NG2 Glia and Cellular Crosstalk in Health and Disease: Focus on Spinal Cord Injury

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review article entitled “NG2 Glia and Cellular Crosstalk in Health and Disease: Focus on Spinal Cord Injury” summarizes current perspectives on the involvement of NG2 glia/OPCs in spinal cord injury (SCI), with particular attention to their interactions with neurons, astrocytes, microglia, and vascular elements. The subject matter is relevant and of considerable interest, and the manuscript appropriately emphasizes that NG2 glia may function not only as oligodendrocyte progenitor cells but also as active modulators of the injured CNS environment. Overall, the manuscript is accessible and informative and should attract readership within the fields of neurobiology and SCI research.
Nevertheless, several points merit further consideration in order to enhance the overall coherence and mechanistic insight of the review.

Major concerns
1. Insufficient consideration of temporal dynamics after SCI
The manuscript describes numerous NG2 glial responses and intercellular interactions following SCI; however, these processes are discussed mostly in a static manner. Because SCI pathology changes substantially over time, the biological roles of NG2 glia are also expected to vary across acute, subacute, and chronic stages. Introducing a clearer stage-dependent perspective would improve the logical flow of the review and help contextualize the relevance of specific cellular interactions and phenotypic changes.
Importantly, this issue could likely be addressed without extensive revision by incorporating a schematic timeline or summary table outlining phase-specific NG2 glial functions and associated cellular events after SCI.

2. Greater integration of the “cellular crosstalk” section would strengthen the manuscript
At present, interactions between NG2 glia and individual cell populations (neurons, astrocytes, microglia, endothelial cells, etc.) are largely presented separately. Although informative, this organization occasionally gives the review a descriptive rather than integrative character. The manuscript may benefit from framing these interactions according to broader functional themes, such as inflammatory modulation, regenerative/remyelinating processes, or vascular/metabolic support.
In addition, a conceptual illustration summarizing the relationships among NG2 glia and surrounding cellular components within the SCI microenvironment would improve readability and provide readers with a more unified overview of the proposed mechanisms.

3. Limited discussion of axon-derived regulatory mechanisms involved in OPC differentiation and remyelination
The review discusses neuronal activity and neuron–NG2 synaptic communication; however, comparatively less attention is given to axon-derived signals that directly influence OPC maturation and remyelination. In particular, contact-mediated axon–glia interactions and regulatory roles of axonal surface-associated molecules are only briefly addressed. Expanding this aspect would provide a more comprehensive mechanistic framework for understanding remyelination failure and repair after SCI.

Minor points
1. The discussion regarding neuronal differentiation potential of NG2 glia would benefit from clearer distinction between in vitro findings, lineage-tracing studies, and definitive in vivo functional evidence.

2. Recent progress in single-cell and spatial transcriptomic approaches is mentioned briefly; however, additional discussion of NG2 glial heterogeneity in the context of SCI may further strengthen the review.

3. The addition of summary figures and/or tables illustrating:
   (i) temporal progression after SCI,
   (ii) reparative versus inhibitory functions of NG2 glia, and
   (iii) principal intercellular signaling networks,
   would greatly improve the clarity and educational value of the manuscript for a broad audience.

In summary, this review addresses an important and evolving area in SCI biology. Consideration of the points above would further enhance the manuscript and increase its value as a comprehensive overview of NG2 glial biology in spinal cord injury.

Author Response

Reviewer 1

The review article entitled “NG2 Glia and Cellular Crosstalk in Health and Disease: Focus on Spinal Cord Injury” summarizes current perspectives on the involvement of NG2 glia/OPCs in spinal cord injury (SCI), with particular attention to their interactions with neurons, astrocytes, microglia, and vascular elements. The subject matter is relevant and of considerable interest, and the manuscript appropriately emphasizes that NG2 glia may function not only as oligodendrocyte progenitor cells but also as active modulators of the injured CNS environment. Overall, the manuscript is accessible and informative and should attract readership within the fields of neurobiology and SCI research.
Nevertheless, several points merit further consideration in order to enhance the overall coherence and mechanistic insight of the review.

Major concerns
1. Insufficient consideration of temporal dynamics after SCI
The manuscript describes numerous NG2 glial responses and intercellular interactions following SCI; however, these processes are discussed mostly in a static manner. Because SCI pathology changes substantially over time, the biological roles of NG2 glia are also expected to vary across acute, subacute, and chronic stages. Introducing a clearer stage-dependent perspective would improve the logical flow of the review and help contextualize the relevance of specific cellular interactions and phenotypic changes.
Importantly, this issue could likely be addressed without extensive revision by incorporating a schematic timeline or summary table outlining phase-specific NG2 glial functions and associated cellular events after SCI.

