PNAS Nexus, 2024, 3, 1–11
https://doi.org/10.1093/pnasnexus/pgad437
Advance access publication 14 December 2023

Research Report

Real-time monitoring of the amyloid β1–42 monomer-tooligomer channel transition using a lipid bilayer system
Yuri Numaguchia,1, Kaori Tsukakoshia,1, Nanami Takeuchia, Yuki Suzukib, Kazunori Ikebukuroaand Ryuji Kawano

a,

*

a

Abstract
This study describes the observation of the transformation of monomeric amyloid β1–42 (Aβ42) into oligomers in a lipid membrane
utilizing a lipid bilayer system for electrophysiological measurement. The relevance of oligomers and protofibrils in Alzheimer's
disease (AD) is underscored given their significant neurotoxicity. By closely monitoring the shift of Aβ42 from its monomeric state toforming oligomeric channels in phospholipid membranes, we noted that this transformation transpired within a 2-h frame. We
manipulated the lipid membrane's constitution with components such as glycerophospholipid, porcine brain total lipid extract,sphingomyelin (SM), and cholesterol (Chol.) to effectively imitate nerve cell membranes. Interesting findings showcased Chol.'s ability
to foster stable oligomeric channel formation in the lipid membrane, with SM and GM1 lipids potentially enhancing channel
formation as well. Additionally, the study identified the potential of a catechin derivative, epigallocatechin gallate (EGCG), in
obstructing oligomerization. With EGCG present in the outer solution of the Aβ42-infused membrane, a noteworthy reduction inchannel current was observed, suggesting the successful inhibition of oligomerization. This conclusion held true in both, prior andsubsequent, stages of oligomerization. Our findings shed light on the toxicity of oligomers, promising invaluable information for
future advancements in AD treatment strategies.

Significance Statement
Given the crucial role of amyloid β1–42 (Aβ42) oligomerization in the progression of Alzheimer's disease (AD), understanding this pro­
cess at the molecular level is paramount. This study provides insights into the modulation of this oligomerization by different lipid
components in the membrane, potentially paving the way for therapeutic strategies. Further, the identification of a compound cap­able of impeding Aβ42 oligomerization could herald a significant meaning in the treatment of AD. These findings underscore the im­portance of continued exploration in this field, as it could lead to useful advances in AD therapeutics.

Introduction
Alzheimer's disease (AD) is a neurodegenerative disease caused bysynaptic loss and neuronal death (1, 2). Amyloid beta (Aβ) is a frag­ment of amyloid precursor protein (APP), a type I single-pass trans­
membrane protein with a large extracellular domain, which is
implicated in the pathogenesis of AD. Two secretases, β-secretaseand ɤ-secretase, cleave an APP to form Aβ fragments with varying
lengths (3–5). Amyloid β1–42 (Aβ42) is considered to be highly cyto­
toxic (6), with the monomers aggregating to form insoluble fibrils
through oligomeric intermediate states (7). Although Aβ42 fibrils
are a major component of senile plaques, their toxicity to neurons
would not be high; the oligomeric structure including the protofibril
is considered to have a high toxicity (8). Intermediate Aβ42 oligom­
ers/protofibrils have been detected in the brain tissue of AD pa­tients, and nanomolar concentrations of this oligomer solution
cause cell death in mature neurons (9). Different types of Aβ assem­
bles, ranging from dimers to large molecular-weight oligomers

consisting of >50 monomers, have been described in detail in sev­
eral studies (10–12). To target this species, lecanemab, a monoclo­
nal antibody that recognizes the protofibrils and prevents the Aβ42
deposition, has recently been approved and used for the treatmentof AD (13). Certain studies have also shown the neurotoxicity of
specific oligomeric species, and their determination is importantfor understanding and treating of AD (14).
Various hypotheses have been reported as to the mechanism ofneuronal toxicity of the Aβ42 oligomer, including channel forma­tion (15), mitochondrial damage (16), and an increase in hyperphos­
phorylated tau protein (17). Among them, the channel formation ofoligomers in cell membranes is a simple and possible mechanism;
the Aβ42 oligomers form channels in the neuronal membrane to in­crease ion influx, leading to neuronal death by disrupting the intra­
cellular ionic equilibrium (18–21). Several methods for observing
the channel formation have been proposed, such as fluorescencemeasurement of the calcium flux through the channel (22, 23), direct

Competing Interest: The authors declare no competing interest.
Received: September 28, 2023. Accepted: December 6, 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences. This is an Open Access article
distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-0011, Japan
Department of Chemistry for Materials, Graduate School of Engineering, Mie University, Mie 514-0102, Japan*To whom correspondence should be addressed: Email: rjkawano@cc.tuat.ac.jp
1
Y.N. and K.T. contributed equally to this work.
Edited By: Josh Wandb

