Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Very well-written and thorough investigation of the interplay between HSV and cholesterol in viral effects on 2 cell lines of neuronal background, one human and the other murine. Figures are clear and discussion is appropriate.

A few issues:

1) In a paper focusing on cholesterol effects in relation to AD, ApoE genotype in the cell model is relevant. While ApoE alleles have been documented for some cell lines (SK-N-SH are ApoE3/E3), the SK-N-MC ApoE genotype is not in the literature. You can easily determine this. It would be interesting to see any modulatory effects of ApoE isoform on your findings.

2) Beta-cyclodextrins are known for their anti-viral activity in general. Please add brief discussion of this (examples: Wudiri GA, Schneider SM, Nicola AV. Herpes Simplex Virus 1 Envelope Cholesterol Facilitates Membrane Fusion. Front Microbiol. 2017;8:2383. Published 2017 Dec 6. doi:10.3389/fmicb.2017.02383 and Jones ST, Cagno V, Janeček M, et al. Modified cyclodextrins as broad-spectrum antivirals. Sci Adv. 2020;6(5):eaax9318. Published 2020 Jan 29. doi:10.1126/sciadv.aax9318).

3) Line 134: Please correct “culured” to “cultured”

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors investigated the role of cellular cholesterol in HSV-1-induced neurodegeneration in neuronal cell models using state-of-the-art methodologies. Through these studies they found that HSV-1 infection triggered cholesterol accumulation in specific subcellular compartments. Decreasing cholesterol levels using MβCD interfered with HSV-1 infection as well as HSV-1-induced neurodegeneration.

Overall, this work identified novel aspects of cholesterol homeostasis during HSV-1 infection in neuroblastoma cell lines. The methodologies used are adequate and the findings are of interest to a broader audience. Below are a few considerations which, I believe, will increase the quality of this work even further. 

Major points

·         Since HSV-1 contains cholesterol and a moi of 10 is very high, this might affect the cellular cholesterol measurements (Fig. 1B). An appropriate control (e.g. inactivated virus) could rule out a contribution of HSV-1 cholesterol.

·         It is not clear whether HSV-1 came in contact with MβCD during the experiments. This is of relevance since it has been suggested that HSV-1 cholesterol is important in the entry process.

·         In Fig. 1D, it would be informative to present the immunostainings for the control as well. In addition, a cytosol, plasma membrane, or at least nuclear stain would make it easier to appreciate the subcellular localization. Some of the colocalizations are not very easy to see and thus I would recommend insets with higher resolution as well as quantifications.

·         Why is there only data from one representative experiment shown in Fig. 3D? The data presented is not very convincing.

Minor points

·         Some of the scale bars seem to have wrong dimensions, please double-check them.

·         In the first paragraph of section 3.1.2. it is confusing to the reader that the “AD-like phenotype” is mentioned.

·         The Amplex red assay detects both, cholesterol and cholesteryl esters. Since the finding that HSV-1 induced cholesterol accumulation is central to the work, it might be worth using an alternative method to quantify cholesterol and cholesteryl esters.

·         The use of “significant” should only be used where significance is shown, e.g. “No significant colocalization with filipin” in the text relating to Fig. 1D.

·         In the text relating to Fig. 1C, it is stated: “Intriguingly, the staining pattern of HSV-1-infected cells closely resembled that observed in U18666A-treated cells”. The staining pattern is quite different and hence “closely resemble” does not reflect the data. Please rephrase.

·         Generally, I recommend avoiding the use of green and red in combination to visualize immunostainings.

·         In Fig. 2B, it is unclear among which groups the statistical analyses were performed. I recommend using a similar approach as in Fig. 5B.

·         I believe Figs. 6A+B would be easier to read if the mock control was presented in the same layout, like it has been done for Figs. 6C.

·         In the discussion, the Aβ secretion data (Fig. 6D) should be discussed.

Comments on the Quality of English Language

Minor language editing will improve the quaility of this manuscript.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed all my concerns and I have no more comments.