Plantainoside B in Bacopa monniera Binds to Aβ Aggregates Attenuating Neuronal Damage and Memory Deficits Induced by Aβ

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-β (Aβ) accumulation and neuronal loss. Particularly, cholinergic neurons in the nucleus basalis of Meynert are some of the first neuronal groups to degenerate; accumulating evidence suggests that Aβ oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aβ-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aβ aggregates, including its oligomers, using Aβ oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aβ attachment on the cell surface of human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. Moreover, it attenuated memory impairment in mice that received intrahippocampal Aβ injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aβ aggregates including its oligomers and brain tissue from a mouse model of Aβ pathology. In addition, plantainoside B could delay the Aβ aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aβ oligomers, thus interrupting the binding of Aβ oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.

INTRODUCTION

Alzheimer’s disease (AD) is the most common form of dementia. Typical pathological features characteristic of AD brain include senile plaques formed by accumulation of amyloid-β (Aβ) protein and neuronal loss. In particular, it has long been suggested that cholinergic neurons in the nucleus basalis of Meynert, a basal forebrain structure, are impaired in the early pathological stages of AD.^1,2) This finding led to the development of several acetylcholinesterase inhibitors for symptomatic treatment of AD, which are now in clinical use. However, these drugs do not provide a fundamental solution; therefore, disease-modifying therapies (DMTs) for AD that can directly affect the pathogenic mechanisms of AD and offer neuroprotection are needed. To develop DMTs, adequate disease models are needed for the elucidation of AD pathophysiology and drug screening. In addition, diagnostic tools that can objectively evaluate disease progression and treatment efficacy by capturing changes at the molecular level are desired.

Differentiation methods from human induced pluripotent stem cells (hiPSCs) toward basal forebrain cholinergic neurons have recently been developed.^3–5) These hiPSC-derived basal forebrain cholinergic neurons could be used as a reasonable AD neuronal model. The hiPSC technology allows us to conduct research on actual human cells, which was previously limited to alternative analyses using transformed cell lines and model animals.⁶⁾ Furthermore, by adding pathogenic substances or using iPSCs derived from patients with a genetic disease, the disease can be reproduced to understand the pathogenesis and effectively screen for drugs in vitro. Therefore, this approach is expected to revolutionize the development of treatment and diagnostic methods for central nervous system (CNS) disorders.^7,8)

In support of the amyloid cascade hypothesis, a vast amount of evidence suggests that Aβ is the responsible for AD pathogenesis.⁹⁾ In fact, lecanemab, an anti-Aβ oligomer antibody, is showing promise for improving cognitive decline by reducing the amount of Aβ in the brain of AD patients.¹⁰⁾ Aβ is produced from Aβ protein precursor (AβPP) through serial cleavage by β-and γ-secretases.¹¹⁾ Aβ_1–42 and Aβ_1–40, are the two major components of amyloid plaques; Aβ_1–42 is characterized by its tendency to aggregate.¹²⁾ Aβ self-assembles during the aggregation process and becomes mature fibrils through oligomers. Various Aβ oligomer forms with demonstrated neurotoxicity^13,14) have been reported,¹⁵⁾ such as Aβ-derived diffusible ligands (ADDLs < 53 kDa),¹⁶⁾ oligomers composed of 15–20 monomers (AβOs < 90 kDa),¹⁷⁾ or protofibrils (24–700-mer).^18,19) Although the detailed mechanisms on Aβ oligomer related neurotoxicity remain unknown, the Aβ oligomer hypothesis is based on the findings of Aβ oligomers in the AD brain, which are key mediators of synaptic impairment and cognitive dysfunction in AD.^13,20–22) Thus, Aβ oligomers are currently considered attractive targets for the development of DMTs and diagnostic tools in AD.¹⁰⁾ However, because Aβ_1–42 is extremely aggregative, it is difficult to handle the transient Aβ oligomers appearing during Aβ aggregation.²³⁾ To resolve this issue, O-acyl isopeptide Aβ_1–42 (isoAβ) can be used to strictly control Aβ aggregation from monomers to fibrils and reproducibly generate Aβ oligomers with high neurotoxicity.^24,25) When applying the depsipeptide method to the synthesis of Aβ_1–42,²⁶⁾ isoAβ can be produced as close to the monomeric seed–free state as possible, without fibrils or aggregates.^27,28) The isoAβ possesses an O-acyl residue at the Gly25–Ser26 sequence and maintains its monomeric state without aggregation under acidic conditions. On changing the pH of the solvent from acidic to neutral, e.g., from trifluoroacetic acid (TFA) to neutral cell culture medium, isoAβ is converted to the native Aβ_1–42 sequence and immediately initiates self–aggregation.

Bacopa monniera (L.) Pennell (Scrophulariaceae) [syn. Bacopa monnieri (L.) Wettst. (Plantaginaceae)] was first described as acting on CNS in Charaka Samhita, one of the oldest texts of Ayurveda, the ancient Indian medical system, and has been used for centuries as a memory enhancer.^29,30) Modern research has demonstrated that B. monniera extract can reduce Aβ levels in the brain of a mouse model of amyloid pathology without altering fibrillar amyloid deposition³¹⁾ and attenuate Aβ_25–35 induced cell death in rat primary cultured cortical neurons due to reduced intracellular oxidative stress.³²⁾ Therefore, there might be some compounds in B. monniera extract that act on Aβ, particularly on its oligomers, to exert neuroprotection.

The present study aimed to identify compounds from B. monniera extract that could bind to Aβ oligomers and show neuroprotective properties. To this end, an Aβ oligomer-induced neurodegeneration model was established using hiPSC-derived cholinergic neurons and isoAβ. We identified plantainoside B, a low molecular weight compound with an affinity for Aβ aggregates, in B. monniera extract. Plantainoside B showed neuroprotection in the Aβ oligomer-induced neurodegeneration model and attenuated Aβ-induced memory deficit in mice. Moreover, the analysis using radioisotope-labeled plantainoside B revealed its binding affinity to Aβ aggregates as well as oligomers and to the brain tissue obtained from a mouse model of amyloid pathology. Thus, the Aβ oligomer binding capacity and neuroprotective effects of plantainoside B indicate its potential for use as a theranostics tool for AD, simultaneously diagnostic and therapeutic drugs, and may accelerate the development of DMTs.

MATERIALS AND METHODS

Cells

Human SH–SY5Y neuroblastoma cells (ATCC, Manassas, VA, U.S.A.) were seeded at density of 1.0 × 10⁴ cells/cm² on 10 cm plates and maintained in Eagle’s minimum essential medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)/Ham’s F–12 (FUJIFILM Wako Pure Chemical Corporation) (1 : 1) containing penicillin–streptomycin solution (1×, FUJIFILM Wako Pure Chemical Corporation), non-essential amino acids solution (NEAA, 1×, FUJIFILM Wako Pure Chemical Corporation), L-glutamine solution (1×, FUJIFILM Wako Pure Chemical Corporation), and 15% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, U.S.A.) in an incubator set at 37 °C, 5% CO₂, and 95% air.

The human induced pluripotent stem cells (hiPSCs, line 1231A3) used in this study were provided by the RIKEN Bioresource Research Center (Tsukuba, Japan).³³⁾ The hiPSCs were maintained as previously described.³⁴⁾ Briefly, a 6-well-plate was coated with iMatrix-511 silk (Nippi, Tokyo, Japan), feeder-free hiPSCs were seeded at a density of 1.5 × 10⁴ cells/well on the plate, and maintained in essential 8 (E8) medium (Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 10 µM Y–27632 (Selleck Chemicals, Houston, TX, U.S.A.) for the first day. Y–27632 was removed from the culture medium from day 2 onwards. The experimental protocols including hiPSCs were approved by the Ethical Review Committee for Medical and Health Research Involving Human Subjects at Kyoto Pharmaceutical University (Approval No. 20-18-26).

Generation of Cholinergic Neurons from hiPSCs

The hiPSCs were seeded at a density of 5.0 × 10⁶ cells/well on iMatrix silk-coated 6-well-plate in E8 medium containing 10 µM Y–27632 and penicillin–streptomycin solution (1×) and cultured for 2 d. Differentiation toward cholinergic neurons began on the next day (day 0) using essential 6 (E6) medium containing a penicillin–streptomycin solution (1×), NEAA (1×), GlutaMAX (1×, Thermo Fisher Scientific), and a 2-mercaptoethanol solution (100 µM, FUJIFILM Wako Pure Chemical Corporation) as a basal medium. From day 0, cells were cultured with the E6 basal medium containing 10 µM Y–27632, 200 nM LDN193189 (Selleck Chemicals), 500 nM A83–01 (FUJIFILM Wako Pure Chemical Corporation), and 2 µM XAV939 (Selleck Chemicals). Purmorphamine (1 µM) was further added from day 1, and Y–27632 was removed from day 3. From days 5 to 11, the E6 basal medium was gradually replaced with neurobasal medium (Thermo Fisher Scientific) supplemented with B27 supplement vitamin A minus (1×, Thermo Fisher Scientific), GlutaMAX (1×, Thermo Fisher Scientific) including penicillin–streptomycin solution (1×), 200 nM LDN193189, 500 nM A83–01, 1 µM purmorphamine, and 2 µM XAV939. A83–01 was removed from the basal medium on day 6. From day 11, the cells were further maintained in culture medium without purmorphamine. On day 16, cells were detached with TrypLE (1×, Thermo Fisher Scientific), re-suspended and replated at a density of 1.0 × 10⁶ cells/cm² on culture plates coated with poly-L-ornithine hydrobromide (Sigma-Aldrich), iMatrix silk, and fibronectin (1 µg/mL; FUJIFILM Wako Pure Chemical Corporation) in neurobasal medium supplemented with B27 supplement vitamin A minus (1×), GlutaMax (1×), penicillin–streptomycin solution (1×), 5 µM SU5402 (Selleck Chemicals), 1 µM PD0325901 (Selleck Chemicals), 200 µM L-ascorbic acid, (Sigma-Aldrich), 10 µM DAPT (Selleck Chemicals), 20 ng/mL brain–derived neurotrophic factor (Peprotech, Rocky Hill, NJ, U.S.A.), and 10 µM Y–27632. Y–27632 was added for the first 24 h after replating. The medium was changed daily until day 15 and then every 3–4 d from day 16 onwards.

