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

Ryohei Tanaka-Kanegae; Koichiro Hamada

doi:10.1371/journal.pone.0258420

. 2021 Oct 12;16(10):e0258420. doi: 10.1371/journal.pone.0258420

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

Ryohei Tanaka-Kanegae ^1,^*, Koichiro Hamada ¹

Editor: Israel Silman²

PMCID: PMC8509891 PMID: 34637466

Abstract

Background

Cholinergic neurons utilize choline (Ch) to synthetize acetylcholine (ACh) and contain a high-affinity Ch transporter, Ch acetyltransferase (ChAT), ACh receptors, and acetylcholinesterase (AChE). As the depletion or malfunction of each component of the cholinergic system has been reported in patients with dementia, many studies have sought to evaluate whether treatment candidates affect each of the cholinergic components. The associated changes in the cholinergic components may be reflected by intra- or extra-cellular ACh levels, with an increase in extracellular ACh levels occurring following AChE inhibition. We hypothesized that increases in intracellular ACh levels can be more sensitively detected than those in extracellular ACh levels, thereby capturing subtle effects in the cholinergic components other than AChE. The objective of this study was to test this hypothesis.

Methods

We developed an in vitro model to measure both extracellular and intracellular ACh levels using the human cholinergic neuroblastoma cell line, LA-N-2, which have been reported to express Ch transporter, ChAT, muscarinic ACh receptor (mAChR), and AChE. With this model, we evaluated several drug compounds and food constituents reported to improve cholinergic function through various mechanisms. In addition, we conducted western blotting to identify the subtype of mAChR that is expressed on the cell line.

Results

Our cell-based assay system was capable of detecting increases in extracellular ACh levels induced by an AChE inhibitor at relatively high doses, as well as increases in intracellular ACh levels following the administration of lower AChE-inhibitor doses and an mAChR agonist. Moreover, increases in intracellular ACh levels were observed even after treatment with food constituents that have different mechanisms of action, such as Ch provision and ChAT activation. In addition, we revealed that LA-N-2 cells expressed mAChR M2.

Conclusion

The findings support our hypothesis and indicate that the developed assay model can broadly screen compounds from drugs to food ingredients, with varying strengths and mechanisms of action, to develop treatments for ACh-relevant phenomena, including dementia and aging-related cognitive decline.

Introduction

Acetylcholine (ACh) is a neurotransmitter that plays crucial roles in both the central and peripheral nervous systems, and central cholinergic transmission is essential for normal cognitive processes [1]. The cholinergic system contains choline (Ch), which is the substrate for ACh synthesis; high-affinity Ch transporter, which carries Ch into cholinergic neurons; Ch acetyltransferase (ChAT), which is responsible for ACh synthesis; muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively); and acetylcholinesterase (AChE). The depletion or malfunction of these components has been reported in individuals experiencing cognitive decline, leading to the evaluation of whether drug candidates can affect the abundance and availability of Ch and the activity of AChE and ChAT, and elicit agonistic effects at ACh receptors [2–6]. From this perspective, numerous food constituents have been studied as therapeutic candidates, as the importance of a nutritional approach to prevent cognitive decline has become apparent [7]. Given that changes in Ch availability and activities of the individual cholinergic components may be reflected in ACh levels inside and/or outside cholinergic cells, determining whether treatment candidates increase ACh levels may represent an index when exploring cholinergic agents.

In vivo, brain fixation [8] and microdialysis [9] techniques are commonly used to measure ACh levels. Although ACh in the synaptic cleft is rapidly hydrolyzed by AChE after its transmission, brain fixation enables the quantification of intracellular and extracellular ACh, while microdialysis quantifies extracellular ACh. Considering that the main sites of ACh biosynthesis and degradation are inside and outside cholinergic cells, respectively [3], the increase in intracellular ACh levels may be detected more sensitively than that in extracellular ACh levels. We, therefore, consider that an in vitro model capable of measuring intracellular ACh separately from extracellular ACh could serve as a promising tool to broadly screen cholinergic agents, especially food components with mild cholinergic activities. However, no such model exists, although a substantial number of in vitro models have been developed focusing on single cholinergic components such as AChE, ChAT, and AChRs [5, 10–13].

We further hypothesized that the detection of increases in extracellular ACh levels could be applied to determine the activity of AChE-inhibiting drugs, whereas the detection of increases in intracellular ACh levels would be sensitive enough to determine the subtle effects elicited by food constituents via various mechanisms other than AChE inhibition. To test this hypothesis, we developed an in vitro model and assessed several drug compounds and food components with a known capacity to enhance cholinergic function through different mechanisms, including AChE inhibition (physostigmine [14], delphinidin [15], and black ginger extract [16]), Ch provision (glycerophosphocholine [17] and lysophosphatidylcholine (LPC) [18]), ChAT activation (luteolin [19] and nobiletin [20]), and AChR activation (muscarine and cytisine [21]).

Materials and methods

Reagents

Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient Mixture F-12 Ham (Ham’s F-12) (D8062), physostigmine, and (+)-muscarine chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cytisine was obtained from LKT Laboratories, Inc. (St. Paul, MN, USA). DMEM/Ham’s F-12 without Ch chloride was obtained from Cell Science & Technology Inst., Inc. (Miyagi, Japan). Fetal bovine serum (FBS) was purchased from Nichirei Biosciences Inc. (Tokyo, Japan). Penicillin and streptomycin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). ACh bromide, Ch chloride, LPC from egg yolk, and luteolin were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Isopropylhomocholine (IPHC) was provided by Eicom Corporation (Kyoto, Japan). Delphinidin chloride was purchased from Tokiwa Phytochemical Co., Ltd. (Chiba, Japan), and black ginger extract was obtained from Maruzen Pharmaceuticals Co., Ltd. (Hiroshima, Japan). Nobiletin was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ, USA). Other food constituents evaluated in the present study are listed in S1 Table. Lysis buffer was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Rabbit anti-mAChR M2 antibody (Ab) (ab109226) was purchased from Abcam (Cambridge, UK) and anti-rabbit Ab horseradish peroxidase (HRP)-linked IgG Ab was obtained from Cell Signaling Technology (Beverly, MA, USA). Amersham enhanced chemiluminescence (ECL) blocking agent and ECL western blotting detection reagents were purchased from Cytiva (Marlborough, MA, USA). Mouse brain tissue lysate was obtained from Abcam. All reagents and chemicals used were of reagent grade.