2 | PNAS Nexus, 2024, Vol. 3, No. 1

observation of the channel using atomic force microscopy (AFM)(24), and electrophysiological measurements of ion flux throughthe channels (15, 25). In addition, NMR (26) and molecular dynamic(MD) simulations (27) have also been reported, which estimate thechannel structure of Aβ42 in membranes. Although various techni­ques have demonstrated that Aβ42 oligomers form channels inmembranes and increase ion influx, the mechanism of Aβ42 mono­
mer aggregation into channels with a lipid membrane remains
incompletely understood. This is due to the limitations of conven­tional studies, which have relied on preprepared Aβ42 oligomers
and have not explored the monomer-to-oligomer transformation
with lipid membranes in detail, primarily due to the difficulties asso­
ciated with kinetic experiments.
In this study, we report the oligomerization of the Aβ42 mono­mers, observed from channel current signals using a microfabri­cated device, which can prepare a stable lipid bilayer (Fig. 1).
The obtained channel current signals are identified as theoligomer- or monomer-induced signals, with the signal classifica­tion in accordance with our previous reports using antimicrobial
peptides (AMPs) (28–30). We also estimate the channel formationin several different types of phospholipid membranes, to imitate
the nerve cell membrane using glycerophospholipid (PC), brain to­tal lipid extract from porcine (BTLE), sphingomyelin (SM), andcholesterol (Chol.). The aggregation state of Aβ42 was confirmed
by additional methods including western blotting (WB) analysis,
thioflavin T (ThT) fluorescence measurement, and AFM observa­
tion. Furthermore, based on the signal estimations of monomerto-oligomer transitions, we aimed to investigate the inhibitory ef­
fects of Aβ42 oligomerization using our system. To achieve this,
we introduced epigallocatechin gallate (EGCG), a well-known in­hibitor of Aβ42 aggregation (31), into the outer solution of the lipid
membrane containing Aβ42 monomers and subsequently ob­
served the effects of EGCG on the channel-forming activity ofAβ42 within the membrane. Our system not only enables us to ob­serve the formation of Aβ42 oligomer channels but also facilitates
the screening of potential inhibitors of Aβ42 oligomerization, evenwhen embedded within the lipid membrane.

Polar Lipids); SM (brain, porcine; Avanti Polar Lipids); BTLE
(Avanti Polar Lipids); n-decane (Wako Pure Chemical Industries,
Ltd., Osaka, Japan); Chol. (Sigma-Aldrich Co., St Louis, MO, USA); di­methyl sulfoxide (DMSO; Wako Pure Chemical Industries);
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Wako Pure Chemical
Industries); sodium chloride (NaCl; Kanto Chemical Co., Inc.,Tokyo, Japan); potassium chloride (KCl; Kanto Chemical); sodiumhydrogen phosphate (Na2HPO4; Kanto Chemical); and potassium
dihydrogen phosphate (KH2PO4; Kanto Chemical). Buffered electro­
lyte solutions were prepared from ultrapure water, which was ob­
tained from a Milli-Q system (Millipore, Billerica, MA, USA). Aβ42
was purchased from Peptide Institute, Inc. (Osaka, Japan). Sample
buffer solution with reducing reagent (6×) for SDS–PAGE (Nacalai
Tesque, Kyoto, Japan) and Tween20 (Kanto Chemical) and skim
milk (Wako Pure Chemical Industries) were used as a solution for
WB. Anti–N terminus of Aβ end-specific, mouse monoclonal anti­
body, 82E1 was purchased from Immuno-Biological Laboratories
Co, Ltd. (Gunma, Japan), and horseradish peroxidase (HRP)conjugated antimouse IgG was obtained from Promega Corporation(Tokyo, Japan). ThT was purchased from Sigma-Aldrich. (-)-EGCG
was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Preparation of Aβ42 monomers and oligomers
Regarding the previous study (32), first, 1 mg of powdered Aβ42 was
dissolved in HFIP to a final concentration of 1 mM and gently agitatedon ice for 10 min. The clear solution containing Aβ42 was quickly ali­quoted in Protein LoBind Tubes (Eppendorf, Germany). Next, HFIP
was removed from the solution containing Aβ42 under vacuum us­
ing a CC-105 centrifugal concentrator and TU-500 cooling trap
(TOMY SEIKO Co., Ltd., Tokyo, Japan), and the resulting Aβ42 wasstored at −20°C. The Aβ42 aliquots were carefully and completely re­suspended to 1 mM in DMSO and sonicated for 3 min (6 cycles of 30 ssonication with 30 s stop) using BIORUPTOR (Cosmo Bio Co., Ltd.,Tokyo, Japan). Samples were stored at −80°C if necessary.
Immediately prior to use, samples were diluted in PBS buffer solution(137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.4 mM KH2PO4, andpH 7.4) to 10 µM and pipetted. The oligomer was prepared by incu­
bating the monomer solution in a tube for 4 h at 37°C.

Experimental
Reagents and chemicals
In this study, we used the following reagents: 1,2-diphytanoyl-snglycero-3-phosphocholine (Avanti Polar Lipids, Inc., Alabaster, AL,USA); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; AvantiPreparation of the microdevice forelectrophysiological measurements
The microdevice was fabricated by machining a 6.0 mm
thick, 10 mm × 10 mm polymethyl methacrylate (PMMA) plate

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Fig. 1. a) An image of channel formation of an Aβ42 oligomer in a planar lipid bilayer membrane. The Aβ42 monomers aggregate into oligomers, forming
channels in the membrane and increasing ion influx into the nerve cell. Ions that pass through the Aβ42 channels in the membrane were detected bychannel current measurements. b) A photograph of the microdevice for the droplet contact method. The microdevice has two chambers and Ag/AgClelectrodes separated by a separator.