Animals

Wild-type C57BL/6 mice (10-week-old) were purchased from Oriental Bio Service (Kyoto, Japan). APdE9 transgenic mice, a mouse model of amyloid pathology, harboring transgenes for mutant Aβ precursor protein (AβPPswe: KM594/5NL) and presenilin 1 (dE9: deletion of exon 9), were originally purchased from the Jackson Laboratory (Bar Harbor, ME, U.S.A.). The transgenic mice were bred by mating with wild-type C57BL/6 mice. All mice were maintained at 25 °C under a 12-h light/dark cycle with free access to food and water at the Bioscience Research Center at Kyoto Pharmaceutical University. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Animal Research at Kyoto Pharmaceutical University (Approval No. DIPS-19-001).

Aβ Preparation and Aggregation

The isoAβ (Peptide Institute Inc., Osaka, Japan) was dissolved in 0.1% TFA (FUJIFILM Wako Pure Chemical Corporation) and centrifuged at 20000 × g, 4 °C for 1 h. The protein concentration in the collected supernatant was determined by measuring absorption spectrum at 280 nm with a micro volume spectrophotometer (NanoPhotometer NP80, Implen, München, Germany). This diluted isoAβ stock (150 µM) was stored at –80 °C until further use. For Aβ aggregation, isoAβ was diluted with the culture medium for SH–SY5Y cells (containing 1% FBS) or phosphate-buffered saline (PBS) to a concentration of 6 µM and incubated at 37 °C.

Plant and Natural Product Material

The methanolic (MeOH) extract of B. monniera was prepared in a previous study.³⁵⁾ As described, dried whole plants of B. monniera cultivated in India were purchased from NTH India Pvt. Ltd. (2013) and identified by author H.M. A plant specimen is on file in Department of Pharmacognosy, Kyoto Pharmaceutical University [BM–NTH–1].

Isolation of an Aβ Binding Compound, Plantainoside B, from B. Monniera

Beads (Cytometric Bead Array Functional Bead A9, BD Biosciences, San Jose, CA, U.S.A.) used for isolation of Aβ binding compounds from the B. monniera extract were prepared as previously reported.³⁶⁾ Briefly, bead activation by labeling with an SH group was performed with 1 M dithiothreitol (DTT). In contrast, isoAβ (6 µM) was preliminarily incubated in PBS for 1 or 24 h and filtered (10 kDa, Amicon Ultra centrifugal filters, Merck Millipore, Darmstadt, Germany) to condense the aggregated Aβ. The free NH₂ group in the aggregated Aβ (100 µL) was bounded to N-2[(4–maleimidomethyl)cyclohexylcarbonyloxy]sulfosuccinimide (sulfo–SMCC) (2 µL, 2 mg/mL in distilled water) at room temperature for 1 h. The SH group-labeled beads were resuspended in Coupling Buffer in Functional Bead Conjugation Buffer Set (20 µL, BD Biosciences, CA, U.S.A.) were mixed with sulfo-SMCC-conjugated Aβ aggregates (100 µL) and shaken for 1 h to allow the beads to bind to the Aβ aggregates. N-Ethylmaleimide was added to mask the remaining SH groups on the beads and shaken for another 15 min at room temperature. The beads conjugated with Aβ aggregates were then washed with Storage Buffer in Functional Bead Conjugation Buffer Set (BD Biosciences) and centrifuged at 900 × g for three min to remove the unbound Aβ. All reactions were performed in the dark.

The MeOH extract (100.0 g) was partitioned into an ethyl acetate (EtOAc)–H₂O (1 : 1, v/v) mixture to obtain an EtOAc–soluble fraction (15.8 g, 15.8%) and H₂O–soluble fraction (84.2 g, 84.2%). The EtOAc–soluble fraction (10 mg) was dissolved in 1 mL distilled water containing 2% DMSO and centrifuged at 10000 × g for 10 min. The resulting EtOAc–soluble fraction was collected and filtered (Cosmonice filter, 0.45 µm, Nacalai Tesque, Kyoto, Jajpan) to remove the insoluble components. The EtOAc–soluble fraction of B. monniera extract and the beads conjugated with Aβ aggregates were mixed and incubated for 1 h in the dark at room temperature; the beads were collected after centrifugation at 900 × g for three min. To separate the bound compounds, ethanol was added to the beads and placed in boiling water for five min. The identification of the compounds binding to Aβ aggregates was performed by LC/MS using a tandem quadrupole mass spectrometer (LCMS–8040, Shimadzu Corporation, Kyoto, Japan) equipped with Cosmosil MS–II column (5 µm particle size, 2.0 mm i.d. × 150 mm, Nacalai Tesque) and LabSolutions LCMS Ver.5.6 software (Shimadzu Corporation). For LC, water (pH adjusted to 3.0 using formic acid) and acetonitrile (MeCN) were utilized as mobile phases; the gradient started at 10% and increased to 100% after 1 h. MS conditions were optimized as follows: nebulizer gas flow, 3 L/min; drying gas flow, 15 L/min; desolvation line temperature, 250 °C; heat block temperature, 400 °C.

The above analysis identified plantainoside B as an Aβ binding compound in the EtOAc–soluble fraction of B. monniera. For isolation, the EtOAc–soluble fraction (14.0 g) was subjected to normal phase silica gel column chromatography [840 g, CHCl₃:MeOH (30 : 1 → 15 : 1 → 7 : 1 → 5 : 1, v/v) → MeOH] resulting in ten fractions [Fr. E1 (2.2 g), Fr. E2 (1.6 g), Fr. E3 (0.3 g), Fr. E4 (0.4 g), Fr. E5 (0.9 g), Fr. E6 (1.5 g), Fr. E7 (4.8 g), Fr. E8 (0.7 g), Fr. E9 (0.8 g), Fr. E10 (0.9 g)]. Fr. E7 (1.1 g) was purified by HPLC [MeCN:H₂O:acetic acid (CH₃COOH) = 14 : 86 : 0.3, Cosmosil MS–II column (5 µm particle size, 10 mm i.d. × 250 mm, Nacalai Tesque)] to produce plantainoside B (45.9 mg, 0.228%). Plantainoside B was identified by comparing the measured physical data (MS and ¹H- and ¹³C-NMR spectra) and reference data.³⁷⁾ NMR was performed using an NMR spectrometer [JEOL JNM–ECA600 (600 MHz), JEOL Ltd., Tokyo, Japan]. All chemicals used in above processes were purchased from Nacalai Tesque.

Blue Native–Polyacrylamide Gel Electrophoresis (BN–PAGE)

The isoAβ aggregation was analyzed by BN–PAGE. Aβ aggregates produced after the incubation of isoAβ (6 µM) in the presence or absence of plantainoside B (100 µM) in culture medium or PBS at 37 °C for 1–72 h were mixed with a NativePAGE Sample Buffer (Thermo Fisher Scientific). The samples were then subjected to BN–PAGE (4–16% Bis–Tris mini protein gels; Thermo Fisher Scientific). After electrophoresis, immunoblotting was performed by transferring the proteins to a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was incubated with Tris-buffered saline containing 0.05% Tween-20 (TBST) and Bullet Blocking One for Western blotting (Nacalai Tesque) to block nonspecific binding. The PVDF membrane was incubated with a primary antibody against Aβ (clone 6E10, 1 : 2000, Biolegend, San Diego, CA, U.S.A.) dissolved in Bullet Immunoreaction Buffer (Nacalai Tesque), followed by a horseradish peroxidase (HRP)-linked secondary antibody against mouse immunoglobulin G (IgG) (1 : 2000, Thermo Fisher Scientific) dissolved in Bullet Immunoreaction Buffer (Nacalai Tesque). HRP activity on the PVDF membrane was visualized with Chemi–Lumi One Super (Nacalai Tesque) and detected by a lumino–image analyzer (ImageQuant LAS500 system, GE Healthcare Life Science, Pittsburgh, PA, U.S.A.).

Aβ Cytotoxicity Assay

SH–SY5Y cells were seeded at a density of 3.0 × 10³ cells/well on a 96–well plate and cultured for 7 d, whereas hiPSC-derived cholinergic neuron progenitors at day 16 were seeded at a density of 1.5 × 10⁵ cells/well on an iMatrix silk-coated 96–well plate and further cultured for 8–12 d. The culture medium of SH–SY5Y cells was replaced by medium containing 1% FBS before isoAβ treatment. Cells were treated with isoAβ (1–6 µM) in the presence or absence of plantainoside B (3–30 µM) for 48 h and Aβ cytotoxicity was analyzed through cell viability with the WST–8 assay and cytotoxicity with lactate dehydrogenase (LDH) assay. Both assays were performed using Cell Count Reagent SF (Nacalai Tesque) and LDH cytotoxicity assay kit (Nacalai Tesque) according to manufacturer’s instruction, respectively. In the LDH assay, the value obtained for the sample with cells lysed using the provided lysate reagent (Triton X-100) was set to 100% as the high control.

