Neurodegenerative Diseases in Children: A Comprehensive Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors synthesize current knowledge regarding the epidemiology, molecular classification, pathophysiology, and emerging therapeutic strategies of major pediatric neurodegenerative disorders. The groups discussed include lysosomal storage disorders, leukodystrophies, mitochondrial encephalopathies, peroxisomal disorders, and other monogenic neurodegenerative syndromes.

The manuscript is well written and provides useful information to clinicians. I have some suggestions to improve the manuscript:

1) 3 (“Step-by-Step Diagnostic Algorithm”) seems to be not very clear. The illustrated flowchart needs to be implemented. Why “comprehensive genetic panel” is alternative to “mitochondrial studies” and “lysosomal enzymes”? Moreover, the relation between WES/WGS and the boxes abos should be better explained.

2) In paragraph 5.1.1. (Clinical evaluation) the manuscript could be improved by underlining also the pivotal role of anamnestic familial anamnesis of patients; this is very important on the light of the emerging knowledge about a possible clinical role of heterozygous variants in gene usually associated with recessive conditions (example: for NPC1 see PMID: 37989569 and 41216805).

3) The section “5.3. Genetic Testing” should also include a clear description of the currently available genetic tests in mitochondrial disorders (see PMID: 36513735)

Author Response

Authors' response to the review report (Reviewer 1)

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have revised the manuscript accordingly and address each point below.

Reviewer’s comment 1:
Figure 3 (“Step-by-Step Diagnostic Algorithm”) does not appear to be sufficiently clear. The illustrated flowchart should be improved. Why is the “comprehensive genetic panel” presented as an alternative to “mitochondrial studies” and “lysosomal enzymes”? Moreover, the relationship between WES/WGS and the boxes above should be better explained.

Response:
We thank the Reviewer for this constructive and helpful comment. In response, we have revised Figure 3 to improve its clarity and overall structure. In particular, we clarified the relationship between biochemical investigations, including mitochondrial studies and lysosomal enzyme testing, and the use of comprehensive genetic panels. We also better explained the position and role of WES/WGS within the diagnostic workflow, in order to make the algorithm more intuitive and clinically applicable.

Reviewer’s comment 2:
In paragraph 5.1.1 (“Clinical evaluation”), the manuscript could be improved by also underlining the pivotal role of family history. This is particularly important in light of emerging knowledge regarding the possible clinical relevance of heterozygous variants in genes usually associated with recessive conditions (for example, for NPC1, see PMID: 37989569 and 41216805).

Response:
We thank Reviewer 1 for this important suggestion. Section 5.1.1 has been revised to emphasize the pivotal role of detailed family history in the clinical evaluation of pediatric neurodegenerative disorders. We also highlighted its relevance in the context of emerging evidence suggesting that heterozygous variants in genes classically associated with recessive conditions, such as NPC1, may in some cases have clinical or disease-modifying significance.

Reviewer’s comment 3:
Section 5.3 (“Genetic Testing”) should also include a clear description of the currently available genetic tests for mitochondrial disorders (see PMID: 36513735).

Response:
We thank Reviewer 1 for this helpful suggestion. Section 5.3 has been revised to include a clearer description of the genetic testing strategies currently available for mitochondrial disorders, including full mtDNA sequencing, nuclear mitochondrial gene panels, combined mtDNA/nuclear testing, and the role of WES/WGS in unresolved or genetically heterogeneous cases. We also clarified that tissue selection may be important, since mtDNA heteroplasmy can vary across different sample types.

Submission Date: 06 March 2026
Date of this review: 11 March 2026, 15:03:51
Date of the response: 17 April 2026

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This review provides a broad overview of pediatric neurodegenerative diseases (NDDs), covering major etiological categories including lysosomal storage disorders, peroxisomal diseases, and mitochondrial disorders. The authors appropriately emphasize that pediatric neurodegeneration occurs in the context of ongoing neurodevelopment, which distinguishes it from adult-onset conditions.The manuscript is generally well organized and informative. However, in its current form, it remains somewhat descriptive rather than analytical, and several sections do not fully reflect the current state of the field, particularly with respect to diagnostic strategies, disease-specific therapies, and translational advances.

In addition, some important emerging concepts (e.g., neurodevelopmental vulnerability, biomarker-driven monitoring, and next-generation therapeutic approaches) are only briefly mentioned or not sufficiently developed. Addressing these points would substantially strengthen the manuscript and improve its value as a comprehensive review.

Major Comments

1. Treatment section requires greater precision and disease-specific detail. In fact, the current treatment section is relatively general and, at times, lacks sufficient technical precision. In particular, different gene therapy approaches are discussed together without clearly distinguishing between platforms, which may lead to conceptual ambiguity. It would further improve clarity if the authors explicitly differentiate between in vivo AAV-based gene therapy (e.g., spinal muscular atrophy; PMID: 33743238), ex vivo lentiviral hematopoietic stem cell gene therapy (e.g., metachromatic leukodystrophy; PMID: 35065785, and cerebral X-linked adrenoleukodystrophy; PMID: 34424497), and enzyme replacement therapy (ERT) including intrathecal delivery approaches (e.g., CLN2 disease; PMID: 29688815; long-term data PMID: 38101904). In addition, the inclusion of a concise disease-specific summary table outlining the disorder, target pathway, therapeutic modality, delivery platform, and optimal treatment window would significantly enhance the clarity and usefulness of this section.
2. Newborn screening and presymptomatic management should be expanded. Although newborn screening is mentioned, the discussion remains relatively brief and does not fully reflect its current clinical importance. More specifically, the manuscript would benefit from a clearer distinction between disorders with established screening frameworks, such as X-linked adrenoleukodystrophy (PMID: 36175155; 33373467), and conditions in which early or presymptomatic treatment is critical for outcome, such as metachromatic leukodystrophy (PMID: 38554683). It would also be helpful to emphasize that early diagnosis often requires structured longitudinal surveillance, including MRI and endocrine monitoring, rather than immediate intervention in all cases. Importantly, highlighting the central role of treatment timing would further strengthen the clinical relevance of this section.
3. The manuscript acknowledges the emergence of genome-first strategies; however, the diagnostic workflow still largely reflects a traditional stepwise approach. In current clinical practice, earlier implementation of genomic testing is increasingly recommended. The authors may therefore consider revising this section to reflect the earlier use of exome or genome sequencing (PMID: 31182824; 34211152), including the role of trio sequencing, as well as the integration of copy-number analysis, mitochondrial DNA testing, and periodic reanalysis. At the same time, targeted metabolic testing remains important in cases where clinical or imaging features suggest potentially treatable conditions. Supporting evidence from genetic white matter disorder cohorts further underscores the value of early genomic testing as a first-line approach (PMID: 32342562).
4. Biomarkers are mentioned only briefly, although they are now central to both clinical management and therapeutic development. In this context, emerging blood-based biomarkers, particularly neurofilament light chain (NfL), have demonstrated utility as indicators of neuroaxonal injury and treatment response. Including disease-specific examples of biomarker application, such as in leukodystrophies or spinal muscular atrophy (PMID: 33753741; 35106137), would further strengthen the translational perspective of the review.
5. The interaction between neurodevelopment and neurodegeneration is an important conceptual framework that could be more clearly articulated. Expanding the pathophysiology section to describe how early developmental disturbances, such as impaired synaptic maturation or neuronal differentiation, may create a vulnerable neural substrate that predisposes to subsequent degeneration would add important depth to the manuscript.
6. While lysosomal dysfunction is discussed, broader aspects of proteostasis are less well represented. A brief expansion to include the role of the ubiquitin-proteasome system, chaperone-mediated autophagy, and protein aggregation across different pediatric neurodegenerative disorders would help unify mechanistic themes across disease categories.
7. The treatment section could also be updated to acknowledge more recent therapeutic approaches, including antisense oligonucleotides (ASOs), gene editing strategies, and individualized or patient-specific therapies. Even a concise discussion of these developments would better reflect the rapidly evolving therapeutic landscape.

Minor Comments

• Please ensure consistent terminology when referring to therapeutic modalities (e.g., gene therapy, AAV-based therapy, HSCT, ERT).
• Some sections, particularly in the pathophysiology discussion, could be slightly condensed to reduce repetition.
• A schematic figure summarizing shared molecular pathways (e.g., mitochondrial dysfunction, oxidative stress, proteostasis) would improve readability.

