Review of the Pathology of Muscle in Amyotrophic Lateral Sclerosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

As a geneticist and evolutionary biologist, I am deeply interested in explaining muscle mechanisms by genotype or gene expression. Therefore, I accepted this review paper and read it carefully. Review papers should offer perspectives on new research areas based on existing knowledge, as well as potential future research opportunities. IJMS, with a 2024 IF of 4.9, is a widely cited and referenced journal. Therefore, the basic mechanisms, evolutionary implications between mouse and human, and therapeutic implications should be presented aggressively. Despite this, I failed to gain insights relevant to ALS research from this review paper, and I recommend submitting it to another journal. To improve this paper, I offer the following suggestions based on my careful reading:

1. There are visible typographic errors (double spaces, etc.) and (perhaps due to my use of a computer with a Chinese character-based East Asian culture) some special characters are broken. Once the final manuscript is completed, it should be printed out and thoroughly reviewed and revised.

2. Section 2 covers muscle-related topics (muscle structure, sarcomere, EC coupling, etc.) for the undergraduate level I teach. This can be easily understood by referring to relevant textbooks or using generative AI like ChatGPT. Considering the readers of IJMS, Section 2, including Figure 1, is unnecessary.

3. Similarly, for Tables 1 and 2, citing overly old papers is inappropriate unless it's a meta-analysis. To illustrate research trends from the 1960s to the present, a schematic diagram or key keywords should suffice.

4. I'm currently researching muscle organoids and applying single-cell RNA-seq and spatial genomics to them. Similarly, I'm developing muscle organoid models for ALS, muscular dystrophy, Parkinson's disease, and Lou Gehrig's disease. Therefore, I expected this information upon carefully reading this paper, but it wasn't there at all. There has been considerable research on omics that can mimic muscle or relate to muscle-related diseases, but this needs significant improvement. A simple review of analysis methods using atropy and H&E techniques is inappropriate at this point.

5. Section 3 simply lists biomarkers such as CK and EMG, and even covers MRI. It should specifically address what molecular and pathological changes in muscle are induced by identifying these markers. As it stands, it feels like a simple textbook summary.

6. Sections 4 and 5, while interesting, remain mere factual summaries. Figure 4 is appropriate, but more model diagrams like this one are needed. I reviewed this paper for two and a half hours, but most IJMS readers will not spend two and a half hours reading it. For this paper to be helpful to readers and generate a large number of citations, it needs compelling diagrams. I found section 5.4 particularly interesting. A schematic diagram of extracellular vesicles is essential.

7. Section 4.2 covers concepts such as animal models and fast-twitch fibers. It would be helpful for readers to see the relationship between animal models and human diseases, especially a comparative anatomical diagram between humans and mice. I believe this would be valuable for my teaching materials.

8. It is necessary to summarize what research has been conducted on therapeutics and how it relates to clinical trials and therapeutic development.

Author Response

Reviewer 1 (R1)

R1 point 1: As a geneticist and evolutionary biologist, I am deeply interested in explaining muscle mechanisms by genotype or gene expression. Review papers should offer perspectives on new research areas based on existing knowledge, as well as potential future research opportunities.

Response: We thank our reviewer for explaining in detail their viewpoint and expectations when reviewing our manuscript. We agree that elucidating ALS muscle mechanisms by genotype and or by gene expression could be a valuable addition to our review. This review was written to start with the findings in skeletal muscle of patients with ALS, and to link these findings to disease mechanisms. ALS subjects are heterogenous, and the aim of starting with findings in muscle biopsies is to identify abnormalities that are common to all subjects with ALS. At this stage, little is known about the variation in muscle pathology according to genotype or differing muscle gene expression. Furthermore, subjects with an ALS linked gene only constitutes ~20% of all ALS cases. We have added these explanations and have worked to complement them with recent details of altered muscle gene and protein expression from ALS muscle biopsies and primary muscles cells created from muscle satellite (stem cells; see lines 51-69, 86-90, 704-728). We have also added a section to state that explorations of genetic variation in muscle biology could be an interesting avenue to pursue when looking for genetic factors that might modify disease course (see lines 70-74).

