The Multi-System Roles of Dp71 Dystrophin Isoforms in Duchenne Muscular Dystrophy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title: The Multi-System Roles of Dp71 Dystrophin Isoforms in Duchenne Muscular Dystrophy (muscles-4256887)

This review article provides the readers with a comprehensive overview of the biological and pathological roles of the dystrophin isoform Dp71 in Duchenne muscular dystrophy (DMD). The authors have efficiently discussed the Dp71 functions in various body systems (nervous system, retina, muscle) along with emerging Dp71-directed therapeutic strategies. The sections on neurological manifestations and retinal biology appear strong. However, the manuscript would benefit from improved critical analysis, better discussion of conflicting findings, improving figure presentation, and careful language editing to improve readability and precision. My comments on the concerns are listed below.

Major Comments

1. The manuscript primarily summarizes the published findings but lacks critical interpretation of published data, for example in the section describing skeletal muscle roles of Dp71 concerns were raised for conflicting evidence, but the authors have not discussed/speculated as to why the conflicts exist in these studies? Is it due to differences in mouse models, overexpression systems, or tissue-specific effects?
2. The ‘Emerging Dp71-based therapies’ section is relatively short compared to the neurological discussion. The expansion could be done by discussing the challenges associated with systemic Dp71 overexpression, isoform-specific targeting approaches, etc.
3. Figures 1 and 2 are conceptually useful but appear somewhat simplistic for a high-quality review article. The figures would benefit from adding a schematic showing Dp71 localization, its interaction with other proteins, downstream physiological effects and associated DMD phenotypes.

Minor Comments

1. Line 243, please correct ‘Dp-71 null’ to Dp71-null
2. Line 384, please correct ‘DMD-Null’ to DMD-null
3. Line no 420, 422, 427, 429, 435, 443 - please correct ' Dp71-Null’ to Dp71-null

 

Author Response

Reviewer 1: (Comments underlined, responses in blue)

Major Comments

1. The manuscript primarily summarizes the published findings but lacks critical interpretation of published data, for example in the section describing skeletal muscle roles of Dp71 concerns were raised for conflicting evidence, but the authors have not discussed/speculated as to why the conflicts exist in these studies? Is it due to differences in mouse models, overexpression systems, or tissue-specific effects?

Thank you for taking the time to review our manuscript. Critical interpretation has been added where appropriate throughout the manuscript, particularly focused on the discrepant results in skeletal muscle. This section now reads “

Several studies have found that transgenic Dp71 expression may exert a detrimental effect in both skeletal and cardiac tissue. It was originally reported that although trans-genic Dp71 can effectively colocalize with the DAPC in muscle tissue, muscle pathology remained severely dystrophic, suggesting minimal function in the Dp71-bound DAPC (74). It was later theorized that skeletal muscle expression of Dp71 may exert a dominant negative effect by competing with full length dystrophin for DAPC binding sites (75). Leibovitz et al. found that transgenic overexpression of Dp71 in healthy mice led to muscle damage similar to a dystrophic mouse, marked by muscle fiber degeneration and elevated serum creatine kinase (75). Although there was no change in isometric tension between healthy and transgenic animals, Dp71-overexpressing mice were found to have a higher risk of sarcolemmal rupture (76). In contrast, it was observed by Lim et al. that healthy mice with transgenic overexpression of human Dp71 demonstrated normal skeletal muscle function, but impaired cardiac muscle function reminiscent of a dilated cardiomyopathy (77). Transgenic mice displayed reduced ventricular ejection fraction and reduced ventricular wall thickness as early as 3 months of age, and by 12 months transgenic mice showed elevated systolic volumes and reduced ejection fraction relative to healthy controls (77). The differing effect of Dp71 overexpression observed by Leibovitz and Lim may arise at least partially from differing methods and magnitudes of overexpression. While Lim used a mouse model overexpressing Dp71 via an integrated human DMD transgene, Leibovitz used a plasmid-based system driven by the CMV promoter that would likely show a much higher overall level of Dp71 expression (75,77). If so, this would suggest that the dominant negative effect of Dp71 on skeletal muscle is dose dependent and may only arise at very high levels of Dp71 expression.”

In addition, summary paragraphs have been added to the end of each subsection to better organize the data presented and provide room for author interpretation and critical analysis.

1. The ‘Emerging Dp71-based therapies’ section is relatively short compared to the neurological discussion. The expansion could be done by discussing the challenges associated with systemic Dp71 overexpression, isoform-specific targeting approaches, etc.

The summary paragraph for this section now better reflects challenges with longer term translation of Dp71-basewd therapies. It now reads:

“Collectively, these studies show that Dp71 can be effectively and selectively overexpressed in the retina leading to restored KiR4.1 expression, restored AQP4 expression, and recovery in ERG deficits, but that expression in the brain fails to address the behavioural phenotype in Dp71-null mice (84,86). This suggests that Dp71 overexpression may have therapeutic value for the retinal defects of DMD, but not for the cognitive defects. However, several barriers exist that must be overcome prior to clinical translation of a Dp71-based therapy. Given the potential dominant negative effects associated with muscular and cardiac expression of Dp71, the risks of systemic or off-target Dp71 expression must be care-fully assessed (75,77). A prior study identified that the BRB in Dp71-null mice remains impermeable to ShH10-GFP despite the known BRB damage in this model, offering some evidence that intravitreal injection will not lead to systemic transgene expression, however this must be validated in humans prior to any translation (82). In addition to the Dp71 transgene, the safety profile of the selected AAV vector must also be explored. Although AAV-based clinical trials have shown high success in numerous recent trials, there have also been a growing number of concerns about the safety of these therapies (87). Although rare, fatal AAV-related toxicity has been observed in five patients across four different trials in recent years (87). Furthermore, treatment with an AAV-based therapy can activate the immunological production of anti-AAV antibodies, precluding the use of additional AAV-based therapies (88). This may conflict directly with AAV-delivered microdystrophin, a different transgenic therapy for DMD that has seen high amounts of research and clinical support in recent years (11,89,90). Continued studies in this field should consider and address these concerns.”