Author Response

Authors' response to the review report (Reviewer 2)

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have revised the manuscript accordingly and address each point below.

This review provides a broad overview of pediatric neurodegenerative diseases (NDDs), covering major etiological categories including lysosomal storage disorders, peroxisomal diseases, and mitochondrial disorders. The authors appropriately emphasize that pediatric neurodegeneration occurs in the context of ongoing neurodevelopment, which distinguishes it from adult-onset conditions.The manuscript is generally well organized and informative. However, in its current form, it remains somewhat descriptive rather than analytical, and several sections do not fully reflect the current state of the field, particularly with respect to diagnostic strategies, disease-specific therapies, and translational advances.

In addition, some important emerging concepts (e.g., neurodevelopmental vulnerability, biomarker-driven monitoring, and next-generation therapeutic approaches) are only briefly mentioned or not sufficiently developed. Addressing these points would substantially strengthen the manuscript and improve its value as a comprehensive review.

Major Comments

1. Treatment section requires greater precision and disease-specific detail. In fact, the current treatment section is relatively general and, at times, lacks sufficient technical precision. In particular, different gene therapy approaches are discussed together without clearly distinguishing between platforms, which may lead to conceptual ambiguity. It would further improve clarity if the authors explicitly differentiate between in vivo AAV-based gene therapy (e.g., spinal muscular atrophy; PMID: 33743238), ex vivo lentiviral hematopoietic stem cell gene therapy (e.g., metachromatic leukodystrophy; PMID: 35065785, and cerebral X-linked adrenoleukodystrophy; PMID: 34424497), and enzyme replacement therapy (ERT) including intrathecal delivery approaches (e.g., CLN2 disease; PMID: 29688815; long-term data PMID: 38101904). In addition, the inclusion of a concise disease-specific summary table outlining the disorder, target pathway, therapeutic modality, delivery platform, and optimal treatment window would significantly enhance the clarity and usefulness of this section.

Response: We thank the Reviewer 02 for this important comment. We have revised the treatment section to improve technical precision and disease-specific clarity by explicitly distinguishing between in vivo AAV-based gene therapy, ex vivo lentiviral hematopoietic stem cell gene therapy, enzyme replacement therapy including CNS-directed delivery, as well as RNA-based therapies and emerging genome editing approaches. We also added a concise disease-specific summary table to clarify the relationship between disorder, target pathway, therapeutic modality, delivery platform, and optimal treatment window.

1. Newborn screening and presymptomatic management should be expanded. Although newborn screening is mentioned, the discussion remains relatively brief and does not fully reflect its current clinical importance. More specifically, the manuscript would benefit from a clearer distinction between disorders with established screening frameworks, such as X-linked adrenoleukodystrophy (PMID: 36175155; 33373467), and conditions in which early or presymptomatic treatment is critical for outcome, such as metachromatic leukodystrophy (PMID: 38554683). It would also be helpful to emphasize that early diagnosis often requires structured longitudinal surveillance, including MRI and endocrine monitoring, rather than immediate intervention in all cases. Importantly, highlighting the central role of treatment timing would further strengthen the clinical relevance of this section.

Response: We thank the Reviewer for this important comment. We agree that the original discussion of newborn screening and presymptomatic management was too brief. In the revised manuscript, we expanded this section to distinguish more clearly between disorders with established newborn screening and surveillance frameworks, such as X-linked adrenoleukodystrophy, and disorders in which presymptomatic or very early treatment is critical for outcome, such as metachromatic leukodystrophy. We also clarified that early diagnosis does not imply immediate intervention in all cases, but often requires structured longitudinal surveillance, including serial brain MRI and endocrine monitoring, so that treatment can be initiated at the optimal stage of disease evolution. Finally, we emphasized that treatment timing is a central determinant of clinical benefit in pediatric neurodegenerative disorders.

1. The manuscript acknowledges the emergence of genome-first strategies; however, the diagnostic workflow still largely reflects a traditional stepwise approach. In current clinical practice, earlier implementation of genomic testing is increasingly recommended. The authors may therefore consider revising this section to reflect the earlier use of exome or genome sequencing (PMID: 31182824; 34211152), including the role of trio sequencing, as well as the integration of copy-number analysis, mitochondrial DNA testing, and periodic reanalysis. At the same time, targeted metabolic testing remains important in cases where clinical or imaging features suggest potentially treatable conditions. Supporting evidence from genetic white matter disorder cohorts further underscores the value of early genomic testing as a first-line approach (PMID: 32342562). 

1. Biomarkers are mentioned only briefly, although they are now central to both clinical management and therapeutic development. In this context, emerging blood-based biomarkers, particularly neurofilament light chain (NfL), have demonstrated utility as indicators of neuroaxonal injury and treatment response. Including disease-specific examples of biomarker application, such as in leukodystrophies or spinal muscular atrophy (PMID: 33753741; 35106137), would further strengthen the translational perspective of the review.

Response: We thank the Reviewer for this important comment. We agree that biomarkers were underrepresented in the original version. In the revised manuscript, we expanded the discussion to emphasize the growing role of fluid biomarkers, particularly neurofilament light chain (NfL), as indicators of neuroaxonal injury and treatment response. We also added disease-specific examples, including blood NfL in cerebral X-linked adrenoleukodystrophy and CSF NfL in CLN2 disease, to strengthen the translational perspective of the review.

1. The interaction between neurodevelopment and neurodegeneration is an important conceptual framework that could be more clearly articulated. Expanding the pathophysiology section to describe how early developmental disturbances, such as impaired synaptic maturation or neuronal differentiation, may create a vulnerable neural substrate that predisposes to subsequent degeneration would add important depth to the manuscript.

Response: We thank the Reviewer for this important conceptual suggestion. We have expanded the pathophysiology section to clarify that, in many pediatric neurodegenerative disorders, early disturbances of neurodevelopment and later neurodegeneration are biologically interconnected rather than fully separate processes. The revised text now emphasizes how impaired neuronal differentiation, synaptic maturation, circuit assembly, and glial support may create a vulnerable neural substrate that predisposes to subsequent degeneration and modifies disease timing and severity.

1. While lysosomal dysfunction is discussed, broader aspects of proteostasis are less well represented. A brief expansion to include the role of the ubiquitin-proteasome system, chaperone-mediated autophagy, and protein aggregation across different pediatric neurodegenerative disorders would help unify mechanistic themes across disease categories.

Response: We thank the Reviewer for this important suggestion. We agree that broader aspects of proteostasis were underrepresented in the original version. In the revised manuscript, we expanded the pathophysiology section to include the role of the ubiquitin–proteasome system, chaperone-mediated autophagy, and protein aggregation, emphasizing that impaired protein quality control represents a unifying mechanistic theme across multiple pediatric neurodegenerative disorders, including lysosomal diseases and leukodystrophies.

1. The treatment section could also be updated to acknowledge more recent therapeutic approaches, including antisense oligonucleotides (ASOs), gene editing strategies, and individualized or patient-specific therapies. Even a concise discussion of these developments would better reflect the rapidly evolving therapeutic landscape.

Response: We thank the Reviewer for this important comment. In the revised manuscript, we expanded the treatment section to include a clearer discussion of ASO-based therapies, CRISPR-based gene editing, and the emerging concept of individualized patient-specific therapies, including mutation-tailored oligonucleotide approaches for ultra-rare neurogenetic disorders. These additions better reflect the rapidly evolving precision-medicine landscape in pediatric neurodegeneration.

Minor Comments

• Please ensure consistent terminology when referring to therapeutic modalities (e.g., gene therapy, AAV-based therapy, HSCT, ERT).

Response: We thank the Reviewer for this helpful observation. We carefully revised the manuscript to ensure consistent terminology throughout the treatment section and elsewhere in the text. In particular, we now use harmonized terms for gene therapy, in vivo AAV-based gene therapy, ex vivo lentiviral hematopoietic stem cell gene therapy, hematopoietic stem cell transplantation (HSCT), and enzyme replacement therapy (ERT) in order to improve clarity and avoid conceptual overlap.

• Some sections, particularly in the pathophysiology discussion, could be slightly condensed to reduce repetition.