 

R1 point 2: IJMS is a widely cited and referenced journal. Therefore, the basic mechanisms, evolutionary implications between mouse and human, and therapeutic implications should be presented.

Response: as mentioned above, the aim of the study was to start with the pathological findings in muscle from subjects with ALS, and to then review the possible mechanisms for this disease. Throughout the manuscript, we have cited studies using ALS model mice (e.g., see lines 58-81, 332-340). We note that many of these studies use inbred strains of mice that carry multiple copies of human transgenes with known ALS mutations, the most common being the SODG93A mutation (human SOD1G93A ALS mouse model Gurney et al, 1994. doi: 10.1126/science.8209258). It is also worth noting that the use of inbred strains of mice to create mammalian ALS models do not show the genetic diversity of humans with ALS. We have added a comment in the introduction to say that mouse models do not show all the features of human ALS. We have added these details (see lines 75-81).

From an evolutionary perspective, we note that the most common genetic form of ALS (due to C9orf72 repeat expansions) is a new disease that arose about 1,500 years ago. When the other variants emerged has not been studied. Other interesting evolutionary issues are the possibility that disease arises in more recently developed areas of the central nervous system, and the possibility that the genes that allowed this increase in brain size predispose neural cells to ALS. We have added some of this information to the introduction, see lines 58-74.

R1 Point 3. Despite this, I failed to gain insights relevant to ALS research from this review paper, and I recommend submitting it to another journal.

Response: We have submitted to IJMS because this is a review that covers areas ranging from clinical to basic science, and the intention of the review is to integrate what is known about human muscle in ALS as a basis for future research. Our expected target audience includes those ALS researchers who focus mainly on the central nervous system and who need to be given some fundamental information about muscle, as noted in our cover letter to the Editor. In our opening statement, we have worked to address our reviewer’s concerns and have taken on board their thoughtful suggestions, to improve our manuscript (see replies to R1 points 1 to 9).

To improve this paper, I offer the following suggestions based on my careful reading:

R1 point 4: There are visible typographic errors (double spaces, etc.) and (perhaps due to my use of a computer with a Chinese character-based East Asian culture) some special characters are broken. Once the final manuscript is completed, it should be printed out and thoroughly reviewed and revised.

Response: We apologize for the typographical errors, some of which appeared upon conversion from the word doc to the PDF file. Most of the double spaces were used to separate sentences. We have corrected these errors.

R1 point 3: Section 2 covers muscle-related topics (muscle structure, sarcomere, EC coupling, etc.) for the undergraduate level I teach. This can be easily understood by referring to relevant textbooks or using generative AI like ChatGPT. Considering the readers of IJMS, Section 2, including Figure 1, is unnecessary.

Response: We chose to include this information because some ALS researchers are neuroscientists who may not be familiar with the physiology of skeletal muscle (see R1 point 3), and clinicians and neurophysiologists who study clinical aspects of muscle in ALS (see lines 82-8, 85-90). We intend our review to be stand alone and not require recourse to other sources. While we agree this background information could be viewed as basic information, we think it allows for a complete review that is accessible to a broad audience, as noted by our other reviewer (Reviewer 2).

R1 point 4: Similarly, for Tables 1 and 2, citing overly old papers is inappropriate unless it's a meta-analysis. To illustrate research trends from the 1960s to the present, a schematic diagram or key keywords should suffice.

Response: the aim of the review is to document all the papers that have studied muscle from human subjects with ALS. As shown in the tables, there are only a limited number of subjects and the techniques varied. This is the basis for our recommendation for further study of muscle from human subjects using modern techniques to confirm the dogma that has arisen from these early studies. To remove old references would lessen the scholarly impact of our review (see reply to R1 point 3).

