Referee 1's Review:

Review of the original submission:

Nogo-66 promotes the differentiation of neural stem cells into astroglial lineage cells through mTOR-STAT3 pathway

Overall claim by the authors: Wang and colleagues report that components of CNS myelin can influence neural stem cell (NSC) fate decisions. Specifically, the presence of CNS myelin or the exogenous addition of extracellular portion of Nogo-A (Nogo-66) causes an increase in the proportion of neural stem cells that adopt an astrocytic phenotype in vitro and a decrease in the proportion of these cells that adopt a neuronal phenotype. The authors suggest that altered fate decisions in the presence of Nogo-66 are mediated by signaling through the Nogo receptor (NgR) and involve mTOR and STAT3 signaling.

Significance: Understanding how NSC fate is controlled by environmental factors is of substantial interest to the CNS injury community since NSCs represent a promising strategy for neuronal replacement. The conclusions are largely supported by the data overall, but the presentation of the work needs further improvement and several key controls are missing in the experiments.

Overall assessment of the paper: One major criticism is that the authors use GFAP as their only marker of astrocytic differentiation rather than supporting their claim with a series of markers (i.e., Connexin-43, S110beta, etc.). Given that activated astrocytes upregulate GFAP expression, it is possible that the effect of Nogo-66 is actually to cause an increase in astrocytic activation rather than differentiation. This could lead to a false conclusion that the number of astrocytes is increased in response to Nogo-66 or myelin when actually the number of activated astrocytes is what increases. This issue needs to be addressed by a more thorough characterization of the astrocytes population at the mRNA and protein level for markers of astrocyte differentiation.
A second major problem with the manuscript is that the proposed model by which Nogo-66 interacts with NgR to promote astrogial differentiation is not compatible with what is known regarding intracellular localization of Nogo-A
A third major problem with the manuscript is that it is not written in an intelligible manner and it is often difficult to understand the authors’ interpretation of the data.

Specific Comments:
Are the experiments, stats and other analyses performed to a sufficient technical standard?
There are several technical deficiencies and oversights that need to be rectified:
1. The materials and methods states that all statistics were done using 2-tailed t tests. However, for experiments comparing multiple treatments an ANOVA and posthoc testing should be used.
2. Figure 2A. The image showing GFAP labeling for the GST control group is clearly taken at a much lower exposure than the other images in the figure (note significantly brighter Hoechst signal in all other panels). There are clearly astrocytes labeled in this image, but they are underexposed. The image does not accurately reflect what is visible in the field and therefore misrepresents the data.
3. Figure 2E. NeuN labeling after exposure to GST at 50 nM seems to have been substantially diminished while GFAP labeling did not appear to increase under these conditions. This suggests the possibility that other factors besides the presence of Nogo-66 may have contributed to the differences in cellular differentiation observed.
4. Figure 3F. The blot in the second panel is difficult to evaluate because of regions of dark spotting that make the bands impossible to compare.

Are the conclusions presented in an appropriate fashion with speculations and hypothesis defined as such?
Overall, some of the conclusions are appropriate given the data but for more importantly there are alternative explanations for their data which are not considered or discussed by the authors. Please note that parts of the manuscript are very difficult to understand and need to be improved before the conclusions will be intelligible to the reader:

1. The authors state that MAG has a similar effect to Nogo-66 fragment in inducing astrocytic differentiation of NSCs. These results are not presented in the manuscript and therefore should not be stated as a conclusion of this study.
2. NSCs can be promoted to adopt a neural phenotype by various manipulations in vivo. The conclusion that Nogo-66 signaling can induce an astrocytic phenotype would be more convincing if the authors showed that when NSCs are promoted to adopt a neuronal phenotype, the NgR, mTOR, STAT3 pathways are NOT activated. Such a result would strengthen the claim that these signaling pathways are directly contributing to glial differentiation. Showing that mature astrocytes do not respond to Nogo-66 via the same pathways AFTER differentiation might also provide support for the conclusion that these pathways are involved specifically in differentiation.
3. The authors suggest that myelin inhibitors present within the niche environment of NSCs grafted to the CNS might be one environmental factor that contributes to the predominately glial phenotype adopted by these cells. Assaying differentiation of NSCs into glial or neuronal phenotypes when NSCs are grown on myelin or on a cell monolayer over-expressing Nogo-A, would indicate whether myelin components could affect NSC differentiation when expressed and localized normally in cells rather than when added exogenously as a soluble fragment extracellularly which is not physiological given that Nogo-A expression is predominantly cytoplasmic and therefore Nogo-66 domain would not be expected to interact with the NgR on the cell surface.
4. Use of GFAP as the only marker of astrocyte differentiation is problematic as it is often upregulated in activated astrocytes; thus addition of Nogo-66 may result in induction of a reactive astrocyte phenotype in a manner analogous to how EGFR stimulation causes activation or formation of cribiform astrocytes which contribute to glial scarring (Erschbamer et al. in J Neurosci. 2007). This possibility should be considered and discussed.
5. A study published by Satoh et al. in 2005 looking at Nogo-A and NgR expression in demyelinating lesions of MS and ischemic lesions of acute and old cerebral infarction reported that Nogo-A was markedly upregulated in surviving oligodendrocytes at the edge of chronic active, whereas NgR expression was greatly enhanced in reactive astrocytes and microglia/macrophages in these lesions when compared with their expression in the brains of neurologically normal controls. The authors should discuss alternative explanations for their findings and consider the possibility that the effect they are seeing with exogenously added Nogo-66 is not a physiologically normal role of Nogo-A on differentiation of NSCs into astrocytes but one in which they have induced aberrant activation of astrocytes.

