PONE-D-21-08197

A novel in vitro assay model to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents

PLOS ONE

Dear Dr. Tanaka,

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: 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: The manuscript, A novel in vitro assay model to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents, represents only one of many attempts to measure Acetylcholine and other cholinergic parameters as means to counter cognitive disorders improving cholinergic neurotransmission.

This model per se may be interesting but, unfortunately, ACh increasing strategy both by cholinergic agonists and by AChE inhibitors looks disastrous. Probably, we should study this system yet but its use as a therapeutic mean to fight neurodegenerative diseases as AD or other disorders, which hit cognitive function doesn't represent a priority!

Therefore, I think this manuscript has some problems with its title. In my opinion, the authors don't present a novel approach, probably the scientific community may not be interested in the measurement of cholinergic agents because they are not useful to successfully treat patients. Besides, honestly, I think we should find other means to fight beta-amyloid and neurofibrillary tangles. Later, we can use also cholinergic enhancers to improve cholinergic function.

Probably, in the meantime, we should also study better other molecules that improve significantly cognitive function in patients with mild cognitive impairment. Here you find some interesting papers that may spark your future researches.

[Choline-containing phospholipids: relevance to brain functional pathways. Clin Chem Lab Med. 2013 Mar 1;51(3):513-21. doi: 10.1515/cclm-2012-0559; Choline-Containing Phospholipids: Structure-Activity Relationships Versus Therapeutic Applications. Curr Med Chem. 2015;22(38):4328-40. doi:10.2174/0929867322666151029104152; Choline alphoscerate (alpha-glyceryl-phosphoryl-choline) an old choline- containing phospholipid with a still interesting profile as cognition enhancing agent. Curr Alzheimer Res. 2013 Dec;10(10):1070-9. doi: 10.2174/15672050113106660173; Volume Analysis of Brain Cognitive Areas in Alzheimer's Disease: Interim 3-Year Results from the ASCOMALVA Trial. J Alzheimers Dis. 2020;76(1):317-329. doi: 10.3233/JAD-190623].

Reviewer #2: ACh is an important neurotransmitter involved in a wide range of physiological processes, and the malfunction of ACh system is associated with many brain disorders. This manuscript established an in vitro model, which used human-derived cholinergic cells LA-N-2 that express several necessary cholinergic components, to measure ACh levels. Combined with HPLC to quantify the extracellular and intracellular ACh levels, such cell line is used to evaluate the effects of some drugs and food constituents on cholinergic function. The authors successfully detected the increased ACh levels affected by some drugs or food constituents. This assay is relatively easy to perform and may be useful for the preliminary screening of drugs or food constituents to treat ACh-related disorders.

It would be good that authors address the below minor critiques before their work to be published.

1. The author could draw a schematic diagram in one figure to illustrate (the principe of) this new assay. This would help readers to understand their manuscript.

2 Many food constituents have been tested in this assay under specific doses. Could authors discuss or mention whether relevant doses are similar to that of normal intake by human?

3. Based on the data you discussed, it seems that the roles of CHT1, ChAT, mAChR, and AChE cannot be distinguished when different drugs were applied (in your assay). Then, what degrees do you think the system you used could simulate the cholinergic metabolic system in physiological condition? Please discuss it.

4. I would suggest to replot at least some tables to graphs, in order for better visualization of your relevant data. There are simply too many tables.

5. In the discussion part, the authors stated, “Given that delphinidin and black ginger extract improve cognitive function in vivo [15, 28], an increase in the intracellular ACh levels could serve as an indicator of cognitive improvement.” It is simply too strong a statement in my opinion. The authors should weaken

Reviewer #3: Tanaka and Hamada use a cell model (LA-N-2 cells) that has some cholinergic properties and treat it with various compounds to screen for cholinergic effects. For this purpose, they incubated the cells with various agents (listed in Figs. 5 and S1) for five hours, then take medium as representative of the extracellular space. Subsequently, they prepared a perchloric acid extract of the cells to represent the intracellular space. Using a sensitive EICOM HPLC system, they report acetylcholine (ACh) and choline levels. They feel that increases of ACh – which were mainly detected in the intracellular space – reflect pro-cholinergic properties of the tested compounds, and that the assays may broadly reflect cholinergic stimulations because both increases of choline acetyltransferase (ChAT), or choline, or inhibition of acetylcholinesterase (AChE) are reflected in the measured values.

The methods are clearly described in this study and data are credible. In my view, however, interpretations of their findings are complicated considerations listed below.