Immunocytochemical Analysis

Cells were fixed in PBS containing 4% paraformaldehyde (PFA) for 30 min at 4 °C, then washed three times with PBS. The primary antibodies used in this study, Aβ (mouse, clone 6E10 1 : 500, Biolegend), NKX2.1 (rabbit, 1 : 500, Abcam, Cambrige, U.K.), FOXG1 (rabbit, 1 : 500, Abcam), ISLET–1 (rabbit, 1 : 500, Abcam), ChAT (goat, 1 : 250, Millipore), MAP2 (chicken, 1 : 1000, Abcam; mouse, 1 : 500, Sigma-Aldrich), and LHX8 (rabbit, 1 : 200, Thermo Fisher Scientific), were diluted in PBS containing 0.1% Triton X (PBST) and 5% donkey serum (Jackson ImmunoResearch) and incubated with fixed cells at 4 °C overnight. After washing with PBST, secondary antibodies (1 : 500 each): Alexa Fluor 488-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific), Alexa Fluor 555-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific), Alexa Fluor 647-labeled anti-rabbit IgG antibody (Abcam), Alexa Fluor 488-labeled anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 555-labeled anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 647-labeled anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 555-labeled anti-goat IgG antibody (Thermo Fisher Scientific), and Alexa Fluor 488-labeled anti-chicken IgY antibody (Abcam) in PBST were incubated with the cells for 2 h at room temperature in the dark. Hoechst 33342 (1 : 4000, Thermo Fisher Scientific) and rhodamine–phalloidin (1 : 3000, Thermo Fisher Scientific) for staining the nuclei and actin cytoskeleton, respectively, were added in the secondary antibody solution. Fluorescence was detected with a confocal laser scanning microscope (LSM800, Zeiss, Oberkochen, Germany).

Calcium Imaging

On day 16, hiPSC-derived cholinergic neuron progenitors were seeded at a density of 3.0 × 10⁵ cells/well in a 4-well plate (Matsunami, Osaka, Japan) coated with iMatrix silk and cultured for another 8–10 d. Cells were treated with fluo–4 AM (10 µM, Thermo Fisher Scientific), a fluorescent calcium indicator dye, incubated at 37 °C for 30 min; then, the culture was replaced with fresh culture medium without fluo-4 AM. The isoAβ (3 µM) with or without plantainoside B (30 µM) was added to the cells. Following incubation for 2 h, the cells were placed in an incubation chamber at 37 °C, 5% CO₂, and 95% air for microscopic analysis, and time–lapse imaging was performed to detect fluo-4 fluorescence with a confocal laser scanning microscope (LSM800). Fluorescent images were acquired every five min for 3 h.

Measurement of Mitochondrial Membrane Potential (MMP) by JC–1 Analysis

MMP was analyzed using the JC–1 dye (JC–1 MitoMP Detection Kit, Dojindo, Kumamoto, Japan). JC–1, as a cationic fluorescent dye, exhibits potential–dependent accumulation in the mitochondria, forming aggregates (red fluorescent). Upon depolarization, JC–1 diffuses across the mitochondria in monomeric state (green fluorescent).³⁸⁾ Thus, a higher ratio of red/green fluorescence indicates that the mitochondria are energetically active, whereas a lower ratio indicates MMP loss.

The hiPSC-derived cholinergic neurons were seeded as in the cytotoxicity assay and treated with isoAβ (3 µM) with or without plantainoside B (3–30 µM) for 48 h. After washing with new culture medium, JC–1 dye (2 µM in final concentration) was added to the cells and incubated at 37 °C, 5% CO₂, and 5% O₂ for 30 min. After several washes, culture medium was replaced by the Imaging Buffer provided in the assay kit, and green and red fluorescence were measured with filter sets of excitation (475 nm)/emission (500–550 nm) and excitation (520 nm)/emission (580–640 nm), respectively using a fluorescence plate reader (GloMAX, Promega, Madison, U.S.A.). The red/green fluorescence ratio was calculated afterwards.

Thioflavin-T (ThT) Fluorescence Assay

The isoAβ (6 µM) was dissolved in PBS in the presence or absence of plantainoside B (100 µM) and incubated at 37 °C, 5% CO₂, and 95% air for 1–24 h. After incubation, ThT (10 µM in final concentration) was added to the samples and the fluorescence spectra obtained under excitation at 485 nm were recorded using a fluorescence spectrophotometer (F–7000, Hitachi, Tokyo, Japan).

Atomic Force Microscopy (AFM)

Aβ aggregation was analyzed by AFM. The isoAβ (6 µM) was dissolved in PBS with or without plantainoside B (100 µM) in Protein LoBind Tubes (Eppendorf, Hamburg, Germany) and incubated at 37 °C, 5% CO₂, and 95% air for 24 h. After incubation, Aβ aggregates were diluted with distilled water (1 : 10). The Aβ solution (20 µL) was spotted onto freshly cleaved mica (Nilaco Corp., Tokyo, Japan) and incubated for 10 min at room temperature. After washing the mica with distilled water, images were obtained under ambient conditions at room temperature using Nanoscope IIIa Tapping Mode AFM (Veeco Instrument, Plainview, NY, U.S.A.) and a single–crystal microcantilever (OMCLAC160TS–R3, Olympus, Tokyo, Japan). At least three regions of each sample were analyzed.

Image Analysis

For the measurement of the Aβ covered area on the cell surface, confocal laser scanning microscope (LSM800) Z-stack images were analyzed, and Aβ attached to the cells was measured using an image analysis software (Photoshop, version 22.2.0., Adobe, San Jose, CA, U.S.A.) using the fluorescence of actin and Aβ detected by rhodamine–phalloidin and anti-Aβ antibody as reference, respectively. The percentage of Aβ covered area on the cell surface was then calculated.

The number of cells expressing cholinergic markers, such as LHX8, ISLET-1, and ChAT, was counted using an image analysis software (Photoshop) for the confocal laser scanning microscopy images (LSM800). Nuclei stained with Hoechst 33342 were counted and considered as the number of total cells. The percentage of cells expressing each cholinergic marker was subsequently calculated using the values indicated above.

Radiosynthesis of ¹²⁵I-Labeled Plantainoside B

Sodium [¹²⁵I]iodide ([¹²⁵I]NaI, carrier free) solution was purchased from PerkinElmer, Inc. (Waltham, MA, U.S.A.) LD-20AD (Shimadzu, Kyoto, Japan) was used for reversed-phase (RP)-HPLC, along with an SPD-20A (Shimadzu) UV detector (220, 254 nm) and RI analyzer Gabi Nova (Elysia-raytest, Liege, Belgium). Radiosynthesis of ¹²⁵I-labeled plantainoside B was performed by electrophilic aromatic substitution reaction. Plantainoside B (500 µg) dissolved in MeOH (10 µL) was mixed with a solvent (MeOH:CH₃COOH = 70 : 20, 90 µL) containing N-chlorosuccinimide (0.5 mg). [¹²⁵I]NaI (15 MBq) was added to the mixture, and then vortexed at room temperature for 30 min. After removing the solvent using N₂ gas, the mixture was purified through RP-HPLC using Cosmosil 5C₁₈-AR-II column (4.6ID × 150 mm, Nacalai Tesque) with H₂O:MeCN (gradient from 10% to 50% MeCN over 30 min) as the mobile phase at a flow rate of 1.0 mL/min, to obtain ¹²⁵I-labeled plantainoside B (3.2–5.9 MBq). MeCN was removed from the sample using N₂ gas and the radio activity was measured using a dose calibrator (IGC-8, Hitachi Ltd., Tokyo, Japan).

Autoradiography and Immunohistochemical Analysis

Brain sections from male 9-month-old APdE9 mice and littermate wild-type mice were used for autoradiography. Mice were euthanized through cervical dislocation and the brains were immediately removed and fixed with 4% PFA at 4 °C for 3 d, followed by dehydration with 30% sucrose for 3 d. Brains were then sectioned (20 µm thickness) using a cryostat. Brain sections were incubated with ¹²⁵I–labeled plantainoside B solution (adjusted to 350 kBq/mL using PBS) on a shaker at room temperature for 2 h, washed three times with PBS, and dried completely on a slide glass (Matsunami, Osaka, Japan). Alternatively, Aβ aggregates resulting from the incubation of isoAβ (6 µM) in PBS at 37 °C for 1 and 6 h were mixed with ¹²⁵I–labeled plantainoside B solution (adjusted to 20 MBq/mL with PBS); the samples were then run on blue native–polyacrylamide gels (4–16% Bis–Tris mini protein gels; Thermo Fisher Scientific). The brain sections and BN–PAGE gels were exposed to a phosphor–imaging plate (FUJIFILM Plate BAS–TR2025, FUJIFILM, Tokyo, Japan) overnight. The imaging plate was scanned using an image analyzer (Typhoon 9410, GE Healthcare, Milwaukee, WI, U.S.A.) at a resolution of 25 μm/pixel.

