A Novel Mouse Model for LAMA2-Related Muscular Dystrophy: Analysis of Molecular Pathogenesis and Clinical Phenotype

Reviewer #1 (Public review):

Strengths:

This work adds another mouse model for LAMA2-MD that re-iterates the phenotype of previously published models. Such as dy3K/dy3K; dy/dy and dyW/dyW mice. The phenotype is fully consistent with the data from others.

One of the major weaknesses of the manuscript initially submitted was the overinterpretation and the overstatements. The revised version is clearly improved as the authors toned-down their interpretation and now also cite the relevant literature of previous work.

Weaknesses:

Unfortunately, the data on RNA-seq and scRNA-seq are still rather weak. scRNA-seq was conducted with only one mouse resulting in only 8000 nuclei. I am not convinced that the data allow us to interpret them to the extent of the authors. Similar to the first version, the authors infer function by examining expression. Although they are a bit more cautious, they still argue that the BBB is not functional in dyH/dyH mice without showing leakiness. Such experiments can be done using dyes, such as Evans-blue or Cadaverin. Hence, I would suggest that they formulate the text still more carefully.

A similar lack of evidence is true for the suggested cobblestone-like lissencephaly of the mice. There is no strong evidence that this is indeed occurring in the mice (might also be a problem because mice die early). Hence, the conclusions need to be formulated in such a way that readers understand that these are interpretations and not facts.

Finally, I am surprised that the only improvement in the main figures is the Western blot for laminin-alpha2. The histology of skeletal muscle still looks rather poor. I do not know what the problems are but suggest that the authors try to make sections from fresh-frozen tissue. I anticipate that the mice were eventually perfused with PFA before muscles were isolated. This often results in the big gaps in the sections.

Overall, the work is improved but still would need additional experiments to make it really an important addition to the literature in the LAMA-MD field.

Reviewer #2 (Public review):

Summary:

This revised manuscript describes the production of a mouse model for LAMA2-Related Muscular Dystrophy. The authors investigate changes in transcripts within the brain and blood barrier. The authors also investigate changes in the transcriptome associated with the muscle cytoskeleton.

Strengths:

(1) The authors produced a mouse model of LAMA2-CMD using CRISPR-Cas9

(2) The authors identify cellular changes that disrupted the blood-brain barrier.

Weaknesses:

(1) The authors throughout the manuscript overstate "discoveries" which have been previously described, published and not appropriately cited.

(2) Alternations in the blood brain barrier and in the muscle cell cytoskeleton in LAMA2-CMD have been extensively studied and published in the literature and are not cited appropriately.

(3) The authors have increased animal number to N=6, but this is still insufficient based on Power analysis results in statistical errors and conclusions that may be incorrect.

(4) The use of "novel mouse model" in the manuscript overstates the impact of the study.

(5) All studies presented are descriptive and do not more to the field except for producing yet another mouse model of LAMA2-CMD and is the same as all the others produced.

(6) Grip strength measurements are considered error prone and do not give an accurate measurement of muscle strength, which is better achieved using ex vivo or in vivo muscle contractility studies.

(7) A lack of blinded studies as pointed out of the authors is a concern for the scientific rigor of the study.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

(1) Some of the figures are of rather poor quality. For example, the H&E and Sirius Red stainings in Figures 3 and 4 are quite poor so it is difficult to see what is going on in the muscles. The authors should take note of another publication on dy3K/dy3K mice of similar age (PMID: 31586140) where such images are of much higher quality. Similarly, the Western blot for laminin-alpha2 (Figure 4B) of the wild-type mouse needs improvement. If the single laminin-alpha2 protein is not detected, there is an issue with the denaturation buffer used to load the protein.

Thank you for the valuable suggestions. We have read the study on dy3K/dy3K mice of similar age (PMID: 31586140) which showed dystrophic changes in dy3K/dy3K muscle throughout the disease course with the whole muscle and representative muscle area. We have generated new figures with higher quality including the whole muscle and representative muscle area for the H&E and Sirius Red stainings. However, due to the large images, we have added them in the new Figure supplement 2 and Figure supplement 3. Also, we have changed the denaturation buffer used to load the protein, and performed Western blot of laminin α2, the result of the laminin α2 protein of the wild-type mice (n =3) and dyH/dyH mice (n =3) detected by Western blot has been showed in Figure 4B.