Authors: We thank the reviewer for this important and constructive suggestion. To address this point, we added a new subsection entitled “Temporal dynamics of NG2 glial responses after spinal cord injury” at the beginning of the SCI-focused part of the manuscript. In this subsection, we now discuss phase-specific NG2 glial responses, including early activation and responsiveness to glutamatergic and inflammatory signals during the acute phase, participation in scar formation, extracellular matrix remodeling, and remyelination attempts during the subacute phase, and persistent context-dependent roles in chronic inflammation, incomplete remyelination, axonal dieback, and long-term tissue remodeling during the chronic phase. In addition, we added a new summary table presenting the stage-dependent roles of NG2 glia after SCI. The table outlines the approximate timing of each phase, dominant pathological events, NG2 glial responses, major cellular interactions, and functional implications.

2. Greater integration of the “cellular crosstalk” section would strengthen the manuscript
   At present, interactions between NG2 glia and individual cell populations (neurons, astrocytes, microglia, endothelial cells, etc.) are largely presented separately. Although informative, this organization occasionally gives the review a descriptive rather than integrative character. The manuscript may benefit from framing these interactions according to broader functional themes, such as inflammatory modulation, regenerative/remyelinating processes, or vascular/metabolic support.
   In addition, a conceptual illustration summarizing the relationships among NG2 glia and surrounding cellular components within the SCI microenvironment would improve readability and provide readers with a more unified overview of the proposed mechanisms.

Authors: We thank the reviewer for this helpful suggestion. In the original manuscript, we included a summary table and a conceptual figure illustrating NG2 glia-centered interactions with neurons, astrocytes, microglia, neutrophils, and extracellular matrix-associated mechanisms. However, we agree that the integrative logic of this section could be made clearer in the text. To address this point, we revised the end of the cellular crosstalk section to explicitly frame NG2 glial interactions according to broader functional axes, including inflammatory modulation, regenerative/remyelinating processes, extracellular matrix and scar remodeling, axonal growth inhibition, and vascular/metabolic support. We also revised the wording around Figure 1 and its title to emphasize that it provides an integrated NG2 glia-centered overview of cellular crosstalk within the perilesional SCI microenvironment. These changes were made to improve the coherence of the section without duplicating an already included conceptual illustration.

3. Limited discussion of axon-derived regulatory mechanisms involved in OPC differentiation and remyelination
   The review discusses neuronal activity and neuron–NG2 synaptic communication; however, comparatively less attention is given to axon-derived signals that directly influence OPC maturation and remyelination. In particular, contact-mediated axon–glia interactions and regulatory roles of axonal surface-associated molecules are only briefly addressed. Expanding this aspect would provide a more comprehensive mechanistic framework for understanding remyelination failure and repair after SCI.

Authors: We thank the reviewer for this important suggestion. To address this point, we expanded the section on neuron-NG2 glia communication by adding a new paragraph focused on axon-derived regulation of OPC maturation and remyelination. In the revised text, we now discuss that remyelination depends not only on the intrinsic capacity and availability of NG2 glia, but also on the state of denuded or spared axons and the signals they provide. We also discuss how, after SCI, inflammatory mediators, scar-associated extracellular matrix components, and NG2/CSPG4-rich inhibitory environments may interfere with productive axon-OPC interactions and contribute to remyelination failure.

Minor points
1. The discussion regarding neuronal differentiation potential of NG2 glia would benefit from clearer distinction between in vitro findings, lineage-tracing studies, and definitive in vivo functional evidence.

Authors: We thank the reviewer for this important clarification. In the revised manuscript, we modified the relevant paragraph to separate:

• in vitro plasticity and experimentally induced neuronal phenotypes
• genetic fate-mapping and lineage-tracing studies showing that NG2 glia are predominantly committed to the oligodendrocyte lineage under physiological conditions
• the limited evidence for spontaneous generation of functionally integrated neurons from endogenous NG2 glia after SCI.

The revised text presents neuronal differentiation as a context-dependent and experimentally inducible phenomenon, while maintaining that the principal and best-established role of NG2 glia in the adult spinal cord is oligodendrocyte lineage progression and remyelination.

2. Recent progress in single-cell and spatial transcriptomic approaches is mentioned briefly; however, additional discussion of NG2 glial heterogeneity in the context of SCI may further strengthen the review.