Response: We thank the Reviewer for this helpful comment. We carefully revised the pathophysiology section to reduce repetition and improve conciseness, while preserving the mechanistic depth and overall conceptual structure of the review.

• A schematic figure summarizing shared molecular pathways (e.g., mitochondrial dysfunction, oxidative stress, proteostasis) would improve readability.

Response: We thank the Reviewer for this helpful suggestion. We added a schematic figure summarizing the major shared molecular pathways underlying pediatric neurodegenerative disorders in order to improve clarity and readability.

Submission Date

06 March 2026

Date of this review

27 Mar 2026 20:10:32

Date of the response: 17 April 2026

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Neurodegenerative Diseases in Children: A Comprehensive Review” addresses an important and clinically relevant topic, covering a wide range of aspects including epidemiology, classification, pathophysiology, diagnosis, and management of paediatric neurodegenerative disorders. The authors compile a substantial body of information and aim to provide an integrated overview of this heterogeneous group of conditions, making the review potentially valuable for clinicians and researchers in paediatric neurology and rare genetic diseases. However, the manuscript requires substantial revision.

In its current form, the text is largely descriptive, with many sections presenting summaries and lists rather than offering a critical comparison of disease groups or a balanced discussion of the limitations and controversies in current approaches. This is particularly evident in the therapeutic sections, where enzyme replacement therapy, haematopoietic stem cell transplantation, gene therapy, RNA-based approaches, and genome editing technologies are discussed together, despite marked differences in their clinical maturity, level of evidence, and applicability. A clearer distinction between established treatments and experimental strategies is needed, along with a more explicit discussion of key limitations, including challenges in central nervous system delivery, dependence on early intervention, and variability in clinical response.

The classification section requires careful reconsideration. In its present form, it combines categories of different conceptual levels, resulting in internal inconsistency. For example, lysosomal storage disorders, peroxisomal and mitochondrial diseases, primary leukodystrophies, autophagy-related disorders, DNA repair disorders, repeat expansion disorders, and ion channel or synaptic disorders are all presented within a single framework. Some of these categories reflect organelle- or molecular-level defects, others represent pathogenic mechanisms, while others correspond to clinical or mixed groupings. This blending of classification principles obscures the underlying structure.

In addition, several conditions are distributed across multiple categories without explicit acknowledgment of this overlap. For instance, metachromatic leukodystrophy and Krabbe disease are presented as lysosomal storage disorders, although they are also prototypical leukodystrophies; X-linked adrenoleukodystrophy is categorised as a peroxisomal disorder, yet it is clinically and pathogenetically one of the major inflammatory leukodystrophies; neuronal ceroid lipofuscinoses are discussed within lysosomal neurodegenerative diseases but later reappear under a broader group of “other genetic neurodegenerative conditions.” Such presentation may give the impression of distinct, non-overlapping classes, whereas in reality these represent intersecting biological and clinical dimensions.

Particularly debatable is the inclusion of ion channel and synaptic disorders (e.g., SCN-related epileptic encephalopathies) as “neurodegenerative epileptic encephalopathies,” as well as their conceptual proximity to conditions such as spinal muscular atrophy. While these disorders may involve severe neurological impairment and developmental regression, their pathogenesis differs fundamentally from that of classical paediatric neurodegenerative diseases driven by progressive neuronal or glial loss due to lysosomal dysfunction, myelin pathology, mitochondrial impairment, or peroxisomal defects. Their inclusion within a unified classification without further clarification is therefore conceptually problematic. The classification would benefit either from adherence to a single organising principle (e.g., molecular-pathogenetic) or from explicitly presenting a layered framework with acknowledged overlap.

The discussion of therapeutic advances is, in several instances, overly generalised. Although significant progress has been achieved for selected conditions – particularly with early molecular diagnosis and timely intervention – these advances remain limited to specific diseases and patient subgroups. The manuscript would benefit from a more nuanced and cautious interpretation, clearly indicating that for the majority of paediatric neurodegenerative disorders, disease-modifying treatment options remain limited.

The reference list also requires careful revision. Although it includes many relevant and up-to-date sources, its overall quality is inconsistent. Notably, reference [52] is a retracted publication, and its inclusion as supporting evidence is inappropriate. In addition, several cited works are older or primarily narrative reviews with limited critical value for supporting specific claims, such as [29], [30], [54], and [80], particularly in sections discussing current molecular mechanisms and therapeutic developments. There are also references whose relevance to paediatric neurodegeneration is indirect, as they address general mechanisms of neurodegeneration or related conditions without clear applicability to paediatric populations. Overall, a more rigorous and selective approach to the literature is needed, prioritising recent high-quality reviews, consensus statements, and key primary studies directly relevant to paediatric neurodegenerative diseases.

In summary, the manuscript addresses an important topic and has a solid foundation, but substantial revision is required. In particular, the authors should strengthen the analytical component, refine the classification framework, present therapeutic advances in a more balanced and evidence-based manner, and improve the quality and relevance of the references.

Author Response

Authors' response to the review report (Reviewer 3)

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have revised the manuscript accordingly and address each point below.

The manuscript entitled “Neurodegenerative Diseases in Children: A Comprehensive Review” addresses an important and clinically relevant topic, covering a wide range of aspects including epidemiology, classification, pathophysiology, diagnosis, and management of paediatric neurodegenerative disorders. The authors compile a substantial body of information and aim to provide an integrated overview of this heterogeneous group of conditions, making the review potentially valuable for clinicians and researchers in paediatric neurology and rare genetic diseases. However, the manuscript requires substantial revision.

In its current form, the text is largely descriptive, with many sections presenting summaries and lists rather than offering a critical comparison of disease groups or a balanced discussion of the limitations and controversies in current approaches. This is particularly evident in the therapeutic sections, where enzyme replacement therapy, haematopoietic stem cell transplantation, gene therapy, RNA-based approaches, and genome editing technologies are discussed together, despite marked differences in their clinical maturity, level of evidence, and applicability. A clearer distinction between established treatments and experimental strategies is needed, along with a more explicit discussion of key limitations, including challenges in central nervous system delivery, dependence on early intervention, and variability in clinical response.

The classification section requires careful reconsideration. In its present form, it combines categories of different conceptual levels, resulting in internal inconsistency. For example, lysosomal storage disorders, peroxisomal and mitochondrial diseases, primary leukodystrophies, autophagy-related disorders, DNA repair disorders, repeat expansion disorders, and ion channel or synaptic disorders are all presented within a single framework. Some of these categories reflect organelle- or molecular-level defects, others represent pathogenic mechanisms, while others correspond to clinical or mixed groupings. This blending of classification principles obscures the underlying structure.

In addition, several conditions are distributed across multiple categories without explicit acknowledgment of this overlap. For instance, metachromatic leukodystrophy and Krabbe disease are presented as lysosomal storage disorders, although they are also prototypical leukodystrophies; X-linked adrenoleukodystrophy is categorised as a peroxisomal disorder, yet it is clinically and pathogenetically one of the major inflammatory leukodystrophies; neuronal ceroid lipofuscinoses are discussed within lysosomal neurodegenerative diseases but later reappear under a broader group of “other genetic neurodegenerative conditions.” Such presentation may give the impression of distinct, non-overlapping classes, whereas in reality these represent intersecting biological and clinical dimensions.

Response:
We thank the Reviewer 03 for this important and constructive observation. We agree that the previous version of the manuscript placed insufficient emphasis on the comparative and critical dimensions of the field. In response, we have substantially revised the manuscript to move beyond a predominantly descriptive overview.

First, we revised the therapeutic section to distinguish more clearly between:
(i) established disease-modifying therapies currently used in clinical practice for selected disorders,
(ii) emerging therapies with early clinical application in highly specific indications, and
(iii) experimental or investigational platforms that remain preclinical or early translational.

We also added a more explicit discussion of major limitations that cut across therapeutic strategies, including limited central nervous system delivery, dependence on presymptomatic or very early treatment, genotype- and stage-dependent variability in response, procedure-related risks, durability of effect, and inequities in access and cost.

Second, throughout the revised manuscript, we introduced more comparative discussion across major disease groups, with particular emphasis on shared pathogenic pathways but also on their clinically relevant differences in reversibility, therapeutic tractability, and timing sensitivity.

