Review Reports - The Calcium Connection: Explaining Motor Neuron Vulnerability in ALS

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review article by Toth et. al entitled “The calcium connection: explaining motor neuron vulnerability in ALS” is a comprehensive review of the role of calcium signaling in neuronal vulnerability in ALS. The review provides a detailed account of the role and contribution of calcium signaling in pathophysiology of ALS. The review explains in detail the role of calcium in hyperexcitability and excitotoxicity as a toxic as well as neuroprotective mechanism. The review further elaborates on calcium mediated endoplasmic reticulum stress as a pathogenic factor in motor neuron health in ALS. The authors touch upon calcium and toxic protein aggregation in ALS, as well as dysregulation of calcium signaling in glial cells in ALS. Importantly, the authors provide an extensive account of tools and methodologies for studying calcium signaling in ALS.

Overall, this is extensive work reviewing status of calcium signaling mediated neurodegeneration in ALS.

There are some minor revisions suggested that will make the review more appealing, and easy read for the people in the of calcium signaling, especially in ALS.

In section 1.1, a summary table can be generated for different genes and their role in ALS. This will help readers grasp key information quickly.
In section 1.2, a diagram of motor neuron circuitry or something similar can be provided. This visual presentation will make the review more appealing.
For sections 2 and 3, again, a table with key information would really help the readers as there is a lot of information in these sections, and it is too heavy on words.
In section 7, maybe a diagram for each technique can be provided. This will help readers understand the technical aspects of each methodology.
There are some reports of the role of calcineurin (PMID: 41485061), which is an important molecule in calcium signaling, in ALS. This information can be included to make the review more attractive.

Author Response

We thank the reviewers for their helpful and constructive comments to improve the manuscript. We have implemented all the suggestions and believe the manuscript has substantially improved. We hope the reviewers find the manuscript acceptable for publication. We have highlighted within the manuscript Reviewer 1 changes in cyan and Reviewer 3 changes are highlighted in green.

Reviewer 1

Relevant text changes highlighted in cyan

In section 1.1, a summary table can be generated for different genes and their role in ALS. This will help readers grasp key information quickly.

We thank the reviewer for this suggestion. A table has been created describing the key ALS causative genes.

In section 1.2, a diagram of motor neuron circuitry or something similar can be provided. This visual presentation will make the review more appealing.

A schematic has been added depicting motor neuron circuitry involving both the motor cortex and the spinal cord (Figure 2) .

For sections 2 and 3, again, a table with key information would really help the readers as there is a lot of information in these sections, and it is too heavy on words.

We appreciate this suggestion and have added Tables for both sections 2 and 3. The first table summarizes the sources of hyperexcitability in ALS. The second table summarizes instances of hyperexcitability being found to be neuroprotective in ALS.

In section 7, maybe a diagram for each technique can be provided. This will help readers understand the technical aspects of each methodology.

We agree with this valid suggestion and have added a diagram, summarizing the different imaging tools and fluorescence-based sensors that can be applied to in vivo imaging of neural activity in human ALS and ALS models (Figure 3).

There are some reports of the role of calcineurin (PMID: 41485061), which is an important molecule in calcium signaling, in ALS. This information can be included to make the review more attractive.

We are grateful for pointing to us regarding this recent research. A brief overview of the role of calcineurin in ALS has been added to section 3 (highlighted in turquoise) and added to a table summarizing targets for new potential ALS treatments.

Reviewer 2 Report

Comments and Suggestions for Authors

In the review titled “The Calcium connection: Explaining Motor Neuron vulnerability in ALS” by Toth et al., the authors had discussed the current evidence on calcium imbalance contribution to motor neuronal vulnerability in ALS. The review is nicely structured by providing current state of studies and discuused very well on the aspect of hyperexcitabiity driven by calcium, in both human iPSC derived and animal models of ALS. Further the authors have discussed the different tools that could be used for analyzing calcium currents in motor neurons.

