A novel cytoskeletal action of xylosides

Caitlin P Mencio; Sharada M Tilve; Masato Suzuki; Kohei Higashi; Yasuhiro Katagiri; Herbert M Geller

doi:10.1371/journal.pone.0269972

. 2022 Jun 28;17(6):e0269972. doi: 10.1371/journal.pone.0269972

A novel cytoskeletal action of xylosides

Caitlin P Mencio ^1,^¤, Sharada M Tilve ¹, Masato Suzuki ², Kohei Higashi ², Yasuhiro Katagiri ¹, Herbert M Geller ^1,^*

Editor: Catherine FAIVRE-SARRAILH³

PMCID: PMC9239447 PMID: 35763520

Abstract

Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. 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. We found a dose-dependent alteration of GAG secretion by Neuro2a cells; however, concentrations of xylosides which were effective in altering neuronal morphology did not cause a large change in the rate of GAG chain secretion. In contrast, both low and high concentrations of xylosides altered HS and CS composition. RNAseq of treated cells demonstrated alterations in gene expression only after treatment with millimolar concentration of xylosides that had no effect on cell morphology. These observations support a novel action of xylosides on neuronal cells.

Introduction

Proteoglycans (PGs) are found in every tissue in the body. Consisting of two major components, a core protein and glycosaminoglycan (GAG) chain(s), they are essential components of the extracellular matrix. There are three major classes, differentiated by their GAG chain composition: heparan sulfate (HS), chondroitin sulfate (CS) and keratan sulfate (KS). PGs are involved in many important biological processes [1], especially in the central nervous system, where they regulate neuronal migration, axon guidance and differentiation [2, 3]. They are also believed to play critical roles in neural de- and re-generation [4].

In the recent past, it has become more apparent that the physiological effects of proteoglycans can be attributed to their GAG chains. Moreover, the wide range of biological function is often attributed to the complexity and diversity of GAG chain modifications, sulfation being the most common. Altering GAG sulfation patterns has been known to change GAG chain receptor binding resulting in modified cell signaling [5–7].

GAG chain biosynthesis is a non-template driven process that begins for all PGs with a common linkage of three sugars (Xyl-Gal-Gal) to a serine on the core protein. The three classes then diverge with addition of disaccharides to the linkage region. For HS, these would be GlcNAc and GlcA, for CS the disaccharides are GalNAc and GlcA, and for KS they are GlcNAc and Gal. Each disaccharide in the chain may undergo several different modifications, primarily sulfation. The impact that modifications to the GAG chains has on neural development as well as other cellular processes remains an active area of research.

One major approach to understanding the function of GAG chains in PGs has been by using chemical modulators of GAG biosynthesis called xylosides which serve as competitive molecules for cellular sugar chain synthesis. Xyloside-induced interference in GAG biosynthesis can lead to changes in GAG concentration, chain type, sulfation pattern, and molecular weight [8–10] as well as biological activity [11–13]. Historically, xyloside research has utilized high (millimolar) concentrations that serve to primarily block endogenous GAG production [14, 15], with relatively few studies using lower than mM concentrations [10, 16]. We therefore conducted a dose-response study to determine the minimal concentration of xyloside that would disrupt GAG chain synthesis in hippocampal pyramidal neurons and Neuro2a cells. To our surprise, we saw major changes in cell morphology when cells were treated with nanomolar xyloside concentrations but not micro- or millimolar treatment. After documenting the cytoskeletal changes, we then determined that treatment of cells with nanomolar concentrations of xyloside altered CS and HS composition with distinct differences found in heparan sulfate (HS) disaccharide composition from both control cells and cells treated with the high concentration of xyloside. This study serves as a first step in our attempt to understand how minor shifts in GAG chain composition or concentration can affect biological processes and continues to support the critical role of sugars in development and cellular function.

Materials and methods

Laboratory animals

Experiments and procedures were performed in accordance with Institutional Animal Care and Use Committee (IACUC) at the National Institutes of Health approved protocols. Pregnant female C57Bl/6 mice (Charles River) were housed in a pathogen free facility with standard 12 h light/dark cycle and unlimited access to food and embryonic (E17-19) pups were utilized for embryonic hippocampal neuron primary cultures.

Cell culture

Primary hippocampal neuron cultures were prepared from embryonic (E17-19) C57Bl/6 mouse brains. Hippocampi were dissected and dissociated into single cell suspensions. Dissociated cells were seeded onto coverslips coated with poly-L-lysine and cultured in 500 μL Neurobasal medium containing B27 supplement (Thermo Fisher) and 24 mM KCl at a density of 8–10 × 10³ cells/well to allow for observation of isolated neurons. After allowing 2 h for neuronal attachment, media was replaced with 1 ml fresh Neurobasal media containing DMSO and concentrations of 4-Methylumbelliferyl-β-D-xylopyranoside (4-MU, SigmaAldrich) between 0.5 nM and 1 mM. Xylosides were dissolved in DMSO and stock solutions of 1 M and 500 μM were made. DMSO and xylosides were added to cells at appropriate dilutions resulting in final concentrations of xyloside and 0.1% DMSO in solution. Cells were incubated for 24–72 h at 37°C and 5% CO2 atmosphere and then fixed and stained for DAPI, βIII-tubulin, and actin (Phalloidin).

Neuro2a (ATCC) cells were cultured in DMEM media containing 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. Cells were seeded onto coverslips, 35 mm glass bottom dishes (MatTek), 6-well plates or T-75 (Corning) flasks depending on experimental design. Cells were seeded at ~8–10 ×10³ cells/well for coverslips, 35mm dishes and 6-well plates and ~1.5 ×10⁶ cells/flask for T-75 flasks.