Authors: We thank the reviewer for this helpful suggestion. In the revised manuscript, we expanded the SCI-focused section by adding a paragraph on recent single-cell and spatial transcriptomic findings. We now discuss evidence that glial progenitor/OPC responses after SCI are heterogeneous and include injury-associated states related to proliferation, inflammatory responsiveness, and oligodendrocyte lineage progression. We also refer to single-cell studies of the injured spinal cord showing that NG2 glia should be interpreted within a broader multicellular injury niche involving gliosis, angiogenesis, fibrosis, and ligand-receptor interactions. In addition, we added a brief discussion of spatial heterogeneity, emphasizing that NG2 glia located in the lesion core, glial border, perilesional white matter, or remote spinal cord regions may be exposed to distinct combinations of inflammatory, axonal, vascular, and extracellular matrix cues.

3. The addition of summary figures and/or tables illustrating:
    (i) temporal progression after SCI,
    (ii) reparative versus inhibitory functions of NG2 glia, and
      (iii) principal intercellular signaling networks,
      would greatly improve the clarity and educational value of the manuscript for a broad audience.

Authors: We thank the reviewer for this useful suggestion.

• The temporal progression of NG2 glial responses after SCI is now addressed in the newly added stage-dependent summary table 1, which outlines acute, subacute, and chronic phases of SCI, including major pathological events, NG2 glial responses, cellular interactions, and functional implications.
• To more clearly distinguish reparative and inhibitory functions of NG2 glia, we revised the existing functional summary table 2. Specifically, we added a separate column indicating the functional orientation of each process. This modification allows readers to distinguish functions related to remyelination, lesion containment, and vascular support from those associated with CSPG-rich extracellular matrix remodeling, axonal growth restriction, and axonal trap formation.
• Regarding principal intercellular signaling networks, the manuscript already included a conceptual figure summarizing NG2 glia-centered interactions within the perilesional SCI microenvironment. We revised the title and explanatory text of this figure to make its integrative purpose clearer and to emphasize that it summarizes experimentally supported and proposed signaling relationships among NG2 glia, neurons/axons, astrocytes, microglia/macrophages, neutrophil-associated matrix remodeling mechanisms, and extracellular matrix components. We believe that these revisions improve the accessibility of the manuscript without adding redundant visual material.

In summary, this review addresses an important and evolving area in SCI biology. Consideration of the points above would further enhance the manuscript and increase its value as a comprehensive overview of NG2 glial biology in spinal cord injury.

Authors: We thank the reviewer for the positive assessment and constructive recommendations. We have revised the manuscript accordingly and believe that the changes have improved its coherence, mechanistic focus, and educational value.

Reviewer 2 Report

Comments and Suggestions for Authors

This review by Ilyas Kabdesh et al. provides a comprehensive overview of the multifaceted roles of NG2 glia in spinal cord injury, with particular emphasis on cellular crosstalk and therapeutic implications. The manuscript is generally well organized and supported by extensive literature. I recommend this manuscript for publication in Pathophysiology after the authors address the following concerns.

Major Issues

1. The authors state in both Table 1 and Figure 1 that “some mentioned mechanisms are derived from related CNS contexts and may not yet be fully validated in the spinal cord.” However, since this review focuses on spinal cord injury, the manuscript would benefit from a clearer distinction between mechanisms that have been directly demonstrated in SCI models and those extrapolated from other CNS contexts. The authors may consider adding an additional row in Table 1 indicating the specific experimental context or disease model in which each mechanism was validated.

2. The authors mentioned the heterogeneity of NG2 cells in both Part 1 and the Conclusion section. From my understanding, the manuscript mainly describes NG2 cell heterogeneity in the normal environment through their interactions with neurons, astrocytes, and microglia.

1) I suggest adding a table in Part 1 to clearly summarize the characteristics and functions of different NG2 cell subtypes under physiological conditions.

2) In the later sections, the manuscript does not clearly discuss the heterogeneity and functional diversity of NG2 cells under pathological conditions. Considering the substantial amount of sequencing data and related studies published over the past five years, the authors may consider expanding the discussion to summarize the characteristics and potential functions of the heterogeneity of NG2 cells in pathological environments, particularly in spinal cord injury and other CNS disorders.

Minor Comments

1. The statement in Line 597-600 saying “Authors should discuss the results...” is confusing. Please delete it.

2. The formatting of Table 1 should be improved for readability, as several words are fragmented and alignment is inconsistent. I recommend reducing the font size to optimize the table layout and improve overall readability.

Author Response

Reviewer 2

This review by Ilyas Kabdesh et al. provides a comprehensive overview of the multifaceted roles of NG2 glia in spinal cord injury, with particular emphasis on cellular crosstalk and therapeutic implications. The manuscript is generally well organized and supported by extensive literature. I recommend this manuscript for publication in Pathophysiology after the authors address the following concerns.

Authors: We sincerely thank the reviewer for the positive evaluation of our manuscript and for recommending it for publication after revision. We appreciate the reviewer’s recognition of the comprehensiveness, organization, and literature support of the review. The comments were helpful for improving the clarity, accuracy, and readability of the manuscript. We have carefully addressed each point and revised the manuscript accordingly.

