellite cells and renewability of cultured myoblasts in hindlimb and diaphragm muscles of G93A mice, but not in EOM, and NaBu treatment restores myoblast renewability. Using RNA-seq analysis, they identify that exon guidance molecules, particularly Cxcl12, are highly expressed in EOM myoblasts, along with more sustainable renewability. Using a neuromuscular co-culture model, they convincingly show that AAV-mediated Cxcl12 expression in G93A myotubes enhances motor axon extension and innervation. Strikingly, NaBu-mediated preservation of NMJ in limb muscles of G93A mice is associated with elevated expression of Cxcl12 in satellite cells and improved renewability of myoblasts. These results together offer molecular insights into genes critical for maintaining satellite cell function and revealing a mechanism through which NaBu ameliorates ALS.
Strengths:
Combination of in vivo and cell culture models. Nice imaging of NMJ and associated satellite cells. Using motoneuron-myotube coculture to establish the mechanism. Tested and illustrated a mechanism through which a clinically used drug ameliorates ALS.
Weaknesses:
Data presentation could be improved (see details in the Recommendation for Authors).
It would have been nice to have included G93A motoneurons in the coculture study.
This is indeed a plan of our future study. In the revised version, we discussed the limitation of not including G93A motor neurons in the coculture assay. (Page 11, Line 445-448)
“However, it is possible that motor neurons carrying ALS mutations will respond differently to Cxcl12 mediated axon guidance than WT motor neurons. This is a limitation of the current study which will be investigated in future co-culture studies.”
Reviewer #2 (Public Review):
Summary:
The work is potentially interesting as it outlines the role of satellite cells in supporting the functional decline of skeletal muscle due to the denervation process. In this context the authors analyze the functional and molecular characteristics of satellite cells in different muscle types differently affected by the degenerative process in the ALS model.
Strengths:
The work illustrates a relevant aspect of the differences in stem cell potential in different skeletal muscles in a mouse model of the disease through a considerable amount of data and experimental models.
Weaknesses:
However, there are some criticisms of the structuring of the results:
It is not clear how many animals were used in each experimental group (Figs 1 and 2, Fig. 2-9). In particular, it is unclear whether the dots in the histograms represent biological or technical replicates. Furthermore, the gender used in experimental groups is never specified. This last point appears to be important considering the gender differences observed in the SOD1G93A mouse model.
The original quantification data and mouse gender specification were actually listed in the corresponding supplementary tables. We now added the gender specification and number of the mice used in all corresponding figure legends. The number of mice used for sorting SCs from different muscles were also specified in the Methods section in the revised manuscript. (Page 12, Line 489-493).
We also added one more supplementary figure (Figure 1-figure supplement 2) to compare the innervation status between male and female mice. The following description has been added in the updated manuscript (Page 3-4; Line 125-130):
“The data shown in Figure 1B has also been replotted to compare the innervation status between male and female mice (Figure 1- figure supplement 2). In terms of well- or partially- innervated ratios, there are no significant gender difference observed in our experimental condition, in which the muscle samples were collected at the end stage of the disease, although there is marginally lower “poorly innervated ratio” in the EDL muscle of G93A female mice compared to G93A male mice.”
However, we acknowledge that the current study has limitations to fully detect cross-gender differences in our experiments due to low “n” numbers per gender. We hope this is understandable as we have to split limited resource of ALS G93A mice between different kinds of experiments, including NMJ integrity assessment, peri-nuclear SC abundance assessment, whole muscle-qPCR, cell sorting for imaging, cell sorting for RNA-Seq, cell-sorting for qPCR, cell-sorting for neuromuscular co-culture, etc., in this pioneer study. However, we do intend to gradually build up “n” numbers for characterization of cross-gender difference in our ongoing studies.
As to what the dots in each plot represent, we have inserted the description in each relevant figure legend as detailed below:
For Fig 1, each dot represents quantification result from a single mouse. Please see Figure 1-figure supplement 1, Figure 1-figure supplement 2 and Figure 1-table supplement 1 for NMJs measured per muscle type per gender. Briefly, EDL, soleus and diaphragm muscles were from 4 male and 6 female mice per group; WT EOM group was from 4 male and 4 female mice; G93A EOM group was from 3 male and 4 female mice; G93A EOM with NaBu feeding group was from 6 female mice.