Cell culture

We used the human neuroblastoma cell line LA-N-2 for the in vitro model as it has a cholinergic phenotype and expresses Ch transporter, ChAT, mAChR, and AChE [18, 22–27], which makes it suitable for evaluating the effects of the candidates on intracellular and extracellular ACh levels. The cell line was purchased from the European Collection of Authenticated Cell Cultures. DMEM/Ham’s F-12 supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) was used for cell culture, whereas DMEM/Ham’s F-12 without Ch chloride supplemented with 2% heat-inactivated FBS was used as an assay medium. The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The number of passages of the cells used for assays ranged from 15 to 25.

Assays

LA-N-2 cells were seeded at 4.0 × 10⁵ cells/well in a 24-well plate and cultured until sub-confluent. During the assay, the culture medium was replaced with 1 mL of the aforementioned assay medium with the test compound dissolved in water, ethanol, or dimethyl sulfoxide (DMSO) (final concentration: 0.1% (v/v) solvent). Test compounds were used in the concentration range at which they did not show any cytotoxicity. After 5 h of incubation at 37°C, the medium was collected as the extracellular fraction, and the cells were rinsed with phosphate-buffered saline (PBS) and scraped with 200 μL of 0.1 M ice-cold perchloric acid to deactivate proteins that could affect Ch and ACh levels. The residual cells and wells were washed with 200 μL of perchloric acid; the rinse solution was mixed with the aforementioned lysate in perchloric acid (the total volume is 400 μL) and stored as the intracellular fraction. The collected culture medium and cell lysates were stored at -30°C until Ch and ACh measurement.

Choline and acetylcholine measurement

We decided to employ electrochemical detection combined with high-performance liquid chromatography (HPLC) to measure Ch and ACh levels owing to its practical advantages, including high sensitivity and selectivity, rapid response, and operational simplicity [28]. We then followed a previously described quantification method [29] with minor modifications. The HPLC system consisted of a pump, column oven, electrochemical detector (ECD) (HTEC-500; Eicom Corporation), data processor (D-7000; Hitachi Ltd., Tokyo, Japan), autosampler (L-7200; Hitachi Ltd.), guard column (CH-GEL, 3.0 mm internal diameter (ID); Eicom Corporation), separation column (Eicompak AC-GEL, 2.0 mm ID × 150 mm; Eicom Corporation), and enzyme column immobilized with AChE and Ch oxidase (AC-ENZYM II, 1.0 mm ID × 4.0 mm; Eicom Corporation). The temperature of the column oven was maintained at 33°C, and a platinum working electrode was used at 450 mV versus a silver (Ag)/silver chloride (AgCl) reference electrode (both electrodes from Eicom Corporation). A mobile phase containing 50 mM potassium bicarbonate, 2.5 mmol/L (M) sodium 1-decanesulfonate, and 134 μM EDTA·2Na was used under isocratic conditions at a flow rate of 150 μL/min.

The collected samples in assays were pretreated for the HPLC analysis. The cell lysates in perchloric acid were sonicated for 3 min (Bioruptor; Tosyo Denki, Kanagawa, Japan) and centrifuged (15,000 × g, 15 min, 4°C). The supernatants were collected and neutralized by adding 1 M potassium bicarbonate. After adding 45 pmol IPHC as an internal standard, the supernatants were mixed with chloroform and vigorously agitated. Delipidation was completed by centrifugation (15,000 × g, 10 min, 4°C) and filtration of the supernatants through a 0.2-μm polytetrafluoroethylene (PTFE) membrane (Millex-LG; Millipore Corporation, Billerica, MA, USA). The culture media were mixed with IPHC and subjected to the same delipidation process as the cell lysates. Filtration was carried out using a 5-kDa cut-off filter (Ultrafree^® MC-PLHCC; Human Metabolome Technologies, Inc., Yamagata, Japan), and the filtrates were injected into the HPLC system. Stock solutions containing Ch and ACh were prepared using 20 mM phosphate buffer containing 20 mM EDTA·2Na, and then aliquoted and preserved at -80°C. For each assay, the stock solution was serially diluted, and IPHC was added to each diluted solution. The ratios of Ch to IPHC and ACh to IPHC were used to construct standard curves; the quantitative range for Ch and ACh was 20 nM to 2 μM. The Ch and ACh content in the samples was recalculated as a percentage of the Ch or ACh level in the test group to that in the vehicle-treated (control) group for interexperimental comparison. For treatments that increased ACh levels, we repeated the assay at least once to confirm reproducibility. A schematic diagram of the new assay system is shown in Fig 1.

Fig 1 — The procedures to measure extracellular and intracellular acetylcholine levels are illustrated.

Western blotting

LA-N-2 cells (4.0 × 10⁶ cells) were seeded into 6-mm dishes and cultured until sub-confluent. Then, cells were treated with or without the assay medium for 5 h. Next, they were washed with PBS twice and lysed in a lysis buffer including 2 mM phenylmethylsulfonyl fluoride. The lysate was sonicated and centrifuged (4,500 × g, 20 min, 4°C), and the supernatant was collected. Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). After blocking with 2% Amersham ECL blocking agent in Tris-buffered saline with Tween-20, the membrane was treated with rabbit anti-mAChR M2 Ab (1:1000) over night at 4°C, followed by the corresponding HRP-conjugated secondary Ab (1:1000) for 2 h at room temperature. The blots were developed using ECL western blotting detection reagents. Mouse brain tissue lysate was used as a positive control.

Statistical analysis

For each assay, significant differences between the control and test groups were evaluated using the unpaired t-test (two-tailed). For multiple comparisons, Dunnett’s post hoc test was performed after one-way analysis of variance (ANOVA). Differences with probability (p) values < 0.05 were considered statistically significant. SAS 9.4 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.

