Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice

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.

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 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.

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.

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.

In the "materials and methods" section the paragraph relating to the methods used for statistical analysis is missing.

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.

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.

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.

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.

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?

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.

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.