Major Issues

1. The authors state in both Table 1 and Figure 1 that “some mentioned mechanisms are derived from related CNS contexts and may not yet be fully validated in the spinal cord.” However, since this review focuses on spinal cord injury, the manuscript would benefit from a clearer distinction between mechanisms that have been directly demonstrated in SCI models and those extrapolated from other CNS contexts. The authors may consider adding an additional row in Table 1 indicating the specific experimental context or disease model in which each mechanism was validated.

Authors: We thank the reviewer for this important and helpful comment. To address this point, we revised the functional summary table 2 by adding a new column entitled “Primary evidence context.” This column specifies whether each mechanism is supported mainly by SCI models, SCI lineage-tracing studies, broader CNS demyelination/remyelination literature, in vitro co-culture systems, related CNS inflammation models, or CNS vascular/BBB studies.

2. The authors mentioned the heterogeneity of NG2 cells in both Part 1 and the Conclusion section. From my understanding, the manuscript mainly describes NG2 cell heterogeneity in the normal environment through their interactions with neurons, astrocytes, and microglia.

Authors: We thank the reviewer for this helpful observation. This point has now been addressed by expanding the SCI-focused section with a paragraph on injury-associated NG2 glial heterogeneity based on recent single-cell and spatial transcriptomic studies. In the revised text, we emphasize that NG2 glial heterogeneity after SCI should be interpreted not only as baseline regional and functional diversity, but also as an injury-associated and spatially dependent phenomenon shaped by lesion compartment, inflammatory environment, extracellular matrix composition, vascular cues, and interactions with spared or dystrophic axons. This revision clarifies the relevance of NG2 glial heterogeneity specifically to SCI.

3. I suggest adding a table in Part 1 to clearly summarize the characteristics and functions of different NG2 cell subtypes under physiological conditions.

Authors: We thank the reviewer for this helpful suggestion. At the same time, because the main focus of the review is spinal cord injury and because new summary elements have already been added to clarify SCI-related temporal dynamics and functional outcomes, we aimed to avoid overloading the manuscript with an additional table focused on the intact CNS. To address the reviewer’s concern while preserving the SCI-centered structure of the review, we revised Part 1 by adding a summary paragraph that synthesizes the major physiological classifications of NG2 glia, including parenchymal, perivascular, intermediate, gray matter-associated, and white matter-associated NG2 glia. The revised text clarifies their main localization, functional features, and limitations, and emphasizes that these categories are not mutually exclusive but reflect anatomical localization, molecular state, and functional specialization. We believe that this modification improves clarity without reducing the focus of the manuscript on SCI.

4. In the later sections, the manuscript does not clearly discuss the heterogeneity and functional diversity of NG2 cells under pathological conditions. Considering the substantial amount of sequencing data and related studies published over the past five years, the authors may consider expanding the discussion to summarize the characteristics and potential functions of the heterogeneity of NG2 cells in pathological environments, particularly in spinal cord injury and other CNS disorders.

Authors: We thank the reviewer for this important suggestion. To address this point without shifting the focus of the review away from SCI, we expanded the SCI-focused section by adding a paragraph on recent single-cell and spatially resolved evidence. The revised text now emphasizes that NG2 glia after SCI do not represent a uniform reactive population, but instead acquire injury-associated and spatially dependent states related to proliferation, inflammatory responsiveness, oligodendrocyte lineage progression, and interactions with the multicellular lesion niche. We also clarify that NG2 glial states may differ between the lesion core, glial border, perilesional white matter, and remote spinal segments depending on exposure to inflammatory, axonal, vascular, and extracellular matrix cues. This revision summarizes the characteristics and potential functions of NG2 glial heterogeneity under pathological conditions, with emphasis on SCI.

Minor Comments

1. The statement in Line 597-600 saying “Authors should discuss the results...” is confusing. Please delete it.

Authors: We thank the reviewer for noticing this issue. The template text was inadvertently retained and has now been deleted.

2. The formatting of Table 1 should be improved for readability, as several words are fragmented and alignment is inconsistent. I recommend reducing the font size to optimize the table layout and improve overall readability.

Authors: We thank the reviewer for this helpful suggestion. The table was expanded in response to comments from another reviewer to include additional information on functional orientation and evidence context. To maintain readability after this expansion, we revised the table layout by increasing spacing between rows and columns, improving alignment, and correcting fragmented words. These formatting changes were made to optimize the table presentation and make the summarized information easier to read.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the authors’ detailed revisions and responses to the reviewers’ comments. The manuscript has been substantially improved and is now suitable for publication in Pathophysiology.