Results

In LA-N-2 cells treated with physostigmine, the intracellular ACh levels increased dose-dependently, with significant differences from those of the control at doses higher than 500 nM (107.8%, p = 0.024 for 500 nM; 109.2%, p = 0.009 for 5 μM; and 113.4%, p < 0.001 for 50 μM), whereas the intracellular Ch levels did not change (Table 1A, Assay 1). Physostigmine at doses higher than 5 μM also led to the detection of ACh in the culture media (extracellular fraction), but not in the control group. In the control group, the extracellular Ch levels were higher than the intracellular Ch levels 5 h after replacing the culture medium with the assay medium (Table 1B, Assay 1). Subsequently, we tested delphinidin and black ginger extract. We observed that 100 μM delphinidin and 100 μg/mL black ginger extract reproducibly increased intracellular ACh levels up to 110.9% and 111.1% compared with the control, respectively. In contrast, the intracellular Ch levels did not significantly change after these treatments (Table 1A, Assays 2 and 3). Further, delphinidin and black ginger extract in the tested concentration ranges did not lead to ACh detection or any change in Ch levels in the extracellular fraction (Table 1B, Assays 2 and 3).

Table 1. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with physostigmine, delphinidin, and black ginger extract, which have been reported to have AChE-inhibiting activity.

(A) Intracellular
		Ch			ACh
		(μM)	(pmol)	(%)	(μM)	(pmol)	(%)
Assay 1	Control	0.181 ± 0.073	72.3 ± 29.4	100.0 ± 40.6	0.331 ± 0.018	132.2 ± 7.3	100.0 ± 5.6
	5 nM physostigmine	0.164 ± 0.060	65.6 ± 24.1	90.7 ± 33.3	0.329 ± 0.008	131.7 ± 3.2	99.6 ± 2.4
	50 nM physostigmine	0.191 ± 0.062	76.6 ± 24.6	105.8 ± 34.0	0.353 ± 0.007	141.1 ± 2.6	106.7 ± 2.0
	500 nM physostigmine	0.173 ± 0.077	69.3 ± 30.8	95.8 ± 42.6	0.356 ± 0.003	142.6 ± 1.2	107.8 ± 0.9 ^*
	5 μM physostigmine	0.203 ± 0.062	81.2 ± 24.7	112.2 ± 34.1	0.361 ± 0.008	144.3 ± 3.3	109.2 ± 2.5 ^**
	50 μM physostigmine	0.175 ± 0.050	70.1 ± 20.2	96.9 ± 27.9	0.375 ± 0.004	150.0 ± 1.6	113.4 ± 1.2 ^**
Assay 2	Control	0.143 ± 0.039	57.4 ± 15.7	100.0 ± 27.3	0.389 ± 0.009	155.7 ± 3.5	100.0 ± 2.2
	25 μM delphinidin	0.165 ± 0.070	66.0 ± 28.0	115.0 ± 48.8	0.415 ± 0.011	165.9 ± 4.6	106.6 ± 2.9
	50 μM delphinidin	0.168 ± 0.055	67.4 ± 22.1	117.4 ± 38.5	0.415 ± 0.007	165.9 ± 2.6	106.6 ± 1.7
	100 μM delphinidin	0.178 ± 0.085	71.1 ± 33.9	124.0 ± 59.1	0.432 ± 0.016	172.6 ± 6.2	110.9 ± 4.0 ^**
Assay 3	Control	0.198 ± 0.092	79.1 ± 36.9	100.0 ± 46.6	0.452 ± 0.022	180.9 ± 8.9	100.0 ± 4.9
	25 μg/mL black ginger	0.205 ± 0.060	81.9 ± 24.1	103.5 ± 30.5	0.454 ± 0.023	181.5 ± 9.1	100.3 ± 5.1
	50 μg/mL black ginger	0.178 ± 0.068	71.0 ± 27.1	89.8 ± 34.3	0.458 ± 0.023	183.3 ± 9.2	101.4 ± 5.1
	100 μg/mL black ginger	0.177 ± 0.018	70.6 ± 7.2	89.4 ± 9.1	0.502 ± 0.013	201.0 ± 5.3	111.1 ± 2.9 ^*
(B) Extracellular
		Ch			ACh
		(μM)	(pmol)	(%)	(μM)	(pmol)	(%)
Assay 1	Control	3.16 ± 0.12	3158 ± 123	100.0 ± 3.9	ND	ND	-
	5 nM physostigmine	3.20 ± 0.08	3204 ± 76	101.4 ± 2.4	ND	ND	-
	50 nM physostigmine	3.24 ± 0.04	3239 ± 45	102.5 ± 1.4	ND	ND	-
	500 nM physostigmine	3.35 ± 0.19	3350 ± 194	106.1 ± 6.1	ND	ND	-
	5 μM physostigmine	3.33 ± 0.15	3326 ± 155	105.3 ± 4.9	0.0310 ± 0.0012	31.0 ± 1.2	-
	50 μM physostigmine	2.86 ± 0.13	2859 ± 126	90.5 ± 4.0	0.0339 ± 0.0026	33.9 ± 2.6	-
Assay 2	Control	3.29 ± 0.11	3289 ± 109	100.0 ± 3.3	ND	ND	-
	25 μM delphinidin	3.23 ± 0.18	3226 ± 181	98.1 ± 5.5	ND	ND	-
	50 μM delphinidin	3.42 ± 0.08	3418 ± 75	103.9 ± 2.3	ND	ND	-
	100 μM delphinidin	3.47 ± 0.31	3469 ± 306	105.5 ± 9.3	ND	ND	-
Assay 3	Control	3.81 ± 0.36	3811 ± 356	100.0 ± 9.3	ND	ND	-
	25 μg/mL black ginger	3.92 ± 0.56	3919 ± 565	102.8 ± 14.8	ND	ND	-
	50 μg/mL black ginger	3.89 ± 0.39	3891 ± 391	102.1 ± 10.3	ND	ND	-
	100 μg/mL black ginger	4.17 ± 0.44	4166 ± 442	109.3 ± 11.6	ND	ND	-

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The values are expressed as absolute (μM and pmol) and relative to vehicle control (%). Data are presented as mean ± standard deviation (SD), n = 3 in all groups

*p < 0.05

**p < 0.01. ACh, acetylcholine; AChE, acetylcholinesterase; Ch, choline; ND, not detected.

Next, we checked the response of LA-N-2 cells to Ch and Ch-containing compounds. After the incubation of LA-N-2 cells with 100 μM Ch, the intracellular Ch and ACh levels increased to 984.7% and 179.5% compared with the control, respectively (Table 2, Assay 1). The extracellular Ch level was above the quantitative limit, and ACh was not detected (S1 Dataset). When the cells were treated with LPC at concentrations between 3.125 and 25 μg/mL, both the intracellular Ch and ACh levels increased in a concentration-dependent manner. Ch levels reached statistical significance when treated with LPC at concentrations higher than 12.5 μg/mL (860.1%, p = 0.014 for 12.5 μg/mL; and 1259.7%, p < 0.001 for 25 μg/mL), and ACh levels at concentrations higher than 6.25 μg/mL (122.0%, p = 0.009 for 6.25 μg/mL; 129.8%, p < 0.001 for 12.5 μg/mL; and 147.6%, p < 0.001 for 25 μg/mL; Table 2, Assay 2). However, ACh was not detected in the extracellular fraction after LPC treatment. In addition, glycerophosphocholine and phosphatidylcholine did not increase the intracellular Ch or ACh levels; ACh was not detected in the extracellular fraction after the treatments (S1 Dataset).

