Abstract

Among the various chemicals that are commonly used as pesticides. organophosphates (OPs), and to a lesser extent. carbamates, are most frequently associated with adverse long-term neurological consequences. OPs and the carbamate, pyridostigmine, used as a prophylactic drug against potential nerve agent attacks, have also been implicated in Gulf War Illness (GWI). which is often characterized by chronic neurological symptoms. While most OP- and carbamate-based pesticides. and pyridostigmine are relatively potent acetylcholinesterase inhibitors (AChEls). this toxicological mechanism is inadequate to explain their long-term health effects, especially when no signs of acute cholinergic toxicity are exhibited. Our previous work suggests that a potential mechanism of the long-term neurological deficits associated with OPs is impairment of axonal transport (AXT); however, we had not previously evaluated carbamates for this effect. Here we thus evaluated the carbamate, physostigmine (PHY). a highly potent AChEI, on AXT using an in vitro neuronal live imaging assay that we have previously found to be very sensitive to OP-related deficits in AXT. We first evaluated the OR diisopropylfluorophosphate (DFP) (concentration range 0.001-10.0 mu M) as a reference compound that we found previously to impair AXT and subsequently evaluated PHY (concentration range 0.01-100 nM). As expected. DFP impaired AXT in a concentration-dependent manner. replicating our previously published results. In contrast. none of the concentrations of PHY (including concentrations well above the threshold for impairing AChE) impaired AXT. These data suggest that the long-term neurological deficits associated with some carbamates are not likely due to acute impairments of AXT.


Authors

Naughton, Sean X.;  Beck, Wayne D.;  Wei, Zhe;  Wu, Guangyu;  Baas, Peter W.;  Terry, Alvin V., Jr.

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  • 1 reviewer
  • pre-publication peer review (FINAL ROUND)
    Decision Letter
    2021/05/07

    07-May-2021

    Dear Dr. Terry,

    It is a pleasure to accept your manuscript entitled "The carbamate, physostigmine does not impair axonal transport in rat cortical neurons" in its current form for publication in Neuroscience Insights. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.

    Thank you for your fine contribution. On behalf of the Editors of Neuroscience Insights, we look forward to your continued contributions to the Journal.

    Sincerely,
    Dr. Elaine Ellerton
    Editor in Chief, Neuroscience Insights
    Elaine.Ellerton@sagepub.com

    Reviewer(s)' Comments to Author:

    Associate Editor(s)' Comments to Author:

    Section Editor: Shetty, Ashok
    Comments to the Author:
    (There are no comments.)

    Decision letter by
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    Author Response
    2021/05/04

    We have addressed the final concern of reviewer 5 about the storage conditions for DFP and have revised the manuscript accordingly (please see the yellow highlighted text in the Materials and Methods section).

    This is the text that was added to the Materials and Methods Section:

    To maintain stability of DFP, the colorless liquid concentrate was stored in a freezer at -70°C. The PHY salt was stored in a refrigerator in a desiccator at 4°C.



    Cite this author response
  • pre-publication peer review (ROUND 2)
    Decision Letter
    2021/05/03

    03-May-2021

    Dear Dr. Terry,

    Manuscript ID EXN-21-0006.R1 entitled "The carbamate, physostigmine does not impair axonal transport in rat cortical neurons" which you submitted to Neuroscience Insights, has been reviewed. The comments of the reviewer(s) are included at the bottom of this letter.

    The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the reviewer(s)' comments and revise your manuscript.

    To revise your manuscript, log into https://mc.manuscriptcentral.com/exn and enter your Author Center, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision.

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    Because we are trying to facilitate timely publication of manuscripts submitted to Neuroscience Insights, your revised manuscript should be uploaded as soon as possible.

    Once again, thank you for submitting your manuscript to Neuroscience Insights and I look forward to receiving your revision.

    Sincerely,
    Dr. Elaine Ellerton
    Editor in Chief, Neuroscience Insights
    Elaine.Ellerton@sagepub.com

    Reviewer(s)' Comments to Author:

    Reviewer: 4

    Comments to the Author
    The authors addressed majority of the queries raised by all reviewers in the revised version. The manuscript can be accepted in the current format.

    Reviewer: 5

    Comments to the Author
    This revised manuscript describes a short series of experiments in a Gulf War Illness context designed to evaluate the carbamate physostigmine on axonal transport using an in vitro live imaging assay previously found by the authors to be sensitive to organophosphate-related deficits in axonal transport. Physostigmine is a potent acetylcholinesterase inhibitor and a lipid-soluble tertiary amine that readily crosses the blood-brain barrier after peripheral administration. The experiments addressed the question of whether carbamate-based acetylcholinesterase inhibitors impair axonal transport that could account for cognitive impairment without encountering the issue surrounding the unlikely BBB penetration of the Gulf War agent pyridostigmine.