For Fig 2, each dot represents quantification result from a single mouse. Please see Figure 2-table supplement 1 for NMJs measured per muscle type per gender. Briefly, WT EDL group was from 2 male and 2 female mice; G93A EDL group was from 3 male and 3 female mice; G93A EDL with NaBu feeding group was from 2 male and 4 female mice; WT soleus group was from 2 male and 3 female mice; G93A soleus group was from 3 male and 2 female mice; G93A soleus with NaBu feeding group was from 1 male and 4 female mice; WT diaphragm group was from 1 male and 4 female mice; G93A diaphragm group was from 1 male and 4 female mice; G93A diaphragm with NaBu feeding group was from 4 female mice; WT EOM group was from 1 male and 3 female mice; G93A EOM group was from 5 female mice; G93A EOM with NaBu feeding group was from 1 male and 3 female mice.
For Fig 3, each dot in the box-and-dot plots represents result from one round of sorting. WT HL SCs were from 8 male and 6 female mice; G93A HL SCs were from 9 male and 5 female mice; WT diaphragm SCs were from 6 male and 3 female mice; G93A diaphragm SCs were from 12 male and 5 female mice. WT EOM SCs were from 6 batches of male and 1 batch of female mice (each batch contains 5-6 mice of the same gender). G93A EOM SCs were from 5 batches of male and 2 batches of female mice.
*Please note these results were from sorting in which the FACS profiles were recorded. Not all rounds of sorting were with FACS profile recorded.
For Fig 4A, each dot in the box-and-dot plots represents one image analyzed. For WT HL SCs, 94 images from 3 rounds of sorting; For WT Dia SCs, 107 images from 3 rounds of sorting; For WT EOM SCs, 75 images from 3 rounds of sorting; For G93A HL SCs, 96 images from 3 rounds of sorting; For G93A Dia SCs, 62 images from 3 rounds of sorting; For G93A EOM SCs, 79 images from 3 rounds of sorting. For the 3 rounds of sorting, 1 was from male and 2 were from female mice.
*Please note that the number of mice used for sorting SCs in different muscles were specified in the Method Section in the revised manuscript. (Page 12, Line 489-493)
For Fig 4B, each dot in the box-and-dot plots represents one image analyzed. For WT HL SCs, 52 images from 3 rounds of sorting; For WT Dia SCs, 51 images from 3 rounds of sorting; For WT EOM SCs, 51 images from 3 rounds of sorting; For G93A HL SCs, 52 images from 3 rounds of sorting; For G93A Dia SCs, 47 images from 3 rounds of sorting; For G93A EOM SCs, 56 images from 3 rounds of sorting. For the 3 rounds of sorting, 1 was from male and 2 were from female mice.
For Fig 5A, each dot in the box-and-dot plots represents one replicate of culture. HL SCs were from male mice.
For Fig 5B, each dot in the box-and-dot plots represents one image analyzed. For G93A HL SCs, 52 images from 3 rounds of sorting; 1-day NaBu treatment, 45 images from 3 rounds of sorting; 3-day NaBu treatment, 51 images from 3 rounds of sorting; For G93A Dia SCs, 47 images from 3 rounds of sorting; 1-day NaBu treatment, 60 images from 3 rounds of sorting; 3-day NaBu treatment, 57 images from 3 rounds of sorting. For the 3 rounds of sorting, 2 were from male and 1 was from female mice.
For Fig 6, all samples used for bulk RNA-Seq were from female mice.
For Fig 7C, each dot in the box-and-dot plots represents one replicate of culture. RNA samples were collected from 3-6 rounds of sorting and sorted cells were seeded into 3 dishes as replicates. WT HL SCs were from 3 male and 1 female mice. WT diaphragm SCs were from 2 male and 2 female mice; WT EOM SCs were from 3 male mice; G93A HL SCs were from 4 male and 2 female mice. G93A diaphragm SCs were from 1 male and 3 female mice; G93A EOM SCs were from 3 male mice.
For Fig 7D, each dot in the box-and-dot plots represents one replicate of culture. RNA samples were collected from 6 rounds of sorting and sorted cells were seeded into 3 dishes as replicates. G93A HL SCs were from 4 male and 2 female mice; G93A diaphragm SCs were from 2 male and 4 female mice.
For Fig 8D, each dot in the box-and-dot plot represents one neurite measured. HL and EOM SCs used for co-culture experiments were all from male mice.