Author Response

Reviewer 2

In the review titled “The Calcium connection: Explaining Motor Neuron vulnerability in ALS” by Toth et al., the authors had discussed the current evidence on calcium imbalance contribution to motor neuronal vulnerability in ALS. The review is nicely structured by providing current state of studies and discuused very well on the aspect of hyperexcitabiity driven by calcium, in both human iPSC derived and animal models of ALS. Further the authors have discussed the different tools that could be used for analyzing calcium currents in motor neurons.

We thank the reviewer for positively summarizing our review and appreciate the reviewer for highlighting the importance of our review to the field of ALS.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Thank you very much for the opportunity to revise this manuscript entitled: "The Calcium Connection: Explaining Motor Neuron Vulnerability in ALS"; this manuscript provides a comprehensive and timely narrative review of the role of calcium dysregulation in selective motor neuron vulnerability in ALS. Overall, the review is well written, logically structured, and covers a wide range of relevant literature, including recent work (2023–2025). The topic is highly appropriate for Cells and of interest to both basic and translational ALS researchers. However, the manuscript would benefit from improved conceptual clarity in several sections. Some sections are overly long and could be streamlined, while others would benefit from clearer figures or summary tables. Below, I outline major and minor comments.

Major comments

1-Clarify the central conceptual framework:

The review presents two partially conflicting models: (i) calcium-driven hyperexcitability as a primary pathogenic mechanism leading to excitotoxicity, and (ii) increased excitability/calcium signaling as an early compensatory and neuroprotective response. While both views are supported by the literature, the manuscript would benefit from a clearer unifying framework. I recommend one of this options: a) Explicitly distinguishing temporal stages (early compensatory vs. late maladaptive calcium signaling), or, b) Clarifying whether these mechanisms differ primarily by disease stage, motor neuron subtype (FF vs. S), genetic background (e.g., SOD1 vs. C9ORF72), or circuit context. In addition, please, try adding a short schematic or summary paragraph that reconciles these models.

2-Some sections (notably Sections 2 and 3) read as sequential literature summaries without sufficient critical comparison. For example: contradictory findings on hyperexcitability in SOD1 models are reported but not fully reconciled. The relative weight of evidence from iPSC models versus in vivo rodent models is not discussed. The authors should more explicitly discuss limitations of each model system and potential reasons for discrepant findings.

3- Section 6 is important but somewhat descriptive. Given the increasing relevance of non–cell-autonomous mechanisms in ALS, I suggest expanding on how glial calcium dysregulation feeds back onto motor neuron excitability and degeneration in a causal manner. In addition, please, clarify whether glial calcium alterations precede, follow, or co-evolve with neuronal dysfunction. Finally, explicitly link glial calcium signaling to therapeutic opportunities (e.g., KCa3.1, SOCE modulation).

4-Therapeutic strategies are mentioned throughout but not systematically discussed. Consider adding a short dedicated subsection or summary table outlining calcium-related therapeutic strategies. In addition, a clear acknowledgment of translational challenges and past failures of calcium-targeting strategies in ALS.

5-The manuscript is very long for a narrative review. Some sections (especially Section 7 on imaging technologies) are detailed to a level that may not be essential for the main biological message. Consider condensing methodological descriptions and focusing more explicitly on how these tools have changed conceptual understanding of ALS pathophysiology.

Minor comments:

1-Ensure consistent use of terms such as “hyperexcitability,” “increased excitability,” and “elevated calcium signaling.”

2-Correct minor typographical issues (e.g., duplicated punctuation, occasional encoding artifacts such as mis-rendered characters, etc).

3-Figure 1 is helpful but quite general. Consider adding labels or temporal annotations (early vs. late ALS) to improve interpretability.

4-A second figure or table summarizing motor neuron subtype vulnerability versus calcium handling properties would be valuable.

5-In addition, a figure in the section 1.2. ALS symptomology would help to improve the flow.

6-A few key claims (e.g., calcium elevation reversing TDP-43 aggregation) would benefit from additional context or cautious wording, as these findings are model-dependent.

Author Response

Reviewer 3:

Relevant text additions highlighted in green.

