PONE-D-20-31683

A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

PLOS ONE

Dear Dr. Lindberg,

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

1) Please address reviewer #1's concern that "the authors should add a short discussion of the fact that this approach does not exclude a possibility for the α-synuclein PFFs with the removed GFPs to interact with the cell, be internalized, and affect cellular processes in parallel with the internalized GFP-labeled PFFs."

2) Reviewer #2 answered "partly" to the questions "Is the manuscript technically sound, and do the data support the conclusions?" This review asked for several controls. Please address major points 1 and 3-5, as well as the minor points. Addressing these issues will improve your study.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting and important study describing a useful methodology for generation of the α-synuclein pre-formed fibrils (PFFs) suitable for the analysis of the cellular processes underlying cell-to-cell transmission of α-synuclein proteopathic aggregates. This is achieved by the incorporation of a tobacco etch virus (TEV) protease cleavage site between α-synuclein and green fluorescent protein. Utilization of this construct allows for the efficient removal of GFP from the non-internalized PFFs. Although the presented data are convincing, the authors should add a short discussion of the fact that this approach does not exclude a possibility for the α-synuclein PFFs with the removed GFPs to interact with the cell, be internalized, and affect cellular processes in parallel with the internalized GFP-labeled PFFs.

Reviewer #2: In the present manuscript, Jarvela et al provided a new tool to detect the endocytosed pre-formed alpha-synuclein fibrils. Given the importance of cell-to-cell transmission of alpha-synuclein Pff in Parkinson’s disease pathophysiology, it is interesting to develop the method visualizing transmitted Pff at the cellular level for further mechanism research. However, it is still many critical flaws to reach the author’s conclusion, in particular, missing many control experiment. Also, considering the relatively short text length, there are many mislabeling everywhere.

Major points:

1. The authors used alpha-synuclein construct inserted with His, TEV-p site, and GFP, and then tested pre- and post-TEV treatment effect in most experiments. Since TEV-p site is critical for this new tool development, it should be included No-TEV-p site construct as a negative control.

2. In Fig3, the authors conclude that TEV-P treatment removes extracellular GFP signal as effectively as trypan blue quenching. However, despite the statistical insignificance in Fig 3-B, it looks like a huge difference between pre- and post-quenching after TEV treatment (Fig 3-A bottom image). It would be necessary to show that the GFP positive signals in the pre-quench (+TEV) is not a membrane-localized signal using a membrane marker antibody.

3. In Fig 4, the authors claim that exposure of cells to TEV-P removes 95% of alpha-syn-TEV-GFP signals using western blot. But it may possible that TEV-P protein can enter the cells and induce proteolysis of the endocytosed proteins. Therefore, it needs to prove that TEV-P treatment is solely working on the extracellular absorbed proteins using a negative control expression. As the authors discussed, if this method can be useful for the endocytosed Pff assay tool, the TEV-Protease should not enter cells freely.

4. In Fig6-A, there is no quantification data.

5. In Fig6-B, considering long exposure of pff on the cultured neurons, authors should include the TEV-P no treatment set as a negative control with quantification data.

Minor points:

-In many places, authors used a-syn or a-synuclein instead of α-syn or α-synuclein

-In many places, the authors used both “ⅹ” and “X” (e.g. 40X oil). Need to make it consistently.

-In Fig 5, there is no statistical marks.

-line 66, need to check “ENLYFQˇG”

-line 74, 50ug/ml change to 50μg/ml

-line 110, 37° C change to 37°C

-line 110, CO2 change to CO2 (lower case)

-line 135, 4 C change to 4°C

-line 141, 35-mM change to 35-mm

-line 159, 164, 260, 37 C change to 37°C

-line 276, incorrect labeling of (top) and (bottom)

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Reviewer #1: Yes: Vladimir N. Uversky

Reviewer #2: No

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A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

PONE-D-20-31683R1

Dear Dr. Lindberg,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Stephan N. Witt, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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.

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No