Particularly debatable is the inclusion of ion channel and synaptic disorders (e.g., SCN-related epileptic encephalopathies) as “neurodegenerative epileptic encephalopathies,” as well as their conceptual proximity to conditions such as spinal muscular atrophy. While these disorders may involve severe neurological impairment and developmental regression, their pathogenesis differs fundamentally from that of classical paediatric neurodegenerative diseases driven by progressive neuronal or glial loss due to lysosomal dysfunction, myelin pathology, mitochondrial impairment, or peroxisomal defects. Their inclusion within a unified classification without further clarification is therefore conceptually problematic. The classification would benefit either from adherence to a single organising principle (e.g., molecular-pathogenetic) or from explicitly presenting a layered framework with acknowledged overlap.

The discussion of therapeutic advances is, in several instances, overly generalised. Although significant progress has been achieved for selected conditions – particularly with early molecular diagnosis and timely intervention – these advances remain limited to specific diseases and patient subgroups. The manuscript would benefit from a more nuanced and cautious interpretation, clearly indicating that for the majority of paediatric neurodegenerative disorders, disease-modifying treatment options remain limited.

Comment:
The classification section combines categories of different conceptual levels, resulting in internal inconsistency. Some categories reflect organelle- or molecular-level defects, others represent pathogenic mechanisms, while others correspond to clinical or mixed groupings.

Response:
We fully agree with the Reviewer 03 that the original classification framework mixed distinct classificatory principles and could therefore obscure the conceptual structure of the field. To address this, we have revised the classification section and clarified that no single scheme fully captures pediatric neurodegenerative disorders.

In the revised manuscript, we now explicitly separate:
(i) a primary etiologic–molecular classification, based on the principal defective cellular system or biochemical pathway (e.g., lysosomal, mitochondrial, peroxisomal disorders);
from
(ii) clinicoradiologic or phenotypic groupings, such as leukodystrophies, which are retained as highly relevant clinical syndromic categories rather than placed on the same conceptual level as organelle-based classes.

We also clarify that categories such as autophagy-related disorders, DNA repair disorders, repeat expansion disorders, and ion channel/synaptic disorders are best understood as cross-cutting mechanistic or genetic groupings rather than equivalent top-level categories within a single linear taxonomy. This change was made both in the text and in the revised summary table.

The reference list also requires careful revision. Although it includes many relevant and up-to-date sources, its overall quality is inconsistent. Notably, reference [52] is a retracted publication, and its inclusion as supporting evidence is inappropriate. In addition, several cited works are older or primarily narrative reviews with limited critical value for supporting specific claims, such as [29], [30], [54], and [80], particularly in sections discussing current molecular mechanisms and therapeutic developments. There are also references whose relevance to paediatric neurodegeneration is indirect, as they address general mechanisms of neurodegeneration or related conditions without clear applicability to paediatric populations. Overall, a more rigorous and selective approach to the literature is needed, prioritising recent high-quality reviews, consensus statements, and key primary studies directly relevant to paediatric neurodegenerative diseases.

In summary, the manuscript addresses an important topic and has a solid foundation, but substantial revision is required. In particular, the authors should strengthen the analytical component, refine the classification framework, present therapeutic advances in a more balanced and evidence-based manner, and improve the quality and relevance of the references.

Response:
We thank the reviewer for this important observation and agree that the reference list required more rigorous curation.

First, we have removed the retracted publication previously cited as reference [52] and replaced its use with more appropriate and valid sources. Second, we carefully re-evaluated the bibliography throughout the manuscript, with particular attention to sections dealing with molecular mechanisms, diagnosis, and therapeutic advances. In doing so, we reduced reliance on older narrative reviews when these were not essential for historical context and prioritized more recent, higher-quality sources, including consensus statements, contemporary disease-focused reviews, and key primary studies directly relevant to pediatric neurodegenerative disorders.

We also reassessed references whose relevance to pediatric neurodegeneration was indirect. Where such citations did not clearly support pediatric-specific claims, they were either removed, replaced, or retained only when used explicitly for broader mechanistic background rather than pediatric clinical inference.

Overall, the revised manuscript now adopts a more selective and evidence-based referencing strategy, with stronger emphasis on recent literature of direct relevance to pediatric neurodegenerative disease.

Submission Date

06 March 2026

Date of this review  09 Apr 2026 07:02:36

Date of the response: 17 April 2026

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Ailioaie et al. present a comprehensive narrative review of neurodegenerative diseases (NDDs) in children, covering epidemiology, molecular classification, pathophysiology, diagnostics, and management. The topic is clinically important and the scope is ambitious. The manuscript succeeds in providing a broad overview accessible to a multidisciplinary audience. However, several conceptual, structural, and factual issues reduce the clarity and scientific rigor of the review. Below I outline specific concerns and constructive recommendations organized by manuscript section.

Epidemiology (Section 2)

Point 1 – The authors state that “genomic diagnostics and new disease registries are improving detection” (lines 67–68). This is true for some monogenic LSDs and leukodystrophies but needs qualification. Most pediatric NDDs are individually ultra‑rare and influenced by modifier genes, epigenetics and variable penetrance; exome/genome sequencing provides a definitive diagnosis in only ~30–50% of cases (as the authors themselves note in Section 5.3). Furthermore, cohorts for many NDD subtypes are still too small for reliable risk prediction or genotype–phenotype associations. The authors should acknowledge these limitations and avoid implying that genomic tools universally resolve the diagnostic challenge.

Point 2 – The Northern Finland cohort (ref. 9, lines 92–97) correctly links newly recognized genetic etiologies to increased incidence, but two issues need mentioning. First, whole-genome sequencing should be extended to familial cases and population analyses to detect founder variants and linked loci without this, incidence estimates are incomplete. Second, some cases labeled as “genetic leukoencephalopathies” may be phenocopies from neuroinflammatory/autoimmune disorders (e.g., ADEM, MOG‑antibody disease, CNS vasculitis); the authors should state how such mimics were excluded and discuss the impact of misclassification on epidemiology.

Point 3 – The NCL description (lines 98–104) notes the triad of epilepsy, neuroregression and vision loss but omits why these co‑occur. The affected CLN genes encode proteins with region-specific expression patterns, for instance, high expression in cortical and retinal neurons. It would strengthen the review to briefly explain that the preferential involvement of visual cortex and retinal neurons accounts for vision loss, while cortical hyperexcitability and progressive neuronal loss underlie epilepsy and regression. The underlying mechanism involves lysosomal substrate accumulation, which triggers ROS production, inflammatory cascades, and ultimately neuronal death. Without this mechanistic link, the clinical description remains purely descriptive.

Point 4 – The statement that mitochondrial disease “often manifests in childhood with multisystem involvement” (lines 110–112) is accurate but raises a diagnostic concern not discussed: mitochondrial disorders can be phenocopied by other conditions causing similar multisystem defects (e.g., organic acidemias, fatty acid oxidation defects, congenital disorders of glycosylation). The clinical overlap means that without rigorous biochemical and genetic confirmation, mitochondrial disease may be overdiagnosed. A brief discussion of differential diagnosis would strengthen the review.

Point 5 – The statement that “early symptoms may mimic more common developmental disorders, epilepsy syndromes, or cerebral palsy-like presentations” (lines 123–125) is problematic. Epilepsy diagnosis in neonates and very young infants is itself challenging and age-dependent—neonatal seizures have distinct semiology, EEG characteristics, and etiological considerations compared to later-onset epilepsy. The implication that NDDs are misclassified as epilepsy in newborns needs qualification, since formal epilepsy diagnosis in this age group follows specific criteria (ILAE neonatal seizure classification). Please clarify the age-dependent diagnostic framework.

Classification (Section 3)

Point 6 – Table 1 is sensibly organized by molecular pathway, but behavioral and psychiatric manifestations (e.g., personality change in juvenile Huntington disease, behavioral decline in cerebral ALD, autism‑like features in some mitochondrial disorders) are not included among the phenotypic descriptors. While these features are non-specific and often polygenic, the same limitation applies to several included descriptors (for example, “cognitive decline”). Their omission reduces the table’s clinical utility, particularly for early recognition. Please either include behavioral/psychiatric signs in the phenotypic schema or discuss why they were excluded and how clinicians should consider them when applying the classification.