For Fig 9D, each dot in the box-and-dot plot represents one image analyzed. HL and EOM SCs used for co-culture experiments were all from male mice.
For Figure 1-figure supplement 1, each dot in the box-and-dot plots represents quantification result from one mouse. Please also see Figure 1-table supplement 2. Briefly, muscles in WT and G93A groups were from 3 male and 3 female mice per group; G93A EDL with NaBu feeding group was from 3 male and 3 female mice. G93A soleus with NaBu feeding group was from 2 male and 3 female mice; G93A diaphragm with NaBu feeding group was from 2 male and 4 female mice; G93A EOM with NaBu feeding group was from 4 male and 2 female mice.
The first paragraph of the results lacks a functional analysis of the motor decline of the animals after the administration of sodium butyrate. The authors, in fact, administered NaBu around 90 days of age while in previous work the drug had been administered at a pre-symptomatic age. It would therefore be useful, to make the message more effective, to characterize the locomotor functions of the treated animals in parallel with the histological evidence of the integrity of the NMJ.
We are still in the process of collecting locomotor function data for G93A mice with and without NaBu treatment. We plan to report them in a future manuscript while this manuscript focuses on the molecular and histological aspect. Additionally, in the revised manuscript, we revised the rationale of the NaBu treatment starting after the disease onset. (Page 4, Line 131-134)
“In the previous study, NaBu treatment initiated at a pre-symptomatic age delayed disease progression in G93A mice. As treatment of ALS patients is initiated after symptoms appear, we further tested whether NaBu treatment started after disease onset (at the age of 3 months, 2% NaBu in water for 1 month) was effective in preserving NMJ integrity.”
Figure 5 should be completed with the administration of NaBu also to the satellite cells isolated from the WT mouse, the same for figure 9 where AAV-CMV-Cxcl12 transduction of WT myotubes is missing.
We appreciate the reviewer’s suggestion of conducting the additional experiment with AAV-delivery of CXCL12 into the myotubes derived from the WT mice. Extensive studies by other investigators have been performed with butyrate on satellite cells derived from WT mice. To name a few here: Fiszman et al., 1980 (DOI: 10.1016/0014-4827(80)90467-X); Johnston et al., 1992 (DOI: 10.1128/mcb.12.11.5123-5130.1992); Lezzi et al., 2002 (DOI: 10.1073/pnas.112218599). To avoid performing redundant experiments, we focus on the effect of butyrate on the proliferation and differentiation of SCs derived from G93A mice. Thanks to the reviewer’s comment, we added additional discussion in the Results section (Page 6, line 216-217).
Regarding the effect of Cxcl12, published studies have demonstrated its role in promoting axon growth. To name a few here: Negro et al., 2017 (DOI: 10.15252/emmm.201607257); Lieberam et al., 2005 (DOI: 10.1016/j.neuron.2005.08.011); Whitman et al., 2018 (DOI: 10.1167/iovs.18-25190). (Page 10, line 434, 440-442).
In the experiment illustrated in Figure 8, treatment of cell cultures with NaBu would improve the outcome as well as the interference of Cxcl12 expression in myotubes derived from G93A EOM SC (Fig.9) would strengthen the specificity of this protein in axon guidance in this NMJ typical of a spared muscle in ALS.
This is a great suggestion. Our study demonstrated the overexpression of CXCL12 in G93A myotube can enhance the axonal guidance and innervation of the co-cultured myotube/moto-neurons. We have also demonstrated the NaBu treatment can enhance the expression of CXCL12 and slow ALS progression. Combining NaBu treatment with CXCL12 overexpression may indeed have additive therapeutic benefits to slow ALS progression. We have added this statement in the revised Discussion. (Page 11, Line 466-468)
In the "materials and methods" section the paragraph relating to the methods used for statistical analysis is missing.
We have added it accordingly. (Page 15, Line 631-636)
Reviewer #3 (Public Review):
Summary:
In their paper, Li et al. investigate the transcriptome of satellite cells obtained from different muscle types including hindlimb, diaphragm, and extraocular muscles (EOM) from wild-type and G93A transgenic mice (end-stage ALS) in order to identify potential factors involved in the maintenance of the neuromuscular junction. The underlying hypothesis is that since EOMs are largely spared from this debilitating disease, they may secrete NMJ-protective factors. The results of their transcriptome analysis identified several axon guidance molecules including the chemokine Cxcl12, which are particularly enriched in EOM-derived satellite cells. Transduction of hindlimb-derived satellite cells with AAV encoding Cxcl12 reverted hindlimb-derived myotubes from the G93A mice into myotubes sharing phenotypic characteristics similar to those of EOM-derived satellite cells. Additionally, the authors were able to demonstrate that EOM-derived satellite cell myotube cultures are capable of enhancing axon extensions and innervation in co-culture experiments.