Table 2. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with choline and lysophosphatidylcholine that have a Ch moiety.

Intracellular
		Ch			ACh
		(μM)	(pmol)	(%)	(μM)	(pmol)	(%)
Assay 1	Control	0.079 ± 0.017	31.6 ± 6.9	100.0 ± 22.0	0.281 ± 0.024	112.4 ± 9.6	100.0 ± 8.5
Assay 1	100 μM choline	0.778 ± 0.154	311.2 ± 61.7	984.7 ± 195.3 ^**	0.504 ± 0.012	201.7 ± 4.7	179.5 ± 4.1 ^**
Assay 2	Control	0.116 ± 0.028	46.4 ± 11.2	100.0 ± 24.2	0.231 ± 0.013	92.4 ± 5.4	100.0 ± 5.8
	3.125 μg/mL LPC	0.249 ± 0.102	99.5 ± 40.8	214.7 ± 87.9	0.265 ± 0.008	105.8 ± 3.2	114.6 ± 3.5
	6.25 μg/mL LPC	0.443 ± 0.236	177.0 ± 94.5	381.9 ± 203.8	0.282 ± 0.015	112.7 ± 5.8	122.0 ± 6.3 ^**
	12.5 μg/mL LPC	0.997 ± 0.366	398.7 ± 146.5	860.1 ± 316.1 ^*	0.300 ± 0.015	119.9 ± 5.8	129.8 ± 6.3 ^**
	25 μg/mL LPC	1.460 ± 0.694	583.9 ± 277.6	1259.7 ± 598.9 ^**	0.341 ± 0.036	136.3 ± 14.6	147.6 ± 15.8 ^**

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The values are expressed as absolute (μM and pmol) and relative to vehicle control (%). Data are presented as mean ± standard deviation (SD), n = 3 in all groups

*p < 0.05

**p < 0.01. ACh, acetylcholine; Ch, choline; LPC, lysophosphatidylcholine.

Subsequently, we assessed luteolin and nobiletin using the cell-based assay system. Although extracellular ACh was not detected, intracellular ACh levels significantly increased after treatment with 50 and 100 μM luteolin (111.6%, p = 0.006 for 50 μM; and 128.3%, p < 0.001 for 100 μM), and 100 μM nobiletin (108.6%, p = 0.025). There were no significant changes in intracellular Ch levels (Table 3).

Table 3. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with luteolin and nobiletin, which have been reported to have ChAT-increasing activity.

Intracellular
		Ch			ACh
		(μM)	(pmol)	(%)	(μM)	(pmol)	(%)
Assay 1	Control	0.202 ± 0.073	80.9 ± 29.2	100.0 ± 36.1	0.450 ± 0.003	180.1 ± 1.2	100.0 ± 0.7
	25 μM luteolin	0.207 ± 0.084	82.6 ± 33.5	102.1 ± 41.5	0.448 ± 0.001	179.0 ± 0.5	99.4 ± 0.3
	50 μM luteolin	0.211 ± 0.102	84.5 ± 40.8	104.4 ± 50.4	0.502 ± 0.027	201.0 ± 10.9	111.6 ± 6.0
	100 μM luteolin	0.223 ± 0.021	89.1 ± 8.4	110.2 ± 10.3	0.578 ± 0.009	231.1 ± 3.6	128.3 ± 2.0 ^**
Assay 2	Control	0.143 ± 0.053	57.1 ± 21.3	100.0 ± 37.3	0.515 ± 0.014	206.1 ± 5.7	100.0 ± 2.8
Assay 2	100 μM nobiletin	0.113 ± 0.014	45.1 ± 5.5	78.9 ± 9.7	0.560 ± 0.017	223.9 ± 6.7	108.6 ± 3.3 ^*

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The values are expressed as absolute (μM and pmol) and relative to vehicle control (%). Data are presented as mean ± standard deviation (SD), n = 3 in all groups

*p < 0.05

**p < 0.01. ACh, acetylcholine; Ch, choline; ChAT, choline acetyltransferase.

Finally, we treated LA-N-2 cells with 100 μM muscarine, an mAChR agonist. Although extracellular ACh was not detected, intracellular ACh levels significantly increased up to 122.5% compared with the control (p = 0.038); intracellular Ch levels did not change after muscarine treatment (Table 4). Other experiments showed that treatment with up to 5 μM cytisine, an nAChR agonist, neither changed ACh nor Ch levels (S1 Dataset). Moreover, western blotting revealed that LA-N-2 cells expressed mAChR M2, even after treatment with the assay medium for 5 h (Fig 2).

Table 4. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with muscarine, a muscarinic acetylcholine receptor agonist.

Intracellular
	Ch			ACh
	(μM)	(pmol)	(%)	(μM)	(pmol)	(%)
Control	0.0652 ± 0.0196	26.1 ± 7.8	100.0 ± 30.0	0.184 ± 0.023	73.6 ± 9.4	100.0 ± 12.8
100 μM muscarine	0.0617 ± 0.0153	24.7 ± 6.1	94.6 ± 23.5	0.225 ± 0.001	90.1 ± 0.2	122.5 ± 0.3 ^*

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The values are expressed as absolute (μM and pmol) and relative to vehicle control (%). Data are presented as mean ± standard deviation (SD), n = 3 in all groups

*p < 0.05. ACh, acetylcholine; Ch, choline.

Fig 2 — LA-N-2 cells were lysed before (-) or after (+) the treatment with the assay medium for 5 h and subjected to SDS-PAGE along with mouse brain tissue lysate as a positive control (PC). Lysates/proteins at 10 μg per lane.