1. Figures 1 and 2 are conceptually useful but appear somewhat simplistic for a high-quality review article. The figures would benefit from adding a schematic showing Dp71 localization, its interaction with other proteins, downstream physiological effects and associated DMD phenotypes.

Both figures have been heavily revised to improve their overall quality and information provided. Figure 1 initially provided a simplistic overview of the four major structural groups of Dp71 characterized by differences in their C-terminal domains. To enhance the complexity of Figure 1 in the manuscript, all major groups and their associated splice variants have been added. In addition, the image now outlines the names and synonyms of the corresponding splice variants, alongside the general location where the major splice variants are found. An additional figures has also been created, illustrating the interaction/localization of Dp71 with other ECM.

 

Minor Comments

1. Line 243, please correct ‘Dp-71 null’ to Dp71-null
2. Line 384, please correct ‘DMD-Null’ to DMD-null
3. Line no 420, 422, 427, 429, 435, 443 - please correct ' Dp71-Null’ to Dp71-null

All minor comments were addressed, and nomenclature for these examples and others was standardized throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a complete overview of the roles of Dp71 dystrophin isoforms in brain, retina and cardiac as well as skeletal muscle, covering multiple aspects from cells, to circuits, organisms (including mouse models), and it is strongly disease and therapy oriented. While this study addresses a timely and very relevant topic, the manuscript would benefit from a stronger integration of the authors’ point of view to improve its impact and clarity. Nevertheless, the information presented is valuable, and the work is of clear interest to the scientific community.

Minor points:

1. Is there a code for the colors in Figure 1 (namely, exon 68, 79f and 77i) that needs to be explained in the legend for clarity?
2. Line 134: rephrase “…producing difficulties in long-term potentiation…”
   
   

   • What is the main question addressed by the research?

   This manuscript provides a complete overview of the roles of Dp71 dystrophin isoforms in brain, retina and cardiac as well as skeletal muscle, covering multiple aspects from cells, to circuits, organisms (including mouse models), and it is strongly disease and therapy oriented.

   • Do you consider the topic original or relevant to the field? Does it
   address a specific gap in the field? Please also explain why this is/ is not
   the case.
   • What does it add to the subject area compared with other published
   material?

   The information presented is valuable, and the work is of clear interest to the scientific community. The focus on the Dp71 is particularly interesting due to its implications in non-muscle symptoms of DMD, considering that the muscle symptoms have been more extensively studied and are more widely covered in the literature. Its roles in the brain are particularly interesting and more innovative.

   • What specific improvements should the authors consider regarding the
   methodology?
   While this study addresses a timely and very relevant topic, the manuscript would benefit from a stronger integration of the authors’ point of view to improve its impact and clarity.

   • Are the conclusions consistent with the evidence and arguments presented
   and do they address the main question posed? Please also explain why this
   is/is not the case.

   This is a review article and the data presented are coherent with the central aim.

   • Are the references appropriate?

   Yes.

   • Any additional comments on the tables and figures.
   Is there a code for the colors in Figure 1 (namely, exon 68, 79f and 77i) that needs to be explained in the legend for clarity?

    

Author Response

Reviewer 2:

Minor points:

1. Is there a code for the colors in Figure 1 (namely, exon 68, 79f and 77i) that needs to be explained in the legend for clarity?

Thank you for taking the time to review our manuscript. The original figure legend did not clearly outline what the different colours of the exons in the image represented. To address this concern, the figure caption now explicitly states the colour code of the corresponding exons. The following statement has been added to the legend “colored regions indicate alternatively spliced sequences: exon 78 (green), alternative exon 79 (purple), and the retained sequence from intron 77 (yellow).”

1. Line 134: rephrase “…producing difficulties in long-term potentiation…”
   
   This section has been rephrased to now read: “When Dp71 is absent, as seen in Dp71-null mice, the DAPC is compromised, leading to reduced initiation and maintenance of long-term potentiation (LTP), a process important for synaptic strengthening and memory formation (32).”

While this study addresses a timely and very relevant topic, the manuscript would benefit from a stronger integration of the authors’ point of view to improve its impact and clarity.

Critical interpretation has been added where appropriate throughout the manuscript, particularly focused on the discrepant results in skeletal muscle. This section now reads “