 

R1 Point 5: I'm currently researching muscle organoids and applying single-cell RNA-seq and spatial genomics to them. Similarly, I'm developing muscle organoid models for ALS, muscular dystrophy, Parkinson's disease, and Lou Gehrig's disease. Therefore, I expected this information upon carefully reading this paper, but it wasn't there at all. There has been considerable research on omics that can mimic muscle or relate to muscle-related diseases, but this needs significant improvement. A simple review of analysis methods using atropy and H&E techniques is inappropriate at this point.

Response: The use of muscle organoids or cells with and without a-MNs has been best developed for muscle diseases with a known genetic mutation, such as Duchenne muscular dystrophy and congenital myasthenia gravis. These two disorders are diseases of the developing neuromuscular system. By contrast, ALS is a neural degenerative disease of the adult neuromuscular system, which even in ALS mammalian models, manifests post the foetal to adult switching of molecular components of the neuromotor system. These include the following. For a-MN synapses, potassium-chloride transporter switches (NKCC1 to KCC2). This developmental switch allows for glycinergic neurotransmission to convert from being depolarizing (excitatory) to inhibitory (hyperpolarizing) (Fogarty, Kanjhan et al. 2016). Thus, during development, a-MNs are largely governed by excitation and then later when pattern output neural activity is required, by a combination of excitation (Glutamatergic and Cholinergic) and inhibition (Glycinergic) (Banks, Kanjhan et al. 2005) (Liu and M.T-T. 2013). There are also foetal to adult neurotransmitter receptors switches of glycinergic receptors that aid in the maturation of glycinergic neurotransmission (Takahashi, Momiyama et al. 1992).

At the NMJ, there is pre- and post-synaptic molecular switching that occurs post birth in mammals (including humans). At the pre-synaptic membrane, N to P/Q voltage gated calcium channels switching allows for enhanced efficacy of synaptic transmission (Ishikawa, Kaneko et al. 2005) (Chand, Lee et al. 2015). During this period in the muscle’s post synaptic region, there is a switch in acetylcholine receptor units, from gamma to epsilon (Missias, Chu et al. 1996). This switch allows for faster modulation of the postsynaptic depolarization that will in turn govern the generation and frequency of muscle action potentials. These events largely occur during the loss of poly-neuronal innervation of muscle post birth. For muscle, this developmental period is accompanied by the specification of muscle fiber type (fast and slow twitch) and stabilization of the motor unit. Human organoid models, which are generated mostly from adult or embryonic pluripotent stem cells (iPSCs), can be of value to model the effect of a defined mutation in ALS (e.g., see (Pereira, DuBreuil et al. 2021) and reviewed by (Yang, Qian et al. 2025)). However, it must be remembered that these models are models of a developing neuromotor system, and not of a mature neural system. The challenge for researchers will be to age these models to display many of the above molecular switches if they are to be translational to ALS (see review by (Yang, Qian et al. 2025)).

In addition, the challenge is to develop a neuromotor system that carries an epi-genetic signature which is acquired by age, a signature that is lost when generating models from stem cells (Rhine, Li R. et al. 2025). This issue is becoming important in ALS research, where researchers such as Kevin Rhine et al have recently shown that direct programming of a-MNs from adult fibroblasts retain an aged epi-genetic signature when compared to aged human brain neurons, and that this signature is lost in a-MNs generated from iPSCs (Rhine, Li R. et al. 2025). Further, as neurons age, they appear to be depleted of RNA binding proteins, including the ALS-linked protein TDP-43 which becomes accumulated in the cytoplasm (Rhine, Li R. et al. 2025). This redistribution of RNA binding proteins such as TDP-43 is likely to be most important in driving the neural pathology of ALS (Mengistu, Terribili et al. 2025). Whether such aged TDP-43 dependent defects also occur in ALS muscle is not known. 

