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Rapid cytotoxicity screening platform for amyloid inhibitors using a membrane-potential sensitive fluorescent probe Jihoon Kim, Yasuhiko Sasaki, Wataru Yoshida, Natsuki Kobayashi, Anthony J. Veloso, Kagan Kerman, Kazunori Ikebukuro, and Koji Sode Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac302442q • Publication Date (Web): 12 Nov 2012 Downloaded from http://pubs.acs.org on November 27, 2012

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Analytical Chemistry

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Rapid cytotoxicity screening platform for amyloid

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inhibitors using a membrane-potential sensitive

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fluorescent probe

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Jihoon Kim†‡, Yasuhiko Sasaki†, Wataru Yoshida†, Natsuki Kobayashi†, Anthony J. Veloso§,

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Kagan Kerman§, Kazunori Ikebukuro† and Koji Sode†

6 †

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Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan ‡

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Department of Biotechnology, Graduate School of Engineering, Tokyo University of

§

i-SENS, Inc. 465-14 Wolgye-dong, Nowon-gu, Seoul 139-845, South Korea

Department of Physical and Environmental Sciences, University of Toronto Scarborough, Toronto, ON, M1C 1A4, Canada.

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Corresponding author email address: [email protected]; Telephone/Fax: +81-42-388-7027

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ACS Paragon Plus Environment

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ABSTRACT

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The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid

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binding and the regulation of membrane potential are critical to the onset and progression of

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neurodegenerative diseases such as Parkinson’s (PD), Alzheimer’s (AD) and prion diseases.

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Advancing the understanding of this field requires the application of varied biophysical and

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biological techniques designed to probe the characteristics and underlying mechanisms of

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membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation

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system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported

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here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-β1-42 (Aβ1-

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42)

to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent

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increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the

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cytotoxicity of α-Syn119 and Aβ1-42 as determined by conventional CC8 and ATP assays.

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Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline

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quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate

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amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We

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envisage that this work would accelerate the development of a rapid and cost-effective high-

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throughput screening platform in drug discovery for AD and PD.

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INTRODUCTION

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Membrane interactions of amyloidogenic proteins and peptides are increasingly considered a

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primary factor of toxicity in the pathological mechanisms underlying several systemic

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amyloidoses, such as Parkinson’s (PD) and Alzheimer’s (AD). A common feature of protein

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misfolding is that the causative proteins change from their natural conformation to ordered β-

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sheet-rich core structures, progressing to an oligomeric state, and subsequently forming

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supramolecular assemblies of extracellular deposits or intracellular inclusions1, 2. During these

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processes, unstable oligomeric species were found to show high cytotoxicity3, 4, 5, 6 (Figure 1a). A

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central area of research on neurodegenerative diseases has recently focused on treatment

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strategies based on maintaining cellular protein homeostasis. Such investigations include the

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assessment of how specific chemical substances affect cellular viability. Investigations of

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cytotoxicity often require the analysis of numerous compounds at different concentrations and

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exposure times using cells in culture. For high-throughput drug screening, we attempted to

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develop a cytotoxicity assay that requires short exposure time with amyloidogenic proteins

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before subjecting the cells to the viability assay. The conventional cytotoxicity assays, such as

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ATP and CC8 assays, generally involve an exposure period of at least 24 h, as these techniques

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are based on the symptoms resulting from cell death by apoptosis or necrosis7, 8.

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In order to develop a rapid in vitro cytotoxicity evaluation system, we focused on the

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mechanism of cytotoxicity caused by amyloidogenic proteins, which may provide early stage

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indication of cell morphological changes during the membrane interactions. There are several

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potential mechanisms of amyloidogenic protein-induced cytotoxicity9, e.g. the destabilization of

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intracellular and extracellular membranes by oligomers (channel theory); the apoptotic cell death


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and receptor-mediated toxicity triggered by the oligomer; the impaired maturation of

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autophagosomes to lysosomes mediated by the oligomer accumulation; the mitochondrial

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dysfunction; the oxidative damage-induced disruption of the cell viability promoted by the

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incorporation of redox metals into amyloid fibrils and the subsequent generation of reactive

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oxygen species (ROS). Among these potential mechanisms, we focused on the channel theory10,

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11, 12, 13

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structures in the membrane that eliminate the ion gradient, causing excessive depolarization,