Several studies have found that transgenic Dp71 expression may exert a detrimental effect in both skeletal and cardiac tissue. It was originally reported that although trans-genic Dp71 can effectively colocalize with the DAPC in muscle tissue, muscle pathology remained severely dystrophic, suggesting minimal function in the Dp71-bound DAPC (74). It was later theorized that skeletal muscle expression of Dp71 may exert a dominant negative effect by competing with full length dystrophin for DAPC binding sites (75). Leibovitz et al. found that transgenic overexpression of Dp71 in healthy mice led to muscle damage similar to a dystrophic mouse, marked by muscle fiber degeneration and elevated serum creatine kinase (75). Although there was no change in isometric tension between healthy and transgenic animals, Dp71-overexpressing mice were found to have a higher risk of sarcolemmal rupture (76). In contrast, it was observed by Lim et al. that healthy mice with transgenic overexpression of human Dp71 demonstrated normal skeletal muscle function, but impaired cardiac muscle function reminiscent of a dilated cardiomyopathy (77). Transgenic mice displayed reduced ventricular ejection fraction and reduced ventricular wall thickness as early as 3 months of age, and by 12 months transgenic mice showed elevated systolic volumes and reduced ejection fraction relative to healthy controls (77). The differing effect of Dp71 overexpression observed by Leibovitz and Lim may arise at least partially from differing methods and magnitudes of overexpression. While Lim used a mouse model overexpressing Dp71 via an integrated human DMD transgene, Leibovitz used a plasmid-based system driven by the CMV promoter that would likely show a much higher overall level of Dp71 expression (75,77). If so, this would suggest that the dominant negative effect of Dp71 on skeletal muscle is dose dependent and may only arise at very high levels of Dp71 expression.”

In addition, summary paragraphs have been added to the end of each subsection to better organize the data presented and provide room for author interpretation and critical analysis. They read as follows:

Neurological section:

“In brief, preclinical studies in rodent and human embryonic brains demonstrate that Dp71 isoforms are temporally regulated and perform distinct roles in neural progenitors, glial cells, mature inhibitory synapses, and in nuclear gene regulation (22,28). Dp71 also displays a role in calcium, water, and potassium homeostasis, helping to regulate neural excitability (36–38,40). Thus, the cellular context and timing of Dp71 loss may influence neurological phenotypes. Mouse studies indicate that the severity of neurobehavioural deficits may correspond with the degree of dystrophin isoform loss. Mdx mice display mild cognitive and behavioural changes, whereas mdx3cv and Dp71-null mice show more pronounced phenotypes, including increased anxiety-like behaviour, impaired spatial learning, altered social interactions, and abnormal vocalization patterns (37,42,43). Interpretation of these outcomes is complicated by the use of multi-isoform mouse models, such as mdx and mdx3cv, which do not distinguish the effects of Dp71 deficiency compared to the absence of other short isoforms. Additional studies using Dp71-null mouse models can help define the precise consequences of Dp71 loss.”

“Collectively, clinical studies have established that Dp71 deficiency is frequently associated with neurocognitive impairments in DMD characterized by lower IQ, difficulties with executive function, and abnormal neurobehavioural assessments (44,45,49–51). However, distal dystrophin gene mutations frequently disrupt several brain-expressed isoforms, such as Dp140, which makes it difficult to isolate the effects of Dp71 alone in human studies. Future longitudinal and genotype-specific studies on Dp71 loss are war-ranted to determine its independent contributions to neurobehavioural outcomes in DMD. Despite this challenge, assessing patterns of Dp71 loss remains translationally relevant for guiding cognitive screening and intervention for patients with distal gene mutations.”

Retina section:

“In summary, Dp71 has an important and distinct role in the retina where it regulates Müller cell homeostasis and vascular permeability. This is mediated through the Dp71-dependent regulation of both KiR4.1 and AQP4, contributing to the transport of wa-ter and potassium (59). Consequently, the lack of Dp71 in the retina leads to retinal dys-function, detectable as reduced B-wave amplitude on ERG, and may contribute to cataract formation (66,68).”

Muscle section:

“Taken together, the multitude of conflicting studies fail to provide a clear picture of the potential roles of Dp71 in muscle. Although recent studies have identified Dp71 in bulk extract from muscle tissue, it is unknown which specific cell populations within might be expressing Dp71. Given that satellite cells have been shown to express Dp71 themselves, further studies should clarify whether the Dp71 signal observed in muscle tissue arises purely from satellite cells, or whether it is also expressed in the mature myo-tubes (79). The lack of a difference in grip strength or treadmill running between mice with or without Dp71 suggests that the primary role of Dp71 in muscle is unlikely to involve interactions with the DAPC in the contractile muscle unit (78). This is further supported by the impaired function and sarcolemmal rupture that arises when Dp71 is overex-pressed at a high rate in muscles (75,76). However, the impaired outcomes in patients lacking Dp71 suggest that Dp71 still has an important role in skeletal muscle, even if it is not directly related to muscle contraction (78,80). Future work should continue to explore this phenomenon, as well as aim to clarify the contrasting results found by many groups at both the basic and clinical levels.”

Therapy section:

“Collectively, these studies show that Dp71 can be effectively and selectively overex-pressed in the retina leading to restored KiR4.1 expression, restored AQP4 expression, and recovery in ERG deficits, but that expression in the brain fails to address the behavioural phenotype in Dp71-null mice (84,86). This suggests that Dp71 overexpression may have therapeutic value for the retinal defects of DMD, but not for the cognitive defects. However, several barriers exist that must be overcome prior to clinical translation of a Dp71-based therapy. Given the potential dominant negative effects associated with muscular and car-diac expression of Dp71, the risks of systemic or off-target Dp71 expression must be care-fully assessed (75,77). A prior study identified that the BRB in Dp71-null mice remains impermeable to ShH10-GFP despite the known BRB damage in this model, offering some evidence that intravitreal injection will not lead to systemic transgene expression, however this must be validated in humans prior to any translation (82). In addition to the Dp71 transgene, the safety profile of the selected AAV vector must also be explored. Although AAV-based clinical trials have shown high success in numerous recent trials, there have also been a growing number of concerns about the safety of these therapies (87). Although rare, fatal AAV-related toxicity has been observed in five patients across four different tri-als in recent years (87). Furthermore, treatment with an AAV-based therapy can activate the immunological production of anti-AAV antibodies, precluding the use of additional AAV-based therapies (88). This may conflict directly with AAV-delivered microdystrophin, a different transgenic therapy for DMD that has seen high amounts of research and clinical support in recent years (11,89,90). Continued studies in this field should consider and address these concerns.”

