PONE-D-22-03467A Novel Cytoskeletal Action of XylosidesPLOS ONE

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: The ms by Mencio et al. describes how the cytoskeleton and the morphology of neural cells (here, hippocampal neurons and N2A cells) are affected by nanomolar doses of xylosides, compounds usually applied in the millimolar range to interfere with GAG chain synthesis. The work is principally interesting since it suggests that very subtle changes in GAG chain composition may affect the actin cytoskeleton and thus, cell morphology and migratory behavior. It is also another example of very low doses of a compound exhibiting different effects than high doses.

The reviewer regrets, however, that some findings of the authors seem contradictory, questionable or to the least, unclear for the reader (see below).

One main concern is that the discussion section, while exhaustively reporting on previous work on GAG chain modification by xylosides, tries hard but does not come up with a sort of theory, let alone an explanation for the low dose effect of xylosides described by the authors. Thus, for the reviewer, the conclusion of the study is already contained in the Introduction lines 69-71 ("this study serves as a first step...").

In the Discussion, any attempts to explain the observed effects are limited to "This is an area of research that needs further exploration", repeated several times. "... [low concentration] xyloside treatment may lean to subtle changes in GAG chains having more distinct biological effects" (lines 389-90), repeated just below (lines 406-408): "This could imply that utilizing lower concentrations that result in distinct, but subtle changes to GAG profiles may produce more unique and useful biological outcomes". Besides "more unique and useful" seeming rather "cryptic" terms in biology, these sentences don't explain, but simply repeat the observed findings. The fact that GAGs may interact for ex. with Sema5A (lines 382-386), which in turn may indeed affect the cytoskeleton, doesn't help either because Sema5A is not present here (as far as we know). Still other arguments are simply not new: (lines305-6): "These findings indicate that xyloside treatment can change GAG chain synthesis and composition, and alterations to HS appear to occur in a concentration dependent manner".

Maybe it could have helped if the authors had produced a model/schematic drawing of what they think happens on the molecular level when using low vs. high xyloside concentrations.

Another critical point may be seen in the fact that the authors used the same "LCX" concentration for primary hippocampus cells and for a "neural" cell line (N2A), with which the majority of the work was performed. Many studies have shown that different cell types react differently to a given concentration of xylosides, so it may have been interesting to redo a dose response curve with these cells.

Finally, one may ask whether the N2A cell line used in this study represents really neural cells (neurons), in view of the images shown that are very different from the hippocampal neurons shown in the beginning.

Major points:

Abstract line 32: "... did not alter GAG chain synthesis rates, nor..." the fact that GAG dissaccharide composition was altered is probably crucial and should be mentioned here.

Introduction, line 61: Other studies than the mentioned ref. 16 already used relatively low xyloside concentrations, for ex. Carrino & Caplan 1994 who used 1µM, which is not that far from 500nM (in comparison to mM).

Materials and Methods: for the reviewer, at least parts of this section are lacking some crucial details.

Controls for xyloside treatment are always termed "DMSO"; however, we do not know how much DMSO was introduced into the cultures, as we do not learn at what stock concentration xyloside was used. Other, similar studies always stated that they used the highest DMSO concentration for controls (corresponding to the highest xyloside conc. applied). So the reader may ask if DMSO addition already modifies something?

Cell culture line 83: cell density 8-10k/well (is this 12-well, 24-well, ...?).

24mM KCl was added to the culture of hippocampal neurons, why ? (usually this is done to depolarize the neurons, but here?).

Line 146- Neurite otgrowth and growth cone analysis: "both longest and total neurite measurements...": "total neurite" doesn't appear anywhere in the ms, and does it mean total length of all neurites ? How were neurite length, and the growth cone area exactly measured (ImageJ plugin?).

Cytoskeleton dynamics: EB3-GFP and Ftractin-mCherry transfections are not detailed (maybe a recent reference would suffice). Also, the reviewer asks whether, since transfections were performed after exposure to xyloside, are cells reacting the same to the transfections as without xyloside, what is the percentage of transfected cells, does transfection itself have other consequences on cell morphology ?