Point 7 – Autophagy disorders are listed as a separate major molecular category in Table 1. However, impaired autophagy is mechanistically downstream of lysosomal dysfunction (LSDs), iron metabolism abnormalities (NBIA), and peroxisomal/mitochondrial ROS production. Listing autophagy as an independent category at the same hierarchical level as its upstream causes creates a taxonomic inconsistency. Autophagy disorders could more accurately be represented as a subgroup or a convergent pathway shared across multiple primary categories. The same concern applies to the ICD classification: G11.4 (Hereditary spastic paraplegia) listed under autophagy disorders overlaps significantly with the clinical spectrum of E75 and G11.3 diseases. Please discuss the rationale for this classification choice or consider restructuring.

Point 8 – Repeat expansion disorders (Table 1) are described as causing a “toxic gain‑of‑function from expanded nucleotide repeats.” This overlooks a key downstream mechanism—pathogenic protein aggregation (e.g., huntingtin in juvenile Huntington disease). Since protein aggregation is a major convergent pathway across neurodegenerative disorders, its omission weakens mechanistic integration; please add or acknowledge this.

Point 9 – In lines 169–171, the authors state: “a child must inherit one defective gene from each parent.” The term “defective gene” is imprecise; “pathogenic allele” or “pathogenic variant” would be more accurate, consistent with current ACMG nomenclature guidelines. Each parent carries one pathogenic allele; the child inherits two. Please update the terminology throughout.

Point 10 – The description of tissue-specific vulnerability through substrate accumulation (lines 182–188) states “neuronal lipid accumulation predominantly results in progressive neurodegeneration” but does not explain the biophysical mechanism. How exactly do lipids accumulate in a way that causes toxicity? Do they form ordered aggregates analogous to amyloid fibrils through hydrophobic interactions? Or is the mechanism primarily through membrane disruption? The literature suggests multiple pathways, including altered membrane fluidity, disruption of lipid raft signaling, and direct cytotoxicity. A brief mechanistic discussion would substantially improve this section.

Point 31 – The claim that Table 2 “facilitates understanding of genotype-phenotype correlations” (line 189) is overstated given the limited phenotypic descriptors and absence of quantitative association data (GWAS scores, odds ratios). The table shows genotype-phenotype associations at a descriptive level, not correlations in the statistical sense.

Point 11 – The clinical description of Pompe disease (Section 3.1.1.4) omits myalgia and exercise intolerance, which are characteristic features of late-onset forms. Similarly, the Niemann-Pick disease section (3.1.1.3) does not mention cognitive decline or ataxia, which are hallmark features of type C. These omissions are significant for a comprehensive review and should be corrected.

Point 12 – Section 3.1.1 begins with an introductory paragraph (“Major lysosomal storage disorders (LSDs) frequently manifest in childhood…”) that essentially repeats information already presented in Section 3.1. This redundancy should be eliminated to improve readability. The transition between sections needs editorial tightening; the sentence fragment “… and include: LSDs are inherited metabolic diseases…” appears to be a formatting or editing artifact.

Point 13 – Section 3.1.1.1 describes Gaucher disease clinical manifestations but provides no specific examples of genotype-subtype correlations (e.g., GBA1 N370S homozygosity in type 1 vs. L444P in neuronopathic forms). Given the heterogeneity mentioned, at least one illustrative genotype-phenotype correlation would be valuable.

Leukodystrophies (Section 3.2)

Point 14 – The table mentions “rare juvenile presentations reported” for adult-onset leukoencephalopathy with axonal spheroids (CSF1R mutations). The authors should clarify whether these rare juvenile cases reflect true early onset due to specific genotype severity, developmental delay from subclinical disease, or ascertainment bias. This distinction has implications for understanding disease mechanism and counseling.

Point 15 – Rosenthal fibers in Alexander disease consist of aggregated GFAP with associated stress proteins (alphaB-crystallin, HSP27). The authors describe these as “glial fibrillary acidic protein aggregation” but do not discuss whether these aggregates have amyloid or amyloid-like structural properties. Some intermediate filament proteins can form amyloid-like structures under stress conditions. A brief comment on the structural nature of Rosenthal fibers would add mechanistic depth.

Mitochondrial Disorders (Section 3.3)

Point 16 – The review does not mention attention-deficit/hyperactivity disorder (ADHD) in the context of mitochondrial dysfunction, despite emerging evidence linking mitochondrial bioenergetic deficits to ADHD pathophysiology. While ADHD is not traditionally classified as a neurodegenerative disorder, its potential mitochondrial component is relevant to a comprehensive classification. At minimum, a brief mention of neurodevelopmental conditions with mitochondrial involvement would broaden the review’s scope.

Point 17 – The statement that “disruptions in mitochondrial dynamics, mitophagy, and ETC activity also drive pediatric neurodegenerative phenotypes” (lines 319–321) presents mitochondrial dysfunction as a primary driver. However, in many NDDs (including LSDs, repeat expansion disorders, and prion-like proteinopathies), protein aggregation is the initiating event that secondarily causes mitochondrial dysfunction through membrane disruption, calcium dysregulation, and ROS generation. The causal directionality aggregation causing mitochondrial dysfunction vs. the reverse should be discussed more carefully, as it has therapeutic implications.

Point 18 – Section 3.3 does not discuss heteroplasmy or somatic mosaicism, which are fundamental concepts in mitochondrial genetics. The threshold effect of heteroplasmy explains the variable penetrance and tissue specificity of mtDNA-linked disorders. This omission is significant for a review that aims to be comprehensive, as heteroplasmy is central to understanding why the same mtDNA mutation produces different phenotypes in different patients and tissues.

Pathophysiology (Section 4)

Point 19 – The introduction to Section 4 (lines 414–420) appears to be formatted in bold text, inconsistent with the rest of the manuscript. This appears to be a formatting or typesetting error that should be corrected.

Point 20 – The discussion of lysosomal dysfunction (Section 4.1) focuses on substrate accumulation and blocked autophagic flux. However, recent literature suggests that accumulated substrates within lysosomes may form condensate-like or amyloid-like structures through liquid–liquid phase separation or ordered aggregation. This concept would add a modern biophysical perspective to the pathophysiology discussion and could explain why lysosomal storage progresses irreversibly.

Point 21 – Section 4.1 does not discuss epigenetic consequences of lysosomal–autophagic insufficiency, such as DNA methylation changes and chromatin reorganization. Impaired autophagy has been linked to altered histone modifications and chromatin accessibility, which may contribute to transcriptional dysregulation in affected neurons. Given the review’s comprehensive scope, this omission is notable.

Point 22 – In Section 4.3, “energy failure at synapses,” “ROS overproduction,” and “defective mitophagy/biogenesis coupling” are presented as three distinct mechanisms. However, defective mitophagy is arguably the upstream cause of both energy failure (accumulation of dysfunctional mitochondria) and ROS overproduction (damaged ETC complexes generating superoxide). Presenting them as parallel rather than hierarchically related obscures the causal chain. Consider restructuring to emphasize the primary defect (mitophagy failure) and its downstream consequences.

Point 23 – The pathophysiology section on mitochondrial bioenergetic failure (4.3) does not discuss mosaicism or heteroplasmy, which directly influence disease severity and tissue selectivity. Somatic mosaicism for mtDNA mutations is typically associated with mitochondrial pathologies through maternal inheritance patterns and random mitotic segregation. This is a significant conceptual gap.

Point 24 – The detailed description of Figure 1 begins within Section 4.8 (Peroxisomal Dysfunction) but covers all four major mechanisms shown in the figure. This content should be placed in a separate section or subsection, as it pertains to the entire pathophysiology chapter, not specifically to peroxisomal disorders.

Diagnosis (Section 5)

Point 25 – Line 596: there is a period instead of a comma between “decline” and “loss” in the list of clinical warning signs (“progressive cognitive decline. loss of motor skills”). Please correct.

Point 26 – The statement “Regression, especially after a period of normal development, is a feature of LSD disorders, mitochondrial diseases, and leukodystrophies” (lines 597–599) is too general. Regression is also seen in non-degenerative conditions (e.g., epileptic encephalopathies, autoimmune encephalitis, Rett syndrome). Without specifying what distinguishes NDD regression from other causes, this clinical pointer loses discriminating value.