To produce conditioned media, Neuro2a cells were grown to about 50% confluency in T-75 flasks (Corning) in DMEM supplemented with 10% FBS. At this point, DMEM containing FBS was removed and cells were washed twice with sterile PBS and then kept in DMEM only overnight. The next day the media was replaced with media containing DMSO, 500 nM xyloside or 1 mM xyloside. Cells were allowed to grow for 48 h, after which time media was removed and placed into 15 ml conical tubes and spun for 10 min at 500 rpm to remove floating cells and debris. The supernatant was transferred into a fresh tube and subject to GAG analysis.

Glycosaminoglycan analysis

Conditioned media was harvested from Neuro2a cells that had been serum starved overnight and then treated for 48 h with xylosides in DMEM culture medium. Approximately 10 ml of conditioned media was collected from each experimental condition: DMSO, 500 nM and 1 mM xyloside. This media was spun for 5 min at 2000 rpm to remove cell debris and then transferred to a new conical tube and frozen until analysis.

GAG extraction was performed as follows. The media (1 mL) was treated with 10% TCA and centrifuged at 12000 rpm for 5 min to remove proteins. GAGs were collected by Amicon Ultra Centrifugal Filter 3K device (Merck Millipore, Billerica, MA, USA) and suspended with 100 μL of H₂O. Fifty μl of GAG solution was moved to new 1.5 ml microcentrifuge tube and lyophilized. Resulting GAG samples were incubated in the reaction mixture (35 μL) containing 28.6 mM Tris-acetate (pH 8.0) and 50 mIU of chondroitinase ABC for 16 h at 37°C. Depolymerized samples were boiled and evaporated, unsaturated disaccharides of CS were collected by Amicon Ultra Centrifugal Filter 30K device (Merck Millipore). The remaining HS samples in filters of spin columns were transferred to new microtubes and incubated in 16 μl of reaction mixture (pH 7.0), containing 1 mU heparinase I (Seikagaku Corp., Tokyo, Japan), 1 mU heparinase II (Iduron, Manchester, UK), 1 mU heparinase III (Seikagaku), 31.3 mM sodium acetate, and 3.13 mM calcium acetate for 16 h at 37°C.

Unsaturated disaccharide analysis using reversed phase ion-pair chromatography with sensitive and specific post-column detection was performed as described previously [17]. Disaccharide composition analysis of CS or HS was performed by reversed phase ion-pairing chromatography with sensitive and specific post-column detection. A gradient was applied at a flow rate of 1.0 ml min^-1 on Senshu Pak Docosil (4.6 × 150 mm; Senshu Scientific Co., Ltd., Tokyo, Japan) at 60°C. The eluent buffers were as follows: A, 10 mM tetra-n-butylammonium hydrogen sulfate in 12% methanol; B, 0.2 M NaCl in buffer A. The gradient program of CS disaccharides analysis was as follows: 0–10 min (1% B), 10–11 min (1–10% B), 11–30 min (10% B), 30–35 min (10–60% B), and 35–40 min (60% B). The gradient program of HS disaccharides analysis was as follows: 0–10 min (1–4% B), 10–11 min (4–15% B), 11–20 min (15–25% B), 20–22 min (25–53% B), and 22–29 min (53% B). Aqueous (0.5% (w/v)) 2-cyanoacetamide solution and 1 M NaOH were added to the eluent at the same flow rates (0.25 ml min^-1) by using a double plunger pump. The effluent was monitored fluorometrically (Ex., 346 nm; Em., 410 nm). Expression levels of HS or CS were expressed as total amounts of unsaturated disaccharides, while the composition was expressed as percent of total GAG.

Microscopy and image processing

Cells were imaged using either a Nikon A1R or Zeiss 880 confocal microscope with 60X and 63X objectives depending on the experiment. Z-stacks were maximally projected onto a single plane using Zeiss or ImageJ [18] image processing software. For images used in fluorescence quantification, image capture settings were held constant, and samples from within each group were imaged at the same time. Fluorescence intensity was measured using ImageJ with identical settings for all samples within each analysis.

Neurite outgrowth and growth cone analysis

After fixation and staining, at least 60 images were taken across two coverslips per condition. Files were analyzed by an experimenter blinded to the experimental conditions. Neurons were measured if they were isolated from other neurons and had distinct nuclei and at least one neurite longer than the diameter of the cell body. At least 60 images were taken for each experimental condition and then randomized using the “Bulk Rename” utility (https://www.bulkrenameutility.co.uk). Duplicates of these files, with all identifying information removed were analyzed. After analysis, results were matched to the treatment condition. Neurite looping at growth cones was counted in a binary yes/no fashion based on the presence of visible circular strands of microtubules (MTs) at the terminal end of the longest neurite. These loops required the presence of an empty center surrounded by one or more continuous MT. If a loop was observed the cell was counted as “having looped MTs”, otherwise it was counted as “not”. Neurite measurements were obtained using the ImageJ trace tool measuring the distance from where the neurite connected to the soma and terminating at the end of the neurite. Both longest and total neurite measurements were obtained for each neuron.

Growth cone measurements were conducted using ImageJ by tracing the end of the neurite from the point it widened from its average diameter of the stalk and where phalloidin staining began to show more intense and spiked appearance as is common with growing ends of neurites. Only the largest growth cone of the neuron was measured. Each experiment was performed in triplicate.

Analysis of cytoskeleton dynamics

EB3 comet analysis: Neuro2a cells were grown in culture until about 30% confluent. Cells were then treated with DMSO, 500 nM xyloside or 1 mM xyloside for 48 h. After a further 24 h, cells were transfected with EB3-GFP using Avalanche^®-Omni transfection reagent [19]. The next day, cells were imaged on a Nikon A1R confocal microscope with a 60X/1.42 N.A. Plan Apochromat oil objective. Transfected cells were imaged every 12.5 sec over 5 min. Images were then processed using u-track software [20] and comets assessed for speed, lifetime and distance travelled.