Results:

Figure 1: An overview image showing more than one neuron would be welcome; here, we just see one well spread growth cone (GC) formed by an LCX treated neuron, while the GC from control and DCX neurons appear rather collapsed. Also, the LCX neurite is much shorter than the other two, so I guess we are dealing with somewhat extreme (so called "typical") examples.

Supp Figure 1: to me, this is the "key" figure of the ms, as it shows the dose response curve for xyloside treatment, on which the rest of the ms, including the work on N2A, is based. The reviewer is puzzled when looking at the images: how many neurons do we see, particularly in the right LCX image? What looks like GC, contains also nuclei? Then, how were "looping MTs" quantified? In a "collapsed" GC like those seen on control neurons (left image) they would be hard to count. This is also not specified in M&M.

Supp Figure 2 is the basis for N2A cell line-based work. (Line 217) "... LCX treated N2A exhibit actin-rich lamellipodia, not observed in DMSO or HCX treated N2A". Besides the fact that in the LCX image the red color appears more saturated to me than in the other two images (see the completely flat yellow zones); this finding appears completely contradictory to Fig. 3 (see below).

Figure 2: Looking at the images, it seems strange to me that at t=0, the tracked cells do not show any extensions, which then develop at t=80 and t=160 min where cells really look like migratory cells (as if someone had given the "start signal"), although cells were kept under the same conditions already for 48h.

Figure 3: In the figure legend it says "phalloidin staining on fixed cells", in the text it says Ftractin mCherry transfection (normally used for live viewing?), what in fact do we see here ?

(lines 243-250) LCX treated N2A lamellipodia had significantly fewer actin bundles...; additionally, ...less robust actin bundles in LCX lammellipodia. How do the authors explain that in Supp Fig 2, DMSO or HCX treated N2A did not exhibit actin-rich lamellipodia, while here they state in some sort the opposite? Moreover, the cells in Fig. 3 do not resemble at all those shown in Supp Figure 2, let alone the (hippocampal) real neurons in Fig. 1. Here and elsewhere, figure legends (M&M) are not precise enough (different culture times between Supp Fig. 2 and Fig. 3, for example?).

To me, the HCX cell looks polarized, much more than the LCX cell; this is even more obvious in Figure 4A.

Figure 4: "These results [...] suggest LCX treatment alters cytoskeleton dynamics, which lead to changes in morphology and movement"; yes, but in Fig. 4 (EB3 comet movement) there is no difference between LCX and HCX treated N2A cells, only between DMSO and xyloside treatment. This is somehow in contrast to the assumption that low, but not high xyloside concentrations lead to major morphology changes (see e.g. Introduction line 64-65; Figs. 1, 2, Supp Fig2; discussion line 405-406: "no visible effect is observed in 1mM xyloside treated neurons or neuro2A cells").

Besides, as in Fig. 3, it looks to me that the cells presented differ in polarity, the HCX cell being the most polarized.

Figure 5: the legend should at least mention what means 0S, NS, 6S, 2SNS, especially for the broad audience of PlosOne who may not all be familiar with these terms.

The figure shows that CS GAG concentration in the medium is sort of xyloside dose dependent, whereas both low and high xyloside doses provoke the same change in CS disaccharide composition. HS GAG concentration in the medium is not altered by LCX, but the relative abundance of N-sulfated GAGs is. This seems interesting to me with regard to the role of N-sulfated HS reported in the literature (see e.g. Grobe et al 2005, Development 132: 3777), and this point could well be analyzed a little more in the Discussion section.

Another point: the difference between LCX effects on CS and HS GAG production may in fact be due to the DMSO controls already producing some HS (~0.1 ng/µL), but no CS.

The problem I see here, is that we do not learn whether the small effect of LCX on CS GAG concentration could have an effect on cell morphology, or whether the altered sulfation of HS GAGs does. These questions may partly be answered by, for example, rising (exogenous) CS concentration in the culture medium (i.e. not through xyloside treatment), or by selectively suppressing N-sulfation.