At the post translational level, proteomic studies using control (non-ALS) and ALS muscle biopsies have revealed that arginine methylation is altered in ALS muscle compared to control muscle (Wong, Blazev et al. 2024), a disturbance that may explain why ALS patients become hypermetabolic (Steyn, Li et al. 2020). These findings provide for future opportunities to explore the function of asymmetric demethylation as a regulator of muscle pathophysiology in ALS. The above descriptions and opinions have been incorporated into a new section 5.6 (see lines 646-697).

 

R1 point 6: Section 3 simply lists biomarkers such as CK and EMG, and even covers MRI. It should specifically address what molecular and pathological changes in muscle are induced by identifying these markers. As it stands, it feels like a simple textbook summary.

Response: We have added more information about the significance of the changes in these biomarkers, linked in with their respective cell and molecular changes (see lines 228-230, 233-241). We have also included a new Figure 4 (lines 268-284) to give this section more impact. This section and its new additions are included to give context to the study of muscle biopsy.

 

R1 point 7: Sections 4 and 5, while interesting, remain mere factual summaries. Figure 4 is appropriate, but more model diagrams like this one are needed. I reviewed this paper for two and a half hours, but most IJMS readers will not spend two and a half hours reading it. For this paper to be helpful to readers and generate a large number of citations, it needs compelling diagrams. I found section 5.4 particularly interesting. A schematic diagram of extracellular vesicles is essential.

Response: We acknowledge the need to be helpful to our readers and recognize that ALS researchers have a wide range of background knowledge. Hence our inclusion of some basic information (see our replies above). We agree that figures are very helpful and have added more. These include the following figures: New Figure 4 (lines, 228, 268-284) that shows the schematic of Human non-invasive assessments (e.g. MRI etc); and New Figure 6 (lines 635, 638-645) that shows a schematic overview of mouse EVs.

 

R1 point 8: Section 4.2 covers concepts such as animal models and fast-twitch fibers. It would be helpful for readers to see the relationship between animal models and human diseases, especially a comparative anatomical diagram between humans and mice. I believe this would be valuable for my teaching materials.

Response: We have added commentary about this in section 4.2. We have stressed that mouse models require inbred strains and that human ALS is heterogenous. We have also noted that mice are smaller and have higher metabolic demands. Nevertheless, the gross anatomical arrangements of most muscles (limb and non-limb) are similar. For example, fibre type 1 grouping seen in ALS muscle (e.g. Ding et al, 2022), is also seen in the ALS SOD1G93A ALS mouse model. We have added these details (see lines 322-331).

R1 point 9: It is necessary to summarize what research has been conducted. on therapeutics and how it relates to clinical trials and therapeutic development.

Response: This topic has been recently reviewed by Gao et al 2024 (DOI: 10.3390/biom14070878) (see lines 710-712). We have extended this review with more recent therapeutic development aimed at stabilizing NMJ muscle proteins Dok7 and MuSK. In the case of MuSK, a first in class human agonist antibody, ARGX-119 is now in Phase 2a clinical trials for ALS (see lines 203-214). See also lines 387-388, 387-388, 543-549, 586-588,593-599 for additional reports on immuno- and molecular-based therapies in ALS.

 

 

References cited in our responses.

Banks, G. B., R. Kanjhan, S. Wiese, M. Kneussel, L. M. Wong, G. O'Sullivan, M. Sendtner, M. C. Bellingham, H. Betz and P. G. Noakes (2005). "Glycinergic and GABAergic synaptic activity differentially regulate motoneuron survival and skeletal muscle innervation." J Neurosci. 25(5): 1249-1259.

Chand, K. K., K. M. Lee, M. P. Schenning, N. A. Lavidis and P. G. Noakes (2015). "Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction." J Physiol. 593(1): 245-265.

Fogarty, M. J., R. Kanjhan, M. C. Bellingham and P. G. Noakes (2016). "Glycinergic Neurotransmission: A Potent Regulator of Embryonic Motor Neuron Dendritic Morphology and Synaptic Plasticity." J Neurosci. 36(1): 80-87.