The brain sections were also incubated with anti-Aβ antibody (clone 6E10, 1 : 2000) and Hoechst 33342 (1 : 4000) overnight at room temperature, followed by incubation with secondary antibody, Alexa Fluor 555-labeled anti-mouse IgG antibody (1 : 500), for 2 h. Fluorescence was detected using a confocal laser scanning microscope (LSM800).

Intrahippocampal Injection of isoAβ

Ten-week-old wild-type male C57BL/6 mice were anesthetized by intraperitoneal injection with a mixture of medetomidine (0.3 mg/kg, Domitor, ZENOAQ, Koriyama, Japan), midazolam (4 mg/kg, Dormicum, Maruishi Pharmaceutical Co., Osaka, Japan), and butorphanol (5 mg/kg, Vetorphale, Meiji Seika Pharma, Tokyo, Japan). Mice were placed on a stereotaxic instrument (51730D; Stoelting, Wood Dale, IL, U.S.A.) and injected with PBS, isoAβ (20 µM), or isoAβ (20 µM) plus plantainoside B (30 µM) at a total volume of 3 µL PBS solution into the bilateral hippocampi (coordinates from bregma: ± 1.5 mm lateral, 2.0 mm posterior, and 2.0 mm ventral) using a 10 µL Hamilton syringe driven by an automated syringe pump (set at 1 µL/min injection rate, Legato130, KD Scientific, Holliston, MA, U.S.A.). After administration, the syringe was slowly removed from the brain and the skin was sutured. Mice received atipamezole (0.3 mg/kg, Antisedan, Orion Co., Espoo, Finland) and returned to the warm box to recover from anesthesia.

Cognitive Assessment with Novel Object Recognition Test (NORT)

Cognitive performance was evaluated with NORT. Mice were placed in the open–field box (length × width × height: 40 × 40 × 30 cm) under a light set at 20 lx and habituated for 10 min. Thirty minutes after habituation, two objects (O1 and O2) were presented to mice for 10 min (trial phase). After a 24 h interval for memory retention, the mice were placed in the same box again where the O2 object was replaced by a novel third object (O3); object preference was analyzed during the test phase for 10 min. Behavioral movements were recorded for 10 min during the test phase with a video camera connected to PC with a tracking system (EthoVision XT 11.5, Noldus Information Technology). An 8 × 8 cm square (target area) centered on each object was set up, and the time spent in the target area (T) was measured. The discrimination index, used as a measure of nonspatial working memory function, was calculated as follows:

  

where T_O1 and T_O3 are the time spent in the O1 or O3 target areas during the test phase, respectively.

Statistical Analyses

GraphPad Prism 5 (GraphPad Software, La Jolla, CA, U.S.A.) was used for all statistical analyses. Results are presented as means ± standard errors of the means (S.E.Ms.). Student’s t-test was used for two-group comparisons. For multiple comparisons, the one-way ANOVA followed by a Bonferroni’s post-hoc multiple comparison test was used for comparing among ≥3 groups. A p–value <0.05 was considered statistically significant.

RESULTS

The isoAβ Aggregation and Cell Toxicity Induced by Aβ Oligomers

We first attempted to construct an Aβ oligomer-dependent neurodegeneration model system in vitro. To this end, isoAβ, which reverts to natural Aβ₁₋₄₂ and initiates self-aggregation under neutral pH conditions, was used for stable and uniform Aβ oligomer formation.²⁴⁾ In the present study, the time-dependent aggregation of isoAβ under neutral pH conditions at 37 °C for 1–72 h was analyzed by BN–PAGE (Fig. 1A). Aβs around 20 kDa (low molecular weight Aβ oligomers and isoAβ monomer) were stably detected up for to 6 h after incubation but not after 9 h. Similarly, Aβ aggregates from about 66 kDa to over 1048 kDa (middle to high molecular weight Aβ oligomers) increased after up to 6 h of incubation, gradually decreasing after 9 h. In contrast, the Aβ fibrils that did not enter the separation gel were clearly detected in the sample incubated for 3 h; their amount increased in a time–dependent manner over incubation.

Fig. 1. Analysis of isoAβ Aggregation and Cytotoxicity in SH–SY5Y Cells

(A) isoAβ (6 µM) was incubated for 1–72 h at 37 °C in culture medium for SH–SY5Y cells; Aβ aggregation was analyzed by blue native–polyacrylamide gel electrophoresis (BN–PAGE) using an anti-Aβ antibody. LMW; low molecular weight, MMW; middle molecular weight, HMW; high weight. (B–C) Cells were treated with isoAβ (1–6 µM) (B) or 1–72 h pre-incubated Aβ (6 µM) (C) for 48 h; cell viability was analyzed by the WST–8 assay. (D) Confocal laser scanning microscopy was performed after treatment with or without 24 h pre-incubation with isoAβ. Cells were stained with antibodies against Aβ (green) and actin (cell morphology marker, red) and Hoechst 33342 (nuclei, cyan). Scale bars, 20 µm. (E) Image analysis was performed to measure the cell surface area covered with Aβ (merged Aβ and actin expression area). Data represents mean ± S.E.M. (B, C; n = 3, E; n = 11). The statistical significance of differences among groups was determined by ANOVA with post-hoc Bonferroni’s/Dunn’s test (B and C) and Student’s t–test (E). *** p < 0.001 vs. control (Cont., B and C) or no pre-incubation (E). One representative experiment is shown; similar results were obtained in three independent experiments.

Subsequently, we examined cell viability after isoAβ treatment with a WST–8 assay using SH–SY5Y cells, a neuroblastoma cell line, cultured under neutral pH conditions. Direct treatment with isoAβ for 48 h significantly decreased SH–SY5Y cell viability in a concentration–dependent manner (Fig. 1B). Next, to narrow down the type of Aβ aggregates responsible for decreased cell viability, aggregates formed after various pre-incubation times were added to SH–SY5Y cells (Fig. 1C). The viability of SH–SY5Y cells was significantly reduced by treatment with Aβ aggregates made by pre-incubation < 12 h whereas those Aβ aggregates resulting from > 24 h pre-incubation caused less or almost no change in cell viability (Fig. 1C). Confocal laser scanning microscopic analysis revealed that Aβ was tightly attached on the surface of SH–SY5Y cells when cells were directly treated with isoAβ without pre-incubation (Figs. 1D, E). In contrast, treatment with Aβ aggregates, produced by pre-incubation in culture medium under neutral pH for 48 h, showed much less binding to the cell surface (Figs. 1D, E). Thus, as shown by this comparison, the non-pre-incubated Aβ adhered strongly to the cell surface, whereas the pre-incubated Aβ adhered weakly to some cells, perhaps also to the bottom of the culture dish, and mostly remained suspended in the cell culture medium. Based on these results, Aβ oligomers (about 66 kDa to over 1048 kDa), but not isoAβ monomers, low molecular weight Aβ oligomers (around 20 kDa), or fibrils, may be involved in the reduced cell viability through tight attachment to the cell surface in this model.

Differentiation of Basal Forebrain Cholinergic Neurons from hiPSCs and isoAβ Induced Neurodegeneration

To develop a specific neurodegeneration model based on the cholinergic hypothesis in AD,²⁾ we investigated the differentiation of cholinergic neurons targeting the basal forebrain from hiPSCs. Recently, several methods for differentiating cholinergic neurons from pluripotent stem cells have been reported.^3–5) Similarly, we independently developed a feeder-free differentiation protocol for basal forebrain and cholinergic neurons using low molecular weight compounds (small molecules) and confirmed the differentiation process (Figs. 2A, B). Confocal laser scanning microscopy revealed the expression of FOXG1 (a telencephalic marker), NKX2.1 (a ventral telencephalic marker),³⁹⁾ and ISLET–1 (a cholinergic neuron maker)⁵⁾ in iPSC-derived cells on day 16 after induction (Fig. 2A). On day 24, the differentiation toward cholinergic neurons further progressed, and expression of ChAT and LHX8 (cholinergic neuron markers)³⁹⁾ and MAP2 (a dendrite marker)³⁴⁾ was detected (Fig. 2B). Based on the immunoreactivity of cholinergic neuron markers such as LHX8, ISLET–1, and ChAT, detected on day 24 of differentiation, the percentage of cholinergic neurons to the total cells (Hoechst33342⁺ cells) was calculated (Fig. 2B). Although the rates of transcription factors and early markers of cholinergic neurons,⁴⁰⁾ such as LHX8 (25.3 ± 7.4%) and ISLET–1 (47.7 ± 5.1%), were relatively low, most cells were positive for ChAT (86.6 ± 3.1%), which is the synthetic enzyme for acetylcholine (Fig. 2B); these results suggest that the cells differentiated beyond the initial stages and are capable of functioning as cholinergic neurons. Thus, we successfully differentiated basal forebrain cholinergic neurons from hiPSCs with a simplified protocol.