Strengths:
The strength of the paper is that the authors successfully isolated and purified different populations of satellite cells, compared their transcriptomes, identified specific factors released by EOM-derived satellite cells, overexpressed one of these factors (the chemokine Cxcl12) by AAV-mediated transduction of hindlimb-derived satellite cells. The transduced cells were then able to support axon guidance and NMJ integrity. They also show that administration of Na butyrate to mice decreased NMJ denervation and satellite cell depletion of hind limbs. Furthermore, the addition of Na Butyrate to hindlimb-derived satellite cell myotube cultures increased Cxcl12 expression. These are impressive results providing important insights for the development of therapeutic targets to slow the loss of neuromuscular function characterizing ALS.
Weaknesses:
Several important aspects have not been addressed by the authors, these include the following points which weaken the conclusions and interpretation of the results.
(a) Na Butyrate was shown to extend the survival of G93A mice by Zhang et al. Na butyrate has a variety of biological effects, for example, anti-inflammatory effects inhibit mitochondrial oxidative stress, positively influence mitochondrial function, is a class I / II HDAC inhibitor, etc. What is the mechanism underlying its beneficial effects both in the context of mouse muscle function in the ALS G93A mice and in the in vitro myotube assay? Cytokine quantification as well as histone acetylation/methylation can be assessed experimentally and this is an important point that has not been appropriately investigated.
Great suggestion by the reviewer.
Our previous publications (DOI: 10.3390/biom12020333; DOI: 10.3390/ijms22147412) have shown the beneficial roles of NaBu in ameliorating mitochondrial function in both motor neuron-like cells and adult muscle fibers. A focus of the current study is to test whether NaBu treatment also affect the SCs by regulating their gene transcription. Regarding the potential on HDAC/acetylation modification, there are previous studies by other investigators. We have added these references in the Discussion (Page 11, line 466-468).
(b) In the context of satellite cell characterization, on lines 151-152 the authors state that soleus muscles were excluded from further studies since they have a higher content of slow twitch fibers and are more similar to the diaphragm. This justification is not valid in the context of ALS as well as many other muscle disorders. Indeed, soleus and diaphragm muscles contain a high proportion of slow twitch fibers (up to 80% and 50% respectively) but soleus muscles are more spared than diaphragm muscles. What makes soleus muscles (and EOMs) more resistant to ALS NMJ injury? Satellite cells from soleus muscles need to be characterized in detail as well.
We agree with the reviewer’s comment that our original statement is misleading regarding the difference between soleus and diaphragm muscles in terms of the content of slow twitch fibers. Our histological studies revealed similar defects in denervation of diaphragm and soleus muscles derived from the G93A mice. Most importantly, the degree of NMJ degeneration and atrophy is less severe in soleus compared to other hindlimb muscles, such as EDL, during ALS progression. We have cited related studies such as Valdez et al., 2012 (DOI: 10.1371/journal.pone.0034640), Atkin et al., 2005 (DOI: 10.1016/j.nmd.2005.02.005). To avoid any confusion, we have removed the original statement and revised the paragraph (Page 4, line 159-162).
“The three groups were determined because they represent the most severely affected, moderately affected and least affected muscles by ALS progression, respectively. Soleus was not included in the hindlimb SCs pool because its less affected than other hindlimb muscles based on our study and others [6,42].”
Furthermore, EOMs are complex muscles, containing many types of fibers and expressing different myosin heavy chain isoforms and muscle proteins. The fact that in mice both the globular layer and orbital layers of EOMs express slow myosin heavy chain isoform as well as myosin heavy chain 2X, 2A, and 2B (Zhou et al., 2010 IOVIS 51:6355-6363) also indicates that the sparing is not directly linked to the fast or slow twitch nature of the muscle fiber. This needs to be considered.