Numaguchi et al. |3(Mitsubishi Rayon, Tokyo, Japan) using computer-aided design andcomputer-aided manufacturing with a 3D modeling machine(MM-100, Modia Systems, Japan), as shown in Fig. 1b (30, 33). Twowells (2.0 mm diameter and 4.5 mm depth) and a chase between
the wells were manufactured on the PMMA plate. However, inthe experiment investigating the effect of EGCG addition, the mi­crodevice was prepared with a well diameter twice as large as usual(4.0 mm), so that when the solution was later added, there were no
sudden changes in osmotic pressure. Each well had a through-holein the bottom and Ag/AgCl electrodes set into this hole. A polymericfilm made of parylene C (polychloro-p-xylylene) with a thickness of
5 μm was patterned with single pores (100 μm diameter) using a
conventional photolithography method and then fixed between
PMMA films (0.2 mm thick) using an adhesive bond (Super X,
Cemedine Co., Ltd., Tokyo, Japan). The films, including the parylene
film, were inserted into the chase to separate the wells.

temperature). Finally, Aβ42 was detected by chemiluminescence
using Immobilon western substrate (Merck Millipore, Tokyo,
Japan) with a Las4000 mini (Cytiva, Tokyo, Japan).

Preparation of planar lipid bilayer
in the microdevice

AFM imaging for Aβ42 aggregation

Channel current measurements and data analysis
The channel current was monitored using a Pico patch clamp amp­
lifier (Tecella, Foothill Ranch, CA, USA) connected to the chambers.
Ag/AgCl electrodes were already present in each droplet when thesolution was added to the chambers. A constant voltage of +100 mV
was applied to the recording chamber, and the ground chamber
was grounded. Pore formation in the lipid bilayer allowed ions topass through the nanopore under the voltage gradient, giving thechannel current signals. The signals were detected using an
8-kHz low-pass filter at a sampling frequency of 40 kHz. An analysisof channel current signals and duration time was performed using
Clampfit ver. 10.7 (Molecular Devices, San Jose, CA, USA) and Excel(Microsoft, Redmond, WA, USA) softwares. Channel current meas­
urements were conducted at 22 ± 2°C.

Western blotting
To assess the aggregation states of Aβ42 in the presence of lipid,
9.4-µL samples of Aβ42 (10 µM in PBS) and 4.6-µL lipid (DOPC,DOCP:Chol., DOPC:SM:Chol., or BTLE) were incubated in Protein
LoBind Tubes for 2 h at room temperature. Next, 3 µL of a sample
buffer solution with reducing reagent (6×) for SDS–PAGE buffer
was added to the Aβ42 solution and incubated on ice for 10 min.
The Aβ42 was separated in Any kD Mini-PROTEAN TGX Precast
Protein Gels (Bio-Rad) at a constant voltage of 150 V for 45 min us­
ing a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad
Laboratories, Inc., Hercules, CA, USA). The separated Aβ42 wastransferred onto nitrocellulose membranes at 21 A and 25 V for
3 min using the Trans-Blot Turbo Transfer System (Bio-Rad
Laboratories). Membranes were blotted for 1 h in a solution of
4% skim milk in PBS-T (phosphate-buffered saline containing
0.05% Tween-20) and washed in PBS-T for 5 min. 82E1 was used
as a primary antibody against Aβ42 (1:2,000 in PBS-T, 1 h atroom temperature), and HRP-conjugated antimouse IgG wasused as the secondary antibody (1:5,000 in PBS-T, 1 h at room

Sixty-microliter samples of Aβ42 (10 µM in PBS) and 30-µL lipid(DOPC, DOPC:Chol., DOPC:SM:Chol., or BTLE) were incubated inProtein LoBind Tubes for 1, 2, 3, 4, 5, 7, and 8 h at room tempera­
ture. The aggregation of Aβ42 was monitored by the ThT assay us­
ing a Varioskan Flash reader (Thermo Fisher Scientific, Inc.,
Waltham, MA, USA). The powdered ThT was dissolved in 0.1 N
HCl to a weight ratio of 0.5% and diluted with PBS buffer to a final
concentration of 20 µM. Fifty microliters of ThT were added to
50-µL samples of Aβ42. Fluorescence at 486 nm was measured
at an excitation wavelength of 450 nm. The blank fluorescence(PBS buffer) was subtracted.

For imaging the aggregation of Aβ42, 20-µL samples of Aβ42
(10 µM in PBS) and 10-µL lipid (DOPC, DOPC/Chol., DOPC/SM/
Chol., or BTLE) were incubated in Protein LoBind Tubes for 2 h atroom temperature. Two-microliter samples of Aβ42 solution
were deposited onto a freshly cleaved mica surface. After incuba­tion for 10 min at room temperature, the mica surface was gently
washed with PBS buffer. The AFM imaging of Aβ42 aggregations
was performed in PBS buffer using a high-speed AFM system(BIXAM, Olympus, Tokyo, Japan) with a silicon nitride cantilever(resonant frequency = 0.8 MHz, spring constant = 0.1 N/m, EBD
tip radius < 10 nm; USC-F0.8-k0.1-T12; Nanoworld, Neuchâtel,Switzerland) (35). The images of 320 × 240 pixels were obtained
at a scan rate of 0.2 fps for static images.