Actin bundle analysis: Neuro2a cells were grown in culture until about 30% confluent. Cells were then treated with DMSO, 500 nM xyloside or 1 mM xyloside for 48 h. After a further 24 h, cells were transfected with Ftractin-mCherry using Avalanche^®-Omni transfection reagent. The next day, cells were imaged using a Zeiss 880 confocal microscope with a 63X/1.4NA apochromat objective. Transfected cells with lamellipodia were imaged in a z-stack. Using ImageJ, z-stacks were max projected, and a line drawn through the lamellipodia and a line scan performed based upon fluorescence. Actin bundles were identified as peaks of increased fluorescence. Bundles were counted and the area under the curve taken to compare quantity and size of the bundles.

RNA-seq

Between 6–15 micrograms per sample of total RNA from three samples each of Neuro2a cells treated with either DMSO, LCX or HCX were sequenced by Illumina at Omega Bioservices (Norcross, GA). Data analysis was performed using the Partek Flow statistical analysis software (Partek Incorporated). For this, raw data from three replicates of the three conditions were imported to Partek Flow for alignment and quality controls. Aligned reads were quantified to transcriptome, filtered out on low expression and normalized. Feature gene lists with at least a two-fold change in gene expression with a false discovery rate of Q < 0.05 using the BH procedure [21] were created by pairwise comparison. Pseudogenes were eliminated from the final list of altered genes and the list plotted as a heatmap. In order to visualize the difference of the expression between DMSO, LCX and HCX, the data were centered by subtracting the mean of the log₂ Fold Change of all samples for each gene from the original log₂ Fold Change value. The centered data of all samples were then plotted into a heat map. The rows of the heatmaps (genes) were ordered by fold change [22].

Statistics

All statistical tests were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA). Neurite lengths in culture were compared using Kruskal-Wallis Analysis of Variance and Mann-Whitney U tests.

Results

Our objective was to determine the effect of xyloside treatment on the morphology of hippocampal neurons in culture. A previous study noted that concentrations of 0.1 and 0.2 mM xyloside perturbed the generation of neuronal polarity [23], but we sought to determine a more complete dose-response curve. We therefore added 4-MU in a range of 0.5 nM to 1 mM to the medium of dissociated embryonic hippocampal neurons, and fixed and stained cultures after 24 or 72 h. At 72 h, we observed a surprising dose-dependent response to 4-MU: neurons treated with lower concentrations showed enlarged growth cones that exhibited looped microtubules as compared to DMSO treated cultures or cultures treated with mM concentrations (Fig 1, S1 Fig). Fig 1A shows representative images taken from the range of concentrations: DMSO (control), 500 nM 4-MU as the low xyloside concentration (LCX) and 1 mM MU as the high xyloside concentration (HCX). As presented in Fig 1B, there was a difference in growth cone size with the different treatments: neurons treated with LCX had significantly larger growth cones than those treated with DMSO or HCX. To determine if the enlarged growth cones was due to neurite stalling, the length of the longest neurite was measured for each neuron in the condition. There was no significant difference in neurite length between all conditions (Fig 1C). The lower end of the dose-response curve is presented in S1 Fig. Moreover, a difference in neurite morphology was apparent as early as 24 hr. after plating (S2 Fig), where neurites of LCX-treated neurons showed splayed microtubules, while neurites of cells treated with either DMSO or HCX show typical compact neurite outgrowth.

Fig 1 — A) Example of a primary mouse hippocampal neuron treated for 72h with DMSO, LCX, or HCX and stained with anti-βIII-tubulin. White arrow indicates enlarged growth cone in LCX treated neuron. B) Quantification of growth cone area and length of longest neurite.in DMSO, LCX and HCX treated neurons. LCX treated neurons have significantly increased growth cone area compared to control or HCX conditions. Scale = 25 μm. **p < 0.01.

As primary neuron culture is often limiting in cell number, we decided to assess if the mouse neural crest-derived cell line Neuro2a would show similar morphological changes and could be used to study any structural or biochemical changes between HCX and LCX treatment. Observation of the cytoskeleton and overall cellular morphology in LCX treated Neuro2a cells shows that these cells exhibit actin-rich lamellipodia that were not observed in Neuro2a cells treated with either DMSO or HCX (S3 Fig). With a confirmed change in cytoskeleton and for the sake of uniformity and economy, we decided to utilize Neuro2a cells for all other experiments in this study.

LCX treatment reduces cellular movement

After observing changes in morphology, we next wanted to check if these changes alter the cells’ ability to migrate. Neuro2a cells were treated with DMSO, LCX or HCX for 48 h, at which point cells were imaged using brightfield microscopy for 4 h with images being taken every 8–10 min (Fig 2). Cells within the image were tracked and velocity and total distance was calculated. There was a significant reduction in both velocity and total distance in LCX treated cells as compared to DMSO and HCX treated Neuro2a cells (Fig 2B). This reduction in movement implicates possible changes in cytoskeleton dynamics.

Fig 2 — Neuro2a cells were observed using time-lapse microscopy. A) Representative images taken at the indicated intervals. Numbers show positions of specific cells. B) Dot plot of velocity of individual cells taken as distance moved in each frame. *p < 0.05. **p < 0.01. Scale = 25 μm.