We greatly appreciate your suggestions and have included these points in the revised Discussion. “It is known that EOMs are complex muscles. Besides the developmental myosin isoforms, EOMs also express both adult fast and slow myosin contractile elements (Zhou et al., 2010 IOVIS 51:6355-6363), suggesting that the sparing may not be solely linked to the fast or slow twitch nature of the muscle fiber, rather the changes in SCs may play a pivotal role in preserving the EOM function during the progression of ALS. ” (Page 9, line 389-392)
(c) In the context of myotube formation from cultured satellite cells on lines 178-179 the authors stained the myotubes for myosin heavy chain. Because of the diversity of myosin heavy chain isoforms and different muscle origins of the satellite cells investigated, the isoform of myosin heavy chain expressed by the myotubes needs to be tested and described. It is not sufficient to state anti-MYH.
We used the pan-anti-MYH antibody (MF20 from DSHB) for the immunostaining of myosin heavy chain for identification of the differentiated myotubes. As described in the commercial website: https://dshb.biology.uiowa.edu/MF-20), FM20 recognizes all myosin heavy chain isoforms. We are happy to examine whether specific myosin heavy chain isoforms may contribute to the differences observed in future studies.
(d) The original RNAseq results have not been deposited and while it is true that the authors have analyzed the results and described them in Figures 6 and 7 and relative supplements, the original data needs to be shown both as an xls list as a Volcano plots (q value versus log2 fold change). This will facilitate the independent interpretation of the results by the readers as some transcripts may not be listed. As presented it is rather difficult to identify which transcripts aside from Cxcl12 are commonly upregulated. Can the data be presented in a more visual way?
We have uploaded the Fastq files and the text files containing TPM values to the Gene Expression Omnibus (GEO) database and included the GEO access number GSE249484 in the revised text. Per recommendation of the reviewer, we have added supplementary tables for Figure 6, to list the top 20 differentially expressed genes (ranked by Log2FC, both the upregulated and downregulated) comparing 1) EOM SCs to their hindlimb and diaphragm counterparts (Figure 6-table supplement 1); 2) G93A SCs to WT SCs of the same muscle origin (Figure 6-table supplement 2); 3) G93A hindlimb and diaphragm SCs with 3 day-NaBu treatment to those without (Figure 6-table supplement 3). (Page 6, Line 237-257)
(e) There is no section describing the statistical analysis methods used. In many figures, more than 2 groups are compared so the authors need to use an ANOVA followed by a post hoc test.
Thank for the comments. We have added it accordingly. (Page 15, Line 631-636)
The authors have achieved their aim in showing that satellite cells derived from EOMs have a distinct transcriptome and that this may be the basis of their sparing in ALS. Furthermore, this work may help develop future therapeutic interventions for patients with ALS.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
The prevailing hypothesis of ALS is that motoneuron degeneration subsequently induces muscle atrophy and wasting. However, evidence also suggests that ALS is a muscle disease independent of motoneuron degeneration. The results from the current study support the latter. The RNA-seq data from cultured myoblasts (without innervation) suggest cell cell-autonomous effect of G93A on muscle cells. While the current analyses in this study identify axon guidance pathways in EOM satellite cells that may underlie their unique gene program that enhances motoneuron function, the powerfulness of the RNA-seq data is underutilized. I suggest that the authors explore the RNA-seq further by comparing genes and pathways altered by G93A in various muscles to better pinpoint how G93A influences satellite cell function.
Thanks for the comments and advice. Further analysis of the RNA-seq data is planned. As our original sequencing provider became unavailable to us since last year, we are currently negotiating with other sequencing providers. We have deposited the raw data files into the GEO database (GSE249484) to foster further analyses by other researcher teams.
To address the reviewer’s concern, we have added three more supplementary tables for Figure 6, which list the top 20 differentially expressed genes (DEG) (ranked by Log2FC, both the upregulated and downregulated) comparing 1) EOM SCs to their hindlimb and diaphragm counterparts (new Figure 6-table supplement 1); 2) G93A SCs to WT SCs of the same muscle origin (new Figure 6-table supplement 2); 3) G93A hindlimb and diaphragm SCs with and without 3 day-NaBu treatment (new Figure 6-table supplement 3). These three DEG lists are discussed in the results section of the revised manuscript as following (Page 6, Line 237-257).