Addition of EGCG to Aβ42 embedded in the lipid
bilayer
The powdered EGCG was dissolved in DMSO to a final concentra­tion of 10 mM and stored at −80°C. Immediately before use, sam­ples were diluted in PBS buffer solution to 100 µM.
For measurements on EGCG addition, the microdevice with lar­ger wells was used, with 4 µL of lipid solution poured into eachchamber and 16 µL of Aβ42 (12.5 µM) solution subsequently added
to form a lipid bilayer. Approximately 30 min after the appearanceof the Aβ42 signal, 4 µL of EGCG (500 µM; control: 4 µL PBS buffer)
was added, and the measurement was continued for another30 min. The final concentrations of Aβ42 and EGCG were 10 and
100 µM, respectively.

Results
Comparison of current signals between the Aβ42
monomer and oligomer
We initially elucidated the difference between the channel current
signals for the Aβ42 monomer and oligomer. The Aβ42 monomer
was prepared, and the monomeric state was confirmed using WB.
A deep band at around the 4–8 kDa region was observed, indicating
the formation of the monomer (Mw = 4.5 kDa) configuration, and thepeak dimer band was also observed at around the 10 kDa region asshown in Fig. 2a. The channel current signals in the DOPC mem­brane represented in Fig. 2a increase gradually with a noise-like cur­rent shape. On the other hand, the oligomer, which was preparedfrom the monomer state (see Experimental section), presented
smeared and ladder-shaped bands in the high-molecular-weight re­gion in the WB (Fig. 2b). The molecular weight of the Aβ42 oligomer,

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Planar lipid bilayers were prepared by the droplet contact method(34) using a microdevice. First, PC, DOPC/Chol. (4:1 [w/w]), DOPC/
SM/Chol. (5:1:2 [w/w]), or BTLE (lipids/n-decane, 10 mg/mL) solu­
tion (2.3 µL) was poured into each chamber. Next, PBS buffer solu­
tion (4.7 µL) with Aβ42 (final concentration of 10 µM) was poured
into both chambers (28, 29). When the planar lipid bilayers rup­
tured, they were reconstituted as planar lipid bilayers by tracingwith a hydrophobic stick between two droplets.

ThT fluorescence measurement

4 | PNAS Nexus, 2024, Vol. 3, No. 1

which has strong cytotoxicity, is reported as 17 kDa (4-mer) to
90 kDa (20-mer). Our prepared oligomer showed similar molecularweight in the WB, and step-like currents were observed in the caseof oligomer current measurements (Fig. 2b).
We have previously proposed the classification of the current
signals of AMPs, which form the α-helical structure, into mainly
four types: spike, erratic, multilevel, and step (Fig. 2c and d) (29).
These four types of current signals were assigned to the mem­brane penetration or pore-formation models that have alreadybeen proposed based on spectroscopic experiments. We also as­
signed four current signals from the Aβ42 monomer/oligomer tofour models: direct penetration model (spike signal), random dis­
ruption model (erratic signal), toroidal model (multilevel signal),
and barrel-stave model (step signal) as depicted in Fig. 2c and d.
Because the Aβ42 and other β-sheet peptides are known to form
the toroidal or barrel-stave model pore in the lipid membrane(36), we consider that the observed step current reflects the rigid
pore formation of Aβ42 oligomers. The average duration of thecurrent step was 9.8 ± 2.7 min, encompassing the time required
for Aβ42 oligomers to reach the membrane and the subsequentpore formation (25). In contrast, we have not observed the current
signals of Aβ40 in our conditions.
Summarizing these results, the monomer mostly shows the
spike and erratic signals, and the oligomer mainly shows the mul­
tiple and step signals, reflecting the rigid channel formation in thelipid membrane. Therefore, in this study, we defined the spike anderratic signals as “monomer-induced signals”; the other two sig­
nals were assigned to “oligomer-induced signals.”