1. LA-N-2 cells are not cholinergic by nature but they adapt some cholinergic properties (especially by up-regulation of ChAT) when differentiated e.g. by retinoic acid or LIF/CDF. It is understandable, therefore, that very little extracellular ACh is detected in most incubations because very little ACh is released by these cells, and the release machinery (SNAPs and SNAREs) may be defective. Cholinergic neurons are characterized by the expression of ChAT, CHT-1 and the vesicular ACh transporter (vAChT), and the manuscript would benefit from information (e.g. cited papers) as to the expression of their genes in LA-N-2 cells. Other cholinergic “features” listed by the authors are not exclusively cholinergic, e.g. choline is used by all cells in the body, muscarinic and nicotinic receptors are expressed by many cell types, and even AChE is expressed by several cell types, e.g. muscle cells. It would be interesting to see how the results change when the LA-N-2 cells were used in a differentiated state.

2. The measurements show intracellular choline levels of 100-200 pmol and extracellular amounts of 4,000 pmol. Unfortunately, data are not given as intra- and extracellular concentrations (µmol/L), and this should be corrected by the authors. Nevertheless, it appears that choline is concentrated much more highly in the medium, and this finding is at variance with physiological conditions: in the live brain, extracellular choline is approximately 3-4 µM whereas intracellular choline is approx. 50 µM. In vivo, this accumulation of choline within the cells is due to (a.) a negative membrane potential inside the cell which attracts the permanent cation choline and (b.) by the sodium-dependence of the high-affinity choline transporter (HACU, CHT-1), with sodium being ten times higher in the extracellular fluid. I wonder why choline is not adequately taken up by LA-N-2 cells: is there a lack of CHT-1 or of negative membrane potential ? In addition to CHT-1, choline is also transported by low-affinity transporters from the family of organic cation transporters. I wonder if these are expressed in LA-N-2 cells ? Also, where does the high choline level in the medium come from ? Fetal calf serum maybe ? Dead cells ?

3. Cholinergic activations are due to an increase of extracellular ACh. Increases of intracellular ACh do not reflect cholinergic activation, in fact they occur in vivo after administration of drugs that decrease ACh release (e.g., phenobarbitone).

4. The next surprise is the poor efficacy of physostigmine in this model. Physostigmine only enhanced ACh levels at 5-50 µM concentration, and only by 10-15%. It must be noted that physostigmine is an AChE inhibitor with a short half-life of about 20 minutes in vivo, and most of its effect may have disappeared after 5 hours of incubation. A stronger response would have been observed with an inhibitor with a longer half-life on the enzyme, e.g. rivastigmine, or with an irreversibly acting organophosphate. I also wonder whether physostigmine (at high concentrations) interfered with the detection of ACh in the HPLC which is dependent on immobilized AChE in the enzyme reactor ?

5. Nevertheless, it is noteworthy that only physostigmine, but none of the plant constituents was able to cause detectable levels of ACh in the medium. This indicates that physostigmine is able to inhibit the small amounts of AChE that may be secreted by LA-N-2 cells. As for intracellular AChE, it is known that the enzyme is processed in the Golgi apparatus and expressed together with the PRiMA anchor, but in a live neuron, the major part of AChE may not interact with ACh until secreted. I do not know how the interaction of intracellular AChE with ACh would be in a neuroblastoma cell line.

6. Choline-containing substances such as choline itself or lyso-phosphatidylcholine (lyso-PC) increased choline levels while PC and glycerophosphocholine (GPC) did not. The fact that choline at 100 µM increased intracellular choline ten-fold shows that high extracellular choline concentrations are required to increase intracellular choline – this speaks for a transport through a low-affinity carrier (see above). The fact that intracellular ACh also increased shows that intracellular choline was rate-limiting for ACh synthesis under basal conditions. It must be kept in mind that the test medium for the LA-N-2 cells was deficient in choline, a situation that does not occur in vivo – hence, addition of choline led to an immediate ACh synthesis. Before the addition of choline, the LA-N-2 cell could only use choline that was recovered from bound choline (PC mostly). To my knowledge, LA-N-2 cells are not able to synthesize choline de novo.

7. It is surprising that lyso-PC increased choline whereas GPC did not because the usual catabolic pathway of lyso-PC in the body is removal of the fatty acid by phospholipase A2 (PLA2) yielding GPC. The only explanation that comes to mind is the one that the authors give: lyso-PC may have been hydrolyzed by a PLD-like enzyme that would produce choline and monoacylglycerol (phosphate). Such PLD-like activities are usually low in the body. Maybe they are present in LA-N-2 cells ? Still, it is surprising that LA-N-2 cells do not express sufficient PLA2-activity to produce GPC and not enough phosphatase to break down GPC. It is less surprising that PC does not work because PC is rather stable, not water soluble and does not enter cells easily.