Figure 4 presentation could be improved by adopting a similar comparison (WT vs G93A) as used in Figure 1-3. The current comparison is not straightforward. In addition, a magnified image of panel A would demonstrate the loss of myoblast homeostasis more clearly. (AKA Figure 2B)
The WT vs G93A comparison was presented in the supplementary figure of Figure 4 (Figure 4-figure supplement 1 in the previous version, and now in Figure 4-figure supplement 2 in the revised version).
As requested, we have added magnified single channel representative images of cultured SCs in the new Figure 4-figure supplement 1 in the revised manuscript.
Co-culture results in Figure 8 are very impressive. It would be nice if the data were quantified. The figure legend states that panel D is the quantification, but I don't see panel D. As the study used rat motoneurons (presumably SOD1 wildtype), it is unknown if G93A motoneurons would respond to muscle-derived CXCL12 similarly to the wildtype motoneurons. This information is crucial for understanding whether the SOD1 mutant ALS1 is a motoneuron disease or muscle disease or both. Some discussion should be provided to reflect the limitation (of not including G93A motoneurons in the coculture).
Panel D (the quantification data) was presented in the original figure setting (but may not be obvious). We have now revised Figure 8 to enlarge panel D to clearly present the quantification data.
We acknowledge the limitation of not including mutant G93A motor neurons in the coculture assay, and have added this important point (and our future plans to do so) in the discussion section of the revised manuscript: (Page 11, Line 445-448)
“However, it is possible that motor neurons carrying ALS mutations may respond differently to Cxcl12 mediated axon guidance than WT motor neurons. This is a limitation of the current study, which will be investigated in future co-culture studies.”
Reviewer #2 (Recommendations For The Authors):
Line 108. The sentence: "Z-stack scans of glycerol-cleared 109 whole muscles were obtained using a high working distance lens in a confocal microscope. The z-stacks were compacted into 2D images by maximal intensity projection" and should be moved to the material and methods section.
Removed from the Result section and added to the Method section as recommended (Page 13, Line 564-568).
Linea 113. The sentence: " In order to quantify the extent of denervation in a categorical manner, NMJs were arbitrarily defined as "well innervated" if SYP staining was present in >60% of the BTX positive area, "partially innervated" if between 60% and 30%, and "poorly innervated" if SYP staining corresponded to less than 30% of the BTX positive area" has already been written in the figure legend.
Thanks for the advice. We have rephrased the sentence to remove the redundant part.
In lines 445-7, it would be better to indicate the enzymatic units instead of the concentrations.
We included enzymatic units for the four enzymes in the Methods Section of revised manuscript (Page 12, Line 497-499).
Reviewer #3 (Recommendations for The Authors):
There are several points that need to be addressed by the authors including:
(a) The authors need to provide experimental evidence as to the mode of action of Na Butyrate and more specifically whether its beneficial effect is mediated by its anti-inflammatory action, inhibition of HDACs, or the combination of several mechanisms. Additionally, it should be clearer why Na Butyrate was administered. The sentence referring to reference 36 is not sufficient and some mechanistic insight needs to be provided in the results section.
Thanks for the great suggestion. We have revised the Results section accordingly to clarify the rationale for NaBu usage (please also see our detailed response to your suggestion above). (Page 4, line 131-134)
(b) Their reason for excluding soleus-derived-satellite cells from the analysis is not valid. Soleus muscles are "more" speared than diaphragm muscles and analysis may help shed light on this observation.
Please see our response to your question (b) in the above public review section.
(c) DATA AVAILABILITY: The RNAseq raw untransformed data has not been provided and Volcano plots are also not shown. I find it quite difficult to follow the results of the RNAseq experiments and this is central to the interpretation of the paper's results. Ideally, one should be able to look at the data and draw his/her own conclusions but as it stands this is difficult to do.
We have uploaded the raw FastQ files and the excel files containing TPM values to the GEO database with the access number GSE249484.
(d) A detailed description of all statistical tests that were used needs to be provided.
Yes, this has been added to the revised manuscript.
(e) Many figure legends are incomplete and some panels are not described appropriately, indicating that the authors need to thoroughly revise all aspects of the manuscript.
We have extensively edited the figure legends to address the issues raised by reviewers.
(f) Line 96-98: it is unlikely that muscles from ALS patients will be biopsied frequently. Furthermore, what biomarkers exactly could be followed in patients in response to therapy? This is unclear.
