Normal Cortical Myelination in Galectin-4-Deficient Mice
, Arancha Mora-Rubio 1,†, Elena Alonso-Calviño 2, Elena Fernández-López 2, Natalia DÃez-Revuelta 1, David Martos-Puñal 1, Juan Aguilar 2, Alonso M. Higuero 1,* and José Abad-RodrÃguez 1,*Round 1
Reviewer 1 Report
The present manuscript submitted by Brocca, Mora-Rubio et al. presents data on the role of Gal-4 in cortical myelination. Previous studies, also by this group, have demonstrated that Gal-4 impacts oligodendrocyte differentiation in vitro and that it is associated with NMS structures in cellular co-cultures in vitro, thus suggesting Gal-4 as a possible regulator of axonal myelination.
The submitted work aims to study the functional consequences of Gal-4 depletion using an LGALS4-KO mouse model. Thus, the study follows a well-argued hypothesis based on a substantial amount of in vitro data. I particularly like the approach by the authors of putting in-vitro data to the test in vivo, and presenting the results in an open, unbiased way. This is indeed of great help to the community and should be explicitly supported. The authors present an elaborate dataset using a comprehensive set of experiments from proteomic and gene expression analyses to functional assays in vivo. The statistics are transparent and adequate, and the necessary controls are considered in all assays.
In fact, I have only a few comments that might help to improve the manuscript.
*) The authors show a mismatch between their in vivo results and data from the literature which had been collected in vitro. Such differences might also be due to inter-species differences. To address this possibility, the authors should add comments on the species from which the mentioned in vitro data had been obtained.
*) It would also be interesting for the general reader to learn if there are any studies using human cells or tissues and whether Gal-4 might play a role in human neurological disease.
*) The authors state that the mouse model “C57BL/6NJ-Lgals4em1(IMPC)J/J” from Jackson was used. However, a search for this model in the literature and on the Jackson website did not provide further information (except for a brief mention in a price list of 2019). Why is this? Is this indeed the first study using this model? Is it still commercially available? Maybe the authors could comment on that.
*) The final phrase of the abstract is not self-explanatory, given the information provided in the abstract.
*) In the paragraph “2.4. Tissue extracts and raft purification”, the temperatures miss the sign for the degree unit (i.e., 40°C instead of 40C).
*) The final phrase of the statistics section (2.12) appears not to be part of the manuscript but rather a copy/paste error.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In this manuscript, the authors used Gal-4-deficient mice to study the role of Gal-4 in cortical myelination in vivo. Indeed, it has been shown that galectin-4 is prominently present in the brain immediately after birth, but is subsequently downregulated in early postnatal life. Downregulation of Gal-4 in the brain is accompanied with the increased expression of myelin-specific proteins such as MBP and PLP (DOI 10.1002/glia.22324). Although the role of Gal-4 in the myelinization of neurons was previously shown, its role in myelinogenesis during mice brain development was not studied so far. Therefore, this work is very interesting but I have some concerns regarding data interpretation:
1. Figure 1A is actually misleading. Why did the authors use cortical neurons from rats to show gal-4 expression by IF in Figure 1A instead of using cortical neurons from mice? Does the cortical neurons isolated from WT mice actually express gal-4?
2. Authors claim that after treatment with TTX (Figure 1A and 1B), the NMS in cortical neurons were formed equally when isolated from Gal-4-KO or WT mice. Why were the authors expecting a difference between the NMS in KO and WT derived neurons? This was not a co-culture experiment and myelinating cells were not present. Therefore, it is not surprising that NMS were produced in vitro even in the absence of Gal-4. Also, this is a condition where the expression levels of gal-4 are not shown. Was the expression of gal-4 monitored in WT-derived neurons before and after TTX treatment in WT mice? Without the appropriate controls such as a western blotting for example, it is hard to believe that indeed gal-4 has any relationship with NMS.