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

This is a timely and potentially useful review of Dp71 dystrophin isoforms in Duchenne muscular dystrophy. The manuscript has several strengths: it addresses an important but relatively specialized aspect of DMD biology; it integrates evidence from neurological, retinal, muscular, and therapeutic studies; and it includes recent literature relevant to Dp71 function and disease mechanisms. The sections on synaptic organization, astrocytic, perivascular roles, retinal Müller cell biology, and AAV-mediated therapeutic approaches are particularly valuable.

Nevertheless, the manuscript requires major revision before it is suitable for publication. The current version is informative but often descriptive. It would benefit from a clearer organizing thesis, stronger critical synthesis, more explicit separation of clinical and preclinical evidence, and a careful audit of the reference list. The authors should also revise the language to reduce informality, repetition, and occasional overstatement.

The manuscript is presented as a review, but the authors do not clearly state whether it is intended as a narrative, scoping, or systematic review. There is no description of the databases searched, the search terms, the date limits, the inclusion/exclusion criteria, or the rationale for study selection. If this is intended as a narrative review, the authors should explicitly state this and briefly describe how the literature was identified. If the journal expects a more structured review, the authors should include a short methods section. This would improve transparency and help readers understand whether the review aims to be comprehensive or selectively interpretative.

The manuscript summarizes many individual studies but does not always integrate them into a clear conceptual framework. The authors should add more synthesis at the end of each major section, for example: What is well established? What remains uncertain? Which findings are model-dependent? Which conclusions are supported by clinical evidence versus animal or cellular models? What are the translational implications?

This is particularly important in the sections on brain function, muscle involvement, and Dp71-based therapy, where the evidence is complex and occasionally conflicting.

Several claims should be phrased more cautiously. For example, the manuscript states that Dp71 loss is the "primary driver" of severe cognitive impairment in DMD. While the evidence strongly supports an association between distal DMD mutations affecting Dp71 and severe intellectual disability, causality should be described carefully because genotype-phenotype correlations may involve overlapping effects of multiple short isoforms, mutation location, and broader disease modifiers.

Similarly, the figure caption stating that Dp71 has been shown to "compete with full-length dystrophin and lead to dysfunction in skeletal and heart muscle" should specify that this is mainly based on transgenic or overexpression models and may not reflect physiological Dp71 expression in human muscle.

The review combines human clinical studies, mouse models, cellular systems, and gene-therapy experiments. This breadth is valuable, but the manuscript should consistently identify the evidentiary level of each claim. For instance, the neurological and behavioral sections include findings from Dp71-null mice, mdx3cv mice, human genotype-phenotype studies, and patient-derived neurons. These should be more clearly separated or summarized in a table. A table distinguishing model/system, Dp71 alteration, main finding, and translational relevance would greatly improve readability.

The manuscript would benefit from at least two tables: Table 1. Dp71 roles by tissue/system; Table 2. Dp71-related therapeutic strategies. These tables would help the review move from a long descriptive format to a more reader-friendly synthesis.

Figure 1 and Figure 2 appear useful but need clearer captions. Figure 1 should define all splice variants shown, clarify exon numbering, and explain what is meant by "major variants (D-G)". Figure 2 should avoid causal overstatement and should visually distinguish well-established roles from hypothesized or model-dependent effects.

The reference list requires substantial revision. There appear to be duplicated entries, incomplete author fields, inconsistent formatting, and publisher and metadata artifacts. Examples include repeated references, malformed entries, and citation records containing extra metadata strings rather than standard bibliographic information.

The Abstract is concise and generally effective, but it should make it clearer that this is a narrative review and briefly state the main conclusion or translational implication.

The conclusion is appropriate but could be more synthetic. It should explicitly identify the most important unresolved questions and translational risks.

Kind regards,

Comments on the Quality of English Language

The English is generally understandable, but the manuscript contains informal phrasing and some awkward constructions. Examples include "ramps up", "sticking to baseline levels", "shed insight", "curiosity about new peers", and "take care to assess safety risks". These should be replaced with more precise scientific language.

The manuscript should use either American or British English consistently. For example, "behavioral" and "behavioural" both appear.

Some paragraphs are very long and should be divided for readability, especially in the neurological section.

Author Response

Reviewer 3:

Dear Authors,

This is a timely and potentially useful review of Dp71 dystrophin isoforms in Duchenne muscular dystrophy. The manuscript has several strengths: it addresses an important but relatively specialized aspect of DMD biology; it integrates evidence from neurological, retinal, muscular, and therapeutic studies; and it includes recent literature relevant to Dp71 function and disease mechanisms. The sections on synaptic organization, astrocytic, perivascular roles, retinal Müller cell biology, and AAV-mediated therapeutic approaches are particularly valuable. Nevertheless, the manuscript requires major revision before it is suitable for publication. The current version is informative but often descriptive. It would benefit from a clearer organizing thesis, stronger critical synthesis, more explicit separation of clinical and preclinical evidence, and a careful audit of the reference list. The authors should also revise the language to reduce informality, repetition, and occasional overstatement.

Thank you for taking the time to review our manuscript. We have addressed all concerns mentioned, and your insight contributed to a substantially improved version of the manuscript. Please find individual changes listed after their specific point below.