LCX treatment affects both actin and microtubule dynamics

To determine if altered cytoskeleton may play a role in LCX induced changes in morphology and migration, we next assessed both actin and microtubules in Neuro2a cells. Neuro2a cells were transfected with Ftractin-mCherry and treated for 48h with DMSO, LCX or HCX. At 48h, visual observation showed a marked difference in lamellipodia of LCX treated cells as compared to DMSO and HCX treated Neuro2a cells. DMSO and HCX-treated cells showed bright and thick actin bundles while LCX treated cells appeared to have fewer bundles which also appeared thinner than their control counterparts (Fig 3A). We quantified both the area and number of actin bundles from these images. LCX treated Neuro2a cells exhibited lamellipodia that had significantly fewer actin bundles per 10μm when compared to controls (Fig 3B). Additionally, the area under the curve for these bundles was significantly reduced in LCX treated cells as compared to DMSO or HCX treated Neuro2a cells (Fig 3C) indicating less robust actin bundles in LCX lamellipodia.

Fig 3 — A) Representative fluorescence images of living Neuro2a cells expressing F-tractin treated with DMSO, LCX, or HCX for 48 h. Insets show line scans of fluorescence intensity across the lamellipodia of indicated cells. B) Plots of area under the curve and the number of peaks for each of the three conditions. Scale = 25 μm. **p < 0.01.

With a measured effect on actin, we next wanted to assess if microtubules, the other major cytoskeleton element, were also affected. To examine microtubule dynamics, Neuro2a cells were treated for 48h with DMSO, LCX or HCX, and transfected with EB3-GFP as outlined in materials and methods. EB3-GFP cells were imaged at 60X with one image taken every 12.5 sec for 5 min (Fig 4A). Images were used to measure speed, persistence and total distance traveled of individual EB3 speckles. Both LCX and HCX treated Neuro2a cells exhibited a higher percentage of faster moving EB3 comets as compared to controls (Fig 4B, left). Additionally, xyloside treatment resulted in longer persistence of EB3 comets compared to DMSO treated Neuro2a cells (Fig 4B, center). There were no significant changes in total distance traveled between all conditions (Fig 4B, right). Thus, while LCX has selective actions on actin dynamics, both LCX and HCX affected microtubule polymerization.

Fig 4 — A) Images of living Neuro2a cells (top) expressing EB3-EGFP were imaged over time. Bottom images show z-projections of 25 frames of fluorescent images of EB3-EGFP. B) Cumulative distribution plots of speed, persistence and total distance traveled by EB3 comets from DMSO, LCX and HCX-treated cells. Scale = 10 μm.

LCX treatment alters GAG chain but not mRNA profile

Xylosides have been known to affect cellular GAG chains, both increasing the rate of synthesis and secretion [24] and altering GAG chain composition [25]. These GAG chains have been linked to cellular signaling that could lead to altered cytoskeleton. To determine if LCX treatment affects GAG chain production or sulfation, Neuro2a cells were treated with DMSO, LCX, or HCX in serum free media for 48h. Conditioned media were collected and the concentration as well as the composition of GAG chains were analyzed. Composition of the GAG addresses the positions of sulfate groups on the individual CS or HS disaccharides. As previously reported, there was a dose dependent increase in GAG chain accumulation for both CS and HS in the medium with xyloside treatment. Both LCX and HCX increased CS chain concentration as compared to control. Notably, HCX caused a much larger increase in CS (Fig 5A). In contrast, there was no difference between HS concentration in CM of DMSO and LCX-treated cells; only HCX treatment led to a large increase in HS secretion as compared to DMSO and LCX (Fig 5B).

Fig 5 — GAG chains were collected from conditioned medium and subject to analysis of total GAG levels as well as disaccharide composition, expressed as percentage of total GAGs. A) Quantification of CS GAG levels present in conditioned media sample. B) CS disaccharides analysis. 0S, ΔHexUA-GalNAc; 4S, ΔHexUA-GalNAc(4-O-sulfate). C) Quantification of HS GAG levels present in conditioned media sample. D) HS disaccharide analysis. 0S, ΔHexUA-GlcNH₂; NS, ΔHexUA-GlcNS; 6S, ΔHexUA-GlcNH₂(6-O-sulfate); 2SNS, ΔHexUA(2-O-sulfate)-GlcNS.

We assayed the disaccharide composition of the secreted GAGs, both CS and HS. Conditioned media from DMSO treated cells contained a close to even split between non-sulfated and 4-sulfated CS GAG chains. Following xyloside treatment with both LCX and HCX, we observed an increase in 4-sulfated CS GAG (Fig 5C). When we examined the disaccharide composition of secreted HS, it was found that all three treatment groups had different disaccharide profiles. Conditioned media from DMSO treated cells showed mostly non-sulfated disaccharides with a small percentage of N-sulfated and even smaller group of 6-sulfated disaccharides (Fig 5D). In contrast, LCX treatment resulted in an increase in N-sulfated disaccharides while HCX treatment led to a larger increase in N-sulfation, loss of 6-sulfation and the presence of a small amount of 2-sulfated, N-sulfated disaccharides (Fig 5D). 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.

Because xyloside effects on the cytoskeleton take several days to develop, it is possible that xylosides altered transcription which may have led to altered protein expression. Therefore, we next performed RNA-seq to determine which, if any, RNAs were altered by xyloside treatment. Cultures of Neuro2a cells were treated with DMSO, LCX and HCX and RNA was extracted after 48 h and pyrosequenced. The raw data were filtered using Partek Flow as noted in materials and methods. The filtered counts were then used to determine those genes whose expression was altered by >2X and statistically significant using q<0.05 FDR. Using these criteria, we found no genes whose change in expression was different between LCX and DMSO, while we did find changes in gene expression when comparing HCX with both DMSO and LCX. Fig 6A presents volcano plots which indicate the differentially expressed genes between HCX and DMSO and HCX and LCX. Fig 6B presents a Venn diagram that indicates the number of genes changed in each condition. Interestingly, majority of genes whose expression was changed were downregulated by exposure to xylosides (Fig 6B). The Venn diagram also indicates that only about 1/3 of the genes were differentially expressed by both HCX vs DMSO and HCX vs LCX. Fig 6C presents a heat map of the level of expression of those genes which are known to be translated into protein as compared to the average level over all conditions. There was no preference in in specific pathways using GO terms. Thus, the changes in cytoskeletal dynamics after LCX treatment are not likely to be due to changes in gene expression.