3. Galectin-4 is prominently present in the brain immediately after birth, but is subsequently downregulated in early postnatal life (almost completely absent after day 23 postnatal, (DOI 10.1002/glia.22324). Since cortices of mice from Figure 2 were isolated from 30-day old mice, I would expect a situation where the levels of gal-4 are pronouncedly reduced if not totally absent from the brain tissue. How were galectin-4 levels in the cortices of WT mice?
4. Experiments from Figure 2-6 were done using 30-day old mice, which might lack gal-4 and mimic a gal-4-KO condition in the brain. Without the appropriate controls this reviewer cannot comment on further results from this manuscript.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
The study is well designed to assess the in vitro findings that indicate a role for galectin 4 in the regulation of myelination in the CNS. The authors appropriately use the Gal4 KO mice and assess oligodendrocyte number/maturation, myelination and CNS function. The images are of high quality and the manuscript is well written. There are a few relatively minor concerns that, if addressed, would strengthen the study.
Materials and Methods: section 2.2; 4th paragraph: What is meant by “a subset of NMS was manually labelled and classified as foreground”? How were the segments manually labelled? How were the segments chosen?
Materials and Methods: section 2.4; 1st paragraph “The extracts were further analysed by WB using antibodies against myelin 160 markers, and GAPDH” Which myelin markers were used and the information for each antibody (source, cat #; concentration) should be provided. Antibody information should be provided for all antibodies used – MBP, PLP1, Olig2, caspr VDAC Gal4, drebrin E, tubulin, MAG, MOG, GAPDH
Why were different ages use for the IHC analyses compared to the NMR analysis compared to the behavioral tests?
What were the ages of the mice used for the WB and EP analyses?
Instead of the term “non-myelinable”, the term "un-myelinated” might be more appropriate. Un-myelinable suggests that the axon segment cannot be myelinated. That has not been established.
In figure 1, the authors use Gal4 as a marker for NMS. Using Gal4 as a marker for these regions seems to be supported by previous work from this group. This could be more clearly stated. In B the authors compare axons from Gal4 KO against WT mice. It is unclear why drebrin e and the other markers used in A were not used in B. The distribution of caspr in the KO image for D14 looks less demarcated than the caspr labeling in the WT axons. It is not clear to this review what is meant by “cumulative proportion”. What is it and how was it calculated? How many segments were measure? How many preps (not cultures) were used for this analysis?
The authors assess the oligodendrocyte population with olig2. Since olig2 is an immature marker for oligodendrocytes, complementing the olig2 assessment with cc1 labeling would add strength. More details explaining how the cell quantitation was conducted should be added. How is “inner” , “middle” and “outer” defined? Are these divisions defined by cortical layers? How were they identified from mouse to mouse? The MBP labeling looks lighter (less immunolabeling) in the KO with less MBP labeling in the cortex and a thinner corpus callosum. This may be the image selection but this should be addressed.
If differences in myelination are expected, looking at young mice, when synaptic plasticity is greater, might provide a greater difference.
Higher level of resolution for myelin analysis is provided by electron microscopy. Analyzing myelin ultrastructure would add strength.
If good antibodies are available for Gal4 and Gal6, it would be informative to show WB data for these proteins in WT and Gal4 KO (c57bl/6nj) mice. checking Gal4 would confirm the model and assessing for Gal6 would address whether Gal6 is expressed in the absence of Gal4. the authors state that Gal6 is not expressed in these mice but that is most likely based on the expression pattern of a WT mouse. Checking Gal8 would also be of interest.
I think the authors mean “sialylated" instead of “syalilated” (line 660)
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The authors have done a great job revising the manuscript. Still, this reviewer has some concerns regarding the expression of galectin-4 in the mouse cortex. Can the authors use the cerebellum as a control for gal-4 western blotting? According to the anti-gal-4 antibody datasheet (Santa cruz, sc-271533) this should be a good positive control for gal-4 expression. In addition, this reviewer strongly disagree with the author's statement: "We have detected Gal-4 mRNA expression in the cortex of P30 WT mice by RT-PCR, a far more sensible and specific technique, as shown in Supplementary Figure S1B." The scientific community is very much aware that mRNA expression does not necessarily mean protein expression.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