(2) My biggest concern is, however, the many overstatements in the manuscript and the over-interpretation of the data. This already starts with the first sentence in the abstract where the authors write: "Understanding the underlying pathogenesis of LAMA2- related muscular dystrophy (LAMA2-MD) have been hampered by lack of genuine mouse model." This is not correct as the dy3K/dy3K, generated in 1997 (PMID: 9326364), are also Lama2 knockout mice; there are also other strains (dyW/dyW mice) that are severely affected and there are the dy2J/dy2J mice that represent a milder form of LAMA2-MD. Similarly, the last two sentences of the abstract "This is the first reported genuine model simulating human LAMA2-MD. We can use it to study the molecular pathogenesis and develop effective therapies." are a clear overstatement. The mechanisms of the disease are well studied and the above-listed mouse models have been amply used to develop possible treatment options. The overinterpretation concerns the results from transcriptomics. The fact that Lama2 is expressed in particular cell types of the brain does not at all imply that Lama2 knockout mice have a defect in the blood-brain barrier as the authors state. If there are no functional data, this cannot be stated. Indications for a blood-brain barrier defect come from work in dy3K/dy3K mice (PMID: 25392494) and this needs to be written like this.

Thank you for your comment and sorry for the overstatements in the manuscript. We have carefully considered our previous statements and corrected them accordingly. We have changed the first sentence in the abstract into "Our understanding of the molecular pathogenesis of LAMA2-related muscular dystrophy (LAMA2-MD) requires improving". Also, we have changed the last two sentences in the abstract with "In summary, this study provided useful information for understanding the molecular pathogenesis of LAMA2-MD".

We also agree that "Lama2 is expressed in particular cell types of the brain does not at all imply that Lama2 knockout mice have a defect in the blood-brain barrier", and the indications for a blood-brain barrier defect come from work in dy3K/dy3K mice (PMID: 25392494). Therefore, we have corrected the overstatement according to the suggestion with "It was reported that the deficiency of laminin α2 in astrocytes and pericytes was associated with a defective blood-brain barrier (BBB) in the dy3K/dy3K mice (Menezes et al., 2014). The defective BBB presented with altered integrity and composition of the endothelial basal lamina, reduced pericyte coverage, and hypertrophic astrocytic endfeet lacking appropriately polarized aquaporin4 channels."

(3) Finally, the bulk RNA-seq data also needs to be presented in a disease context. The authors, again, mix up changes in expression with functional impairment. All gene expression changes are interpreted as direct evidence of an involvement of the cytoskeleton. In fact, changes in the cytoskeleton are more likely a consequence of the severe muscle phenotype and the delay in muscle development. This is particularly possible as muscle samples from 14-day-old mice are compared; a stage at which muscle still develops and grows tremendously. Thus, all the data need to be interpreted with caution.

Thank you for your comment. We have changed the over-interpretation of the bulk RNA-seq data, and have corrected the last sentence in the Result with "These observations important data for the impaired muscle cytoskeleton and abnormal muscle development which were associated with the muscle pathology consequence of severe dystrophic changes in the dyH/dyH mice.".

(4) In summary, the authors need to improve data presentation and, most importantly, they need to tone down the interpretation and they must be fully aware that their work is not as novel as they present it.

Thank you for your comments and valuable suggestions, and we have changed the previous overstatements and interpretation of the results. We are sorry that we failed to clearly present our rational of making this mouse model. Indeed, there were many existing mouse models, which were all important to the research in the field. One of the reasons why we wished to create dyH/dyH is to make a mouse model without any trace of engineering (e.g., inserted bacterial elements for knockout). By doing so, we were hoping to provide a novel model suited for gene-editing-based gene therapy development. To this end, dyH/dyH was created to reflect the hot mutation region in the Chinese population. Hopefully, you will agree with our points and see that we were not trying to belittle previous models but were simply trying to provide a different option. The overstatements were largely rooted from language barriers, and we have tried to make our statements more cautious and acceptable to the readers.

Reviewer #2 (Public Review):

(1) The major weakness is the manuscript reads like this was the first-ever knockout mouse model generated for LAMA2-CMD. There are in fact many Lama2 knockout mice (dy, dy2J, dy3k, dyW, and more) which have all been extensively studied with publications. It is important for the authors to comment on these other published studies that have generated these well-studied mouse lines. Therefore, there is a lack of background information on these other Lama2 null mice.

Thank you for your comment. We have added background information on these other Lama2 null mice with the sentences "The most common mouse models for LAMA2-MD are the dy/dy, dy3k/dy3k, dyw/dyw and dy2J/dy2J mice (Xu et al., 1994; Michelson et al., 1995; Miyagoe et al., 1997; Kuang et al., 1998; Sunada et al., 1995). Among them, the dy/dy, dy3k/dy3k, dyw/dyw mice present severe muscular dystrophy, and dy2J/dy2J mice show mild muscular dystrophy and peripheral neuropathy (Gawlik and Durbeej, 2020). The mutation of the dy/dy mice has been still unclear (Xu et al., 1994; Michelson et al., 1995). The dy3k/dy3k mice were generated by inserting a reverse Neo element in the 3' end of exon 4 of Lama2 gene in 1997 (Miyagoe et al., 1997), and the dyw/dyw mice were created with an insertion of lacZ-neo in the exon 1 of Lama2 gene in 1998 (Kuang et al., 1998). The dy2J/dy2J mice were generated in 1970 by a spontaneous splice donor site mutation which resulted in a predominant transcript with a 171 base in-frame deletion, leading to the expression of a truncated laminin α2 with a 57 amino acid deletion (residues 34-90) and a substitution of Gln91Glu (Sunada et al., 1995). They were established in the pre-gene therapy era, leaving trace of engineering, such as bacterial elements in the Lama2 gene locus, thus unsuitable for testing various gene therapy strategies. Moreover, insufficient transcriptomic data of the muscle and brain of LAMA2-CMD mouse models limits the understanding of disease hallmarks. Therefore, there is a need to create new appropriate mouse models for LAMA2-CMD based on human high frequently mutated region using the latest gene editing technology such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9."

(2) The phenotypes of dyH/dyH are similar to, if not identical to dy/dy, dy2J/dy2J, dy3k/dy3k, dyW/dyW including muscle wasting, muscle weakness, compromised blood-brain barrier, and reduced life expectancy. This should be addressed, and a comparison made with Lama2 deficient mice in published literature.

Thank you for your comment. We have added Table supplement 3 to make a comparison between dyH/dyH with other Lama2 deficient mice. We aslo have added the statement in Discussin with "Compared with other Lama2 deficient mice including dy/dy, dy2J/dy2J, dy3k/dy3k and dyW/dyW, the phenotype of the dyH/dyH mice presented with a very severe muscular dystrophy, which was similar to that of the dy3k/dy3k mice (Table supplement 3)."

(3) Recent published studies (Chen et al., Development (2023), PMID 36960827) show loss of Itga7 causes disruption of the brain-vascular basal lamina leading to defects in the blood-brain barrier. This should be referenced in the manuscript since this integrin is a major Laminin-211/221 receptor in the brain and the mouse model appears to phenocopy the dyH/dyH mouse model.

Thank you for your great suggestion. We have cited the published studies (Chen et al., Development (2023), PMID 36960827) and added statements in Discussion with "As reported, the aberrant BBB function was also associated with the adhesion defect of alpha7 integrin subunit in astrocytes to laminins in the _Itga_7-/- mice (Chen et al., 2023). In this study, loss of communications involving the laminins’ pathway between laminin α2 and integrins were predicted between vascular and leptomeningeal fibroblasts and astrocytes in the dyH/dyH brain, providing more evidence for the impaired BBB due to laminin α2 deficiency."

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Improve the data presentation (as mentioned above). Make a new picture of the histology; repeat the Western blots. Discuss the RNA-seq data with more caution and present it in a more attractive way. Tone down the wording.

Thank you for your recommendations. We have revised the overstatements and improved the RNA-seq data interpretation as suggested. Also,we have made a new picture of the histology, and repeated the Western blots.

Reviewer #2 (Recommendations For The Authors)

(1) There are many grammatical errors within the manuscript. The manuscript should be carefully proofread.

Thank you for your recommendations. We have carefully corrected the grammatical errors within the manuscript.

(2) Figure 2: The animal numbers used in this analysis were not indicated. Please include this number in the figure legend.

Thank you for your recommendations. We have added animal numbers in the figure legends wherever applicable.

(3) Figure 2: The forelimb grip strength is informative but has limitations. Ex vivo or in vivo muscle contractility is the gold standard for measuring muscle strength.

Thank you for your recommendations. We do agree that the ex vivo or in vivo muscle contractility is the gold standard for measuring muscle strength, and we really want to finish this experiment. However, we feel sorry that this test has not been finished due to the following reasons: (1) The forelimb grip strength for measuring muscle strength is a classic method and remains a commonly used method for measuring mouse muscle strength in the studies of different muscular dystrophies, such as LAMA2-MD (Amelioration of muscle and nerve pathology of Lama2-related dystrophy by AAV9-laminin-αLN linker protein. JCI Insight. 2022;7(13):e158397. PMID: 35639486), Duchenne muscular dystrophy (Investigating the role of dystrophin isoform deficiency in motor function in Duchenne muscular dystrophy. J Cachexia Sarcopenia Muscle. 2022;13(2):1360-1372. PMID: 35083887), facioscapulohumeral muscular dystrophy (Systemic delivery of a DUX4-targeting antisense oligonucleotide to treat facioscapulohumeral muscular dystrophy. Mol Ther Nucleic Acids. 2021;26:813-827. PMID: 34729250), and etc. (2) The forelimb grip strength for measuring muscle strength is also used in the human studies (PMID: 32366821; PMID: 29313844; PMID: 34499663, and etc). In view of reasons above, for measuring muscle strength, we used the forelimb grip strength, and have not finished the supplementary experiment of ex vivo or in vivo muscle contractility.

(4) Figure 3: Muscle fibrosis should be measured with a hydroxyproline assay.

Thank you for your recommendations. We do agree that the hydroxyproline assay is one of the most classic method to evaluate collagen content for measuring muscle fibrosis. However, we performed Sirius Red staining for measuring muscle fibrosis due to the following reasons: (1) Muscle fibrosis measured by Sirius Red staining can be observed more directly, and the other pathological features also can be observed, and compared through muscle pathology. (2) Sirius Red staining is also a classic method and remains a commonly used method for measuring muscle fibrosis, which has been previously reported in the mouse studies of muscle disorders, such as PMID: 22522482 (Losartan, a therapeutic candidate in congenital muscular dystrophy: studies in the dy(2J) /dy(2J) mouse. Ann Neurol. 2012;71(5):699-708.), PMID: 34337906 (Aging-related hyperphosphatemia impairs myogenic differentiation and enhances fibrosis in skeletal muscle. J Cachexia Sarcopenia Muscle. 2021;12(5):1266-1279.), PMID: 28798156 (Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy. FASEB J. 2017;31(12):5307-5320.), and etc. Therefore, we used Sirius Red staining to measure muscle fibrosis in this study.

(5) Figure 8: The N=3 is very low which could result in type I or II statistical errors. A larger sample size will reduce the chance of statistical errors.

Thank you for your recommendations. We have increased the number of animals to reduce the chance of statistical errors. We have performed the supplementary experiment, the number of animals for each group has been increased to 6 (3 male and female each). The results were consistent with previous data in Figure 8.

(6) Power analysis to estimate experimental animal numbers should be reported in the manuscript.

Thank you for your recommendations. Refer to previous study (Power and sample size. Nature Methods. 2013;10:1139–1140), “The distributions show effect sizes d = 1, 1.5 and 2 for n = 3 and α = 0.05. Right, power as function of d at four different a values for n = 3”, and “If we average seven measurements (n = 7), we are able to detect a 10% increase in expression levels (_μ_A = 11, d = 1) 84% of the time with α = 0.05.”, the experimental animal numbers estimated were 3 to 7. Moreover, if the increased number of experimental animals could be available, we would retain data.

(7) It is unclear if the studies were performed with adequate rigor. Were those scoring outcome measures blinded to the treatment groups?

Thank you for your recommendations. We performed the studies with those scoring outcome measures not blinded to the treatment groups, the groups were based on their genotype. Actually, it was easy to discriminate the dyH/dyH groups from the WT/Het mice due to their small body shape.

(8) Authors should appropriately cite previous studies that have generated Lama2 null mice.

Thank you for your recommendations. We have cited previous studies that have generated Lama2 null mice with the sentence “The most common mouse models for LAMA2-MD are the dy/dy, dy3k/dy3k, dyw/dyw and dy2J/dy2J mice (Xu et al., 1994; Michelson et al., 1995; Miyagoe et al., 1997; Kuang et al., 1998; Sunada et al., 1995)”.

(9) The number of animals should be increased to reduce the chance of statistical error.

Thank you for your recommendations. We have performed the supplementary experiment, the number of animals for each group has been increased to reduce the chance of statistical error.

(10) A power analysis should be performed to determine the number of experimental animals.

Thank you for your recommendations. We have performed a power analysis to determine the number of experimental animals as mentioned above.

(11) There are many grammatical errors within the manuscript. The manuscript should be carefully proofread.

Thank you for your recommendations. We have carefully corrected the grammatical errors within the manuscript.