We have listed drug compounds and food constituents that increased ACh levels in our cell-based assay in Table 5 with their reported mechanisms of action on the cholinergic system, and the other drug compounds and food constituents, which did not, in Table 6.

Table 5. Drug compounds and food constituents that increased extracellular and/or intracellular ACh levels in our cell-based assay.

Reported mechanism of action on the cholinergic system	Name	Drug/food	Concentration used in assay	Fraction where the increase was detected
AChE inhibition	Physostigmine	Drug	5 nM–50 μM	Extracellular and intracellular
	Delphinidin chloride	Food	25–100 μM	Intracellular
	Black ginger extract	Food	25–100 μg/mL	Intracellular
Ch (ACh precursor) provision	Ch chloride	Food	100 μM	Intracellular
Ch (ACh precursor) provision	LPC	Food	3.125–25 μg/mL	Intracellular
ChAT activation	Luteolin	Food	25–100 μM	Intracellular
ChAT activation	Nobiletin	Food	100 μM	Intracellular
mAChR activation	(+)-Muscarine chloride	Drug	100 μM	Intracellular

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Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; Ch, choline; ChAT, choline acetyltransferase; LPC, lysophosphatidylcholine; mAChR, muscarinic acetylcholine receptor.

Table 6. A drug compound and food constituents that did not increase extracellular or intracellular ACh levels in our cell-based assay.

Name	Drug/food	Concentration used in assay
Cytisine	Drug	50 nM–5 μM
Glycerophosphocholine	Food	100 μg/mL
Arachidonic acid	Food	20 μg/mL
Astaxanthin	Food	100 μM
L-Carnitine	Food	100 μM
Citrulline	Food	100 μM
Curcumin	Food	20 μM
Cyanidin chloride	Food	100 μM
Cyanocobalamin	Food	100 μM
Docosahexaenoic acid	Food	20 μg/mL
Eicosapentaenoic acid	Food	20 μg/mL
Ferulic acid	Food	100 μM
Glycine	Food	100 μM
Lutein	Food	100 μM
Methylcobalamin	Food	100 μM
Octanoic acid	Food	100 μg/mL
Phosphatidylcholine from egg yolk	Food	100 μg/mL
Phosphatidylethanolamine, dimyristoyl	Food	100 μg/mL
Phosphatidylethanolamine, dioleoyl	Food	100 μg/mL
Phosphatidylethanolamine, dipalmitoyl	Food	100 μg/mL
Phosphatidylethanolamine, distearoyl	Food	100 μg/mL
Phosphatidylserine	Food	100 μg/mL
Pyrroloquinoline quinone	Food	100 μM
L-Serine	Food	100 μM
cis-15-Tetracosenoic acid	Food	100 μg/mL
Zeaxanthin	Food	100 μM

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Discussion

When LA-N-2 cells were incubated in the assay medium for 5 h as the control, the concentration of extracellular Ch was quantified to be 3–4 μM (Table 1B). Tucek reported that the concentration of extracellular Ch in the human brain is similar to that in the cerebrospinal fluid, within the range of 0.4–5.1 μM, and this notion has well been supported by microdialysis studies [30, 31]. Importantly, the concentration of extracellular Ch observed in our study fell within this range, which indicates the ability of this system to simulate the cholinergic metabolic system in physiological conditions.

ACh was not detected in the extracellular fraction in the control group (Table 1B). This supports the finding that this cell type expresses AChE [24]. Although the cholinergic phenotype of LA-N-2 cells is known to be enhanced when differentiated [24, 26], the cell line without differentiation seemed to produce a sufficient amount of ChAT for intracellular ACh to be produced and AChE for extracellular ACh to be degraded. Therefore, we prioritized a quick screen system set up and decided to perform assays without differentiation.

When LA-N-2 cells were treated with physostigmine, a commonly used AChE inhibitor, at doses higher than 5 μM, extracellular ACh was successfully detected. Given its short half-life [14], it could be possible to use an even lower concentration of physostigmine to detect extracellular ACh if the incubation time is optimized. However, 5 μM physostigmine is commonly used when performing microdialysis to increase the basal ACh level up to a detectable magnitude [32]. In addition, Lau et al. reported that their in vitro model using lung cancer cells could detect the increase in extracellular ACh levels after neostigmine treatment. Given that they successfully detected this increase using 50 μM neostigmine but not 20 μM [33], and that the half-maximal inhibitory concentration of neostigmine toward AChE is similar to that of physostigmine [32], we consider that our model can sensitively detect the extracellular ACh following AChE inhibition. Interestingly, using physostigmine, the detection of extracellular ACh coincided with the increase in intracellular ACh levels; this increase was observed at a ten times lower dose of physostigmine (Table 1, Assay 1).

Delphinidin, an anthocyanidin commonly present in pigmented fruits and vegetables [15], and black ginger (Kaempferia parviflora) extract have also been reported to have an inhibitory effect on AChE [15, 16]. Treatments with these two food components increased intracellular ACh levels, although ACh was not detected in the extracellular fraction (Table 1, Assays 2 and 3). These findings support the possibility that the increase in intracellular ACh levels is more easily detected than that in extracellular ACh levels. Moreover, the findings suggest that our cell-based assay model is sensitive enough to detect the increase in intracellular ACh levels induced by food components. Given that delphinidin and black ginger extract improve cognitive function in vivo [15, 34], an increase in the intracellular ACh levels may serve as an indicator of cognitive improvement. For these reasons, we decided to proceed with the assays with a focus on intracellular ACh.

When Ch was added to the assay medium, it was taken up and converted to ACh in the cells (Table 2, Assay 1); this supports the existence of Ch transporter and ChAT in LA-N-2 cells. LPC, which constitutes enzyme-modified lecithin and is used as a food emulsifier owing to its amphipathic nature [35], has been shown to be taken up by and converted to ACh via Ch in LA-N-2 cells [18]. This was confirmed with 3.125 to 25 μg/mL LPC in our experimental model, and 25 μg/mL LPC evoked higher intracellular Ch levels than 100 μM Ch (Table 2, Assays 1 and 2). This finding can be explained by the intermediate-affinity uptake of Ch by LA-N-2 cells [27] and the detergent-like property of LPC; 50 μg/mL LPC caused cytotoxicity and decreased intracellular ACh levels (S1 Dataset).