Point 27 – Section 5.4 states that transcriptomics can “detect splicing defects.” This is imprecise. Transcriptomics (RNA-seq) primarily provides gene expression profiles and identifies differentially expressed genes. Detection of splicing defects requires specific analysis pipelines for alternative splicing events, which is a specialized subfield. Standard transcriptomic analysis would not detect splicing abnormalities unless specifically designed to do so. Please clarify this distinction.

Point 28 – On page 15, there is a missing period between two sentences in the paragraph describing axonal transport defects: “…KIF1A (kinesin family member 1A) mutations Axonal transport is essential…” should read “…KIF1A (kinesin family member 1A) mutations. Axonal transport is essential…”

Point 29 – The paragraph at lines 570–575 describing convergent mechanisms leading to synaptic dysfunction does not mention developmental delays and failures as clinical consequences. For a review of pediatric NDDs, the impact on ongoing brain development (synaptogenesis, myelination, circuit formation) is arguably more important than in adult-onset disease and should be explicitly discussed.

Minor Comments

Several abbreviations are defined multiple times (e.g., HSCT is abbreviated as both HCST and HSCT in different sections). Please standardize.

Reference formatting should be checked for consistency (some references use abbreviated journal names, others do not).

Overall Recommendation

The manuscript addresses an important topic and provides a useful broad overview. However, it requires major revision to address: (1) mechanistic depth in key sections (lipid accumulation biophysics, epigenetic consequences, heteroplasmy); (2) conceptual precision in classification (autophagy as downstream vs. primary mechanism, behavioral symptoms); (3) multiple terminology and formatting errors; and (4) more careful discussion of the limitations of genomic diagnostics for rare, heterogeneous disorders. The review would also benefit from integrating modern concepts such as phase separation biology and amyloid-like aggregation in lysosomal/mitochondrial contexts. With these revisions, the manuscript could make a valuable contribution to the field.

Author Response

Authors' response to the review report (Reviewer 4)

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have revised the manuscript accordingly and address each point below.

The topic of this article addresses an issue of great importance from both a medical and social perspective. Neurodegenerative diseases in children, although relatively rare, pose a significant burden not only to their families but also to the public health system. Therefore, it is fitting that the authors conducted a comprehensive review that synthesizes current knowledge regarding the epidemiology, molecular classification, pathophysiology, and emerging therapeutic strategies of major pediatric neurodegenerative disorders.

The structure of the article is typical of review articles. However, it seems that the "Introduction" section is too superficial and requires expansion. I suggest that the authors supplement it with elements such as the aim of the study and the basic assumptions of the literature review.

Response to Reviewer 4

We thank the reviewer for this constructive comment. In response, we revised and expanded the Introduction to better define the aim of the review and to include the main assumptions underlying our literature analysis. We also clarified the rationale for focusing on the major categories of pediatric neurodegenerative diseases within a molecular and clinical framework. We believe these changes have improved the clarity and scope of the manuscript.

As a result of the extensive literature review, the authors identified the following thematic groups: Epidemiology of Neurodegenerative Diseases in Children, Pathophysiological Mechanisms in Neurodegenerative Diseases in Children, and Diagnosis in Pediatric Neurodegenerative Diseases. This division organizes the medical knowledge within the discussed group of diseases, which is a significant convenience for physicians, especially practitioners, interested in this topic. This is undoubtedly a strength of the reviewed manuscript.

A weakness of the article is the lack of a methods section. This section should contain basic information regarding the research assumptions, the inclusion and exclusion criteria used in the literature selection, etc. It would also be advisable for the authors to include a 'Limitations' section at the end of the article.

Response to Reviewer 4:

We thank the reviewer for this important comment. In response, we have added a dedicated Materials and Methods section describing the review assumptions, literature search approach, and the inclusion and exclusion criteria used for study selection. We have also added a separate Limitations section near the end of the manuscript to clarify the scope and constraints of this narrative comprehensive review. These additions have improved the transparency and methodological clarity of the article.

After making the changes and additions suggested above, I support the publication of the manuscript.

Submission Date

06 March 2026

Date of this review

09 Apr 2026 12:24:38

Date of the response: 17 April 2026

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The topic of this article addresses an issue of great importance from both a medical and social perspective. Neurodegenerative diseases in children, although relatively rare, pose a significant burden not only to their families but also to the public health system. Therefore, it is fitting that the authors conducted a comprehensive review that synthesizes current knowledge regarding the epidemiology, molecular classification, pathophysiology, and emerging therapeutic strategies of major pediatric neurodegenerative disorders.

The structure of the article is typical of review articles. However, it seems that the "Introduction" section is too superficial and requires expansion. I suggest that the authors supplement it with elements such as the aim of the study and the basic assumptions of the literature review.

As a result of the extensive literature review, the authors identified the following thematic groups: Epidemiology of Neurodegenerative Diseases in Children, Pathophysiological Mechanisms in Neurodegenerative Diseases in Children, and Diagnosis in Pediatric Neurodegenerative Diseases. This division organizes the medical knowledge within the discussed group of diseases, which is a significant convenience for physicians, especially practitioners, interested in this topic. This is undoubtedly a strength of the reviewed manuscript.

A weakness of the article is the lack of a methods section. This section should contain basic information regarding the research assumptions, the inclusion and exclusion criteria used in the literature selection, etc. It would also be advisable for the authors to include a 'Limitations' section at the end of the article. After making the changes and additions suggested above, I support the publication of the manuscript.

Author Response

Authors' response to the review report (Reviewer 5)

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have revised the manuscript accordingly and address each point below.

The topic of this article addresses an issue of great importance from both a medical and social perspective. Neurodegenerative diseases in children, although relatively rare, pose a significant burden not only to their families but also to the public health system. Therefore, it is fitting that the authors conducted a comprehensive review that synthesizes current knowledge regarding the epidemiology, molecular classification, pathophysiology, and emerging therapeutic strategies of major pediatric neurodegenerative disorders.

The structure of the article is typical of review articles. However, it seems that the "Introduction" section is too superficial and requires expansion. I suggest that the authors supplement it with elements such as the aim of the study and the basic assumptions of the literature review.

Response to Reviewer 5

We sincerely appreciate the reviewer’s insightful suggestion. In accordance with this recommendation, we have revised and expanded the **Introduction** to better articulate the purpose of the review and to present the main conceptual assumptions underlying our analysis of the literature. Furthermore, we have strengthened the introductory framework by clarifying the justification for concentrating on the principal categories of pediatric neurodegenerative diseases from both molecular and clinical perspectives. We believe that these modifications have significantly improved the depth, clarity, and contextual grounding of the manuscript.

As a result of the extensive literature review, the authors identified the following thematic groups: Epidemiology of Neurodegenerative Diseases in Children, Pathophysiological Mechanisms in Neurodegenerative Diseases in Children, and Diagnosis in Pediatric Neurodegenerative Diseases. This division organizes the medical knowledge within the discussed group of diseases, which is a significant convenience for physicians, especially practitioners, interested in this topic. This is undoubtedly a strength of the reviewed manuscript.

A weakness of the article is the lack of a methods section. This section should contain basic information regarding the research assumptions, the inclusion and exclusion criteria used in the literature selection, etc.

Response to Reviewer 5

We greatly appreciate the reviewer’s valuable observation. In accordance with this suggestion, we have incorporated a dedicated **Materials and Methods** section that outlines the methodological assumptions underlying the review, the strategy used for the identification and selection of relevant literature, and the inclusion and exclusion criteria applied during the screening process. We believe that this revision has enhanced the transparency, reproducibility, and overall methodological robustness of the manuscript.

It would also be advisable for the authors to include a 'Limitations' section at the end of the article. After making the changes and additions suggested above, I support the publication of the manuscript.

Response to Reviewer 5:

We thank the reviewer for this constructive recommendation. In accordance with this suggestion, we have added a dedicated **Limitations** section at the end of the manuscript to explicitly address the scope and inherent constraints of this comprehensive narrative review. This addition improves the transparency and methodological clarity of the manuscript.

Submission Date

06 March 2026

Date of this review

09 Apr 2026 12:24:38

Date of the response: 17 April 2026

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have provided satisfactory responses to the majority of the points raised in the prior review, resulting in a clear improvement of the manuscript.

Author Response

Comments 1

The authors have provided satisfactory responses to the majority of the points raised in the prior review, resulting in a clear improvement of the manuscript.

Response 1

We thank Reviewer 2 for his positive assessment of our revised manuscript and for acknowledging the improvements made in response to the previous review. We are pleased that the responses and revisions satisfactorily addressed to the majority of the points raised.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

After reviewing the authors’ responses and the revised manuscript, I believe the authors have adequately addressed the main concerns raised in the previous round of review. The manuscript has improved in clarity, structure, and overall balance, and in its current form may be considered suitable for publication.

Author Response

Comments 1

After reviewing the authors’ responses and the revised manuscript, I believe the authors have adequately addressed the main concerns raised in the previous round of review. The manuscript has improved in clarity, structure, and overall balance, and in its current form may be considered suitable for publication.

Response 1

We are grateful to Reviewer 3 for the careful reassessment of our revised manuscript and for the positive evaluation. We appreciate the reviewer’s recognition that the manuscript has improved in clarity, structure, and balance, and we thank them for considering it suitable for publication in its current form.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have responded conscientiously to the first-round comments and made substantive revisions across nearly all points raised. The manuscript is considerably improved in mechanistic depth, terminological precision, and structural coherence. The addition of a Materials and Methods section, a dedicated Limitations section, a diagnostic workflow figure, and expanded discussions of heteroplasmy, differential diagnosis, and the developmental - degenerative continuum represent meaningful improvements. The classification framework is now more logically organized, with autophagy correctly repositioned as a cross-cutting mechanism and repeat expansion disorders linked to protein aggregation pathology.

However, several issues remain that should be addressed before publication. These include one factual error, a duplicated paragraph, mechanistic additions that lack supporting references, and editorial inconsistencies. None of these require a full additional revision round; they can be resolved through minor revision.

Substantive Issues Requiring Correction

1. Duplicated paragraph in Section 4.2 (Mitochondrial Disorders)

Lines 319 - 326 contain a verbatim duplication. The sentence beginning “Because the developing brain has exceptionally high metabolic demands, mitochondrial dysfunction commonly manifests in childhood with neurodevelopmental delay, developmental regression, epilepsy, encephalopathy, movement disorders, ataxia, and multisystem involvement” appears twice consecutively within the same paragraph. This is clearly a copy-paste artifact introduced during revision and must be removed.

2. Factual error: POLG misclassified as a lysosomal gene (line 850)

In the description of Figure 2, the text reads: “mitochondrial dysfunction, associated with pathogenic variants in lysosomal genes such as POLG…” (emphasis added). POLG encodes the catalytic subunit of mitochondrial DNA polymerase gamma and is a mitochondrial gene, not a lysosomal gene. This appears to be an editing error introduced during revision. The word “lysosomal” should be replaced with “mitochondrial.”

3. Figure 2 description placement (Point 24 follow-up)

In the first-round review, Point 24 noted that the detailed description of Figure 2 was misplaced within Section 5.8 (Peroxisomal Dysfunction), despite covering all four major pathophysiological mechanisms. The authors’ response states that this description has been “moved to a more appropriate position within the general pathophysiology section.” However, in the revised manuscript the Figure 2 description still appears at lines 838 - 873, immediately following the peroxisomal dysfunction text and before Section 6 (Diagnosis). It does not appear to have been relocated. Please verify and correct the placement.

4. Mechanistic additions without supporting references

Two mechanistic additions made in response to first-round comments are presented without any supporting citations:

• Lysosomal condensate/aggregate-like states (lines 751 - 755): The text now states that “lysosomal storage may, in some disorders, involve formation of condensed or aggregate-like substrate states that further impair lysosomal homeostasis and proteostasis.” This is an appropriate and well-hedged addition, but no references are provided. At least one or two citations to recent work on lysosomal condensate biology or substrate phase behavior should be added.
• Epigenetic consequences of lysosomal - autophagic insufficiency (lines 756 - 758): The statement that impaired autophagy - lysosome function may be associated with “altered histone modifications, chromatin reorganization, and transcriptional dysregulation” is a single sentence with no supporting references. This represents an important emerging concept that warrants at least minimal citation support.

5. Lipid toxicity mechanism (Point 10 follow-up)

The authors’ response letter provides a detailed mechanistic explanation of how lipid accumulation causes neuronal toxicity, including membrane fluidity disruption, lipid raft signaling perturbation, calcium dysregulation, and psychosine-mediated cytotoxicity. However, the corresponding text in the manuscript (Comment AC18, surrounding Table 2) remains comparatively compressed. While the revision is an improvement, incorporating one or two additional sentences from the response into the manuscript body would better reflect the mechanistic depth described in the authors’ reply.

6. Semantic duplication in the Introduction

The final two paragraphs of the Introduction (lines 78-88) contain semantic overlap that weakens the concluding focus. The penultimate sentence of the assumptions paragraph states: “Finally, an integrated review of epidemiology, pathophysiology, diagnosis, and treatment is necessary to better contextualize these disorders within modern precision medicine.” The subsequent aim paragraph then restates: “We focus on their epidemiology, molecular classification, key pathophysiological mechanisms, diagnostic approaches, and current as well as emerging therapeutic strategies.” These two statements are semantically equivalent - the first framing the need, the second the scope - but in their current proximity the effect is redundant. The authors should either merge these into a single concise statement of aim or differentiate them more clearly to avoid repetition.

7. Sentence length in Section 3.1 (Epidemiology)

The sentence at lines 168 - 172 describing the Northern Finland cohort runs to approximately 60 words and contains a semicolon pivot with an embedded relative clause: “More granular regional epidemiology comes from a longitudinal population-based cohort in Northern Finland, where the cumulative childhood incidence of childhood-onset GWMDs was 30 per 100,000 live births, spanning both classic leukodystrophies and a broader spectrum of genetic leukoencephalopathies; notably, a substantial share involved newly described or recently recognized genetic etiologies, underscoring the impact of improved molecular diagnostics on measured incidence.” For a review addressing a multidisciplinary audience, splitting this into two sentences at the semicolon would improve readability without altering meaning.

8. Residual bold-text formatting artifacts

Despite correction of the formatting issue identified in the first round (Point 19), at least one additional bold-text artifact remains. At line 302, the phrase “of their” appears in bold, inconsistent with the surrounding paragraph. A systematic check for residual formatting inconsistencies is recommended.

9. CSF1R table entry readability

The revised CSF1R entry in Table 3 is now more nuanced but also substantially longer and somewhat convoluted. Consider tightening the phrasing for readability, for example: “Rare early-onset presentations have been described and may reflect a broader disease spectrum, though the roles of variant severity and ascertainment remain incompletely defined.”

10. Abbreviation list inconsistency

The abbreviation list includes “PCDs - Post-convulsive disorders,” but this abbreviation does not appear to be used anywhere in the manuscript text. Please either remove the unused entry or confirm its usage.

Overall Recommendation

The revised manuscript represents a substantial improvement over the original submission. The authors engaged seriously and constructively with the first-round review. The remaining issues are correctable through minor revision. The most critical items requiring attention are: (1) the factual error at line 850 (POLG misclassified as a lysosomal gene); (2) the duplicated paragraph in Section 4.2; and (3) the addition of references for the two unsupported mechanistic claims (lysosomal condensates and epigenetic consequences). Once these corrections are made, the manuscript should be suitable for acceptance.

Author Response

Responses to Reviewer 4

1. Duplicated paragraph in Section 4.2 (Mitochondrial Disorders)

Lines 319 - 326 contain a verbatim duplication. The sentence beginning “Because the developing brain has exceptionally high metabolic demands, mitochondrial dysfunction commonly manifests in childhood with neurodevelopmental delay, developmental regression, epilepsy, encephalopathy, movement disorders, ataxia, and multisystem involvement” appears twice consecutively within the same paragraph. This is clearly a copy-paste artifact introduced during revision and must be removed.

Response to reviewer

We thank the reviewer for identifying this duplication. The repeated sentence in Section 4.2 was an unintended copy-paste artifact introduced during revision and has now been removed.

Thank you very much!

2. Factual error: POLG misclassified as a lysosomal gene (line 850)

In the description of Figure 2, the text reads: “mitochondrial dysfunction, associated with pathogenic variants in lysosomal genes such as POLG…” (emphasis added). POLG encodes the catalytic subunit of mitochondrial DNA polymerase gamma and is a mitochondrial gene, not a lysosomal gene. This appears to be an editing error introduced during revision. The word “lysosomal” should be replaced with “mitochondrial.”