Real-time observation of Aβ42 oligomerizationin the lipid membrane
We next attempted to observe the oligomerization process of theAβ42 monomer in the DOPC membrane by the current signaltransition. To determine the time of measurement for the oligo­
merization, we performed fluorescence measurements using
ThT, which fluoresces when binding to the Aβ42 fibrils. The fluor­
escence intensity of ThT was measured in samples incubated at
37°C for 1, 2, 3, 4, 5, 7, and 8 h, respectively, and the intensity be­came higher in samples incubated for >4 h (Fig. S1). These results
imply that Aβ42 forms mature fibrils via the oligomerization state
after 4 h in these conditions. Next, we investigated the transitionof the current signal from the monomer-induced signal to theoligomer-induced signal, with a typical current recording shownin Fig. 3a. The monomer-induced signals were observed at theearly stage (0 to 60 min), and thereafter the oligomer-induced sig­
nals, such as step signals, appeared in the current trace (Fig. 3b).
After the measurements, we took the recording solution andchecked the oligomerization of the Aβ42 monomer in the record­ing chamber by WB, which resulted in gels with bands in thehigh-molecular-weight region (over 250 kDa, calculated to be>55 mer, at stacking gel; Fig. 3c). The aqueous solution in the re­cording chamber is surrounding the lipid monolayer, and it has
been reported that such a lipid environment may induce the oligo­
merization of the Aβ42 monomer. We also checked the oligomer­
ization of the Aβ42 monomer in a protein low-bind tube that hasaqueous and lipid/oil phases, similar to the recording condition.
After incubating for 2 h, several bands appeared between 75 and250 kDa, giving a similar result to the samples extracted fromthe recording chamber (Fig. S2).
The entire current signals were classified into the “monomerinduced” or “oligomer-induced” signal in accordance with previ­
ous classification methods (28, 29), and the appearance ratio of
each signal was calculated every 30 min, as shown in Fig. 3b.
The monomer-induced signal was observed mainly in the initialhour; after that, most of the signals suddenly transitioned to theoligomer-induced signal. This rapid transition occurred at around50 min (Fig. S3). This time was not contradictory to the report by

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Fig. 2. a) Typical signal for the measurement of the Aβ42 monomer (left) and WB analysis of the Aβ42 monomer (right). b) Typical signal for themeasurement of the Aβ42 oligomer (left) and WB analysis of the Aβ42 oligomer (right). Both current measurements were performed using 10 µM of Aβ42,a PC membrane, and an applied voltage of 100 mV. The typical signal shapes of the c) spike and erratic signals and d) multilevel and step signals. The
spike and erratic signals were assigned to monomer-induced signals, and the multilevel and step signals were assigned to oligomer-induced signals.

Numaguchi et al. |5

Choucair et al. (37) that the small aggregates of Aβ42 started to ac­
cumulate on the domains approximately after 85–90 min of incu­
bation with DOPC/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) bilayers. Next, the ion flux of Aβ42 channels/defects wasestimated. This value reflects the peak activity of ions throughthe transmembrane channel. The ion flux is the number of ionsflowing into the channel per unit time, and it is calculated fromthe area of the signal up to the limiting current value (5,000 pA;
Fig. 3d) (30). Figure 3e shows the results of the ion flux every 30

min. The value increased with time, suggesting that many or large
channels/defects were formed.

The channel-forming and aggregation activities
of Aβ42 in different lipid compositions
We next investigated the channel formation and aggregation ac­
tivities of Aβ42 (10 µM) in the four different compositions of lipid
membrane: [PC], [PC:Chol.], [PC:SM:Chol.], and [BTLE (porcine
brain extraction)]. The [PC] and [PC:Chol.] (=4:1 w/w) membranes
imitate a mammalian cell membrane. The [PC:SM:Chol.] (=5:2:1

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Fig. 3. a) Signal obtained from the channel current measurements of Aβ42 (10 µM) in a PC membrane for 2 h at room temperature (applied voltage:
100 mV). b) The ratio of monomer-induced and oligomer-induced signals is shown every 30 min. In each time zone, the number of oligomer-induced andmonomer-induced signals was counted, and the ratio of the number of each signal was calculated (n = 5; n, the number of 2-h measurements). c) The WB
analysis of Aβ42 before measurement (before) and Aβ42 collected after 2 h of measurement in the droplet chamber (after). The measurement conditions
were as follows: 10 µM of Aβ42 in a PC membrane and an applied voltage of 100 mV. d) Definition of ion flux: ion flux was calculated from the area between
the baseline and the current amplitude as colored in yellow. e) Value of ion flux every 30 min (n = 5; n, the number of 2-h measurements).

6 | PNAS Nexus, 2024, Vol. 3, No. 1

w/w) and [BTLE] membranes mimic the nerve cell membrane, andthe [PC:SM:Chol.] membrane should form lipid raft (domain)
structures in artificial lipid membranes (38). The effect of the lipid
raft can be examined using this system because Aβ42 is prone toaggregate around a lipid raft structure (39). We estimated thechannel-forming and aggregation activities using the channel
current recordings, WB, AFM, and the fluorescent intensity ofThT.
For the channel current recordings, the appearance ratio of theoligomer-induced signals from all observed signals over 2 h for thefour different membrane compositions is shown in Fig. 4a. The ra­tio follows the order: [PC:Chol.] ≃ [PC:SM:Chol.] > [BTLE] >> [PC].
To confirm the aggregation state of Aβ42 in each membranecomposition, the Aβ42 monomer was incubated for 2 h in a protein
low-bind tube with the aqueous/lipid oil solution of each mem­brane composition. The oligomerization/aggregation states werechecked by WB, AFM, and fluorescent measurement using ThT.
In the WB for each lipid membrane composition, smeared bands
were mainly observed ranging from 75 to 250 kDa, which is anoligomer region (Fig. 4b). Among them, the stronger bands inthis oligomer region were observed in the [PC:Chol.] composition
when using a semiquantitative analysis of the images (using
ImageJ software as shown in Fig. S4). In the ThT measurements,
the order of the intensity was [PC] ≃ [BTLE] > [PC:Chol.] ≃ [PC:
SM:Chol.] (Fig. 4c). The lower intensity in [PC:Chol.] and [PC:SM:
Chol.] conditions may suggest reduced protofibril formation. In