Figure 6C: there seem to be at least some genes whose expression is changed between DMSO and LCX ? The formulation (lines 314-15) "we found no genes whose change in expression was different between LCX and DMSO..." is not very clear.

Discussion:

When reading the discussion, two more points came to my mind: i) is the observed low dose effect on cell morphology specific for "neural" cells, or may it affect also the other cell types (muscle etc.) that had already been used in xyloside experiments? ii) It may have been interesting to include HS/CS staining of the cells after LCX/HCX treatment, as seen for ex. in the Nishimura et al. work (ref. 20).

Minor points:

The abbreviations for HS, CS (and KS) need be introduced (Introduction line 39).

Figure legends are often too short. Ex., Figure1: "altered cytoskeleton", the label is not indicated (tubulin?). Figure 2, "letters" should rather read "numbers". Figures 3 and 4, see before. Also in Fig.4, "distribution plots of speed, persistence and comets" ("lifetime" missing?), and in the histograms themselves is marked "200715_Speed" etc. which doesn't tell us anything. Figure 5, add abbreviations.

Supp Fig. 1, the bar is missing.

Some typo etc. errors, such as "DMOS" (line 104), "in in" (line 323), "on at" (line 344), "1C" (1µ?; line 346), "the change...were..." (line 356).

Reviewer #2: PONE-D-22-03467

This paper described that a nM range concentration of xyloside (4-methyl-umbelliferyl-�-D-xylopyranoside) had effects in cellular morphology of primary neurons and Neuro2A cells, while a mM range does not. Authors report changes in growth cone size with an increase in microtubules looping in primary neuronal cultures, reduced cell migration and altered actin bundling in Neuro2A cell line after 48h in low concentration of xyloside (LCX). Even though these observations have not been made previously, the data does not offer a mechanistic explanation for the observations, nor it does establish a clear link between the morphological changes and changes in proteoglycans (PGs) and/or glycosaminoglycans (GAGs) synthesis.

Evaluation of this paper has been difficult because of a lack of some technical details, incongruent description of the data in figure legends and text and a somewhat limited discussion of the data.

Major points:

- Xyloside treatment impairs the incorporation of GAGs in the PGs core proteins and increases the secretion of free GAG chains into the media. Thus, these changes in morphology could be due to partially modified core proteins at LCX conditions, and analyzing changes in GAG composition of cell and matrix associated proteoglycans could be important to evaluate here. Alternatively, if the partially glycosylated core protein are responsible, similar morphological changes could be obtained by knocking-down expression of Xylosyltransferase 1 and 2 in these cells.

- In the discussion the possibility that an intracellular function for GAGs should also be discussed considering the recent publication of Fang et al. (https://doi.org/10.1016/j.immuni.2021.03.011), where he found the participation of GAGs chains in polymerization and activation of STING at the Golgi level in immune cells. Still undiscovered intracellular functions for GAGs in neuronal cells could be responsible for the morphological changes described here.

-Line 48. Please clarify that only CS/DS and HS have the common Xyl-Gal-Gal linkage to core protein. KSPGs are bound to PGs core proteins by N- or O- linkage sugars and as such are not influenced by xyloside treatment.

- Line 96. Please clarify the “normal cell culture conditions”; Is this with or without FBS? This is important to evaluate your GAGs composition results.

- Line 114. Methods indicates that a 30K filter was used to separate CS disaccharides from HS chains. Could this be a typo? HS chains will go through a 30K filter.

- Line 241-255. Text accompanying Figure 3 explained an F-tractin-mCherry experiment while the figure 3 legend described a phalloidin staining experiment. Which one is it? Or the wrong figure was included?

- Line 269. It is unclear how many cells were evaluated for microtubules dynamics in Figure 4B.

- Line 285. It is unclear the number of samples quantified per treatment group and the statistical analysis used to assess significance (see line 283).