Glycerophosphocholine and phosphatidylcholine caused no significant changes in intracellular Ch or ACh levels (Table 6, S1 Dataset). As LA-N-2 cells have been reported to have glycerophosphodiester phosphodiesterase activity [24], which hydrolyzes glycerophosphocholine to Ch, it seems that glycerophosphocholine was not incorporated into the cells nor degraded by the enzyme. Moreover, LA-N-2 cells have been shown to express phospholipase D to utilize phosphatidylcholine in the cell membrane [18]. However, as the cell membrane was impermeable to the phosphatidylcholine in the medium, its utilization as an ACh precursor was prevented. Similarly, we assume that phosphatidylserine, which has been shown to reverse ACh levels in aged animals [36], failed to increase ACh levels in our cell-based assay (Table 6, S1 Dataset) owing to the lack of related metabolic enzymes and its inability to cross the cell membrane from the medium.

The intracellular ACh levels increased after treatment with luteolin and nobiletin (Table 3). Luteolin is a flavonoid that is present in fruits and vegetables such as celery, chrysanthemum flower, sweet bell pepper, carrot, onion leaf, broccoli, and parsley [37]; the oral administration of luteolin has been reported to increase ACh levels in the brain of amyloid β-infused rats through ChAT activation [19]. Nobiletin, a citrus flavonoid known to improve cognitive function in vivo [38], has also been reported to increase ACh levels via the upregulation of ChAT expression [20]. These findings suggest that the increase in intracellular ACh levels observed after treatments with these flavonoids resulted from the upregulation of ChAT.

The cell line showed a significant increase in intracellular ACh levels when incubated with muscarine (Table 4). To the best of our knowledge, previous studies have suggested the existence of mAChR on LA-N-2 cells but have not identified the subtype expressed [25]. Therefore, we performed western blotting and showed the constant expression of mAChR M2 on the cells (Fig 2). The increase in intracellular ACh levels following muscarine stimulation may be partly mediated by mAChR M2 because the activation of this subtype leads to the feedback inhibition of ACh release and accumulation of ACh in neurons [39]. Importantly, our result demonstrated that an increase in the intracellular ACh levels owing to stimulation by mAChR agonists could be detected by our assay system. In contrast, another ACh receptor type, nAChR, may not be expressed on LA-N-2 cells, which was supported by our data; cytisine, a nicotinic agonist [21], did not alter ACh or Ch levels (Table 6, S1 Dataset).

As shown above, our cell-based assay system could detect an increase in extracellular ACh levels by an AChE-inhibiting drug at relatively high doses. In addition, the developed model could detect an increase in intracellular ACh levels by lower doses of the AChE-inhibiting drug and an mAChR agonist, and the increase was observed even when treated with food constituents that have different mechanisms of action, such as Ch provision and ChAT activation. We consider that compared with previously reported in vitro models that evaluate a single component of the cholinergic system, the developed model makes it possible to more broadly screen lead compounds that have different strengths of activity and mechanisms of action by evaluating both extracellular and intracellular ACh. Although the development of cholinergic drugs has been highly focused on ACh inhibition and many AChE inhibitors have been developed [10, 11, 40], food constituents that are expected to counteract cognitive decline have various mechanisms besides AChE inhibition, as shown above [15, 16, 19, 20, 34, 38]. Therefore, the developed assay model may be particularly effective to screen food constituents with mild cholinergic activities.

According to the amyloid hypothesis, the accumulation of amyloid β in the brain is the primary factor driving the pathogenesis of Alzheimer’s disease. Although a treatment approach based on this hypothesis is one of the most convincing approaches [41], multidirectional approaches may be needed for the management of dementia given the complex pathogenesis [42]. In addition, the interplay between amyloid β and the cholinergic system has been studied; the results showed that the activation of mAChR can shift the processing of amyloid β precursor protein toward the nonamyloidogenic pathway [3]. From these perspectives, the practical applicability of the developed assay model for screening cholinergic agents is evident.

Our study had the following limitations. First, we did not measure the activity of components of the cholinergic system (e.g., AChE) or investigate the interactions between the test molecules and cholinergic markers. Therefore, we cannot exclude the possibility that a test compound may have different mechanisms of action (e.g., inhibiting AChE and activating ChAT) simultaneously. Second, we used doses of drugs and food constituents up to 100 μM or 100 μg/mL because our main objective was to compare the response of intracellular and extracellular ACh. Given the metabolic process and existence of the blood–brain barrier, it is unlikely for some drugs and food constituents evaluated in this study to reach the brain at such doses [43]. The assay model described here should serve as a preliminary in vitro screen, and further in vivo and clinical studies are needed to clarify whether the candidates selected using this screening model could improve ACh transmission, neural plasticity, and even cognitive function.

In conclusion, this is the first study to report an assay model using LA-N-2 cells where both extracellular and intracellular ACh can be quantified. The simple and quick method enabled us to screen cholinergic agents, and the values were comparable and reproducible between assays when expressed as levels relative to those of the control. Notably, the increase in ACh levels by food constituents with different mechanisms of action on the cholinergic system was sensitively detected in the intracellular fraction. We propose that the developed screening system can be used in the first step of the development of new functional foods, as well as drugs, to treat ACh-relevant phenomena such as dementia and aging-related cognitive decline.

Supporting information

S1 Table. List of drug compounds and food constituents evaluated in our cell-based assay.

(DOCX)

Click here for additional data file.^{(50.8KB, docx)}

S1 Dataset. A dataset for choline and acetylcholine quantification.

(XLSX)

Click here for additional data file.^{(278.1KB, xlsx)}

S1 Raw image. The raw image of western blot.

Fig 1 was generated from this raw image. We evaluated different lysates of LA-N-2 cells that were harvested another day, and confirmed the reproducibility of the expression of mAChR M2.

(TIF)

Click here for additional data file.^{(421.7KB, tif)}

S2 Raw image. The raw image of western blot.

The same membrane as S1 Raw image but with longer exposure time to increase the intensity of bands of the molecular weight marker.

(TIF)

Click here for additional data file.^{(156.2KB, tif)}

Acknowledgments

We thank our colleagues, especially Mr. Kiyohiko Magata, for helpful discussions. The main author is also grateful to Dr. Koichiro Hamada for managing the project.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The funder provided support in the form of salaries for authors [RT and KH], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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PLoS One. doi: 10.1371/journal.pone.0258420.r001

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PONE-D-21-08197

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

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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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PLoS One. 2021 Oct 12;16(10):e0258420. doi: 10.1371/journal.pone.0258420.r002

Author response to Decision Letter 0

10 Aug 2021

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We apologize for the insufficient formatting. We have carefully read the Journal’s style templates again and revised the format.