Ishikawa, T., M. Kaneko, H.-S. Shin and T. Takahashi (2005). "Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice." J Physiol. 568(Pt 1): 199-209.

Liu, Q. and W.-R. M.T-T. (2013). "Postnatal development of Na+-K+-2Cl− co-transporter 1 (NKCC1) and K+-Cl−co-transporter 2 (KCC2) immunoreactivity in multiple brain stem respiratory nuclei of the rat." Brain Res. 1538: 1-16.

Mengistu, D. Y., M. Terribili, C. Pellacani, L. Ciapponi and M. Marzullo (2025). "Epigenetic regulation of TDP-43: potential implications for amyotrophic lateral sclerosis." Front Mol Med. 5: 1530719.

Missias, A. C., G. C. Chu, B. J. Klocke, J. R. Sanes and J. P. Merlie (1996). "Maturation of the acetylcholine receptor in skeletal muscle: regulation of the AChR gamma-to-epsilon." Dev Biol. 179(1): 223-238.

Pereira, J. D., D. M. DuBreuil, A.-C. Devlin, A. Held, Y. Sapir, E. Berezovski, J. Hawrot, K. Dorfman, V. Chander and B. J. Wainger (2021). "Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions." Nat Commun. 12(1): 4744.

Rhine, K., Li R., H. M. Kopalle, K. Rothamel, X. Ge, E. Epstein, O. Mizrahi, A. A. Madrigal, H.-L. Her, T. A. Gomberg, A. Hermann, J. L. Schwartz, A. J. Daniels, U. Manor, J. Ravits, R. A. J. Signer, E. J. Bennett and G. W. Yeo (2025). "Neuronal aging causes mislocalization of splicing proteins and unchecked cellular stress." Nat Neurosci. 28(6): 1174-1184.

Steyn, F. J., R. Li, S. E. Kirk, T. W. Tefera, T. Y. Xie, T. J. Tracey, D. Kelk, E. Wimberger, F. C. Garton, L. Roberts, S. E. Chapman, J. S. Coombes, W. M. Leevy, A. Ferri, C. Valle, F. René, J. P. Loeffler, P. A. McCombe, R. D. Henderson and S. T. Ngo (2020). "Altered skeletal muscle glucose-fatty acid flux in amyotrophic lateral sclerosis." Brain Commun 2(2): fcaa154.

Takahashi, T., A. Momiyama, K. Hirai, F. Hishinuma and H. Akagi (1992). "Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels." Neuron. 9(6): 1155-1161.

Wong, J. P. H., R. Blazev, Y.-K. Ng, C. A. Goodman, M. K. Montgomery, K. I. Watt, C. S. Carl, M. J. Watt, C. T. Voldstedlund, E. A. Richter, P. J. Crouch, F. J. Steyn, S. T. Ngo and P. B.L. (2024). "Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in amyotrophic lateral sclerosis." FASEB J 38(10): e23647.

Yang, J.-L., S.-Y. Qian, M.-L. Chen, L.-X. Wang, Y. Wang, J.-J. Liu, C.-S. Xi, Y.-X. Yang, Y. Li, C. Gao and G.-Q. Zheng (2025). "Skeletal muscle, neuromuscular organoids and assembloids: a scoping review." EBioMedicine. 118: 105825.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The review is a comprehensive body of work, describing both clinical and laboratory based analysis of ALS muscle. The authors should be commended on the quality and the coverage of the literature.