Fig. 2. Analysis of isoAβ Neurotoxicity in hiPSC-Derived Cholinergic Neurons

(A, B) Confocal laser scanning microscopy was performed during differentiation of human induced pluripotent stem cells (hiPSCs) toward basal forebrain cholinergic neurons using antibodies against NKX2.1 [maker for ventral telencephalon, red in (A) and green in (B)], FOXG1 [telencephalon, red in (A)], ISLET–1 [cholinergic neurons, red in (A) and green in (B)], MAP2 [dendrites, white in (B)], LHX8 [cholinergic neurons, white in (B)], ChAT [cholinergic neurons, red in (B)], and Hoechst 33342 [nuclei, blue in (B)]. Scale bars, 50 µm. The graph shows the rate of cells expressing cholinergic markers, such as LHX8 (an early marker), ISLET–1 (an early marker), or ChAT (a functional marker), in relation to the total number of cells (Hoechst 33342⁺ cells). The data represent the mean ± S.E.M. (n = 3). (C, D) Cells were treated with isoAβ (1–6 µM) for 48 h; cell viability and cytotoxicity were analyzed by the WST–8 (C) and LDH assays (D), respectively. Data represents mean ± S.E.M. (C, D; n = 3). The statistical significance of differences among groups was determined by ANOVA with a post-hoc Bonferroni’s/Dunn’s test. * p < 0.05, *** p < 0.001, vs. control (Cont.). One representative experiment is shown; similar results were obtained in three independent experiments. (E–F) Confocal laser scanning microscopy was performed using antibodies against Aβ [green in (E) and magenta in (F)], actin [cell morphology marker, red in (E)], and MAP2 [dendrites, green in (F)]. Representative images of cell bodies and neurites are shown in (E) (Z–stack) and (F), respectively. (F) Nuclei are stained with Hoechst 33342 (blue) and arrow heads show damaged dendrites. Scale bars, 20 µm.

We next examined isoAβ induced neurodegeneration using hiPSC-derived cholinergic neurons on day 24. Cell viability and cytotoxicity were analyzed by the WST–8 (Fig. 2C) and LDH assays (Fig. 2D), respectively. Similar to SH–SY5Y cells, the isoAβ treatment induced neurodegeneration of hiPSC-derived cholinergic neurons in a concentration-dependent manner. Confocal laser scanning microscopy revealed that Aβ was tightly bound to the surface of both cell bodies (Fig. 2E) and neurites (Fig. 2F) of hiPSC-derived cholinergic neurons generated after 24 d of differentiation. Thus, using iPSC-derived cholinergic neurons and isoAβ, we constructed a neurodegeneration model based on the cholinergic and Aβ oligomer hypothesis.

Neuroprotection by Plantainoside B, an Aβ Oligomer–Binding Small Molecule Isolated from B. Monniera Extract

We next attempted to identify Aβ oligomer binding small molecules with potential neuroprotective activity (Fig. 3A). Beads conjugated with Aβ aggregates produced after 1 or 24 h of isoAβ incubation were immersed in the EtOAc fraction of B. monniera extract. LC/MS revealed that several compounds derived from B. monniera extract were bound to Aβ aggregates. Of these, small molecules (molecular weight < 500) that bound more to the Aβ aggregates produced by 1 h incubation than that of 24 h were chosen, and plantainoside B was identified (molecular weight; 478.45) by NMR and MS analysis (Fig. 3B).

Fig. 3. Analysis of the Neuroprotective Effects of Plantainoside B against Aβ Neurotoxicity in hiPSC-Derived Cholinergic Neurons

(A) Schematic diagram indicating the process of identifying low molecular weight compounds with binding affinity to Aβ aggregates from B. monniera extract. (B) Chemical structure of plantainoside B (Plant. B). (C–I) Cells were treated with isoAβ (3 µM) in the presence or absence of Plant. B (3–30 µM) for 48 h. Cell viability and cytotoxicity were analyzed by the WST–8 (C) or LDH assay (D), respectively. (E, F) Confocal laser scanning microscopy (Z–stack) was performed using antibodies against Aβ (green) and actin (cell morphology marker, red) (E). Cell surface area covered with Aβ (merged Aβ and actin area) was measured by image analysis (F). Scale bars, 20 µm. (G, H) Time–lapse calcium imaging with fluo–4 was performed using a confocal laser scanning microscope (G). Image analysis was performed to measure the increase of fluorescent intensity 180 min after Aβ treatment (H). Scale bars, 5 µm. (I) The mitochondrial membrane potential was assessed by JC–1 analysis and the red/green fluorescence ratio calculated. Data represent means ± S.E.M.s. The statistical significance of differences among groups was determined by ANOVA with a post-hoc Bonferroni’s/Dunn’s test (C, D, and I; n = 3) or Student’s t–test [F; n = 10 and H; n = 19 (Aβ), n = 15 (Aβ + Plant. B)]. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (Cont., C, D, and I) or Aβ (F and H), ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001 vs. Aβ only

We subsequently analyzed the neuroprotective activity of plantainoside B in the neuronal cell death model described above using isoAβ and hiPSC-derived cholinergic neurons. Plantainoside B significantly attenuated the decreased cell viability (Fig. 3C) and increased the LDH release (Fig. 3D) induced by Aβ oligomers in a concentration-dependent manner.

To explore the neuroprotective mechanism of plantainoside B, we performed confocal laser scanning microscopy. Plantainoside B treatment reduced Aβ binding to hiPSC-derived cholinergic neurons (Figs. 3E, F). On the other hand, Aβ oligomers induce Ca²⁺ influx into the cytoplasm.^41–44) We, therefore, examined Ca²⁺-imaging using a fluorescent dye (fluo–4) and found that plantainoside B suppressed the Aβ-induced Ca²⁺ influx in hiPSC-derived cholinergic neurons (Figs. 3G, H). Moreover, as MMP dysregulation by Aβ oligomers has been reported,⁴⁵⁾ we further evaluated MMP using the indicator dye, JC–1.³⁸⁾ We found that plantainoside B significantly attenuated Aβ-induced MMP dysregulation in hiPSC-derived cholinergic neurons (Fig. 3I). Taken together, these findings suggest that plantainoside B may attenuate neurodegeneration by blocking the binding of Aβ oligomers to the cell surface, thereby suppressing subsequent cytotoxic events such as dysregulation of the cytoplasmic Ca²⁺ level and MMP.

Inhibitory Effect of Plantainoside B on Aβ Aggregation and Binding Affinity to Aβ Oligomers

We next examined the effects of plantainoside B on Aβ aggregations. A ThT assay revealed that isoAβ markedly self-aggregated after 6 h of incubation and that plantainoside B strongly suppressed aggregation (Fig. 4A). We further examined the inhibitory effect of plantainoside B on Aβ aggregation by AFM (Fig. 4B) and BN–PAGE (Fig. 4C); both confirmed the suppressive effect of plantainoside B on Aβ aggregation.

Fig. 4. Effect of Plantainoside B on Aβ Aggregation and Binding of ¹²⁵I-Labeled Plantainoside B to Aβ Aggregates

(A, B) The isoAβ (6 µM) was incubated at 37 °C for 1–24 h with or without plantainoside B (Plant. B, 100 µM) in PBS. Thioflavin T (ThT) assay (A) and AFM (B) were performed to analyze the effect of Plant. B on Aβ aggregation. AFM shows Aβ aggregates 24 h after pre-incubation. Scale bars, 2 µm. (C, D) The isoAβ (6 µM) was self–aggregated at 37 °C for 1–24 h with or without Plant. B (30 µM) in cell culture medium (C) or PBS (D). Aβ aggregates were analyzed by blue native–polyacrylamide gel electrophoresis with an anti-Aβ antibody. (E) Aβ aggregates pre-incubated at 37 °C for 1 h or 6 h in PBS were mixed with ¹²⁵I–labeled Plant. B and run on a blue native–polyacrylamide gel; the radioactivity on the gel was detected by autoradiography. Blank indicates that ¹²⁵I–labeled Plant. B without Aβ aggregates was loaded on the gel.

The binding affinity of plantainoside B to Aβ aggregates was analyzed using BN–PAGE (Figs. 4D, E). Aβ aggregation was first confirmed by BN–PAGE with anti-Aβ antibody (Fig. 4D). A smear band of Aβ aggregates (from around 20 kDa Aβs to high molecular fibrils) was detected 1 h after isoAβ incubation in PBS whereas a band of fibrils was detected 6 h after incubation (Fig. 4D). Subsequently, ¹²⁵I–labeled plantainoside B was run on blue native–polyacrylamide gels with or without Aβ aggregates made by pre-incubation of isoAβ for 1 or 6 h in PBS. In autoradiography, despite background radioactivity of ¹²⁵I–labeled plantainoside B on the blank (¹²⁵I–labeled plantainoside B only), Aβ pre-incubated for 1 h at the position with Aβ aggregates (Aβs from around 20  to 242 kDa and fibrils over 1048 kDa) (Fig. 4E) produced a stronger signal. On the other hand, weaker and stronger signals were similarly detected with Aβ pre-incubated for 6 h with Aβ aggregates (around 20  to over 242 kDa) and fibrils, respectively (Fig. 4E). Because of the high sensitivity of radioactivity, ¹²⁵I–labeled plantainoside B could be detected in Aβ aggregates that could not be detected with the anti-Aβ antibody in the sample with Aβ pre-incubated for 6 h. Accordingly, plantainoside B appears to have binding affinity to Aβ aggregates (especially, from around 20 to over 242 kDa) and fibrils.