The manuscript is presented as a review, but the authors do not clearly state whether it is intended as a narrative, scoping, or systematic review. There is no description of the databases searched, the search terms, the date limits, the inclusion/exclusion criteria, or the rationale for study selection. If this is intended as a narrative review, the authors should explicitly state this and briefly describe how the literature was identified. If the journal expects a more structured review, the authors should include a short methods section. This would improve transparency and help readers understand whether the review aims to be comprehensive or selectively interpretative.

This article is a narrative review without strict search terms. This has been better reflected, and the introduction now reads: “This narrative review compiles and summarizes current knowledge regarding Dp71, with a focus on its roles in neuro-logical, retinal, and muscular systems, and evaluates emerging therapeutic approaches. Publications involving Dp71 and Dp71-deficient mouse models were manually curated through the U.S National Library of Medicine’s PubMed database”

The manuscript summarizes many individual studies but does not always integrate them into a clear conceptual framework. The authors should add more synthesis at the end of each major section, for example: What is well established? What remains uncertain? Which findings are model-dependent? Which conclusions are supported by clinical evidence versus animal or cellular models? What are the translational implications? This is particularly important in the sections on brain function, muscle involvement, and Dp71-based therapy, where the evidence is complex and occasionally conflicting.

 

Summary paragraphs have been added to the end of each subsection to better organize the data presented and provide room for author interpretation and critical analysis. They read as follows:

Neurological section:

“In brief, preclinical studies in rodent and human embryonic brains demonstrate that Dp71 isoforms are temporally regulated and perform distinct roles in neural progenitors, glial cells, mature inhibitory synapses, and in nuclear gene regulation (22,28). Dp71 also displays a role in calcium, water, and potassium homeostasis, helping to regulate neural excitability (36–38,40). Thus, the cellular context and timing of Dp71 loss may influence neurological phenotypes. Mouse studies indicate that the severity of neurobehavioural deficits may correspond with the degree of dystrophin isoform loss. Mdx mice display mild cognitive and behavioural changes, whereas mdx3cv and Dp71-null mice show more pronounced phenotypes, including increased anxiety-like behaviour, impaired spatial learning, altered social interactions, and abnormal vocalization patterns (37,42,43). Interpretation of these outcomes is complicated by the use of multi-isoform mouse models, such as mdx and mdx3cv, which do not distinguish the effects of Dp71 deficiency compared to the absence of other short isoforms. Additional studies using Dp71-null mouse models can help define the precise consequences of Dp71 loss.”

“Collectively, clinical studies have established that Dp71 deficiency is frequently associated with neurocognitive impairments in DMD characterized by lower IQ, difficulties with executive function, and abnormal neurobehavioural assessments (44,45,49–51). However, distal dystrophin gene mutations frequently disrupt several brain-expressed isoforms, such as Dp140, which makes it difficult to isolate the effects of Dp71 alone in human studies. Future longitudinal and genotype-specific studies on Dp71 loss are war-ranted to determine its independent contributions to neurobehavioural outcomes in DMD. Despite this challenge, assessing patterns of Dp71 loss remains translationally relevant for guiding cognitive screening and intervention for patients with distal gene mutations.”

Retina section:

“In summary, Dp71 has an important and distinct role in the retina where it regulates Müller cell homeostasis and vascular permeability. This is mediated through the Dp71-dependent regulation of both KiR4.1 and AQP4, contributing to the transport of wa-ter and potassium (59). Consequently, the lack of Dp71 in the retina leads to retinal dys-function, detectable as reduced B-wave amplitude on ERG, and may contribute to cataract formation (66,68).”

Muscle section:

“Taken together, the multitude of conflicting studies fail to provide a clear picture of the potential roles of Dp71 in muscle. Although recent studies have identified Dp71 in bulk extract from muscle tissue, it is unknown which specific cell populations within might be expressing Dp71. Given that satellite cells have been shown to express Dp71 themselves, further studies should clarify whether the Dp71 signal observed in muscle tissue arises purely from satellite cells, or whether it is also expressed in the mature myo-tubes (79). The lack of a difference in grip strength or treadmill running between mice with or without Dp71 suggests that the primary role of Dp71 in muscle is unlikely to involve interactions with the DAPC in the contractile muscle unit (78). This is further supported by the impaired function and sarcolemmal rupture that arises when Dp71 is overex-pressed at a high rate in muscles (75,76). However, the impaired outcomes in patients lacking Dp71 suggest that Dp71 still has an important role in skeletal muscle, even if it is not directly related to muscle contraction (78,80). Future work should continue to explore this phenomenon, as well as aim to clarify the contrasting results found by many groups at both the basic and clinical levels.”

Therapy section:

“Collectively, these studies show that Dp71 can be effectively and selectively overex-pressed in the retina leading to restored KiR4.1 expression, restored AQP4 expression, and recovery in ERG deficits, but that expression in the brain fails to address the behavioural phenotype in Dp71-null mice (84,86). This suggests that Dp71 overexpression may have therapeutic value for the retinal defects of DMD, but not for the cognitive defects. However, several barriers exist that must be overcome prior to clinical translation of a Dp71-based therapy. Given the potential dominant negative effects associated with muscular and car-diac expression of Dp71, the risks of systemic or off-target Dp71 expression must be care-fully assessed (75,77). A prior study identified that the BRB in Dp71-null mice remains impermeable to ShH10-GFP despite the known BRB damage in this model, offering some evidence that intravitreal injection will not lead to systemic transgene expression, however this must be validated in humans prior to any translation (82). In addition to the Dp71 transgene, the safety profile of the selected AAV vector must also be explored. Although AAV-based clinical trials have shown high success in numerous recent trials, there have also been a growing number of concerns about the safety of these therapies (87). Although rare, fatal AAV-related toxicity has been observed in five patients across four different tri-als in recent years (87). Furthermore, treatment with an AAV-based therapy can activate the immunological production of anti-AAV antibodies, precluding the use of additional AAV-based therapies (88). This may conflict directly with AAV-delivered microdystrophin, a different transgenic therapy for DMD that has seen high amounts of research and clinical support in recent years (11,89,90). Continued studies in this field should consider and address these concerns.”