- Line 328 and 176. Significance stated in Materials and methods is different that the one stated in the figure legend. Data appear to be adjusted by false discovery rate (FDR) but no threshold was stated in the figure legend. Also, the genes listed in figure 6C are impossible to read. A data file with the list of genes and fold changes should be supplied as supplemental data. Please explain the -10 to 10 color scale used.

- Raw and processed RNA-seq data should be make available in the National Center for Biotechnology Information Gene Expression Omnibus.

Minor points:

- Antibodies used should be specified in Materials and Methods.

- Line 66. Xiloside is misspelled.

- Line 104. Typo, it should read DMSO.

- Line 171. First mention of LCX and HCX, please define here.

- Line 181 and 210. Punctuation should be corrected.

- Line 249. There is no Figure 2C so this probably should read Figure 3C.

- Line 346. …with concentrations down to 1 C? Unclear, Typo?

- Line 378. Defined DS.

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PONE-D-22-03467R1A Novel Cytoskeletal Action of XylosidesPLOS ONE

Dear Dr. Geller,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================First, there is a problem with the PDF of the revised manuscript, which does not include anymore Figure 3 and Figure 6 !!! Since the legends of these two Figs are still present in the text, I assume that reviewer 1 took into account Fig 3 and Fig 6, whereas reviewer 2 found the revised version very problematic.Please, take into account all the precise comments of reviewer 1 including: 1-adding HCX image in Supp Fig.1, 2-adding for Fig. 2  the total distance for cell migration and significance values, please read carefully the text and correct the typos. As mentioned by Reviewer 2, you are indicating in the point-by-point answer: "we have added images of several HC neurons at low power”, no changes were made to Figure 1, is it shown in a suppl Fig ?Both reviewers acknowledge that the revised manuscript has been reworked and improved and we hope that you could now submit a final revised version of the manuscript.

==============================

Please submit your revised manuscript by Jun 12 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

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Reviewer #1: also uploaded as PDF file.

The reviewer acknowledges that the revised ms has been profoundly reworked and greatly improved, although I am still not completely convinced by all arguments provided.

- I agree that the GAG chain synthesis and composition studies, and transfections, are easier to perform with N2A cells, but for the rest I personally do not see why hippocampal (or cortical? or maybe even adult DRG) neuron cultures are "too variable" as you say. Since your group is working on axon guidance, regeneration, etc., and you show here at the beginning of your ms how low dose xyloside treatment affects MT looping, growth cone stalling and neuronal morphology, I wonder how you will be able to "translate" results from your in vitro work with N2A cells lacking growth cones and even morphologically not really resembling neurons, for use in your other studies. Sure this is a personal "regret" of the reviewer, not relevant for publication in PLOSOne.

- You say that for Fig.1 you added images of several neurons at low power, but those are not found in the revised ms? Could be a Supplemental Figure.

- Supp Fig.2: Overall, there is now more similarity with Fig.3. However, the caption on the fig. does not correspond to the legend in the ms (line 584). At the same time, the caption on the fig. describes the facts better than the ms legend ("increased levels of interior phalloidin actin staining" vs. "increased levels of actin"; "actin" should at least read F-actin by the way). I think the legend to Supp Fig.2 should be more precise to not induce us into erroneous interpretation of Fig.3, which has obviously not changed (?).

- The discussion has been indeed improved. It does however not deal with the question where the effect on the actin cytoskeleton of LCX vs. HCX treatment may attack. Could there be an effect already during GAG synthesis in Golgi/cytosol and subsequent PG transport, or is there only an effect on (extracellular) signaling via secreted or membrane-bound PGs? (Let's say that is a question that would interest me personally, but you need not answer it).

- There are still some errors and typos, see below.

Line numbers refer to the Word (.docx) document "Final Revision" !

Abstract: the reformulated lines 29-31 (-32) are not very "elegant", and the end may be misleading ("higher concentrations had minor effects"). I'd propose something like:

To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology of hippocampal neurons as well as of Neuro2a cells, affecting both actin and tubulin cytoskeletal dynamics. Such effects/morphological changes were not observed with higher xyloside concentrations.