    1) In the previous review the authors were requested to specify the storage conditions for diisopropylfluorophosphate (DFP), but they responded with a description of efforts to maintain stability of this compound in the experimental media. The storage conditions should be reported as DFP does not have an extended shelf-life.

    The other concerns of this reviewer were adequately addressed in the revised manuscript.

    Associate Editor(s)' Comments to Author:

    Section Editor: Shetty, Ashok
    Comments to the Author:
    Please address the comment of the second reviewer

    "The storage conditions should be reported as DFP does not have an extended shelf-life"

    Decision letter by
    Cite this decision letter
    Reviewer report
    2021/04/30

    This revised manuscript describes a short series of experiments in a Gulf War Illness context designed to evaluate the carbamate physostigmine on axonal transport using an in vitro live imaging assay previously found by the authors to be sensitive to organophosphate-related deficits in axonal transport. Physostigmine is a potent acetylcholinesterase inhibitor and a lipid-soluble tertiary amine that readily crosses the blood-brain barrier after peripheral administration. The experiments addressed the question of whether carbamate-based acetylcholinesterase inhibitors impair axonal transport that could account for cognitive impairment without encountering the issue surrounding the unlikely BBB penetration of the Gulf War agent pyridostigmine.

    1) In the previous review the authors were requested to specify the storage conditions for diisopropylfluorophosphate (DFP), but they responded with a description of efforts to maintain stability of this compound in the experimental media. The storage conditions should be reported as DFP does not have an extended shelf-life.

    The other concerns of this reviewer were adequately addressed in the revised manuscript.

    Reviewed by
    Cite this review
    Reviewer report
    2021/04/27

    The authors addressed majority of the queries raised by all reviewers in the revised version. The manuscript can be accepted in the current format.

    Reviewed by
    Cite this review
  • pre-publication peer review (ROUND 1)
    Decision Letter
    2021/04/15

    15-Apr-2021

    Dear Dr. Terry,

    Manuscript ID EXN-21-0006 entitled "The carbamate, physostigmine does not impair axonal transport in rat cortical neurons" which you submitted to Neuroscience Insights, has been reviewed. The comments of the reviewer(s) are included at the bottom of this letter.

    The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the reviewer(s)' comments and revise your manuscript.

    To revise your manuscript, log into https://mc.manuscriptcentral.com/exn and enter your Author Center, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision.

    You may also click the below link to start the revision process (or continue the process if you have already started your revision) for your manuscript. If you use the below link you will not be required to login to ScholarOne Manuscripts.

    PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm.

    https://mc.manuscriptcentral.com/exn?URL_MASK=42711e3b71ae4ba889fa1d6d631da8a4

    You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript using a word processing program and save it on your computer.

    PLEASE HIGHLIGHT ALL CHANGES TO YOUR MANUSCRIPT USING TRACK CHANGES MODE IN MS WORD OR BY USING BOLD OR COLORED TEXT.

    Once the revised manuscript is prepared, you can upload it and submit it through your Author Center.

    When submitting your revised manuscript, you will be able to respond to the comments made by the reviewer(s) in the space provided. You can use this space to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the reviewer(s).

    IMPORTANT: Your original files are available to you when you upload your revised manuscript. Please delete any redundant files before completing the submission.

    Because we are trying to facilitate timely publication of manuscripts submitted to Neuroscience Insights, your revised manuscript should be uploaded as soon as possible. If it is not possible for you to submit your revision by the deadline, please contact the editorial office to arrange a new deadline.

    Once again, thank you for submitting your manuscript to Neuroscience Insights and I look forward to receiving your revision.

    Sincerely,
    Dr. Elaine Ellerton
    Editor in Chief, Neuroscience Insights
    Elaine.Ellerton@sagepub.com

    Reviewer(s)' Comments to Author:

    Reviewer: 1

    Comments to the Author
    Summary: Gulf War Illness is thought to be due to exposure to pesticides and to pyridostigmine bromide, a carbamate drug taken by soldiers to protect from the toxicity of nerve agents. Neurotoxicity can be measured by effects on axonal transport. This report confirmed previous reports from the authors’ laboratory that organophosphorus toxicants decrease the rate of axonal transport. Neurons and their connections to other neurons can be destroyed when the axonal transport system malfunctions. This work was undertaken with the goal of determining the effect on axonal transport of physostigmine, a carbamate inhibitor of acetylcholinesterase activity. It was concluded that physostigmine does not impair axonal transport in neurons. Since organophosphates and physostigmine both inhibit the activity of acetylcholinesterase, but only organophosphates impair axonal transport, it was concluded that adverse effects on axonal transport are due to organophosphate targets other than acetylcholinesterase.
    The authors are congratulated for their outstanding skill in measuring axonal transport.