While it is true that it is not generally part of the diagnostic workup for ALS, muscle biopsy is increasingly being used pre- and post-treatment in ALS clinical trials to examine responses to potential new therapies. Muscle biopsy is also being explored in several ongoing studies as a potential ALS-relevant peripheral tissue amenable to biopsy (as opposed to brain or spinal cord) for predictive, pharmacodynamic, and prognostic biomarkers. This includes studies attempting to recapitulate pathophysiological patient clusters observed in CNS autopsy tissues and studies to detect aberrant TDP-43 aggregates in intramuscular nerve twigs, among others. Indeed, Dr. Ostrow’s clinical duties include performing muscle biopsies and interpreting muscle pathology, and he is involved in several ongoing studies attempting to correlate postmortem CNS and muscle analyses for these purposes.
To avoid potential controversy on the feasibility of multiple biopsies, we rephrased the sentence as follows (Page 3, Line 96-98)
“Characterizing the distinct EOM SC transcriptomic pattern could provide clues for identifying potential biomarkers in therapeutic trials in both ALS patients and animal models, in addition to identifying therapeutic targets.”
(g) Line 388-389. What do the authors mean by this sentence? It is not clear.
Thanks for the comment, we have expended the discussion to make it clearer in the revision. (Page 10, Line 428-431)
“It is possible that the more frequent self-renewal and spontaneous activation of EOM SCs contribute to higher rate of mitochondrial DNA replication, leading to accelerated spreading of mitochondrial DNA defects, resulting in higher proportion of COX-deficient myofibers than other muscles”.
(h) Were the experimenters blinded as to the results shown in Figures 2, 7, 8, and 9?
We endeavored to blind experiments whenever possible. Not all experiments were blinded due to logistic complexity and the clear difference in microscopic and gross appearances of wild-type and mutant muscle. The differences observed in Figures 2, 7, 8, 9 are qualitative (ie more than just quantitative), which should minimize the impact of possible human bias. Additionally, we employed multiple different experimental approaches to assess our hypotheses.
For Fig 2, the physical appearance is notably different between G93A and WT muscles. The different innervation status (Fig 2A) is also not amenable to blinding.
For Fig 7, the expression level of Hmga2, Notch3 and Cxcl12 detected by the qPCR assay are substantially greater in EOM derived SCs than counterparts from other muscles, and these results are also consistent with RNA-Seq, immunofluorescence assays.
For Fig 8, the overexpression of Cxcl12 and the coculture with EOM SC derived myotubes not only increased the length of the longest neurites but also promoted axon branching, which can be easily observed.
For Fig 9, only the EOM SC derived myotubes were capable of aligning the neurites along with them on a global scale. This qualitative difference is easy to appreciate, even under low magnification.
(i) Line 64 -65 The authors refer to a very old paper by Fischer et al in 2002 for the expression profile of EOMs. There are more recent papers including that of Eckhardt et al. (eLife 2023, 12:e83618) showing the differences in proteome between EOMs and soleus and EOMs and EDL muscles. There are more than
2000 (and not 300!!) differentially expressed proteins.
Thank you for the newly published reference. We have revised the Introduction section to include this new proteomic study. (Page 2, Line 64-69)
(j) Figure 7 C. The Y axis is mislabeled as they should be log2 fold change and not the growth conditions.
Thank you for catching this. We have fixed it.
(k) In all figures, if each symbol represents the results obtained on 1 mouse, this needs to be clearly stated. What do the panels on the right of Figures 4 and 5B show?
Thanks for the comments. For Figure 1B and 2C, as well as Figure 1-figure supplement 1B, one dot in the box-and-dot plots represents result obtained from 1 mouse. For Figure 3B, one dot represents one round of sorting. Generally, one mouse was euthanized for each round of sorting for HL and diaphragm SCs. But the sorting of EOM SCs could take up to 6 mice (as the EOMs are much smaller). For Figure 4 and 5B, each dot represents one image analyzed. All images were collected from three rounds of sorting. For Figure 5A, each dot represents one replicate of culture. For Figure 5B, each dot represents one image analyzed. All images were collected from three rounds of sorting. We have indicated those details in the revision.
Please also see our response to the 1st question of Reviewer 2 in the public review section.
(l) Figure 6 Table supplement 3 does NOT show the FDR but only the log2 fold change. Please amend.
We have amended the supplementary table accordingly.