Response to reviewer

We appreciate the reviewer’s careful observation. This was an editing error introduced during revision. We have corrected the sentence by replacing “lysosomal genes” with “genes involved in mitochondrial function.”

Thank you very much!

3. Figure 2 description placement (Point 24 follow-up)

In the first-round review, Point 24 noted that the detailed description of Figure 2 was misplaced within Section 5.8 (Peroxisomal Dysfunction), despite covering all four major pathophysiological mechanisms. The authors’ response states that this description has been “moved to a more appropriate position within the general pathophysiology section.” However, in the revised manuscript the Figure 2 description still appears at lines 838 - 873, immediately following the peroxisomal dysfunction text and before Section 6 (Diagnosis). It does not appear to have been relocated. Please verify and correct the placement.

Response to reviewer

We thank the reviewer for this important clarification. We agree that the detailed description of Figure 2 is intended to synthesize the major mechanisms discussed across the entire pathophysiology chapter and should not appear within the peroxisomal dysfunction subsection. In the revised manuscript, Figure 2 and its explanatory text have now been relocated to a separate integrative subsection at the end of Section 5, immediately before Section 6 (Diagnosis). This restructuring better reflects the figure’s scope and resolves the previous placement inconsistency.

Thank you very much!

4. Mechanistic additions without supporting references

Two mechanistic additions made in response to first-round comments are presented without any supporting citations:

• Lysosomal condensate/aggregate-like states (lines 751 - 755): The text now states that “lysosomal storage may, in some disorders, involve formation of condensed or aggregate-like substrate states that further impair lysosomal homeostasis and proteostasis.” This is an appropriate and well-hedged addition, but no references are provided. At least one or two citations to recent work on lysosomal condensate biology or substrate phase behavior should be added.
• Response to reviewer 

We thank Reviewer 4 for this helpful observation. In response, we have added appropriate supporting references and slightly refined the text to reflect that this remains an emerging concept rather than a universally established mechanism. Thank you very much!

• Epigenetic consequences of lysosomal - autophagic insufficiency (lines 756 - 758): The statement that impaired autophagy - lysosome function may be associated with “altered histone modifications, chromatin reorganization, and transcriptional dysregulation” is a single sentence with no supporting references. This represents an important emerging concept that warrants at least minimal citation support.

• Response to reviewer

We thank the reviewer for this important observation. During revision, the original sentence on the epigenetic consequences of lysosomal–autophagic insufficiency was removed as part of a broader restructuring of the paragraph. The current version instead focuses on the emerging concept of condensed or aggregate-like lysosomal substrate states, which we have now supported with appropriate references [75,76]. Thank you very much!

5. Lipid toxicity mechanism (Point 10 follow-up)

The authors’ response letter provides a detailed mechanistic explanation of how lipid accumulation causes neuronal toxicity, including membrane fluidity disruption, lipid raft signaling perturbation, calcium dysregulation, and psychosine-mediated cytotoxicity. However, the corresponding text in the manuscript (Comment AC18, surrounding Table 2) remains comparatively compressed. While the revision is an improvement, incorporating one or two additional sentences from the response into the manuscript body would better reflect the mechanistic depth described in the authors’ reply.

• Response to reviewer

We thank the reviewer for this valuable suggestion. In the revised manuscript, we expanded the paragraph preceding Table 2 by adding concise mechanistic clarification that accumulated lipids may disrupt membrane fluidity, perturb lipid raft–dependent signaling, impair calcium homeostasis, and, in selected disorders such as Krabbe disease, exert direct cytotoxic effects through psychosine. Thank you very much!

6. Semantic duplication in the Introduction

The final two paragraphs of the Introduction (lines 78-88) contain semantic overlap that weakens the concluding focus. The penultimate sentence of the assumptions paragraph states: “Finally, an integrated review of epidemiology, pathophysiology, diagnosis, and treatment is necessary to better contextualize these disorders within modern precision medicine.” The subsequent aim paragraph then restates: “We focus on their epidemiology, molecular classification, key pathophysiological mechanisms, diagnostic approaches, and current as well as emerging therapeutic strategies.” These two statements are semantically equivalent - the first framing the need, the second the scope - but in their current proximity the effect is redundant. The authors should either merge these into a single concise statement of aim or differentiate them more clearly to avoid repetition.

Response to reviewer

We appreciate this observation and agree that the final sentences of the Introduction contained semantic overlap. To improve focus and avoid repetition, we removed the sentence referring to the need for an integrated review and retained a single, streamlined aim paragraph that clearly defines the scope of the review.

Thank you very much!

7. Sentence length in Section 3.1 (Epidemiology)

The sentence at lines 168 - 172 describing the Northern Finland cohort runs to approximately 60 words and contains a semicolon pivot with an embedded relative clause: “More granular regional epidemiology comes from a longitudinal population-based cohort in Northern Finland, where the cumulative childhood incidence of childhood-onset GWMDs was 30 per 100,000 live births, spanning both classic leukodystrophies and a broader spectrum of genetic leukoencephalopathies; notably, a substantial share involved newly described or recently recognized genetic etiologies, underscoring the impact of improved molecular diagnostics on measured incidence.” For a review addressing a multidisciplinary audience, splitting this into two sentences at the semicolon would improve readability without altering meaning.

Response to reviewer

We thank the reviewer for this helpful suggestion. We agree that the original sentence was overly long and could affect readability. It has now been divided into two sentences to improve clarity while preserving the original meaning. Thank you very much!

8. Residual bold-text formatting artifacts

Despite correction of the formatting issue identified in the first round (Point 19), at least one additional bold-text artifact remains. At line 302, the phrase “of their” appears in bold, inconsistent with the surrounding paragraph. A systematic check for residual formatting inconsistencies is recommended.

Response to reviewer

We thank the reviewer for this careful observation. The residual bold-text artifact has been corrected, and the manuscript was systematically rechecked for additional formatting inconsistencies. Thank you very much!

9. CSF1R table entry readability

The revised CSF1R entry in Table 3 is now more nuanced but also substantially longer and somewhat convoluted. Consider tightening the phrasing for readability, for example: “Rare early-onset presentations have been described and may reflect a broader disease spectrum, though the roles of variant severity and ascertainment remain incompletely defined.”

Response to reviewer

We thank the reviewer for this helpful suggestion. We agree that the revised CSF1R entry was somewhat overly detailed for a table format. It has now been condensed to improve readability while retaining the necessary caution regarding interpretation.

Thank you very much!

10. Abbreviation list inconsistency

The abbreviation list includes “PCDs - Post-convulsive disorders,” but this abbreviation does not appear to be used anywhere in the manuscript text. Please either remove the unused entry or confirm its usage.

Response to reviewer

We thank the reviewer for noting this inconsistency. We confirmed that the abbreviation “PCDs” was not used in the manuscript text and have therefore removed it from the abbreviation list.  Thank you very much!

Overall Recommendation

The revised manuscript represents a substantial improvement over the original submission. The authors engaged seriously and constructively with the first-round review. The remaining issues are correctable through minor revision. The most critical items requiring attention are: (1) the factual error at line 850 (POLG misclassified as a lysosomal gene); (2) the duplicated paragraph in Section 4.2; and (3) the addition of references for the two unsupported mechanistic claims (lysosomal condensates and epigenetic consequences). Once these corrections are made, the manuscript should be suitable for acceptance.

Response to reviewer

We sincerely thank the reviewer for the careful re-evaluation of our manuscript and for the constructive overall assessment. We appreciate the acknowledgment that the revised version represents a substantial improvement. In response to the remaining comments, we have corrected the factual error regarding POLG, removed the duplicated paragraph in Section 4.2, and addressed the unsupported mechanistic additions by adding appropriate references to the revised statement on lysosomal condensate/aggregate-like states; the original sentence on epigenetic consequences was removed during paragraph restructuring and is no longer included in the manuscript. We believe these revisions have addressed the remaining concerns and further strengthened the manuscript. Thank you very much

Submission Date

06 March 2026

Date of this review

22 Apr 2026 15:08:16

We sincerely appreciate Reviewer 4’s time, careful reading, and constructive feedback. The comments provided were highly valuable and helped us strengthen the manuscript in both clarity and scientific precision.