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Fig. 4. For each lipid composition, a) the ratio of oligomer-induced signals. Each signal obtained from the 2-h measurement was classified into a
monomer-induced or oligomer-induced signal. The average value of the percentage of oligomer signals analyzed for each signal was calculated (n = 5–6;
n, the number of 2-h measurements). Statistical analysis was performed using the Dunnett test. b) The WB analysis of Aβ42. c) Fluorescence intensity of
ThT bound to Aβ42 fibrils (n = 3). Statistical analysis was performed using the Holm test. d) The AFM image of Aβ42. The white areas are Aβ42. Scale bar:
200 nm. The concentration of Aβ42 was unified to 10 µM in all experiments.

Numaguchi et al. |7

Table 1. The estimated diameter of the Aβ42 channel under
membrane conditions for each lipid composition.
MembraneDiameter main peak (40)

Diameter subpeak (40)

[PC:Chol.][PC:SM:Chol.][BTLE]

1.3
1.2
1.3

1.9
3.7, 6.6
Not determined

The diameter was calculated from the Hille equation.

the AFM observations, nanosized aggregated particles were ob­served for each condition (Fig. 4d), and the number of particles
and their area were analyzed in each image (using ImageJ as
shown in Fig. S5). The average area of spherical Aβ42 aggregates
was not contradictory with a previous study with DOPC/DPPC bi­
layers (see more detail in Fig. S5) (37). Although a few fibril-likestructures were observed in the [PC] composition, Aβ42 incubatedwith the other compositions showed only spherical aggregates.
The number of Aβ42 aggregates in 1 µm² was the largest for the[PC:Chol.] composition. In the [BTLE] composition, the numberof aggregates and their area were larger than that for the othercompositions.

Analysis of the channel size and ion flux of Aβ42
oligomers
We next estimated the size of channels formed by the Aβ42
oligomers using the channel conductance of the step signals.
The diameter of the channel was calculated using the Hille equa­
tion:
􏼒
􏼓
V
πd 4ρ
R= = L+I
4 πd2
where R is resistance, V is applied voltage (V), I is current (A), L is
the length of the channel (m), ρ is ion conductivity (Ω m), and dis the diameter of the channel (m). Figure 5a shows the histogramof the channel diameter of Aβ42 for the [PC:Chol.] composition: 1.3
and 1.9 nm are the main peak and subpeak, respectively. The
channel size in the other membrane compositions is listed inTable 1. In the case of the [PC] composition, the channel diameter
could not be calculated because the step signal was not observed.

Next, we estimate the number of assembling monomers of Aβ42,
combined with the data from WB. The observed bands in the [PC:Chol.] composition were analyzed by their color intensity, result­ing in a single large peak at around 100 kDa (Fig. 5b). The numberof the assembling monomers can be calculated by the molecularweight of the monomer (4.5 kDa); this gives a 22-mer.
The ion flux in each membrane composition is shown in Fig. 5c.
The order of the ion flux is [PC:SM:Chol.] > [PC:Chol.] > [PC]> [BTLE].

Inhibition of the oligomerization of Aβ42 in lipid
membranes using EGCG
We next attempted to estimate the inhibition effect of the Aβ42
oligomerization in our lipid bilayer system. EGCG, which is themain catechin in green tea, was used in this experiment because
the high activity of the aggregation inhibition of Aβ42 has previ­
ously been reported (36, 41). Based on the previous experimental
conditions, we conducted and measured the current signal of
the Aβ42 monomer with 100 µM EGCG, incubating for 2 and 72 h
in the [PC:Chol.] membrane composition. The ion flux after incu­
bation with EGCG decreased dramatically, as depicted in Fig. 6a.
Particularly, for the 72 h incubation, the ion flux was reduced byaround 10% compared with the control experiment. EGCG itself
did not show any current signal (Fig. S8). To confirm that this phe­nomenon is also observed when Aβ42 is embedded in the lipid
membrane, we added EGCG after observing (around 30 min) the
oligomer-induced current signals. The current signal also de­creased after adding EGCG, as shown in Fig. 6b. In addition, theion flux was also reduced upon EGCG addition vs. the control ex­periment (Figs. 6c and S8).

Discussions
Real-time and long-term observation of Aβ42
oligomerization using the lipid bilayer system
which provides a useful tool for observing Aβ42
dynamics with lipid membranes
Oligomerization, or crystallization prenucleation, is notoriously
difficult to detect experimentally. They are expected to have

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Fig. 5. a) Histogram of diameter values of the Aβ42 channel in the [PC:Chol.] membrane calculated from the Hille equation. Histograms under all lipid
conditions are shown in Fig. S6. b) The WB analysis. The band of Aβ42 (10 µM) in [PC:Chol.] (left) and the plot of band density using ImageJ (right). The photoof the whole band is shown in Fig. S7. c) Ion flux value of Aβ42 (10 µM) for each lipid composition; the average of the ion flux values was calculated from thesignals obtained over a 2-h measurement (applied voltage: 100 mV). The ion flux of each signal was the average of the ion flux values every 5 min (n = 5–6;
n, the number of 2-h measurements). Statistical analysis was performed using the Steel–Dwass test.