Binding Affinity of Plantainoside B to Brain Sections from Mice with Aβ Pathology and Attenuation of Memory Impairment in IsoAβ Injected Mice

We further examined the binding affinity of ¹²⁵I–labeled plantainoside B to brain sections from a wild-type mouse and a model mouse of Aβ pathology (APdE9). Confocal laser scanning microscopic analysis revealed Aβ accumulation in the brain tissue from model mice but not from wild-type mice (Fig. 5A). In the autoradiography, diffuse and much stronger ¹²⁵I–labeled plantainoside B signals were detected in the brain sections of model mice compared with those of wild-type mice (Fig. 5B). In contrast, treatment with ¹²⁵I⁻ did not get clear radioisotope signals in the brain tissues from a wild-type mouse and a mouse model of amyloid pathology (Supplementary Fig. S1). Moreover, blocking with an excess amount of non-labeled plantainoside B compared with that of ¹²⁵I-labeled plantainoside B reduced the radioactivity in the brain tissues from a mouse model of amyloid pathology (Supplementary Fig. S1). Thus, the high radioactivity in brain tissues from a mouse model of amyloid pathology obtained after incubation with ¹²⁵I-labeled plantainoside B (Fig. 5B) was considered to be specific to the binding affinity of plantainoside B rather than ¹²⁵I.

Fig. 5. Binding Affinity of Plantainoside B on Mouse Brain Tissues and Attenuation of Aβ Induced Memory Impairments by Plantainoside B in Mice

(A, B) Confocal laser scanning microscopy with anti-Aβ antibody (red) and Hoechst 33342 (nuclei, blue) (A) and autoradiography with ¹²⁵I–labeled plantainoside B (Plant. B) (B) were performed using brain tissues from wild–type mice and a mouse model of amyloid pathology (APdE9 mice). Scale bars, 1 mm and 50 µm in insets. (C) Schematic diagram indicating the coordinates of intrahippocampal injection of PBS, isoAβ, or isoAβ plus Plant. B in wild–type mice and schedule of novel object recognition test (NORT) (D) Bars indicate the discrimination index analyzed by NORT. Data represent means ± S.E.M.s (PBS injection; n = 10, Aβ injection; n = 8, Aβ + Plant. B injection; n = 10). The statistical significance of differences among groups was determined by ANOVA with post-hoc Bonferroni’s/Dunn’s test. * p < 0.05 vs. PBS injection, ^† p < 0.05. (E) Representative trajectories of mice in the NORT test phase. O1 and O3 indicate the position of familial object and novel object, respectively.

Finally, we examined the effects of plantainoside B on Aβ-induced memory deficits in wild–type mice. Mice were bilaterally injected with PBS or isoAβ with or without plantainoside B into the hippocampus and NORT was performed to investigate memory function (Fig. 5C). The intrahippocampal injection of isoAβ induced memory deficits as indicated by the discrimination index (Fig. 5D) and trajectories (Fig. 5E). In contrast, simultaneous injection with plantainoside B attenuated the memory deficits induced by the intrahippocampal injection with isoAβ (Figs. 5D, E). Thus, plantainoside B improved the Aβ-induced memory deficits in mice.

DISCUSSION

In familial AD (FAD) with the Arctic mutation (E693G) in AβPP, there is a high production of Aβ protofibrils.⁴⁶⁾ In FAD with the Osaka variant (E693Δ) of AβPP, the mutant Aβ (E22Δ) primarily produces Aβ oligomers rather than Aβ fibrils. Thus, the genetic analysis of FAD strongly supports that the neurotoxicity of Aβ oligomers is responsible for the neurological symptoms in AD.^47,48) In the present study, we found that the neurotoxicity of isoAβ decreases as the amount of Aβ monomers and oligomers decreased and fibrils gradually formed (Figs. 1A, C). Furthermore, significant cytotoxicity was detected (Fig. 1C) when monomers and low molecular weight Aβ oligomers < 20 kDa were barely detectable after 9 h of incubation (Fig. 1A). However, these results do not rule out the neurotoxicity of monomers and low molecular weight Aβ oligomers, rather suggest that middle and high molecular weight Aβ oligomers (about 66 kDa to over 1048 kDa) such as ADDLs, AβO, and protofibrils are the primary neurotoxic forms in the present neurodegeneration model.

In the present study, we further detected tight binding of Aβ to the cell surface, significant increase of cytosolic Ca²⁺ levels, and MMP disruption in hiPSC-derived cholinergic neurons after treatment with Aβ oligomers, confirming that human basal forebrain cholinergic neurons are indeed impaired by Aβ oligomers. The increase of cytosolic Ca²⁺ levels and mitochondrial dysfunction caused by Aβ oligomers was previously demonstrated in primary cultured-human fetal cortical neurons and rat cortical neurons.^17,49) Regarding the mechanisms of increased cytosolic Ca²⁺ levels by treatment with Aβ oligomers, Ca²⁺ channels produced by Aβ, called amyloid channels,^50,51) and activation of N-methyl-D-aspartate receptors^44,52) have been implicated. Moreover, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors are involved in the Aβ oligomer-induced Ca²⁺ influx into the cytoplasm.⁵³⁾ However, these studies were performed in immortalized cell lines and primary cultured rodent cortical and hippocampal neurons. Therefore, the Aβ oligomer-induced neurodegeneration model constructed in the present study is advantageous in that the above mechanism could be analyzed using human basal forebrain cholinergic neurons in the future.

Impairment of cholinergic neurons in the nucleus basalis of Meynert is an early pathology of AD.^1,2) A study using a mouse model of Aβ pathology showed that the activation of the cholinergic circuit from the basal forebrain to the hippocampus can improve the cholinergic metabolism and attenuate memory impairment in mice.⁵⁴⁾ Therefore, early protection of cholinergic neurons in the basal forebrain is key to cure AD and elucidating the pathological mechanism underlying the cholinergic neurodegeneration induced by Aβ, especially Aβ oligomers, is expected to contribute to the development of DMTs in AD. However, the molecular mechanisms of Aβ oligomer–induced neurodegeneration in human cholinergic neurons are not fully understood due to the difficulty of handling unstable Aβ oligomers⁵⁵⁾ and the lack of appropriate models for human cholinergic neurons.^56,57) In the present study, we attempted to overcome this problem by combining the isoAβ^27,28) with hiPSC technologies. The isoAβ reproduces well Aβ oligomers and has been used to analyze the toxicity of Aβ oligomers.²⁵⁾ Furthermore, FAD studies using isoAβ revealed abnormal Aβ oligomerization with the A673V mutation of AβPP.²⁴⁾ With respect to the hiPSC technologies, differentiation protocols of cholinergic neurons from mouse and human pluripotent stem cells have been reported.^3–5) In the present study, we also independently developed a method for the differentiation of hiPSCs into cholinergic neurons targeting the basal forebrain using small molecules under the feeder-free condition. While previous reports have confirmed the expression of ChAT, a cholinergic neuronal marker, at more than 40 d after differentiation, in our protocol, ChAT was detected at 24 d after differentiation. In addition to the time savings, replacing humoral factors with small molecules in differentiation process could have resulted in a cost-effective and more stable protocol. Thus, the elucidation of the cell death mechanism using isoAβ with hiPSC-derived basal forebrain cholinergic neurons can assist the development of diagnosis methods and DMTs based on the Aβ oligomer and cholinergic hypothesis.

Plantainoside B has been shown to reduce plasma triglycerides levels in mice.⁵⁸⁾ However, its effects on neurons have not been previously reported. In the present study, we found for the first time that plantainoside B has an affinity for a broad range of Aβ aggregates (Aβs from around 20 kDa to 242 kDa and fibrils over 1048 kDa) and neuroprotective activity. This binding affinity of plantainoside B to Aβ oligomers may critically contribute to the inhibition of Aβ binding to the cells and slow the rate of Aβ aggregation. The inhibition of Aβ oligomer binding to the cell surface may be a primary mechanism of neuroprotection as indicated by the attenuated increase of cytosolic Ca²⁺ level and MMP dysregulation. Previously, a B. monniera extract was shown to significantly reduce the total levels of Aβ_1–42 in the brain of a mouse model of amyloid pathology.³¹⁾ It is possible that the binding affinity of plantainoside B in B. monniera extract to Aβ aggregates and its aggregation–delaying action may decrease the levels of Aβ in the brain, but the extent of this contribution is unknown. Therefore, research on the administration of a single component of plantainoside B to mouse models of Aβ pathology is needed in the future.

While technological development toward in vitro diagnosis for AD by the detection of Aβ in blood and cerebral spinal fluid continues,^59,60) a significance of bioimaging is that it can detect pathogenic factors in the body as well as modulate them in the host. This makes bioimaging a useful tool in theranostics. Theranostics aims to influence the pathogenesis mechanism by initiating early treatment along with diagnosis, delaying the onset of disease or reducing symptoms, thereby enhancing treatment effectiveness and improving the patient’s QOL; this concept has already been adopted in oncology.^61,62) In a similar, for neurodegenerative diseases and neurological disorders including AD, where early diagnosis and treatment are required, introducing theranostics (neurotheranostics) as a method of intervention would be highly beneficial.^63,64) Since the development of Pittsburgh Compound-B, a positron emission tomography tracer for amyloid imaging,⁶⁵⁾ research has been conducted to develop further non-invasive diagnostic tools for AD, such as probes for magnetic resonance imaging (MRI). Curcumin, a polyphenol derived from the natural herb turmeric, directly inhibits fibrillation of Aβ and destabilizes Aβ aggregates,⁶⁶⁾ and curcumin derivatives are being developed as Aβ oligomer–specific probes for MRI with neuroprotective properties.^67,68) Furthermore, rutin, a natural flavonol glycoside in various plants, has been combined with Congo red and magnetic nanoparticles to develop MRI probes for amyloid plaques with antioxidant properties.⁶⁹⁾ In the present study, plantainoside B showed neuroprotective and inhibitory effects on memory deficits induced by Aβ. Furthermore, radioisotope-labeled plantainoside B detected Aβ aggregates including its oligomers on blue–native polyacrylamide gels and produced a diffuse radioactive signal in the brain sections of a mouse model of amyloid pathology. Of note, the diffuse nature of the signal detected in the brain sections may be attributed to the fact that plantainoside B detected Aβ oligomers distributed throughout the brain sections, rather than only the Aβ fibrils that constitute Aβ plaques. Taken together, these results suggest that plantainoside B may be a pioneer compound that provides an opportunity for the development of theranostics in AD research. Accordingly, this study may provide valuable information for the practical application of theranostics, which is a new concept in AD treatment.