    Minor comments
    1. Page 8 line 10. Please define ARAC
    2. Please define APP and MBO
    3. Page 10 line 56. Typing error Fig 3C should be Fig 1C

    Reviewer: 2

    Comments to the Author
    Well executed experimental work, clear writing, established group.

    Reviewer: 3

    Comments to the Author
    This article entitled as: "The carbamate, physostigmine does not impair axonal transport in rat cortical neurons" is well written and presents meaningful research in the field of GWI and mechanisms behind development of toxic syndrome. Authors examined effects of physostigmine (carbamates) on acute impairment of axonal transport and as result development of long-term neurological deficits similar to those known as GWI syndrome in humans. Authors used range of concentrations of physostigmine to test main hypothesis and could successfully, methodically address the question using imaging assay previously found effective and sensitive to test OP-related deficits in AXT. I recommend this article for publication. This well designed study will positively contribute to existing literature in this area of research.

    Reviewer: 4

    Comments to the Author
    In this manuscript, the authors report that long-term neurological deficits associated carbamates like are not likely due to acute impairments of axonal transport. This invitro study on cortical neurons compared the effect of the physostigmine (PSY) on axonal transport in reference with DFP an OP potential mechanism of the long-term neurological deficits associated with OPs is impairment of axonal transport (AXT). While the results are of interest there few concerns on data analysis

    1) The transfection of GFP-APP in rat primary cortical neurons and anterograde and retrograde movements of MBOs was nicely demonstrated. These results suggested that carbamates like PSY does not impair axonal transport. Nevertheless, whether similar results replicate in invivo conditions would be an interesting query
    2) This study indicates that mechanism of action of PSY is not through impairment of axon transport. The possible mechanism involved in reversible cholinesterase inhibition by PSY should be discussed elaborately.
    3) The specificity of anterograde and retrograde movements of MBOs should be discussed more for information to readers
    4) The results are perplexing to draw a conclusion as lower concentration (0.01 – 10 nM) has no effect on velocity while the highest concentration (100 nM) increased the velocity of MBOs. Therefore, the statement PSY does not impair axonal transport may be reconsidered.

    Reviewer: 5

    Comments to the Author
    This manuscript describes a short series of experiments in a Gulf War Illness context designed to evaluate the carbamate physostigmine on axonal transport using an in vitro live imaging assay previously found by the authors to be sensitive to organophosphate-related deficits in axonal transport. Physostigmine is a potent acetylcholinesterase inhibitor and a lipid-soluble tertiary amine that readily crosses the blood-brain barrier after peripheral administration. The experiments addressed the question of whether carbamate-based acetylcholinesterase inhibitors impair axonal transport that could account for cognitive impairment without encountering the issue surrounding the unlikely BBB penetration of the Gulf War agent pyridostigmine. Nonetheless, there are important shortcomings present in the paper that must be addressed by the authors.

    1) This study is largely an offshoot of earlier work examining organophosphate compounds using the same methodology to assess axonal transport. Only Table 1 and Figure 4 present new data. While physostigmine did not alter the velocity of anterograde or retrograde transport at most concentrations, the drug produced multiple measurable effects that were not emphasized or pursued by the authors. Consequently, in its current form the paper offers little new information of apparent significance.

    2) Since concentrations to affect acetylcholinesterase and axonal transport are not necessarily related, the authors must indicate how the physostigmine levels were chosen for study.

    3) In the Introduction the authors should briefly mention how axonal transport deficits might be translated into cognitive/neurobehavioral impairments.

    4) What are Glutamax and ARAC (page 8)? The latter abbreviation, GFP-APP, and MBO must be defined as standard practice.

    Other points:
    ---The authors should describe in Methods the storage of diisopropylfluorophosphate to maintain its stability and prevent degradation.
    ---The last line on page 9 should read “the boxes in Fig 1C” rather than “the arrows in Fig 3C”.

    Associate Editor(s)' Comments to Author:

    Section Editor: Shetty, Ashok
    Comments to the Author:
    (There are no comments.)