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Our response to Reviewers

Reviewer #1

Thank you for your valuable comments and for suggesting some interesting research papers related to choline-containing phospholipids. We agree that the strategy targeting beta-amyloid and neurofibrillary tangles seems to be effective. However, we consider that adopting a multidirectional approach increases the possibility to successfully cure neurodegenerative disorders, a notion that is supported by a paper you suggested, which showed the enhanced effect of donepezil when combined with choline alphoscerate. The cholinergic agents found by our in vitro system could possibly be combined with other types of treatment agents, such as anti-amyloid-beta antibody. Further research is warranted to assess the effectiveness of such a combination.

Regarding the title of our manuscript, the approach we adopted has sufficient novelty in the space of phytomedicine and method development as no other screening assays that utilized this method have been reported. Therefore, we consider the original title appropriate.

Reviewer #2

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

Thank you for your helpful advice. We drew and added a schematic diagram as Fig 1.

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?

We consider that concentrations adopted for some food constituents may be higher than those observed in the human blood and brain after their normal intake. However, we believe that the developed model can be used for preliminary screening because it could detect the change in ACh levels after treatment with some agents, the cholinergic activities of which have already been shown in in vivo or clinical studies.

Nevertheless, this limitation has been described in the Discussion (page 25, line 379-83).

We believe that the discrete contributions of CHT1, ChAT, mAChR, and AChE on changes in ACh levels should be addressed in another study. The concentration of extracellular Ch in the assay was maintained between 3 and 4 µM over a 5-h incubation, which is consistent with the physiological concentration.

Relevant sentences have been added in the Discussion (page 21, line 271-7).

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.

Given the different concentration ranges of ACh and Ch, we found that it was difficult to put these values in a single graph. In addition, we have added a concentration unit (µM) considering a comment by Reviewer #3, which made it more difficult to illustrate these results in graphs. Thus, we decided to leave them as tables. We hope you are satisfied with our opinion.

We have weakened the statement (page 22, line 308).

Reviewer #3

Cholinergic neurons are characterized by the expression of ChAT, CHT-1 and the vesicular ACh transporter (vAChT). Other cholinergic “features” listed by the authors are not exclusively cholinergic.

We appreciate your detailed and constructive comments. As you pointed out, differentiation agents enhance cholinergic characteristics of LA-N-2. However, the cell line shows a partial cholinergic phenotype by nature and have been described as cholinergic elsewhere (ref. [24, 26]).

We also wondered if the results would change when LA-N-2 cells were differentiated. However, the cell line without differentiation seemed to produce a sufficient amount of ChAT for intracellular ACh to be produced and AChE for extracellular ACh to be degraded. Therefore, we prioritized a quick screen system set up and decided to perform assays without differentiation. This background has been added in the Discussion (page 21, line 279-84).

We apologize for the misleading description about “cholinergic” and corrected some sentences (page 2, line 11 and 22; page 4, line 40; and page 5, line 102 and 103).

2. 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. 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?

We have recalculated Ch and ACh levels and expressed them as concentrations (µM). During this process, we found a mistake in our previous calculations, and have updated the values and reconfirmed statistical significance. Although critical aspects of the results did not change, we sincerely apologize for these necessary minor changes. Given the corrected values, we can observe that the concentrations of extracellular Ch are in good agreement with those in earlier studies (ref. [29, 30]). Regarding the concentration of intracellular Ch, the cellular contents were diluted as a result of lysing with 400 µL of perchloric acid as described in the Methods. Therefore, it is difficult to compare the values obtained in this study with a previously reported one (50 µM), and to judge whether Ch was not adequately taken up. Nevertheless, given that the concentration range of extracellular Ch obtained from the assays was similar to that in physiological conditions, we assumed that the transportation system for Ch worked in our model and did not conduct further research into it.

Although FBS contains Ch, the assay medium we used contained only 2% FBS. Therefore, we consider that the quantity of Ch derived from FBS is not significant. Dead cells were not observed under a microscope after the 5-h incubation without Ch.

3. Cholinergic activations are due to an increase of extracellular ACh. Increases of intracellular ACh do not reflect cholinergic activation.

We completely agree that the increase in intracellular ACh is not always parallel to cholinergic activation. Therefore, as a next step, an in vivo study should assess whether the treatment candidates chosen by this screening system affect cholinergic transmission.

Thank you again for your insightful comment. As a sentence has been added in the Discussion (page 21, line 288-90), 5-50 µM physostigmine is commonly used in microdialysis studies and we do not consider the efficacy of physostigmine observed in this study to be low. However, we agree that the amount of ACh detected in the extracellular fraction may increase if the incubation time is optimized for physostigmine or other AChE inhibitors with a longer half-life are applied. We consider that these possibilities should be assessed in another study.

We doubt the possibility that physostigmine interferes with AChE in the enzyme column because (a) the two columns preceding the enzyme column are packed with porous polymer, which would trap contaminants, including physostigmine in analytes, and (b) if so, we would observe an inverted concentration-response against ACh levels, which we did not.

I do not know how the interaction of intracellular AChE with ACh would be in a neuroblastoma cell line.

Although we did not investigate the interaction of intracellular AChE with ACh in LA-N-2 cells, the activity of intracellular AChE in another neuroblastoma cell line (N18TG2) was reported by Melone et al. (Int J Dev Neurosci. 1987;5(5-6):417-28), as well as the ability of the cell to release a considerable amount of the enzyme in the culture medium.

6. 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.

It is indeed intriguing to further explore the Ch transport system in LA-N-2 cells. However, our focus was to develop a screening system to broadly catch cholinergic agents with several mechanisms of action. Hence, we generated a Ch-deficient environment on purpose to screen agents that can be a Ch source to the cell. We consider that it is worth screening Ch-containing compounds because positive clinical results have been reported for some of them.

7. 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.

PLD is present in synaptosomal membranes and it has been shown that LA-N-2 cells express PLD and PLA2 (ref. [24]). In the study, the authors found that the activities of GPC-phosphodiesterases in LA-N-2 cells do not change over incubation time, which suggests that the cell line is not able to use GPC as an ACh precursor.