8 | PNAS Nexus, 2024, Vol. 3, No. 1very short lifetimes, and the populations are inherently limited.
To date, although ThT and AFM experiments have used alter­nate approaches to monitor the kinetics or dynamics of oligo­
merization, it has remained a challenge (42, 43). Several
studies have reported the channel current measurement ofAβ42 using a planar lipid bilayer, which is formed with theMontal–Mueller or painting methods (15, 25). Although thesemethods are well known as the conventional methods for lipid
bilayer formation, the bilayer mechanical stability has been aserious issue for long-term experiments. The previous reports
therefore showed mainly the channel conductance anddid not mention the kinetic behavior of Aβ42, such as themonomer-to-oligomer transition. This is probably due to the is­sue of the low mechanical stability of the lipid membrane. On
the other hand, in this study, we conducted all the channel cur­rent measurements for 2 h and compared them with each data­set, including the ion flux and signal classification. The ratio of
the oligomer-induced signals and the ion flux values increased
with time (Fig. 3b and d), suggesting that Aβ42 formed channels
upon oligomerization in the membrane. This result is also sup­ported by the WB; the bands appeared in the oligomer region
after the 2-h measurement (Fig. 3c). These results suggest that
we observed the process of aggregation of the Aβ42 monomer
into the oligomer and its channel formation around 60 min.

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Fig. 6. a) Ion flux value of Aβ42 (10 µM) for each condition; the average of the ion flux values was calculated from the signals obtained over 2-hmeasurements. The ion flux of each signal was the average of the ion flux values every 5 min (n = 4–5; n, the number of 2-h measurements). Statisticalanalysis was performed using the Steel–Dwass test. b) The signals were observed when the solution (PBS buffer or 100 µM of EGCG solution) was added
after the appearance of the Aβ42 signal. “before” was the signal before the solution was added, and “after” was the signal after the solution was added.
Less than 5% of the DMSO solution (final conc.) did not interfere with the lipid membrane in the current measurements (Fig. S9). c) Comparison of ion flux
values of Aβ42 before and after the addition of solution with Welch's t test. The ion flux value of the signal was calculated for 25 to 40 min before and after
adding the solution, respectively. The ion flux of each signal was the average of the ion flux values every 2 min (n = 3–4; n, the number of measurements).

Numaguchi et al. |Moreover, the lipid molecules that surround the aqueous phaseshould also have a strong effect on the oligomerization, as pre­viously mentioned.

Chol. induces Aβ42 oligomerization in the lipid
membrane

The channel size and ion flux of the Aβ42 oligomer
The Aβ42 monomers assemble to form the oligomer channel in aplanar lipid bilayer, resulting in the appropriate current signals(53). The size of the channel can be estimated by the conductanceof the step signals for each membrane condition (Fig. 5). In thecase of the PC + Chol. membrane, the diameter is 1.3 nm, withmonomers assembling as the 22-mer. Although the histogram of
the channel diameter showed several different peaks dependingon the lipid composition (Table 1), the highest peak was common­ly observed around 1.3 nm. This channel size may be the most sta­
ble state to assemble the Aβ42 monomers in the lipid membrane.
Besides, this value corresponds well with previous reports esti­
mated by patch clamping of living nerve cells (1.7, 2.1, and
2.4 nm) (25) and MD simulation (1.9 nm, 18-mer) (27).
The ion flux may directly reflect the membrane disruption–in­
duced cytotoxicity. The value of the ion flux in the [PC:Chol.] and[PC:SM:Chol.] membranes was relatively high (Fig. 6c). Also, theamount of the oligomer-induced current signals increased inthese systems. Combining these results, it seems that thechannel-forming activity of Aβ42 is high in the [PC:Chol.] and[PC:SM:Chol.] membranes. On the other hand, the ion flux in the[BTLE] membrane was low, while the binding affinity between
Aβ42 and the membrane may be high due to [BTLE] consisting ofa negatively charged PS. While the reason for the low ion flux inthe [BTLE] membrane is still under consideration, Aβ42 formed
the channel with only a 1.3-nm diameter; therefore, the amountof ion flux was relatively low.