Fig 6 — Neuro2a cells were treated with the indicated levels of xylosides for 72h. RNA was extracted and sequenced. A) Volcano plots showing significant changes in gene expression (>50% change, P < 0.5) between HCX and DMSO-treated cells and HCX and LCX-treated cells. B) Venn diagram showing numbers of genes changed with HCX compared to DMSO and LCX treatment. C) Heat map of expressed genes that were changed by xyloside treatment. Color represents deviation from the average expression over all conditions.

Discussion

Xylosides have been used in research as a GAG-biosynthesis inhibitor since the 1970s [14], and continue to be used today. Virtually all published work has used these small molecules in the millimolar range as originally published by Schwartz et al. [24]. Our work reveals a novel action of xylosides on cell morphology that occurs at sub-micromolar concentrations, well below any previously reported to be active. Interestingly, LCX treatment only slightly raised the secretion of CS and HS into the medium, though we did confirm a large increase in GAG secretion by HCX, a major effect of these compounds when used in culture. We did not identify any changes in gene expression in cells exposed to the low concentrations of 4-MU that produced the morphological and cytoskeletal changes, though we did find changes after exposure to HCX.

Several studies have investigated dose-response curves with xylosides on GAG chain production and composition at concentrations in the micromolar range. We found that LCX produced a small change in CS secretion into the medium, with no change in HS, while HCX produced large increases in CS and HS GAG production. In contrast, both LCX and HCX changed GAG chain composition both for CS and HS. For CS, xyloside treatment caused an increase in sulfation of the GalNAc sugars at the 4-position. Our research has found an increase in 4-S sugars to be associated with injury to the CNS [26, 27], and that CS enriched in 4-S GAG acts to reduce neurite outgrowth [26]. These results can be compared to those of Carrino and Caplan [10], who found increased incorporation of ³⁵S into the medium of chick muscle cultures with concentrations down to 1 μM, with minimal effect on the structure of CS GAG chains, while mM concentrations resulted in GAG chains that were >90% enriched in 6-sulfated GalNAc. Similarly, Weinstein, et al. [28] found increased ³⁵S secretion into the medium by human chondrocytes treated with 25 μM xylosides, with a maximum attained at 100 μM; higher concentrations then suppressed GAG synthesis. More recently, Persson, et al. [25], systematically looked at GAG chain secretion and composition after treatment with different xylosides in several cell lines. Consistent with other observations, they found an increase in GAG production with 10 μM 4-MU in all cell lines. However, in contrast to our results, they found a decrease in the proportion of ΔHexUA-GalNAc-4S and an increase in the proportion of ΔHexUA-GalNAC-6S in the secreted CS. Because they observed divergent responses in different cell lines, it may be that Neuro2a cells respond differently.

Treatment of Neuro2a cells with HCX, but not LCX, caused an increase in HS secretion into the medium. In contrast, both concentrations of 4-MU caused changes in HS GAG composition. LCX increased ΔHexUA-GlcNS, while HCX further increased the percentage of ΔHexUA-GlcNS, and also increased the percentage of ΔHexUA-2SGlcNS. Knockout of the Ndst1 gene, which controls N-sulfation of HS chains alters many developmental processes, including brain development, in both mice [29] and humans [30]. The knockout mice also have altered responses to both FGF [31] and VEGF [32], which likely contribute to the developmental phenotypes in these animals. It is therefore possible that the increase in ΔHexUA-2SGlcNS could have contributed to the phenomena we observed.

The major cellular change we found with low concentration of 4-MU were in the cytoskeleton. Interestingly, we observed a change in actin dynamics in Neuro2a cells with LCX, but not HCX, while both concentrations changed microtubule dynamics. However, the major phenotype we observed in hippocampal neurons with LCX treatment was enlarged growth cones with microtubule looping. Several previous studies have examined the effect of high concentrations of xylosides on cytoskeleton. Adding 1 mM xyloside to vascular smooth muscle cells lead to a reduction in the number of α-actin containing cytoskeletal filaments [33, 34]. Treatment with 2 mM xyloside treatment impeded tubule formation during nematocyst development, implicating CS in the stabilization of membrane protrusions [35]. However, our observations suggest that there may be specific alterations of the cytoskeleton after exposure to much lower concentrations of xylosides. This suggests that, although LCX has specific morphological actions on both cells types, the underlying mechanism could be different.

Previous research in our lab as well as others has shown that neurons and neuronal cell types are sensitive to GAG chains and changes in sulfation patterns [26, 36, 37]. This sensitivity is linked to several processes that are regulated by cytoskeletal rearrangements such as neural migration, polarity and axon guidance. For example, degradation of GAG chains by enzyme or disruption of biosynthesis by xylosides led to altered neuronal migration [38]. The addition of HS and CS to embryonic rat neurons primarily resulted in neurons with a single long axon. Conversely the addition of DS resulted in neurons with increased dendritic growth that maintained higher levels of microtubule-associated protein 2 expression [39]. In terms of axon guidance, many studies have shown the bifunctionality of HS and CS as guidance cues. HS is commonly associated with permissive substrates while CS, especially 4-sulfated CS, is seen as inhibitory [40]. These actions clearly depend upon alterations in the cytoskeleton.