Several claims should be phrased more cautiously. For example, the manuscript states that Dp71 loss is the "primary driver" of severe cognitive impairment in DMD. While the evidence strongly supports an association between distal DMD mutations affecting Dp71 and severe intellectual disability, causality should be described carefully because genotype-phenotype correlations may involve overlapping effects of multiple short isoforms, mutation location, and broader disease modifiers.

The causality of statements has been generally lessened throughout the manuscript, and confounding effects such as multi-isoform deficiency have been discussed as limiting factors where appropriate.

Similarly, the figure caption stating that Dp71 has been shown to "compete with full-length dystrophin and lead to dysfunction in skeletal and heart muscle" should specify that this is mainly based on transgenic or overexpression models and may not reflect physiological Dp71 expression in human muscle.

This figure has been overhauled to improve its overall quality and more clearly and accurately summarize relevant studies.

The review combines human clinical studies, mouse models, cellular systems, and gene-therapy experiments. This breadth is valuable, but the manuscript should consistently identify the evidentiary level of each claim. For instance, the neurological and behavioral sections include findings from Dp71-null mice, mdx3cv mice, human genotype-phenotype studies, and patient-derived neurons. These should be more clearly separated or summarized in a table. A table distinguishing model/system, Dp71 alteration, main finding, and translational relevance would greatly improve readability.

In each section, studies have been sub-sectioned as either “Preclinical” or “Clinical” to aid in organization. Specifically for the neurological and behavioural section, a new table has been created to better visualize this information-dense section.

The manuscript would benefit from at least two tables: Table 1. Dp71 roles by tissue/system; Table 2. Dp71-related therapeutic strategies. These tables would help the review move from a long descriptive format to a more reader-friendly synthesis. Figure 1 and Figure 2 appear useful but need clearer captions. Figure 1 should define all splice variants shown, clarify exon numbering, and explain what is meant by "major variants (D-G)". Figure 2 should avoid causal overstatement and should visually distinguish well-established roles from hypothesized or model-dependent effects.

The image and captions for Figure 1 have been substantially revised. Figure 1 now depicts all of the fourteen known splice variants, in addition to their names/synonyms, and location found. The original figure caption stated “Figure 1. Structural Characterization of Dp71 Splicing Variants. The Dp71 isoform exhibits C-terminal diversity, with alternative splicing generating distinct variants. Exon organization of the major variants (D–G) are presented.” The caption has been modified as follows to improve depth and clarity: “Figure 1. Schematic representation of Dp71 splice variants, corresponding nomenclature, and location. Alternative splicing of the Dp71 transcript generates four major C-terminal variant groups (D–G), distinguished by differences in exon composition. Exon numbering corresponds to the human DMD gene transcript. Group D contains exons 78 and 79, whereas Group F lacks exon 78, thereby altering the reading frame and producing an alternative exon 79 sequence (79f). Group E lacks exons 78 and 79 and retains part of intron 77 (i77), while Group G contains a premature stop codon in exon 77. Colored regions indicate alternatively spliced sequences: exon 78 (green), alternative exon 79 (purple), and the retained sequence from intron 77 (yellow). To date, Dp71d and Dp71f represent the best characterized Dp71 isoforms in the brain and retina, as evidence supporting expression of the remaining isoforms remains limited.”

Figure 2 has been overhauled to improve its overall quality and more clearly and accurately summarize relevant studies, providing a more visually intuitive option for the information proposed by the reviewer as Table 1. For a table on Dp71-related therapeutic strategies, given that all studies in this field come from a single group and represent progressive steps on the same therapy, a table comparing them may not be appropriate. A clearer summary paragraph has instead been added to concisely convey the current status of Dp71-based therapy.

A new table has instead been generated to better visualize the information-dense neurological and behavioural section.

 

 

The reference list requires substantial revision. There appear to be duplicated entries, incomplete author fields, inconsistent formatting, and publisher and metadata artifacts. Examples include repeated references, malformed entries, and citation records containing extra metadata strings rather than standard bibliographic information.

The reference list has been cleaned to ensure no duplicates and consistent formatting.

The Abstract is concise and generally effective, but it should make it clearer that this is a narrative review and briefly state the main conclusion or translational implication.

The abstract now reads” The DMD gene is well known for its product dystrophin, a large rod-shaped protein that plays a critical role in muscular membrane strength and integrity. Mutations affecting dystrophin lead to Duchenne muscular dystrophy, a fatal X-linked disease characterized by muscular weakness and breakdown. In addition to the full-length dystrophin product that is most often associated with disease, the DMD gene also encodes for multiple shorter isoforms of dystrophin with diverse functions. One isoform in particular, Dp71, has been increasingly found to play a wide variety of roles throughout the body. In this narrative review, we consolidate the numerous studies on Dp71 to provide a comprehensive foundation for future work. We outline and summarize the current state of knowledge on the role of Dp71 in the brain, the retina, and skeletal muscles, identifying current knowns and unknowns in the field. We also explore Dp71-based therapies currently being tested in the pre-clinical landscape and identify potential limitations for clinical translation”

The conclusion is appropriate but could be more synthetic. It should explicitly identify the most important unresolved questions and translational risks.

