1. Reviewer #3 (Public Review):

    This is a very interesting and well conducted study that addresses a question of crucial importance and will make a very valuable contribution to the literature. The question of the vulnerability of newly generated oligodendrocytes in an inflamed environment has not previously been examined with anything like the sophistication of the current series of experiments. The paper is excellent and the data convincing. I only have a few relatively minor issues that the authors might want to consider.

    The first results section on sephin1 in EAE is a little confusing. If I have understood the rationale correctly, it is to activate the ISR to protect oligodendrocytes, newly generated from OPCs, in the face of a hostile inflammatory environment. If that is correct, then perhaps this could be explained more explicitly, and the concluding sentence re-worded so as not to give the impression that sephin-1 is able to enhance remyelination (which I realise is not what is stated but is the conclusion that might be drawn).

    The effect of the BZA-sephin combination of g ratio of remyelinated axons is very interesting. This could, of course, be because the process is accelerated with this combination rather than enhanced given that g ratios in the CC will eventually return to normal after cuprizone induced demyelination (eg Stidworthy et al., Brain Pathology 2003). This could perhaps be addressed in the discussion.

    The authors could make the point in the discussion that regenerative medicines are very unlikely to be given in the absence of effective drug-mediated suppression of aggrieved inflammation.

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  2. Reviewer #2 (Public Review):

    This is an interesting paper showing that prolonging the integrated stress response provides protection to oligodendrocytes in the presence of an inflammatory cytokine. For their experiments, the authors use the cuprizone model in transgenic mice overexpressing IFNg in an inducible manner in combination with a genetic and pharmacological approach to enhance the integrated stress response. The experiments are well conducted and the results clearly presented in the text. The Popko lab has previously demonstrated in a series of papers the importance of the integrated stress response for oligodendrocyte function. The novel aspect of this work is that targeting the integrated stress response requires a neuroinflammtory environment for the protective effects to occur.

    It is important to improve the introduction. As written it is not clear what was known before and how this paper goes beyond the existing literature.

    The rational for combining for combining BZA and Seph needs to be explained.

    The figures and legends could be improved according to the following suggestions:

    The evidence that Sephin1 promotes remyelination in the EAE model shown in Figure 1 is only based on differences in g-ratio with the overall number of myelinated axons being unchanged. It is difficult to make conclusion based on these results. It is difficult to obtain accurate g-ratios in lesions. Maybe the authors could extend the analysis by performing histology and counting the number of oligodendrocytes.

    Figure 2 contains only a scheme. Figure 2 should be combined with Figure 3. In addition, a scheme showing the time line of the cuprizone treatment and recovery from the treatment would be helpful. I assume W0 is at the time of treatment, W5 after 5 weeks of cuprizone and W8 represents 5 weeks of cuprizone and 3 weeks of recovery. If yes, it is not clear why the ASPA cell count shown it not reduced between W0 and W5. The numbers seem to be similar for W0, W5 and W8 in the absence of IFNg. In addition, the comparison shown in Figure 3 are incomplete. W0 is only shown without IFNg but not with. Does IFNg affect ASPA number in the absence of cuprizone?

    Panel B and C in Figure 5 could be combined to be able to compare the analyses and to evaluate the recovery of cell number by Seph at W8. The number of mice per group is borderline (only 3 mice).

    Same issue as above: Panel B and C in Figure 6 should be combined and a multiple comparison should be performed between W0, W5 and W8.

    The rational for combining BZA and Seph as shown in Figure 8 should be explained in the text. The figure and legends should be improved to clarify at which time point the analyses were performed. The panel number stated in the legends do not match with what is shown in the figure. I assume the analyses were done at W8. Only g-ratios change, whereas the number of ASPA cells and amount of myelinated axons are not affected by the combined treatment. The interpretation of this result is not easy, and the emphasis of this result should be removed from the abstract.