Major comments

Clarify the central conceptual framework:

The review presents two partially conflicting models: (i) calcium-driven hyperexcitability as a primary pathogenic mechanism leading to excitotoxicity, and (ii) increased excitability/calcium signaling as an early compensatory and neuroprotective response. While both views are supported by the literature, the manuscript would benefit from a clearer unifying framework. I recommend one of this options: a) Explicitly distinguishing temporal stages (early compensatory vs. late maladaptive calcium signaling), or, b) Clarifying whether these mechanisms differ primarily by disease stage, motor neuron subtype (FF vs. S), genetic background (e.g., SOD1 vs. C9ORF72), or circuit context. In addition, please, try adding a short schematic or summary paragraph that reconciles these models.

We thank the reviewer for this insightful comment. The text has been updated to make clearer the framework of how calcium-driven hyperexcitability has been shown to act as both an early-stage compensatory mechanism and a later stage maladaptive mechanism and a summary section reconciling the models has been added.

2 Some sections (notably Sections 2 and 3) read as sequential literature summaries without sufficient critical comparison. For example: contradictory findings on hyperexcitability in SOD1 models are reported but not fully reconciled. The relative weight of evidence from iPSC models versus in vivo rodent models is not discussed. The authors should more explicitly discuss limitations of each model system and potential reasons for

We agree with the valid comment and changes to the text have been made to comment on conflicts between different studies and a table summarizing the different advantages and disadvantages of in vitro iPSC vs in vivo rodent models has been added.

Section 6 is important but somewhat descriptive. Given the increasing relevance of non–cell-autonomous mechanisms in ALS, I suggest expanding on how glial calcium dysregulation feeds back onto motor neuron excitability and degeneration in a causal manner. In addition, please, clarify whether glial calcium alterations precede, follow, or co-evolve with neuronal dysfunction. Finally, explicitly link glial calcium signaling to therapeutic opportunities (e.g., KCa3.1, SOCE modulation).

We have taken note of this suggestion by expanding on the connection between glial cell calcium dysregulation and neuron excitability and degeneration. The temporal order of glial dysregulation and neural dysregulation has been further discussed. Therapeutic opportunities linked to glial calcium signaling have been included in the text and the final table.

Therapeutic strategies are mentioned throughout but not systematically discussed. Consider adding a short dedicated subsection or summary table outlining calcium-related therapeutic strategies. In addition, a clear acknowledgment of translational challenges and past failures of calcium-targeting strategies in ALS.

A summary table of potential therapeutic strategies targeting calcium has been added. The challenges of existing drugs that affect calcium activity in ALS like riluzole lomerizine, pimozide have been added/expanded.

The manuscript is very long for a narrative review. Some sections (especially Section 7 on imaging technologies) are detailed to a level that may not be essential for the main biological message. Consider condensing methodological descriptions and focusing more explicitly on how these tools have changed conceptual understanding of ALS pathophysiology.

Section 7 has had a flow-chart diagram added that condenses the information on imaging tools and technologies into an easy-to-read format. Where possible, we have attempted to streamline the text without compromising the content of the manuscript or losing essential information.

Minor comments:

1 Ensure consistent use of terms such as “hyperexcitability,” “increased excitability,” and “elevated calcium signaling.”

Thank you for pointing out this discrepancy. We have improved consistency with these terms and changed to hyperexcitability throughout the manuscript.

2 Correct minor typographical issues (e.g., duplicated punctuation, occasional encoding artifacts such as mis-rendered characters, etc).

We have read the manuscript to correct minor typographical errors

3 Figure 1 is helpful but quite general. Consider adding labels or temporal annotations (early vs. late ALS) to improve interpretability.

We have added those tie lines in the figure and it is currently Fig. 2

4 A second figure or table summarizing motor neuron subtype vulnerability versus calcium handling properties would be valuable.

Thank you for this comment, we have inserted a table no. 2 explaining motor neuro subtype vulnerability versus calcium handling properties.

5 In addition, a figure in the section 1.2. ALS symptomology would help to improve the flow.

A figure has been added to this section

A few key claims (e.g., calcium elevation reversing TDP-43 aggregation) would benefit from additional context or cautious wording, as these findings are model-dependent.

We have attempted to provide additional context to claims in different research studies discussed in the manuscript and those are highlighted in green