Because the morphological changes in neurons take several days to develop, we tested the hypothesis that there would be changes in gene expression. However, our RNAseq analysis did not find any RNAs whose level was significantly changed by exposure to LCX as compared to the control (DMSO alone) situation. However, we did find a subset of genes whose expression was significantly increased by HCX over either control or LCX. A large group of these code for proteins in the extracellular matrix, including Tenascin-C, Collagen 14, MMP9 and astrotactin 2, while another group, including α-actin, advillin, protein tyrosine phosphatase receptor type T and ABI family member 3 are involved in controlling cytoskeletal dynamics. However, none of these were changed at concentrations that produced the morphological changes. Treatment of HeLa cells with mM xylosides caused the upregulation of several RNAs related to proteoglycan synthesis [41], but we did not find any overlap with our results. Thus, it is not likely that the morphological changes we observe are due to changes in gene expression.

The question then arises as to what mechanism is mediating these changes in cellular morphology due to LCX treatment. Changes in cytoskeleton are most often induced by changes in cell signaling. Because many signaling pathways are modulated by interactions with cell-surface GAG chains, it is possible that signaling was altered. However, while short-term changes in signaling are mediated through post-translational modifications (PTMs), longer exposure normally produces changes in transcription. As noted above, our RNA-sequencing data shows little to no change in transcription after LCX treatment, indicating that any changes to transcription are not robust and thus alterations in signaling pathways controlling gene expression are not likely the main culprit behind changes in cell morphology. We found that LCX only influenced actin dynamics, while both concentrations caused changes in microtubules. While this might suggest a predominant effect on actin dynamics, it is possible that the larger concentration of secreted GAGs under HCX treatment activated pathways to alter intracellular signaling, antagonizing the effects of LCX.

This leaves changes in PTMs due to xyloside treatment as the most likely driving force behind the altered cytoskeleton. PTMs include phosphorylation, methylation, acetylation and glycosylation. Changes in PTMs are known to alter protein degradation, kinase activity, and intracellular protein localization. This study has shown that both high and low concentration xyloside treatment can alter glycosylation in a dose dependent manner as evidenced by the different disaccharide profiles in the primed GAGs. Previous work has shown that receptors in many signaling pathways are sensitive to changes in GAG sulfation [29]. The differences in changes in glycosylation between LCX and HCX along with the significant increase in CS after HCX treatment which is not found after LCX treatment could explain why the phenomenon is dose dependent.

Supporting information

S1 Fig. Microtubule looping in xyloside-treated growth cones.

A) Images of hippocampal neurons treated with either DMSO or LCX. LCX-treated neurons have large growth cones with extensive microtubule looping (arrow). B) Dose response curve for xyloside treatment. Percentage of neurons with looped microtubules at the end of the growth cones increased and peaked at 500 nM xyloside treatment.

(PDF)

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S2 Fig. LCX treatment alters early neurite outgrowth.

Images of hippocampal neurons treated with either DMSO, LCX or HCX 24 h after plating. Arrows point to splayed tubulin at the ends of growing neurites in LCX-treated cultures. Scale bar = 25 μm.

(PDF)

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S3 Fig. Altered Neuro2a morphology in cells treated with LCX.

Cells were transfected with F-tractin (red) and fixed and stained with DAPI (blue) 48 h later. (Left) DMSO-treated Neuro2a cells show typical morphology irregular shape and intense actin staining at the periphery. (Center) LCX-treated cell shows large lamellipodia (arrows) with centripetal actin organization. (Right) HCX-treated cells resemble DMSO-treated cells with irregular shape and peripheral actin staining. Scale = 25 μm.

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S1 Data

(XLSX)

Click here for additional data file.^{(336.5KB, xlsx)}

Acknowledgments

Images in this manuscript were acquired in the Light Microscopy Core of the Division of Intramural Research of the National Heart, Lung, and Blood Institute, NIH. We very much appreciate the comments and suggestions of the referee.

Data Availability

All relevant data for Figs 1–5 are within the paper and its Supporting Information files. Data supporting Fig 6 are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE206057.

Funding Statement

This work was funded by the NHLBI Division of Intramural Research (DIR) with project number 1Z01HL006021. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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

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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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PLoS One. 2022 Jun 28;17(6):e0269972. doi: 10.1371/journal.pone.0269972.r002

Author response to Decision Letter 0

1 Apr 2022

Response to Reviewer :

We thank the reviewer for their thorough and incredibly valuable comments and suggestions on the manuscript. We appreciate that this is a novel observation and have tried to answer each of the reviewers’ comments with appropriate changes in either the text or the figures. We have organized this response such that each comment or suggestion has a reply in different font from the comment. We hope that this submission is suitable for publication.

Herbert M. Geller, for the authors.

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).

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.

We agree that the discussion was not as informative as we would all like. Unfortunately, we could not conduct the mechanistic studies that would be important to answer this question, and so any interprétations we have are speculative. We did not include a model/schematic, as we think that any figure would be based on these speculations, and a reader who simply looked at figures (as many do) could be misled. Instead, we have replaced the verbiage cited by the reviewer with a more focussed discussion about what our data do support and do not.

While we began these studies with hippocampal neurons, we realized that certain data on biochemistry would be difficult to determine using primary cultures which can be highly variable. For consisency, we therefore evaluated the two concentrations of xylosides that were used in hippocampal neurons on Neuro2a cells and found that they had differential concentration-dependent effects at the same concentrations used in neurons, and therefore continued with these concentrations.