Related sentences have been added in the Discussion (page 22, line 317-22).

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

Given your comment, we conducted western blotting and found muscarinic AChR M2 to be expressed on LA-N-2 cells.

Related sentences have been added in the Abstract, Methods, Results, and Discussion.

As you pointed out, luteolin, delphinidin, and other flavonoids are unlikely to reach the brain at a concentration of 100 µM after normal intake. However, we consider that it is still worth evaluating these food constituents even at relatively high concentrations as a preliminary screen. As stated in our manuscript, orally administered luteolin and delphinidin have been shown to improve the cholinergic system in the brain (ref. [15, 19]). Moreover, a technique to improve the bioavailability of unstable plant constituents has been recently developed. Using this technique, curcumin has been successfully used to improve AD pathology in a clinical trial (Ma Z, et al. J Control Release. 2019), and attempts to enhance the bioavailability of luteolin and other flavonoids are being made (Ali F, et al. CNS Neurol Disord Drug Targets. 2019).

We have added sentences to discuss the doses of food constituents we used in our study (page 25, line 379-83).

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PLoS One. doi: 10.1371/journal.pone.0258420.r003

Decision Letter 1

Israel Silman

7 Sep 2021

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,

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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?

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Reviewer #3: Partly

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

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Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

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 #3: No

PLoS One. 2021 Oct 12;16(10):e0258420. doi: 10.1371/journal.pone.0258420.r004

Author response to Decision Letter 1

24 Sep 2021

Reviewer #2

Thank you for your helpful advice. When the cells were treated with some chemicals including LPC at high concentrations, cell death and a decrease in ACh levels were observed. The related sentence and data have been added (page 22, line 318, and S1 dataset).

2. Discussing the expression of receptor subtypes on the membrane based on relevant RNA-seq data will be helpful.

We searched for the RNA-seq and array data regarding the expression of ACh reseptors in LA-N-2 cells but could not find any (only short RNA-seq data is available). We consider that the expression of the other subtypes of mAChR should be investigated in another study.

Reviewer #3

Although many clinical trials that administered Ch sources failed to improve the clinical status of patients with dementia, some Ch sources combined with an AChE inhibitor have succeeded and showed better outcomes than the AChE inhibitor alone (Traini E, et al. 2020, Piamonte BLC, et al. 2020). Therefore, we consider that detecting the Ch providing property of test compounds is of value and that our screening system enables it, at least for some Ch containing compounds. We consider this as one of the advantages of our system. Therefore, we did not delete the sentences you pointed out, but modified them (page 2, line 11 and page 3, line 40). In addition, we have changed the preposition from “against” to “at” (page 3, line 47).

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.

We believe that you may be thinking of the microdialysis study with neostigmine like reviewed in [32]. In the setting, 1 µM neostigmine is enough to detect ACh because ACh is protected from AChE once it goes through the semi-permeable membrane of the microdialysis probe. However, in Lau’s and our in vitro system, ACh is subjected to hydrolysis by AChE unless the enzyme is fully inhibited or deactivated (we added perchloric acid to the medium).

We admit that we could not clearly distinguish CHT-1 from other types of Ch transporters. According to a previous study [27], the choline-uptake mechanism of LA-N-2 involves intermediate-affinity transporters (Ch transporter-like protein 1) rather than high-affinity ones (CHT-1) (they used hemicholinium-3 in their experiments). This finding explains why a relatively low incorporation of extracellular Ch was observed in our experimental setting. The related sentence has been added to the Discussion (page 22, line 316).

We have revised the sentence (page 23, line 321). Additionally, we agree that LPC is a membrane detergent and that this property may have contributed to the higher ACh accumulation after treatment with LPC than after treatment with Ch. Related sentences have been added to the Discussion (page 22, line 317).

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

We have corrected the sentence (page 22, line 323). We appreciate all of your excellent comments and suggestions.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0258420.r005

Decision Letter 2

Israel Silman

28 Sep 2021

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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Israel Silman

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0258420.r006

Acceptance letter

Israel Silman

4 Oct 2021

PONE-D-21-08197R2

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

Dear Dr. Tanaka-Kanegae:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. List of drug compounds and food constituents evaluated in our cell-based assay.

(DOCX)

Click here for additional data file.^{(50.8KB, docx)}

S1 Dataset. A dataset for choline and acetylcholine quantification.

(XLSX)

Click here for additional data file.^{(278.1KB, xlsx)}

S1 Raw image. The raw image of western blot.

Fig 1 was generated from this raw image. We evaluated different lysates of LA-N-2 cells that were harvested another day, and confirmed the reproducibility of the expression of mAChR M2.

(TIF)

Click here for additional data file.^{(421.7KB, tif)}

S2 Raw image. The raw image of western blot.

The same membrane as S1 Raw image but with longer exposure time to increase the intensity of bands of the molecular weight marker.

(TIF)

Click here for additional data file.^{(156.2KB, tif)}

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(73.5KB, doc)}

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(48.5KB, doc)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

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

Ryohei Tanaka-Kanegae

Koichiro Hamada

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Reagents

Cell culture

Assays

Choline and acetylcholine measurement

Fig 1. A schematic diagram of the new in vitro assay system.

Western blotting

Statistical analysis

Results

Table 1. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with physostigmine, delphinidin, and black ginger extract, which have been reported to have AChE-inhibiting activity.

Table 2. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with choline and lysophosphatidylcholine that have a Ch moiety.

Table 3. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with luteolin and nobiletin, which have been reported to have ChAT-increasing activity.

Table 4. Changes in the choline and acetylcholine levels in LA-N-2 cells after treatment with muscarine, a muscarinic acetylcholine receptor agonist.

Fig 2. The expression of muscarinic acetylcholine receptor M2 on LA-N-2 cells.

Table 5. Drug compounds and food constituents that increased extracellular and/or intracellular ACh levels in our cell-based assay.

Table 6. A drug compound and food constituents that did not increase extracellular or intracellular ACh levels in our cell-based assay.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Israel Silman

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Author response to Decision Letter 0

Decision Letter 1

Israel Silman

Roles

Author response to Decision Letter 1

Decision Letter 2

Israel Silman

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Acceptance letter

Israel Silman

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Associated Data

Supplementary Materials

Data Availability Statement

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