Line32: Xylosides ... produces...

Line33: ...large change in GAG chain synthesis rate

Finally, you did not include the effect on GAG composition in the Abstract, why? You have done so in the Introduction, where you state that your study may contribute to understanding "how a minor shift in GAG composition can affect biological processes..."

Cell culture: Stock solutions are now described, but it would not have been necessary to do it two times (lines 93, 107).

Line108: 10 m should probably read 10 min.

Growth cone analysis:

Lines164-167: this is a bit unclear for the reader. "The randomized files were then numbered sequentially and saved for reference. Duplicates of these files had all identifying information removed and then the numbered files were analyzed."

I guess that the duplicate files without information were analyzed (to make for 'double blind')? Here it sounds as if the reference files (the "numbered" ones) were analyzed. I'd prefer a simple: "Analysis was then performed on duplicates of these files from which all identifying information had been removed".

Lines162- : this does not really answer my question how "collapsed" GC were counted? (as seen in Fig.1: LCX shows a neat, large GC, DMSO an almost collapsed, and HCX no visible GC at all, making it impossible to count/evaluate microtubules).

Results:

Not very "elegant" beginning: "we sought to..., but we sought to..."; and the first sentence is not really true since you did not want to inhibit GAG synthesis here. Maybe you could start with something like "Previous studies on GAG chain synthesis had used...

Here, we wanted to establish a dose-response curve... to determine...".

Fig.1: You could have at least added F-actin staining of those "real" neurons since the rest of the paper is mostly about lamellae and F-actin on N2A cells. F-actin is shown in Supp Fig.1, but there an HCX image is missing.

Fig.2: As in the text you say that velocity and total distance were significantly different, this should be shown in the figure (that shows only velocity), or at least the significance values for total distance mentioned in the text.

Fig.3: the image selected in Supp Fig.2 is a bit closer in comparison now, but I'm still not convinced: what exactly do you designate lamellipodium here in Fig.3 (clearly identifiable in the Supp. Fig.2 for the LCX cell). We should see (if I get it right??) that in LCX treated cells there are well-formed lamellipodia (reminiscent of neural growth cone), but less and thinner actin bundles than in HCX cells.

Several typos in Supp Fig.2 legend on the figure itself (but not in the manuscript).

Line 273: (Figure 2C) should read Fig. 3C.

Discussion:

Lines 303-4: "Treatment with xyloside treatment..."

Line 408: I don't see how you can suggest a different action of LCX on hippocampal neurons and N2A cells based on MT looping in growth cones, since the latter don't form a growth cone (at least not in your study).

Line 442: ...caused changed...

Line 443: predominant effect of actin, or on actin?

Line 452: full stop missing.

Line 454: "...the phenomenology is dose dependent". Normally, phenomenology is a science (sort of) and cannot be dose dependent.

Reviewer #2: PONE-D-22-03467R1

Even though the paper’s text and figures have been extensively changed, the lack of further crucial experiments to clarify the function of CS/HS in this phenomenon is disappointing. As suggested previously by the reviewers, this paper needs additional experimental approaches before it is ready for publication.

Furthermore, changes stated by the authors were not made. For example for Figure 1 “we have added images of several HC neurons at low power”, but no changes were made to Figure 1. Also, the new version does not include Figure 3 or 6. In particular for Figure 3 since the figure legend changed substantially, it is unclear if the figure did too.

As for the swapping of images in supplemental Figure 2 to match the look of Figure 3, it is problematic to me. The cell morphology is so radically different that I wonder what the authors consider to be a representative image. Now the cells in new Figure 2A looks different than cells in supplemental Figure 2 and original Figure 3. Why? A suitable explanation should be offered to the readers in particular when the whole paper is based on cytoskeleton differences between cell treatments.

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Reviewer #2: No

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Submitted filename: Re-review Mencio et al.pdf

A Novel Cytoskeletal Action of Xylosides

PONE-D-22-03467R2

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