In summary, we established a neurodegeneration model specialized to the Aβ oligomer and cholinergic hypothesis in AD using isoAβ and hiPSC-derived basal forebrain cholinergic neurons. Using this model, we found that plantainoside B, a compound in B. monniera, shows neuroprotective effects by having binding affinity to Aβ aggregates. Plantainoside B further showed a high binding affinity to brain tissue from a mouse model of amyloid pathology and attenuated Aβ induced memory deficits in mice. Thus, the binding capacity to Aβ aggregates and neuroprotective effects of plantainoside B indicate its potential as a novel neurotheranostics tool for AD that could accelerate the development of new diagnostic methods and DMTs.

Acknowledgments

We thank Yuya Araki and Eri Hosoda (Kyoto Pharmaceutical University) for technical assistance. This study was supported by Grants-in-Aid from the Private University Research Branding Project for Ministry of Education, Culture, Sports, Science and Technology, the Japan Society for the Promotion of Science (JSPS) KAKEN (21K06637 to S. Nakashima., 19K07854 and 22K07382 to K.N., 20H03569 to K.T.), the Hoansha Foundation (K.T.), Kobayashi Foundation (K.T.), and the Smoking Research Foundation (K.T.), and Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan (A.F.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, DeLong MR. Alzheimer’s disease and senile dementia: loss of neurons in the basal forebrain. Science, 215, 1237–1239 (1982).
• 2) Giacobini E, Cuello AC, Fisher A. Reimagining cholinergic therapy for Alzheimer’s disease. Brain, 145, 2250–2275 (2022).
• 3) Yue W, Li Y, Zhang T, Jiang M, Qian Y, Zhang M, Sheng N, Feng S, Tang K, Yu X, Shu Y, Yue C, Jing N. ESC-derived basal forebrain cholinergic neurons ameliorate the cognitive symptoms associated with Alzheimer’s disease in mouse models. Stem Cell Reports, 5, 776–790 (2015).
• 4) Hu Y, Qu ZY, Cao SY, Li Q, Ma L, Krencik R, Xu M, Liu Y. Directed differentiation of basal forebrain cholinergic neurons from human pluripotent stem cells. J. Neurosci. Methods, 266, 42–49 (2016).
• 5) Krajka V, Naujock M, Pauly MG, Stengel F, Meier B, Stanslowsky N, Klein C, Seibler P, Wegner F, Capetian P. Ventral telencephalic patterning protocols for induced pluripotent stem cells. Front. Cell Dev. Biol., 9, 716249 (2021).
• 6) Liu Q, Waltz S, Woodruff G, Ouyang J, Israel MA, Herrera C, Sarsoza F, Tanzi RE, Koo EH, Ringman JM, Goldstein LS, Wagner SL, Yuan SH. Effect of potent gamma-secretase modulator in human neurons derived from multiple presenilin 1-induced pluripotent stem cell mutant carriers. JAMA Neurol., 71, 1481–1489 (2014).
• 7) Fujimori K, Ishikawa M, Otomo A, Atsuta N, Nakamura R, Akiyama T, Hadano S, Aoki M, Saya H, Sobue G, Okano H. Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent. Nat. Med., 24, 1579–1589 (2018).
• 8) Kondo T, Imamura K, Funayama M, Tsukita K, Miyake M, Ohta A, Woltjen K, Nakagawa M, Asada T, Arai T, Kawakatsu S, Izumi Y, Kaji R, Iwata N, Inoue H. iPSC-based compound screening and in vitro trials identify a synergistic anti-amyloid beta combination for Alzheimer’s disease. Cell Reports, 21, 2304–2312 (2017).
• 9) Selkoe DJ, Hardy J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol. Med., 8, 595–608 (2016).
• 10) Swanson CJ, Zhang Y, Dhadda S, Wang J, Kaplow J, Lai RYK, Lannfelt L, Bradley H, Rabe M, Koyama A, Reyderman L, Berry DA, Berry S, Gordon R, Kramer LD, Cummings JL. A randomized, double-blind, phase 2b proof-of-concept clinical trial in early Alzheimer’s disease with lecanemab, an anti-Abeta protofibril antibody. Alzheimers Res. Ther., 13, 80 (2021).
• 11) Zhang C, Browne A, Divito JR, Stevenson JA, Romano D, Dong Y, Xie Z, Tanzi RE. Amyloid-beta production via cleavage of amyloid-beta protein precursor is modulated by cell density. J. Alzheimers Dis., 22, 683–694 (2010).
• 12) Jarrett JT, Berger EP, Lansbury PT Jr. The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer’s disease. Biochemistry, 32, 4693–4697 (1993).
• 13) Figueiredo CP, Clarke JR, Ledo JH, Ribeiro FC, Costa CV, Melo HM, Mota-Sales AP, Saraiva LM, Klein WL, Sebollela A, De Felice FG, Ferreira ST. Memantine rescues transient cognitive impairment caused by high-molecular-weight Abeta oligomers but not the persistent impairment induced by low-molecular-weight oligomers. J. Neurosci., 33, 9626–9634 (2013).
• 14) Yasumoto T, Takamura Y, Tsuji M, Watanabe-Nakayama T, Imamura K, Inoue H, Nakamura S, Inoue T, Kimura A, Yano S, Nishijo H, Kiuchi Y, Teplow DB, Ono K. High molecular weight amyloid beta1-42 oligomers induce neurotoxicity via plasma membrane damage. FASEB J., 33, 9220–9234 (2019).
• 15) Ono K. Alzheimer’s disease as oligomeropathy. Neurochem. Int., 119, 57–70 (2018).
• 16) Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. U.S.A., 95, 6448–6453 (1998).
• 17) Deshpande A, Mina E, Glabe C, Busciglio J. Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. J. Neurosci., 26, 6011–6018 (2006).
• 18) Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, Benedek GB, Selkoe DJ, Teplow DB. Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates. J. Biol. Chem., 274, 25945–25952 (1999).
• 19) Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J. Biol. Chem., 272, 22364–22372 (1997).
• 20) Selkoe DJ. Alzheimer’s disease is a synaptic failure. Science, 298, 789–791 (2002).
• 21) Balducci C, Beeg M, Stravalaci M, Bastone A, Sclip A, Biasini E, Tapella L, Colombo L, Manzoni C, Borsello T, Chiesa R, Gobbi M, Salmona M, Forloni G. Synthetic amyloid-beta oligomers impair long-term memory independently of cellular prion protein. Proc. Natl. Acad. Sci. U.S.A., 107, 2295–2300 (2010).
• 22) Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature, 416, 535–539 (2002).
• 23) Nirmalraj PN, List J, Battacharya S, Howe G, Xu L, Thompson D, Mayer M. Complete aggregation pathway of amyloid beta (1-40) and (1-42) resolved on an atomically clean interface. Sci. Adv., 6, eaaz6014 (2020).
• 24) Messa M, Colombo L, del Favero E, Cantù L, Stoilova T, Cagnotto A, Rossi A, Morbin M, Di Fede G, Tagliavini F, Salmona M. The peculiar role of the A2V mutation in amyloid-beta (Abeta) 1-42 molecular assembly. J. Biol. Chem., 289, 24143–24152 (2014).
• 25) Stravalaci M, Bastone A, Beeg M, Cagnotto A, Colombo L, Di Fede G, Tagliavini F, Cantù L, Del Favero E, Mazzanti M, Chiesa R, Salmona M, Diomede L, Gobbi M. Specific recognition of biologically active amyloid-beta oligomers by a new surface plasmon resonance-based immunoassay and an in vivo assay in Caenorhabditis elegans. J. Biol. Chem., 287, 27796–27805 (2012).
• 26) Coin I. The depsipeptide method for solid-phase synthesis of difficult peptides. J. Pept. Sci., 16, 223–230 (2010).
• 27) Sohma Y, Hirayama Y, Taniguchi A, Mukai H, Kiso Y. “Click peptide” using production of monomer Abeta from the O-acyl isopeptide: application to assay system of aggregation inhibitors and cellular cytotoxicity. Bioorg. Med. Chem., 19, 1729–1733 (2011).
• 28) Taniguchi A, Sohma Y, Hirayama Y, Mukai H, Kimura T, Hayashi Y, Matsuzaki K, Kiso Y. “Click peptide”: pH-triggered in situ production and aggregation of monomer Abeta1-42. ChemBioChem, 10, 710–715 (2009).
• 29) Russo A, Borrelli F. Bacopa monniera, a reputed nootropic plant: an overview. Phytomedicine, 12, 305–317 (2005).
• 30) Roodenrys S, Booth D, Bulzomi S, Phipps A, Micallef C, Smoker J. Chronic effects of Brahmi (Bacopa monnieri) on human memory. Neuropsychopharmacology, 27, 279–281 (2002).
• 31) Holcomb LA, Dhanasekaran M, Hitt AR, Young KA, Riggs M, Manyam BV. Bacopa monniera extract reduces amyloid levels in PSAPP mice. J. Alzheimers Dis., 9, 243–251 (2006).