 

There are only a few minor comments that should be addresssed before publication:

1. The Greek letters have not transferred across throughout the manuscript - and instead been replaced with random symbols (spiral e.g. lines 35, 38, 47, 50 etc) and fleur de lis (line 34).
2. The current figures are blurred as included in the pdf - this could be a conversion issue though (same as point 1).
3. The sarcomere should be labelled as such in figure 1.
4. Line 64 reads "As first conceived by Sherrington, a motor unit is the anterior horn cell (-MN), its axon and all the muscle fibres that it innervates [12, 13]." which could be clarified as "As first conceived by Sherrington, a motor unit is composed of the anterior horn cell (-MN), its axon and all the muscle fibres that it innervates [12, 13]."
5. Why did the authors describe the motor unit type as "Type FR (fast, fatigue resistant)" but then use the abbreviation FFR from that point on? Should FFR be changed to FR?
6. Line 106, AMPK, SIRT1 and PGC1(spiral spiral) abbreviations should be in full. 
7. Line 330 and 331 there appears to be some issues with the referencing. Line 330 has only "[2" and line 331 has {Soraru, 2010 #133]. 
8. Line 452 (and potentially other places), the author includes SOD1^{G93A & G86R} which should be written as "SOD1^G93A and SOD1^G86R" as the current form suggests a double mutant, rather than multiple strains from different studies. 
9. Lines 537 - 545 touch on EVs. This could be elaborated slightly to include or compare to prion-like spread of misfolded proteins. (This point can be disregarded if authors prefer as it is not the foucs of the review, and is instead a thought).

Author Response

Reviewer 2 (R2). The review is a comprehensive body of work, describing both clinical and laboratory based analysis of ALS muscle. The authors should be commended on the quality and the coverage of the literature. There are only a few minor comments that should be addressed before publication:

 

R2 point 1: The Greek letters have not transferred across throughout the manuscript - and instead been replaced with random symbols (spiral e.g. lines 35, 38, 47, 50 etc) and fleur de lis (line 34).

            Response: These errors have been corrected, a result of Word doc to PDF conversion (see R1 point 4)

 

R2 point 2: The current figures are blurred as included in the pdf - this could be a conversion issue though (same as point 1).

            Response: We thank our reviewer for pointing this issue out. The poor Figure quality resulted from a conversion issue as our word doc contained sharp images. We have also uploaded high resolution TIFF files in the hope these will be used in the final manuscript assembly.

 

R2 point 3: The sarcomere should be labelled as such in Figure 1

Response: This fix has been done, revised Figure 1.

 

R2 point 4: Line 64 reads "As first conceived by Sherrington, a motor unit is the anterior horn cell (a-MN), its axon and all the muscle fibres that it innervates [12, 13]." which could be clarified as "As first conceived by Sherrington, a motor unit is composed of the anterior horn cell (a-MN), its axon and all the muscle fibres that it innervates [12, 13]."

            Response: We have made this improvement (See line 112)

 

R2 point 5: Why did the authors describe the motor unit type as "Type FR (fast, fatigue resistant)" but then use the abbreviation FFR from that point on? Should FFR be changed to FR?

            Response: We apologize for these typographical mistakes. These have been corrected (see lines 117-121)

 

R2 point 6: Line 106, AMPK, SIRT1 and PGC1alpha abbreviations should be in full. 

Response: We thank our reviewer for pointing out these errors and have now placed these abbreviations in our abbreviation table (line 736) and spelt out these abbreviations when first used (see lines 153-155).

 

R2 point 7: Line 330 and 331 there appears to be some issues with the referencing. Line 330 has only "[2" and line 331 has {Soraru, 2010 #133]. 

            Response: This has been corrected

 

R2 point 8: Line 452 (and potentially other places), the author includes SOD1^{G93A & G86R} which should be written as "SOD1^G93A and SOD1^G86R" as the current form suggests a double mutant, rather than multiple strains from different studies. 

            Response: We apologize for this confusion and corrected this mistake (see lines 430,540,545).

 

R2 point 9: Lines 537 - 545 touch on EVs. This could be elaborated slightly to include or compare to prion-like spread of misfolded proteins. (This point can be disregarded if authors prefer as it is not the foucs of the review and is instead a thought).

            Response: We thank our reviewer for this thought; we have included a figure that shows the details of EVs from muscle and how they may operate on the innervating motor neuron (See our reply to R1 point 7).

 

Author Response File: Author Response.pdf