Neuro2a cells are mouse neuroblastoma cells and have been used in many different settings to examine signal transduction and biochemistry. Because they are a tumor cell line, they may exhibit various morphologies depending upon treatment, as we show here. There are many published papers that present data from both hippocampal neurons and Neuro2A cells, but we do not presume that all measurements will be the same in both.

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.

We agree and have modified the manuscript both in the introduction and discussion to emphasize these results.

We did cite this paper and its results in the discussion, but we have now included it in the introduction as well.

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

We have edited the methods to show the stock concentrations and the dilution factor for DMSO and xylosides used in the paper. Additionally, we had compared DMSO treatment to untreated cells and saw no noticable difference in morphology. No previous studies have shown any changes in the cell at the percentage of DMSO we utilize.

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

We have clarified which cell culture dishes were used as well as cell densities for each type of dish.

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

This concentration of KCl added to neurobasal medium has been found to promote neuronal health and survival of cultured neurons, and we do this in our lab protocols (Pearson CS, Mencio CP, Barber AC, Martin KR, Geller HM. Identification of a critical sulfation in chondroitin that inhibits axonal regeneration. Elife. 2018 May 15;7:e37139. doi: 10.7554/eLife.37139. PMID: 29762123; PMCID: PMC5976435.) A literaturer search shows that there are many other publications using high KCl to promote neuronal culture survival.

Line 146- Neurite outgrowth 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?).

We have added more detail into the materials and methods section on the analysis and data collection for neurites and growth cones.

The tranfection order was determined based on optimization of both cell survival and transfection efficiency. We found routinely about 40% of cells were transfected. We have used this transfection protocol to express GFP (without xylosides) in other experiments with Neuro2A cells (Agbaegbu Iweka C, Hussein RK, Yu P, Katagiri Y, Geller HM. The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory molecules. J Neurochem. 2021 May;157(3):494-507. doi: 10.1111/jnc.15271. Epub 2021 Jan 3. PMID: 33320336; PMCID: PMC8106640) (now cited in the manuscript) and have not observed changes in morphology.

Results:

We have added images of several HC neurons at low power. Unfortunately, it is difficult to display the large growth cônes and looping at that magnification, so we have retained the higher res images.

Indeed, there were several neurons in each image as indicated by the DAPI staining, making it difficult to interpret. We have therefore replaced image in the figure with one of a single neuron with the representative phenotype. We hope that these images are more easily interpreted.

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.

We have edited our materials and methods to provide clarity on how we determined the presence of looped MTs.

We appreciate this comment. N2A cells have various morphologies and our goal was to select representative images from cells grown at the same time, and so the cells in Supp. Fig. 2 were somewhat different from those in Fig. 3. We have now selected images of f-tractin transfected cells for Supp. Fig. 2 which are contemporaneous with those in Fig. 3. These images more clearly display the morphologies quantified in N2A cells.

These images are included as examples of how we can track movement. Not all the cells are migrating all the time. However, we have replaced the former images with images showing the various morphologies.

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 ?

We thank the referee for pointing this out. We have corrected the text to correspond with the figure.

We hope that we have answered this comment with the images in the new Supp. Fig. 2 which clearly shows the actin bundles in the LCX-treated cells. We also provide more details on the culture and transfection time in the figure legends.

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

We did not observe consistent changes in polarity. We have selected new representative images that show cells in a more similar state of polarization across all three experimental conditions.

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

Thank you for pointing out this issue. We have now modified the text in both the results and discussion to point out the difference in the response of the actin and tubulin cytosekeletons to xylosides, i.e., that both concentrations alter MT dynamics, but only LCX alters actin.

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.

We agree that the these findings were not adequately discussed. We have altered the discussion of the GAG sulfation to also indicate biological implications We have also included the definitions in the figure legend as requested.

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.

We agree that these are possibilities. Unfortunately, we are unable to conduct these experiments within a reasonable time frame, as the NIH has been quite strict about lab occupancy. We did think about adding the conditioned medium from HCX cells to the LCX condition, but this would still be contaminated by xylosides. In response to this and the suggestion above, we have included these possibilities in the discussion for potential future experiments.

According to our analysis using the Partek Flow pipeline, there were no genes whose expression was significantly changed (FDR < 0.05) between the LCX and DMSO conditions. Fig. 6C included all genes whose expression was significantly different between either LCX and HCX or DMSO and HCX, but we put in the relative change in expression for all conditions. Thus, the top one, Gstp3, was only signficantly changed between HCX and LCX.

Discussion:

It is clearly possible that other cell types may respond similarly. As noted in the manuscript, few papers used comparable concentrations of xylosides in their experiments, so there is no point of reference. As to the staining with HS/CS, be believe that the biochemical experiments are more informative, as they looked at both production and composition which is not possible with immunocytochemistry.

Minor points:

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

We have introduced these abbreviations

Figure legends are often too short. Ex., Figure1: "altered cytoskeleton", the label is not indicated (tubulin?).

Thanks, we have now indicated that this is tubulin staining, and added additional text to the other legends as well.

Figure 2, "letters" should rather read "numbers".

Thanks again. Corrected.

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.

We thank the reviewer for catching these errors. They have been corrected.

Figure 5, add abbreviations.

Done

Supp Fig. 1, the bar is missing. FIxed

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).

Thank you. We hope this version is free of typos.

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Submitted filename: Response to Reviewers.docx

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PLoS One. doi: 10.1371/journal.pone.0269972.r003

Decision Letter 1

Catherine FAIVRE-SARRAILH

28 Apr 2022

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

Dear Dr. Geller,

==============================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.