    Decision letter by
    Cite this decision letter
    Reviewer report
    2021/04/15

    This manuscript describes a short series of experiments in a Gulf War Illness context designed to evaluate the carbamate physostigmine on axonal transport using an in vitro live imaging assay previously found by the authors to be sensitive to organophosphate-related deficits in axonal transport. Physostigmine is a potent acetylcholinesterase inhibitor and a lipid-soluble tertiary amine that readily crosses the blood-brain barrier after peripheral administration. The experiments addressed the question of whether carbamate-based acetylcholinesterase inhibitors impair axonal transport that could account for cognitive impairment without encountering the issue surrounding the unlikely BBB penetration of the Gulf War agent pyridostigmine. Nonetheless, there are important shortcomings present in the paper that must be addressed by the authors.

    1) This study is largely an offshoot of earlier work examining organophosphate compounds using the same methodology to assess axonal transport. Only Table 1 and Figure 4 present new data. While physostigmine did not alter the velocity of anterograde or retrograde transport at most concentrations, the drug produced multiple measurable effects that were not emphasized or pursued by the authors. Consequently, in its current form the paper offers little new information of apparent significance.

    2) Since concentrations to affect acetylcholinesterase and axonal transport are not necessarily related, the authors must indicate how the physostigmine levels were chosen for study.

    3) In the Introduction the authors should briefly mention how axonal transport deficits might be translated into cognitive/neurobehavioral impairments.

    4) What are Glutamax and ARAC (page 8)? The latter abbreviation, GFP-APP, and MBO must be defined as standard practice.

    Other points:
    ---The authors should describe in Methods the storage of diisopropylfluorophosphate to maintain its stability and prevent degradation.
    ---The last line on page 9 should read “the boxes in Fig 1C” rather than “the arrows in Fig 3C”.

    Reviewed by
    Cite this review
    Reviewer report
    2021/04/05

    In this manuscript, the authors report that long-term neurological deficits associated carbamates like are not likely due to acute impairments of axonal transport. This invitro study on cortical neurons compared the effect of the physostigmine (PSY) on axonal transport in reference with DFP an OP potential mechanism of the long-term neurological deficits associated with OPs is impairment of axonal transport (AXT). While the results are of interest there few concerns on data analysis

    1) The transfection of GFP-APP in rat primary cortical neurons and anterograde and retrograde movements of MBOs was nicely demonstrated. These results suggested that carbamates like PSY does not impair axonal transport. Nevertheless, whether similar results replicate in invivo conditions would be an interesting query
    2) This study indicates that mechanism of action of PSY is not through impairment of axon transport. The possible mechanism involved in reversible cholinesterase inhibition by PSY should be discussed elaborately.
    3) The specificity of anterograde and retrograde movements of MBOs should be discussed more for information to readers
    4) The results are perplexing to draw a conclusion as lower concentration (0.01 – 10 nM) has no effect on velocity while the highest concentration (100 nM) increased the velocity of MBOs. Therefore, the statement PSY does not impair axonal transport may be reconsidered.

    Reviewed by
    Cite this review
    Reviewer report
    2021/04/05

    This article entitled as: "The carbamate, physostigmine does not impair axonal transport in rat cortical neurons" is well written and presents meaningful research in the field of GWI and mechanisms behind development of toxic syndrome. Authors examined effects of physostigmine (carbamates) on acute impairment of axonal transport and as result development of long-term neurological deficits similar to those known as GWI syndrome in humans. Authors used range of concentrations of physostigmine to test main hypothesis and could successfully, methodically address the question using imaging assay previously found effective and sensitive to test OP-related deficits in AXT. I recommend this article for publication. This well designed study will positively contribute to existing literature in this area of research.

    Reviewed by
    Cite this review
    Reviewer report
    2021/03/23

    Well executed experimental work, clear writing, established group.

    Reviewed by
    Cite this review
    Reviewer report
    2021/03/16

    Summary: Gulf War Illness is thought to be due to exposure to pesticides and to pyridostigmine bromide, a carbamate drug taken by soldiers to protect from the toxicity of nerve agents. Neurotoxicity can be measured by effects on axonal transport. This report confirmed previous reports from the authors’ laboratory that organophosphorus toxicants decrease the rate of axonal transport. Neurons and their connections to other neurons can be destroyed when the axonal transport system malfunctions. This work was undertaken with the goal of determining the effect on axonal transport of physostigmine, a carbamate inhibitor of acetylcholinesterase activity. It was concluded that physostigmine does not impair axonal transport in neurons. Since organophosphates and physostigmine both inhibit the activity of acetylcholinesterase, but only organophosphates impair axonal transport, it was concluded that adverse effects on axonal transport are due to organophosphate targets other than acetylcholinesterase.
    The authors are congratulated for their outstanding skill in measuring axonal transport.

    Minor comments
    1. Page 8 line 10. Please define ARAC
    2. Please define APP and MBO
    3. Page 10 line 56. Typing error Fig 3C should be Fig 1C

    Reviewed by
    Cite this review
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