The conclusion now reads: “Dp71 has been shown to play a steadily increasing number of diverse roles through-out the body. It is critical for neuronal development and synaptic function, and the absence of Dp71 is associated with both cognitive and behavioural deficits. It also plays a key role in retinal health and function, and the loss of Dp71 is a contributing factor to im-paired vision and retinal vascular health. Finally, Dp71 is implicated in a growing number of interactions with skeletal muscle, however the data is mixed on whether the presence of Dp71 in muscle is a net benefit or a detriment to muscle strength and function. Future studies should aim to clarify this discrepancy, particularly exploring the expression and contributions of Dp71 to muscle satellite cells versus the muscle contractile unit itself. Lastly, Dp1-based therapies have been explored and validated at the preclinical stage, but consideration must be given to the potential safety concerns associated with systemic Dp71 expression and competition with AAV-delivered microdystrophin.”

The English is generally understandable, but the manuscript contains informal phrasing and some awkward constructions. Examples include "ramps up", "sticking to baseline levels", "shed insight", "curiosity about new peers", and "take care to assess safety risks". These should be replaced with more precise scientific language. The manuscript should use either American or British English consistently. For example, "behavioral" and "behavioural" both appear.

Informal phrases and inconsistent spelling/capitalization have been addressed throughout the manuscript.

Some paragraphs are very long and should be divided for readability, especially in the neurological section.

Long paragraphs have been broken up and reorganized to improve readability in the neurological and musculoskeletal sections.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

See attached.

Comments for author File: Comments.pdf

Author Response

Additional minor comments to be addressed in the revised copy-

1. (52)Overall – Correct extra reference 52 in p11, line 329

Thank you for taking the time to review the revised version of our manuscript. The erroneous extra reference has been deleted.

2. Please make sure line alignment is consistent throughout the manuscript

Alignment and spacing of paragraphs, titles, and subtitles have been standardized and verified throughout the document.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The revised manuscript is substantially stronger than the previous version. The added synthesis, clearer evidence-level separation, revised figures, and new table have improved the manuscript’s scientific value and readability. The topic remains important, and the review has the potential to be a useful contribution to the DMD and dystrophin-isoform literature.

The manuscript is now suitable for consideration after minor revision. The remaining changes are specific and correctable. I encourage the authors to focus on four priorities before resubmission: correcting residual overstatements, revising Figure 3 and its caption, completing a rigorous reference-list audit, and polishing residual language and citation artefacts.

Relatively to the reference list has been cleaned, but the revised manuscript still contains multiple visible problems. These include malformed section headings, apparent layout artifacts, inconsistent formatting, incomplete or unusual author formatting, and residual publisher template artifacts. Because this is a review article, reference accuracy and presentation are especially important. The authors should perform a complete reference-management audit before publication. Every in-text citation should be checked against the final reference list, and every reference should be formatted consistently according to journal style.

Figure 3 is visually useful, but the caption is potentially misleading. The caption states that "in the ECM, Dp71 is suggested to associate with dystroglycan". Dp71 is not an extracellular matrix protein, it should be described as being localized at the cytoplasmic, submembranous side of the membrane, interacting through the dystrophin-associated protein complex and dystroglycan-associated structures.

Should revise the figure caption to avoid implying that Dp71 itself is located in the ECM. A better formulation would be: “At the perivascular astrocytic endfoot membrane, Dp71-associated complexes support the localization or clustering of AQP4 and Kir4.1 through interactions with dystroglycan-associated membrane complexes". 

There were improvements in the causal framing, but some statements still read as stronger than the evidence supports. For example, phrases such as "Dp71 is essential", "directly disrupts", and "leads to" should be checked throughout the manuscript. In a narrative review integrating mouse, cellular, and human genotype-phenotype evidence, cautious language is preferable unless direct causality is experimentally established. Suggested alternatives include "is associated with", "contributes to", "supports", "is required in this model for", or "may participate in".

Now is state that this is a narrative review and that publications were manually curated through PubMed. This is acceptable, but the manuscript would benefit from one additional sentence clarifying the approximate search period, whether only English-language publications were considered, and whether reference lists of key articles were also screened. This does not need to become a systematic review methods section, but a little more transparency would improve reproducibility and reader confidence.

Table 1 is a valuable addition. However, some entries still risk overinterpretation. For example, statements such as "strong evidence that Dp71 disruption is a major predictor of cognitive impairment" should be phrased more carefully as "distal DMD mutations predicted to affect Dp71 are associated with increased risk of cognitive impairment". This distinction matters because human mutations may affect multiple brain-expressed dystrophin isoforms, including Dp140.  The table should also use consistent terminology for "behavioural/behavioral", "genotype-phenotype", and "Dp71 region".

Figure 2 is clearer than before and provides a helpful visual summary. However, the column "Effects of transgenic Dp71 expression" combines therapeutic re-expression, transgenic overexpression, and AAV-mediated expression across different tissues and experimental contexts. This may be confusing. It should be clarified whether this column refers to "Dp71 overexpression/re-expression in experimental models" rather than "transgenic Dp71 expression" in general. The figure should also avoid implying that outcomes observed in one model necessarily apply across tissues or delivery strategies.

Several residual line-level errors remain, including duplicate citations, spacing issues, typographical errors, awkward wording, and inconsistent capitalization. These should be corrected before publication. Provided these corrections are made, the manuscript should be suitable for publication.