8. Muscarine increases ACh, this is surprising. Cytisin, a nicotinic agonist, does not. Which ACh receptors are expressed on LA-N-2 cells ?

9. The relevance of the data with plant constituents (e.g., flavonoids) is arguable. This reviewer does not believe that increases of ACh as observed in this study with luteolin or delphinidin would have a beneficial influence on a neurodegenerative disease such as Alzheimer´s dementia (AD). First, the extent of ACh increase is too low to affect cholinergic transmission to any measurable extent. Second, it is not known whether some of these constituents reach the blood or the brain in sufficient concentrations to exert an effect – flavonoids, for example, are extensively metabolized in the liver upon first pass. Experiments in cell culture do not reveal if substances are capable of crossing the blood-brain barrier. Taken together, it is very unlikely that delphinidin or luteolin would reach the high micromolar concentrations required to increase ACh in the brain in vivo.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-21-08197R1A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agentsPLOS ONE

Dear Dr. Tanaka,

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. In your revised manuscript, please address, as fully as possible, the remaining comments and criticisms of Reviewer 3.

Please submit your revised manuscript by Oct 22 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Israel Silman

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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

Reviewer #3: (No Response)

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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

Reviewer #3: Partly

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

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: 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

Reviewer #3: Yes

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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: Having edited and added some results based on previous comments, the manuscript becomes clearer and more accurate. There are still some minor critiques which can be addressed by the authors.

1. The author applied many chemicals to the cell system using a series of concentrations. Will some high concentrations of the chemicals have toxicities towards cells and affect their normal metabolism? It would be better if you can discuss it in your manuscript.

2. The author mentioned that LA-N-2 cells expressed mAChR M2 using western blotting. However, the author just showed the result using the antibody of mAChR M2, probably there are more than one subtype. Discussing the expression of receptor subtypes on the membrane based on relevant RNA-seq data will be helpful.

Reviewer #3: Tanaka and Hamada have submitted a revised version of their manuscript that is clearly improved over the first version. A Western blot of m2 receptors has been added, the new Fig. 1 is useful, some values have been re-calculated, and the discussion is more to the point. Overall, my points were answered well, and several of my comments were incorporated in the revised discussion. I still think that several questions about the assay system remain, but maybe some of them can be addressed in future studies. For instance, I still do not understand (a.) why the extracellular choline level is now 3-4 µM and (b.) how the intracellular choline level can remain at 0.2 µM in this situation (Table 1), and not even increase beyond 1 µM when 100 µM choline is added to the dish (Table 2). I also do not understand how lyso-PC can evoke higher choline levels that the addition of choline itself (also Table 2). These may be the author´s measurements, but the interpretation of these findings remains a mystery to me.

In the revised version, there are some points that still need attention:

1. Line 40, the point that “the cholinergic system contains choline” is trivial. Choline is present all over the body, in blood plasma and in all cells. Please delete. It would be more reasonable to include acetylcholine as a characteristic of the cholinergic system (which was named cholinergic system out of reluctance to write “acetylcholinergic system”).

2. Line 46. Similarly, there is no evidence that lack of choline plays a role in cholinergic dysfunction or dementia, except in experimental systems. Please write “agonistic effects at/on receptors”, not “against”.

3. Line 289: The Lau et al. study does not seem to make much sense. An increase of ACh with neostigmine at 50 µM, but not at 20 µM ? Something is wrong there, in almost all labs neostigmine is active even at 1 µM.

4. Line 310: the fact that there is an increase of intracellular choline after addition of choline does not mean that there is CHT-1. Any choline transporter could do that. The authors may check the presence of CHT-1 by Western blot, or they can use hemicholinium-3 to get more information.

5. Line 316: Since there is GPC diesterase, GPC should be useable by the cells; however, the enzyme is intracellular, and GPC is not taken up by cells. A change of GPC diesterase activity is not necessary to break down GPC, its presence is enough. Please rephrase the discussion of Singh et al. so that it makes sense. Lyso-PC is a membrane detergent and may have entered the cell by unspecific mechamisms, requiring no transporters.

6. Line 317: The esterase does not “hydrate” GPC, it “hydrolyzes” GPC. Please change.

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Reviewer #2: No

Reviewer #3: No

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A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents

PONE-D-21-08197R2

Dear Dr. Tanaka,

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,

Israel Silman

Academic Editor

PLOS ONE

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Reviewers' comments: