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

Reviewer # 1 (Public Review):

Summary:
The paper nicely confirms the phenotype of Lama2 knockout mice and extends the phenotypic description with a set of new molecular studies (transcriptomics) that might serve as a resource for other scientists interested in the LAMA2-MD.

Strengths:
Set of new molecular studies (transcriptomics) that might serve as a resource for other scientists interested in the LAMA2-MD.

Weaknesses:
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.

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.

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.

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.

Reviewer #2 (Public Review):

Summary:
This manuscript describes the production of a mouse model for LAMA2-CMD. This mouse was produced using CRISPR-Cas9 and deleted exon 3 of the Lama2 gene. The mice exhibit reduced life expectancy, muscle pathology, and disruption of the gliovascular basal lamina assembly leading to defects in the blood-brain barrier. Single-cell RNAseq was used to explore the effect that loss of Laminin-211/221 had on gene expression.

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

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

Weaknesses:
(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, dy4k, 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.

(2) The phenotypes of dyH/dyH are similar to, if not identical to dy/dy, dy2J/dy2J, dy4k/dy4k, 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.

(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.