Have the techniques been documented in sufficient detail to allow replication?
There are several deficiencies and lack of experimental detail which need to be given to the reader so he/she may be able to evaluate the findings:

1. How was myelin-basic protein (MBP) purified from myelin preparations or where was it obtained?
2. For all procedures involving antibodies, the dilutions used should be stated and non-immune IgG or IgM should be used to establish the specificity of the staining.
3. The method for culturing NSCs is only explained up to 8 days/neurosphere formation. How were the cultures maintained after this point?
4. Figure 1B. The presence of full length GST-Nogo-66 in inclusion bodies is shown along with a band representing broken fragments in the soluble fraction. Which GST-Nogo-66 was used? Was the full-length GST-Nogo-66 fragment derived after re-solubilization of the inclusion bodies used? The authors need to provide clarification. What was the procedure for refolding the protein and what tests were performed to validate its activity?
5. Figure 1C. Molecular weight markers should be included in the figure.
6. Figure 1D. The same fields should be shown for both betaIII Tubulin and GFAP so that equivalent areas can be compared.
7. Figure 3J. Under what conditions was this experiment performed? Are these cells treated with Nogo-66? The authors claim in their discussion that, “mTOR-STAT3 complex formation is essential for phosphorylation of STAT3 induced by Nogo-66 in NSCs.” The data show that mTOR/STAT3 complexes are present in NSCs and that complex formation can be inhibited by rapamycin, but there is no direct evidence that complex formation is required for phosphorylation of STAT3 unless cultures were treated with Nogo-66.
Is the report presented in an intelligible fashion and written in English?
This is a serious problem with the manuscript and authors are advised to obtain editing assistance to improve the quality and clarity of their ideas:
1. The text is extremely difficult to follow and many words are mis-spelled (meylin instead of myelin) or chosen poorly (underline instead of underlying). Authors are advised to obtain editing assistance.
2. Perhaps a minor point but there is no introduction. The authors should introduce the main facts pertaining to NSC differentiation. For example, what factors are already known to be involved in fate decisions in vitro and in vivo? What is known about the role of mTOR and JAK/STAT signaling in differentiation of NSC or other cell types?
3. Several figures are missing axis labels: Figure 1E-H x-axis labels needed, Figure 2B-D, F x-axis labels needed.
4. Figure 3B. What proteins are indicated by green and red fluorescent markers? Labels should be checked throughout the manuscript.

Have the exact results reported been published elsewhere?
As far as I know, these results are novel.

Does the research meet applicable ethics and integrity standards?
No concerns.

Review of the first revised manuscript:
Nogo-66 promotes the differentiation of neural stem cells into astroglial lineage cells through mTOR-STAT3 pathway

The authors have addressed the majority of the issues raised in the initial review and in particular the major criticisms:
1) Additional markers of glial differentiation have been added and their expression is consistent with the GFAP results.
2) The issue of topology/localization was addressed with a citation showing that it may be possible for the interaction between Nogo-66 and NgR to occur in injured CNS rather than experimentally, but is satisfactory. This issue and their explanation and citation should be included in the discussion.
3) The grammar is significantly improved but there are still a few grammatical errors (lack of subject verb agreement) and typographical errors. Please edit further.

Other minor points:

1) For Question #3: The authors state that ANOVAs were performed, but the post-hoc tests for significance are not stated. Please indicate what tests was used (Tukey's? Neuman-Keuls?) to find which groups were different from each other.
2) For Question #8: Please include this possibility in the discussion.
3) For Question # 13: Please include this in the manuscript
4) Please add page numbers to the manuscript.
5) For all procedures involving antibodies, the dilutions used should be stated and indicate whether non-immune IgG or IgM was used to establish the specificity of the staining.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.