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  3. Reviewer #1 (Public Review):

    Drs. Chen and colleagues report that augmentation of the integrated stress response (ISR) increases the oligodendrocytes and myelination during recovery after experimental demyelination in the presence of inflammation. Homozygous GADD43 KO mice or Sephin1 are used, respectively, to genetically and pharmacologically augment the ISR. Sephin1 treatment in mice with experimental autoimmune encephalomyelitis (EAE) shows increased remyelination in the spinal cord after inflammatory demyelination. Cuprizone administration to GFAP/tre;TRE/IFN-gamma double transgenic mice produced corpus callosum demyelination and CNS inflammation, with release of interferon-gamma initiated by removal of doxycycline from the drinking water. GADD43 KO did not change overall severity of cuprizone demyelination based on loss of oligodendrocytes and demyelination in corpus callosum after 5 weeks of cuprizone with ectopic interferon-gamma. The authors state that GADD43 KO enhanced the recovery of oligodendrocytes and remyelination during the 3 weeks after removal of cuprizone from the diet, but an incorrect figure prevents evaluation of this result. In double transgenic mice, with initiation of CNS inflammation, but without the GADD43 null mutation, pharmacologically enhancing the ISR with Sephrin1, increased recovery of oligodendrocytes and remyelination at 3 weeks after removal of cuprizone from the diet. These effects of genetically or pharmacologically enhancing ISR were not observed in the absence of ectopic interferon-gamma. Genetic and pharmacologic enhancement of the ISR did not appear to significantly alter the progenitor or microglial response to cuprizone demyelination. The combination of Sephin1 with bazedoxifene (BZA) enhanced the oligodendrocyte density and remyelination during the recovery period to a similar extent as either treatment alone. The authors provide several results supporting their interpretation that augmenting the ISR can overcome inhibitory effects of inflammation to enhance oligodendrocyte density and remyelination. Clarifications of the methods, correction of missing data, and additional experiments are needed to support the authors' conclusions that the potentially significant findings that combination of Sephin1 and BZA protects remyelinating oligodendrocytes and promotes remyelination even in the presence of inflammation.

    Major concerns:

    1. The experimental design and interpretation of the results would be strengthened by examining an indicator of the ISR to allow the reader to interpret the extent of ISR activation and the effect of the genetic and pharmacologic modulators of the ISR. This analysis would be particularly helpful in the corpus callosum in conditions with and without cuprizone.

    2. Cuprizone is started at 6 weeks of age which is designated as week 0 (W0). The studies use W0 for comparison to the treatment groups that are analyzed at W5 or W8. The authors refer to W0 as pre-lesion or baseline levels, which is appropriate. The authors' statements related to the vehicle condition are appropriate as is. However, it is not clear why the W8 age-match (non-cuprizone and non-IFN-gamma) was not used to more directly interpret the extent of recovery. Using W0, the comparison is 6 versus 14 weeks of age. Myelinated axons continue to significantly increase during this age interval in mice.

    3. The data graphed in panel 3C for the KO genetic prolongation of the ISR is exactly the same and the data graphed in panel 5C for the Seph pharmacologic enhancement of the ISR. The graph in 3C is actually labeled for Seph and so must have been inadvertently inserted when the graph of the KO data was intended.

    4. The combined Sephin1/BZA treatment does not appear to work through remyelination, based on the definition of thinly myelinated axons (g-ratio >0.8) as used by the authors. The authors state that the data shows the after cuprizone demyelination, mice treated with Sephin1/BZA "reached myelin thickness levels comparable to pre-lesion levels" and "restored myelin thickness to baseline levels". To support this interpretation, the authors would need to include analysis of the Sephin1/BZA mice at 5 weeks of cuprizone to show that the combined treatment, which is initiated at 3 weeks of cuprizone, did not protect oligodendrocytes or reduce demyelination during weeks 3-5 of cuprizone and Sephin1/BZA treatment.

    5. Conditions during which augmenting ISR is protective of mature oligodendrocytes or protecting remyelinating oligodendrocytes should be more clearly presented in the Discussion. The prior EAE results are reported as protecting mature oligodendrocytes. The results (Figures 3B and 5B) show that genetically or pharmacologically augmenting the ISR did NOT protect from mature oligodendrocyte loss at 5W cuprizone. The results (Figure 5B) show increased oligodendrocytes at 8W cuprizone. The current results are interpreted as protecting remyelinating oligodendrocytes, which are presumably mature as well.

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  4. Evaluation Summary:

    This is an elegantly conducted study showing integrated stress response (ISR) contributes to protection of oligodendrocytes in the remyelination process in the setting of an inflammatory environment. The authors use both genetic (GADD43KO) and pharmacological approaches (Sephin1) to study ISR in demyelination animal models. The data are convincing and have important clinical implications for multiple sclerosis and other diseases. The reviewers agree that revisions are needed for the sake of presentation, clarity, rationale, and interpretation of datasets.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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