Inhibition of the oligomerization when adding
EGCG
Since the ion flux decreased with the prolonged incubation ofAβ42 with EGCG (Fig. 6a), EGCG may inhibit the channel formationof the Aβ42 oligomers. DMSO, used as a solvent for EGCG, was alsopresent in the measuring solution prior to the addition of EGCG,
suggesting that DMSO is less likely to inhibit the channel forma­tion of Aβ42. Furthermore, the flux also decreased even when
the EGCG was added after inserting the Aβ42 monomers into thelipid membrane (Fig. 6b and c). These results suggest that EGCG
inhibits the oligomerization of Aβ42 not only in the aqueous phase
but also in the membrane. We therefore considered the configur­
ation of Aβ42 in the lipid bilayer (54, 55).
The EGCG molecule binds to H14, Y10, and K28 of a tetramer ofAβ42 with π–π (H14, Y10) or cation–π (K28) interaction in solution,
resulting in the inhibition of aggregation (31, 56). On the other
hand, in the case of inhibition in the membrane phase, EGCG
may bind the outer-membrane region of Aβ42 from the aqueous
phase, because EGCG is relatively insoluble in the hydrophobic
lipid phase. The MD simulation of Aβ42 in the lipid membrane de­picts that Aβ42 monomers assemble to form β barreled structures,and the hydrophobic N termini are exposed to the outermembrane region (57). Also, the H14 and Y10 are set near the N
terminus. Therefore, EGCG may bind around the N terminus tocollapse the oligomerization state.

Conclusion
In summary, we prepared lipid bilayers that imitate neuronal cell
membranes and evaluated the channel-forming activity of Aβ42
in the membranes by electrophysiological methods. Current
measurements demonstrate that the Aβ42 aggregated frommonomers to oligomers and formed channels in the membrane.
In addition to the current measurements, the results of WB ana­
lysis, ThT fluorescence intensity, and AFM observation suggestthat Chol. was a key component that promoted the Aβ42 oligomer­
ization, resulting in the formation of the channel structure. The
current measurements and WB bands showed that the Aβ42
oligomers formed channels with a diameter of ∼1.3 nm (22 mer),consistent with previous studies (27). This size of oligomer forms
a relatively stable channel in the lipid membrane and would in­duce the cytotoxicity. Furthermore, current measurements using
the inhibitor EGCG showed that the addition of EGCG from outsideof the membrane decreased the channel-forming activity of both
the Aβ42 monomer and oligomer.
These results shed light on the monomer-to-oligomer transi­tion and the channel formation of Aβ42 with several different
types of lipid membranes, such as a composition imitating thenerve cell membrane. Although the relationship between AD
and Aβ42 oligomerization is still unclear and involves more com­plex factors, the current measurement used in this study is apromising method for understanding the molecular assembly
mechanism while imitating several types of lipid systems.

Acknowledgments
The authors thank K. Shimizu and A. Tada for their help in per­forming some experiments.

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Using our 2-h measurements, we can estimate the real-time Aβ42
oligomerization with a lipid membrane. We next investigatedwhat the most significant molecules were for Aβ42 oligomeriza­tion by using different membrane compositions: [PC], [PC:Chol.],[PC:SM:Chol.], and [BTLE (porcine brain extraction)]. Through sev­
eral experiments such as current recording, WB, ThT, and AFM
observation, we conclude that Chol. is the most significant factor
for the Aβ42 oligomerization in the membrane. The ratio of theoligomer-induced signal in both [PC:Chol.] and [PC:SM:Chol.] con­ditions are high (Fig. 4a), as in previous studies, which reported
that Chol. may induce the frequency of channel formation (44–46). It has previously been reported that the cytotoxicity induced
by Aβ42 drastically increased in the presence of Chol. in lipid
membranes (47, 48). Similarly, the ion flux, which reflects thepore-forming activity, was increased in the presence of Chol.
The other experiments also support the strong effect of Chol.; inthe [PC:Chol.] condition, bands are observed ranging from 75 to
250 kDa in the WB (Figs. 4b and S4), and there are many aggrega­tion particles under AFM observation (Figs. 4d and S5) (24, 49).
Besides, the intensity of ThT fluorescence in the [PC:Chol.] condi­tion is lower than that for the other conditions (Fig. 4c), suggesting
reduced protofibril formation (50).
The kinetic model of the oligomerization and fibrillization ofAβ42 has recently been proposed as [monomer] ⇆ [oligomer] ⇆[fibril] (51). Also, it has already been reported that the Aβ42 has
a Chol.-binding domain (Aβ22–35) (52). Combining these facts,
the Aβ42 monomer may bind to Chol. and form the “oligomer
channel”; however, it may be difficult for the formed oligomers
to proceed to form the fibril structure when the lipid membrane
has Chol., as summarized below:[PC:Chol.] or [PC:SM:Chol.]: [monomer] → [channel oligomer][PC] or [BTLE]: [monomer] → (protofibril) → [fibril]

9

10 |

PNAS Nexus, 2024, Vol. 3, No. 1

Supplementary Material
Supplementary material is available at PNAS Nexus online.

Funding
This work was partially supported by Ministry of Education,
Culture, Sports, Science and Technology KAKENHI [Grant
Number JP21H05229] and Japan Science and Technology Agency,
CREST [Grant Number JPMJCR21B2].

Y.N., K.T., and R.K. conceived the original idea. Y.N. and K.T. con­ducted the experiments, with the exception of the AFM measure­ments. Y.S. performed the AFM measurement. Y.N., K.T., N.T.,
Y.S., and R.K. wrote the manuscript. K.I. and R.K. supervised thisproject.

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Data Availability
All data are included in the manuscript and/or supplementarymaterial.

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