Kind regards,

Comments on the Quality of English Language

The English is generally understandable, but the manuscript contains informal phrasing and some awkward constructions. Examples include "ramps up", "sticking to baseline levels", "shed insight", "curiosity about new peers", and "take care to assess safety risks". These should be replaced with more precise scientific language.

The manuscript should use either American or British English consistently. For example, "behavioral" and "behavioural" both appear.

Some paragraphs are very long and should be divided for readability, especially in the neurological section.

Author Response

 

Reviewer 3: (Comments underlined, responses in blue)

The manuscript is now suitable for consideration after minor revision. The remaining changes are specific and correctable. I encourage the authors to focus on four priorities before resubmission: correcting residual overstatements, revising Figure 3 and its caption, completing a rigorous reference-list audit, and polishing residual language and citation artefacts.

Thank you for taking the time to review the revised version of our manuscript. Please find responses to your specific points below.

Relatively to the reference list has been cleaned, but the revised manuscript still contains multiple visible problems. These include malformed section headings, apparent layout artifacts, inconsistent formatting, incomplete or unusual author formatting, and residual publisher template artifacts. Because this is a review article, reference accuracy and presentation are especially important. The authors should perform a complete reference-management audit before publication. Every in-text citation should be checked against the final reference list, and every reference should be formatted consistently according to journal style.

Thank you for your attention to detail. Alignment, spacing, and general quality control for paragraphs, titles, and subtitles have been standardized and verified throughout the document.  Please note that having the “show changes” function toggled on can create artificial errors in the formatting of the manuscript, and that the manuscript is best viewed with this setting toggled off. Please also note that publisher template artifacts are a required part of the manuscript to aid the Muscles editorial team but will be removed prior to final publication. All references have been verified, and the duplicate in-text “52” from line 329 was removed.

 

Figure 3 is visually useful, but the caption is potentially misleading. The caption states that "in the ECM, Dp71 is suggested to associate with dystroglycan". Dp71 is not an extracellular matrix protein, it should be described as being localized at the cytoplasmic, submembranous side of the membrane, interacting through the dystrophin-associated protein complex and dystroglycan-associated structures. Should revise the figure caption to avoid implying that Dp71 itself is located in the ECM. A better formulation would be: “At the perivascular astrocytic endfoot membrane, Dp71-associated complexes support the localization or clustering of AQP4 and Kir4.1 through interactions with dystroglycan-associated membrane complexes". 

The recommended figure caption has been used to avoid a potentially misleading caption.

 

There were improvements in the causal framing, but some statements still read as stronger than the evidence supports. For example, phrases such as "Dp71 is essential", "directly disrupts", and "leads to" should be checked throughout the manuscript. In a narrative review integrating mouse, cellular, and human genotype-phenotype evidence, cautious language is preferable unless direct causality is experimentally established. Suggested alternatives include "is associated with", "contributes to", "supports", "is required in this model for", or "may participate in".

Causal statements have been replaced with more cautious language as per reviewer suggestion. Individual instances are highlighted throughout the manuscript.

 

Now is state that this is a narrative review and that publications were manually curated through PubMed. This is acceptable, but the manuscript would benefit from one additional sentence clarifying the approximate search period, whether only English-language publications were considered, and whether reference lists of key articles were also screened. This does not need to become a systematic review methods section, but a little more transparency would improve reproducibility and reader confidence.

This section has been updated and now reads “This narrative review compiles and summarizes current knowledge regarding Dp71, with a focus on its roles in neurological, retinal, and muscular systems, and evaluates emerging therapeutic approaches. Publications in English involving Dp71 and Dp71-deficient mouse models were manually curated through the U.S National Library of Medicine’s PubMed database. Reference lists from key articles were also screened. No publication date cutoffs were set.”

 

Table 1 is a valuable addition. However, some entries still risk overinterpretation. For example, statements such as "strong evidence that Dp71 disruption is a major predictor of cognitive impairment" should be phrased more carefully as "distal DMD mutations predicted to affect Dp71 are associated with increased risk of cognitive impairment". This distinction matters because human mutations may affect multiple brain-expressed dystrophin isoforms, including Dp140.  The table should also use consistent terminology for "behavioural/behavioral", "genotype-phenotype", and "Dp71 region".

The table has been updated with the recommended changes to avoid causal overstatement and to maintain consistency. As this table only provides a brief overview of each study, references are also provided so that viewers can easily look up the original studies for more details.

 

Figure 2 is clearer than before and provides a helpful visual summary. However, the column "Effects of transgenic Dp71 expression" combines therapeutic re-expression, transgenic overexpression, and AAV-mediated expression across different tissues and experimental contexts. This may be confusing. It should be clarified whether this column refers to "Dp71 overexpression/re-expression in experimental models" rather than "transgenic Dp71 expression" in general. The figure should also avoid implying that outcomes observed in one model necessarily apply across tissues or delivery strategies.

To prevent overstatement and oversimplification, references have been added to the bottom righthand corner of each segment so that interested readers can explore further experimental details. The caption has also been updated and now reads

 “Effects are organized by affected system: brain, retina, and skeletal/cardiac muscle. Given the presence of some species-specific findings, effects observed in mice can not be assumed to exist in humans until experimentally confirmed, and vice versa. This figure references data from a variety of studies with different methodologies, and references can be found in the bottom right corner of each segment for further experimental details.”

Several residual line-level errors remain, including duplicate citations, spacing issues, typographical errors, awkward wording, and inconsistent capitalization. These should be corrected before publication. Provided these corrections are made, the manuscript should be suitable for publication.

Alignment, spacing, and general quality control for paragraphs, titles, and subtitles has been standardized and verified throughout the document. The duplicate in-text “52” from line 329 was removed.