• 32) Limpeanchob N, Jaipan S, Rattanakaruna S, Phrompittayarat W, Ingkaninan K. Neuroprotective effect of Bacopa monnieri on beta-amyloid-induced cell death in primary cortical culture. J. Ethnopharmacol., 120, 112–117 (2008).
• 33) Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, Yoshida Y, Toyoda T, Osafune K, Sekiguchi K, Yamanaka S. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci. Rep., 4, 3594 (2014).
• 34) Amimoto N, Nishimura K, Shimohama S, Takata K. Generation of striatal neurons from human induced pluripotent stem cells by controlling extrinsic signals with small molecules. Stem Cell Res., 55, 102486 (2021).
• 35) Ohta T, Nakamura S, Nakashima S, Oda Y, Matsumoto T, Fukaya M, Yano M, Yoshikawa M, Matsuda H. Chemical structures of constituents from the whole plant of Bacopa monniera. J. Nat. Med., 70, 404–411 (2016).
• 36) Nakashima S, Oda Y, Morita M, Ohta A, Morikawa T, Matsuda H, Nakamura S. Analysis of active compounds using target protein cofilin-cucurbitacins in cytotoxic plant Bryonia cretica. Toxins (Basel), 14, 212 (2022).
• 37) Chakravarty AK, Sarkar T, Nakane T, Kawahara N, Masuda K. New phenylethanoid glycosides from Bacopa monniera. Chem. Pharm. Bull., 50, 1616–1618 (2002).
• 38) Sivandzade F, Bhalerao A, Cucullo L. Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio Protoc., 9, e3128 (2019).
• 39) Zhou C, Yang G, Chen M, He L, Xiang L, Ricupero C, Mao JJ, Ling J. Lhx6 and Lhx8: cell fate regulators and beyond. FASEB J., 29, 4083–4091 (2015).
• 40) Allaway KC, Machold R. Developmental specification of forebrain cholinergic neurons. Dev. Biol., 421, 1–7 (2017).
• 41) Bhatia R, Lin H, Lal R. Fresh and globular amyloid beta protein (1-42) induces rapid cellular degeneration: evidence for AbetaP channel-mediated cellular toxicity. FASEB J., 14, 1233–1243 (2000).
• 42) Kawahara M, Kuroda Y, Arispe N, Rojas E. Alzheimer’s beta-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium elevations by a common mechanism in a hypothalamic GnRH neuronal cell line. J. Biol. Chem., 275, 14077–14083 (2000).
• 43) Demuro A, Mina E, Kayed R, Milton SC, Parker I, Glabe CG. Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J. Biol. Chem., 280, 17294–17300 (2005).
• 44) Caballero E, Hernando-Pérez E, Tapias V, Calvo-Rodríguez M, Villalobos C, Núñez L. Amyloid beta oligomers-induced Ca²⁺ entry pathways: role of neuronal networks, NMDA receptors and amyloid channel formation. Biomedicines, 10, 1153 (2022).
• 45) Mattson MP. Pathways towards and away from Alzheimer’s disease. Nature, 430, 631–639 (2004).
• 46) Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Näslund J, Lannfelt L. The ‘Arctic’ APP mutation (E693G) causes Alzheimer’s disease by enhanced A beta protofibril formation. Nat. Neurosci., 4, 887–893 (2001).
• 47) Tomiyama T, Matsuyama S, Iso H, Umeda T, Takuma H, Ohnishi K, Ishibashi K, Teraoka R, Sakama N, Yamashita T, Nishitsuji K, Ito K, Shimada H, Lambert MP, Klein WL, Mori H. A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo. J. Neurosci., 30, 4845–4856 (2010).
• 48) Tomiyama T, Nagata T, Shimada H, Teraoka R, Fukushima A, Kanemitsu H, Takuma H, Kuwano R, Imagawa M, Ataka S, Wada Y, Yoshioka E, Nishizaki T, Watanabe Y, Mori H. A new amyloid beta variant favoring oligomerization in Alzheimer’s-type dementia. Ann. Neurol., 63, 377–387 (2008).
• 49) Sanz-Blasco S, Valero RA, Rodríguez-Crespo I, Villalobos C, Núñez L. Mitochondrial Ca²⁺ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs. PLoS ONE, 3, e2718 (2008).
• 50) Bode DC, Baker MD, Viles JH. Ion channel formation by amyloid-beta42 oligomers but not amyloid-beta40 in cellular membranes. J. Biol. Chem., 292, 1404–1413 (2017).
• 51) Arispe N, Rojas E, Pollard HB. Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. Proc. Natl. Acad. Sci. U.S.A., 90, 567–571 (1993).
• 52) Bigi A, Cascella R, Fani G, Bernacchioni C, Cencetti F, Bruni P, Chiti F, Donati C, Cecchi C. Sphingosine 1-phosphate attenuates neuronal dysfunction induced by amyloid-β oligomers through endocytic internalization of NMDA receptors. FEBS J., febs.16579 (2022).
• 53) Sivanesan S, Tan A, Rajadas J. Pathogenesis of Abeta oligomers in synaptic failure. Curr. Alzheimer Res., 10, 316–323 (2013).
• 54) Liu W, Li J, Yang M, Ke X, Dai Y, Lin H, Wang S, Chen L, Tao J. Chemical genetic activation of the cholinergic basal forebrain hippocampal circuit rescues memory loss in Alzheimer’s disease. Alzheimers Res. Ther., 14, 53 (2022).
• 55) Shen CL, Murphy RM. Solvent effects on self-assembly of beta-amyloid peptide. Biophys. J., 69, 640–651 (1995).
• 56) de Medeiros LM, De Bastiani MA, Rico EP, Schonhofen P, Pfaffenseller B, Wollenhaupt-Aguiar B, Grun L, Barbé-Tuana F, Zimmer ER, Castro MAA, Parsons RB, Klamt F. Cholinergic differentiation of human neuroblastoma SH-SY5Y cell line and its potential use as an in vitro model for Alzheimer’s disease studies. Mol. Neurobiol., 56, 7355–7367 (2019).
• 57) Kovalevich J, Langford D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol. Biol., 1078, 9–21 (2013).
• 58) Nakashima S, Ohta T, Nakamura S, Oda Y, Koumoto M, Kashiwazaki E, Kado M, Shimada A, Akita R, Matsuda H. Caffeic acid derivatives from Bacopa monniera plants as inhibitors of pancreatic lipase activity and their structural requirements. Nat. Prod. Commun., 11, 1855–1858 (2016).
• 59) Liu L, Kwak H, Lawton TL, Jin SX, Meunier AL, Dang Y, Ostaszewski B, Pietras AC, Stern AM, Selkoe DJ. An ultra-sensitive immunoassay detects and quantifies soluble Abeta oligomers in human plasma. Alzheimers Dement., 18, 1186–1202 (2022).
• 60) Georganopoulou DG, Chang L, Nam JM, Thaxton CS, Mufson EJ, Klein WL, Mirkin CA. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A., 102, 2273–2276 (2005).
• 61) Janib SM, Moses AS, Mackay JA. Imaging and drug delivery using theranostic nanoparticles. Adv. Drug Deliv. Rev., 62, 1052–1063 (2010).
• 62) Okamoto S, Shiga T, Tamaki N. Clinical perspectives of theranostics. Molecules, 26, 2232 (2021).
• 63) Moorthy H, Govindaraju T. Dendrimer architectonics to treat cancer and neurodegenerative diseases with implications in theranostics and personalized medicine. ACS Appl. Bio Mater., 4, 1115–1139 (2021).
• 64) Kevadiya BD, Ottemann BM, Thomas MB, Mukadam I, Nigam S, McMillan J, Gorantla S, Bronich TK, Edagwa B, Gendelman HE. Neurotheranostics as personalized medicines. Adv. Drug Deliv. Rev., 148, 252–289 (2019).
• 65) Klunk WE, Engler H, Nordberg A, et al. Imaging brain amyloid in Alzheimer’s disease with Pittsburgh Compound-B. Ann. Neurol., 55, 306–319 (2004).
• 66) Ono K, Hasegawa K, Naiki H, Yamada M. Curcumin has potent anti-amyloidogenic effects for Alzheimer’s beta-amyloid fibrils in vitro. J. Neurosci. Res., 75, 742–750 (2004).
• 67) Yanagisawa D, Ibrahim NF, Taguchi H, Morikawa S, Hirao K, Shirai N, Sogabe T, Tooyama I. Curcumin derivative with the substitution at C-4 position, but not curcumin, is effective against amyloid pathology in APP/PS1 mice. Neurobiol. Aging, 36, 201–210 (2015).
• 68) Yanagisawa D, Kato T, Taguchi H, Shirai N, Hirao K, Sogabe T, Tomiyama T, Gamo K, Hirahara Y, Kitada M, Tooyama I. Keto form of curcumin derivatives strongly binds to Abeta oligomers but not fibrils. Biomaterials, 270, 120686 (2021).
• 69) Hu B, Dai F, Fan Z, Ma G, Tang Q, Zhang X. Nanotheranostics: Congo red/rutin-MNPs with enhanced magnetic resonance imaging and H2O2-responsive therapy of Alzheimer’s disease in APPswe/PS1dE9 transgenic mice. Adv. Mater., 27, 5499–5505 (2015).