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

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

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**********

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

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

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 #1: No

Reviewer #2: No

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PLoS One. 2022 Jun 28;17(6):e0269972. doi: 10.1371/journal.pone.0269972.r004

Author response to Decision Letter 1

26 May 2022

We really appreciate the time the reviewer has taken with this manuscript. We have made additional changes in response to the comments and questions, and have attempted to answer each of the points made by the referee. Because we consider these comments to have greatly improved the manuscript, we have added an acknowledgment of the referee. We hope that these changes have made the manuscript acceptable for publication in PLOS One.

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.

I would not argue that the questions you raise are important, just that we don’t have the resources to address them at this time. While the cells are quite different, the result that low concentrations of xylosides have specific actions on the cytoskeleton is likely to be a general effect on many different cell types (though we have not looked a many).

- 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.

Unfortunately, low power images of 72-h treated neurons do not have adequate resolution for fine tubulin or actin filaments, as we plated at low density to avoid neurite overgrowth. However, we have added a supplementary figure showing that differences in neurite morphology could be observed as early as 24 h after plating. Because these cells had much shorter neurites, we are able to present images with more than one cell at a time at a lower resolution than in the other figures. I am also including a low power reviewers’ figure which does not have the adequate resolution to demonstrate the microtubule morphology, but does show general morphology. We hope that these additional images will help interpret the phenomenon.

- 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 original images that were used to illustrate the technique in Fig. 3 were not representative of the morphology under LCX treatment. We have now replaced the Fig. 3 images with ones that are more representative. In addition, the supplemental figure is now Supp. Fig. 3 and we have corrected the figure legend. We think these should adequately illustrate the phenomenon.

- 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).

This is also a great question. I actually lean towards the idea that the xylosides might be acting on the cytoskeleton totally independentely of GAGs, since the effects of LCX are modest on GAG secretion (though they do change GAG composition). However, proving this is beyond the scope of the current manuscript, and I did not want to put idle speculation in the text.

- 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.

Thanks for taking so much time for this. We have reworded using your suggestions.

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).

We now include GAG composition I the abstract.

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".

We again thank the reviewer for suggesting better wording.

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).

We divided neurons into categories based on whether they contained looped microtubules or not in any of their growth cones, and did not focus on any other morphological features, including collapse. Low power images show that most growth cones had some actin staining suggesting that they are not collapsed, but these images do not have the resolution for further evaluation. We hope that the current presentation focusing on microtubules in growth cones is sufficient to demonstrate the phenomenon.

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...".

I’ve tried to rewrite this sentence, but it seems that being accurate is not entirely compatible with being elegant. So I changed it to “xyloside treatment”, rather than GAG chain inhibition.

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.

Unfortunately, we concentrated on MT looping when we took these images, and so we did not have suitable images that contain F-actin for HCX at the equivalent power. I am including a reviewer’s figure that does have f-actin staining of hippocampal neurons, but these images were taken at 10X, such that microtubule looping is poorly imaged. But it does show f-actin p-domains in most neurites. We would be happy to include this figure if the reviewer thought it will help.

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.

We have now included the total distance in the results, though the result is not really independent since the persistence was not significantly different.

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.

We agree that the images were not optimal As noted above, we have replaced the images in Fig. 3 with ones more representative. They closely align with what we find in the supplementary figure (Now Supp. Fig. 3).

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).

Thanks for pointing this out. I have reworded the paragraph.

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.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(21.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0269972.r005

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2 Jun 2022

A Novel Cytoskeletal Action of Xylosides

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PLoS One. doi: 10.1371/journal.pone.0269972.r006

Acceptance letter

Catherine FAIVRE-SARRAILH

20 Jun 2022

PONE-D-22-03467R2

A Novel Cytoskeletal Action of Xylosides

Dear Dr. Geller:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Microtubule looping in xyloside-treated growth cones.

(PDF)

Click here for additional data file.^{(7MB, pdf)}

S2 Fig. LCX treatment alters early neurite outgrowth.

Images of hippocampal neurons treated with either DMSO, LCX or HCX 24 h after plating. Arrows point to splayed tubulin at the ends of growing neurites in LCX-treated cultures. Scale bar = 25 μm.

(PDF)

Click here for additional data file.^{(4.9MB, pdf)}

S3 Fig. Altered Neuro2a morphology in cells treated with LCX.

(PDF)

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S1 Data

(XLSX)

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Data Availability Statement

All relevant data for Figs 1–5 are within the paper and its Supporting Information files. Data supporting Fig 6 are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE206057.

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PERMALINK

A novel cytoskeletal action of xylosides

Caitlin P Mencio

Sharada M Tilve

Masato Suzuki

Kohei Higashi

Yasuhiro Katagiri

Herbert M Geller

Roles

Abstract

Introduction

Materials and methods

Laboratory animals

Cell culture

Glycosaminoglycan analysis

Microscopy and image processing

Neurite outgrowth and growth cone analysis

Analysis of cytoskeleton dynamics

RNA-seq

Statistics

Results

Fig 1. Altered cytoskeleton seen in cells treated with 500 nM (LCX) but not 1 mM (HCX) xyloside.

LCX treatment reduces cellular movement

Fig 2. LCX reduces cell migration.

LCX treatment affects both actin and microtubule dynamics

Fig 3. LCX alters actin bundling.

Fig 4. Xyloside treatment alters microtubule dynamics.

LCX treatment alters GAG chain but not mRNA profile

Fig 5. Analysis of glycosaminoglycan chains in conditioned media from Neuro2a cells after xyloside treatment.

Fig 6. RNAseq analysis of xyloside-treated Neuro2a cells.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Catherine FAIVRE-SARRAILH

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Author response to Decision Letter 0

Decision Letter 1

Catherine FAIVRE-SARRAILH

Roles

Author response to Decision Letter 1

Decision Letter 2

Catherine FAIVRE-SARRAILH

Roles

Acceptance letter

Catherine FAIVRE-SARRAILH

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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