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consequently leading to cell death. In general, cells exposed to toxic oligomers display a

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remarkable increase in the levels of reactive oxygen species (ROS). Such modification of the

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intracellular redox state can result in a chain of events including lipid peroxidation,

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overstimulation of excitatory glutamate receptors, and the up-regulation of heme oxygenase-114,

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15

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as the toxic oligomers are formed at an early stage of aggregation16, 17. Some studies have

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employed the patch clamping technique to monitor the initial alterations of fundamental cellular

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processes associated with membrane perturbation12. However, the patch clamping technique is

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time-consuming, technically demanding, and excessively labour-intensive for primary screening

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purposes. Alternatively, membrane potential-sensitive fluorescent probes, such as DASPEI (2-(4-

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(dimethylamino)styryl)-N-ethylpyridinium iodide)18 and DiBAC4(3) (bis-(1,3-dibutylbarbituric

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acid)-trimethine oxonol)19, 20 have also been utilized for detecting and evaluating changes in ion

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flux during cell death. DiBAC4(3) has been widely used in flow cytometry21. The usefulness of

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DiBAC4(3) in the detection of ion channel modulators has been reported recently in the

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measurements of membrane hyperpolarization induced by the activation of ATP-dependent K+

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channels20, 22, 23. When the membrane depolarization occurs, the membrane potential-sensitive

(Figure 1b). According to this theory, oligomers form non-selective ion-channel-like

. Therefore, it has become increasingly critical to monitor the cell membrane permeabilization,


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probe localizes from the extracellular to intracellular compartment, wherein it binds to

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intracellular proteins. Fluorescence intensity would subsequently increase due to biomolecular

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interactions of the probe with the hydrophobic environment. Therefore, the cytotoxicity assay

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system using DiBAC4(3) could evaluate cytotoxicity with short cell exposure time of before

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viability evaluation compared to conventional cytotoxicity assays, such as ATP and CC8 assays,

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because the system enables us to evaluate cytotoxicity before cell death.

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In this study, we monitored the membrane interactions of C-terminal truncated α-synuclein (α-

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Syn119) and amyloid-β1-42 (Aβ1-42) using DiBAC4(3) as the fluorescent probe. α-Syn119 has

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been reported to have a higher propensity for aggregation and cytotoxicity compared to wild-

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type α-Syn in the pathology of PD24, 25. Aβ1-42 is central to the pathology of AD. It has been

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established that a 5% difference in the primary structure of the two predominant all forms, Aβ1-40

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and Aβ1-42, results in distinct assembly pathways and toxic properties26,

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suggested that our novel biosensing technique could aid in accelerating the screening of sizable

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compound libraries, leading to an improved drug-development process for future therapies of PD

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and AD.

27, 28

. The results

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Figure 1. (a) Scheme of the conformational changes of amyloidogenic proteins depicting their

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transformation into pathological species. (b) Scheme of the formation of an amyloid ion channel.

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In cells exposed to toxic oligomeric species, intracellular ROS elevation can be an immediate

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consequence of the intracellular Ca2+ increase following the formation of ion channels. The

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resulting oxidative stress can increase free Ca2+ levels by modifying the physicochemical and

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functional features of the cell membrane.

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Experimental section

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Chemicals. Aβ1-42 (human, 1-42) (trifluoroacetate form) was purchased from Peptide Institute,

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Inc. (Japan). The membrane potential-sensitive probe DiBAC4(3) ([Bis-(1,3-dibutylbarbituric

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acid) trimethine oxonol], Ultrapure grade) was purchased from Dojindo Molecular Technologies,

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Inc. (Kumamoto, Japan). Pyrroloquinoline quinone (PQQ) was kindly donated by Mitsubishi Gas

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Chemical Company, Inc. Baicalein (98%), epigallocatechin gallate (EGCG, >95%), and

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myricetin (>96%), and thioflavin T (TfT) were purchased from Sigma.

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Preparation of recombinant protein and amyloid formation. The expression and

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purification of C-terminal-truncated α-Syn119 was prepared as previously described24,

29

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Purified α-Syn119 was concentrated to approximately 5-6 mg/mL using Amicon Ultra-15 filters

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(Millipore) in PBS buffer (pH 7.3), centrifuged (at a speed of 150,000 × g for 1 h at 4°C) to

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remove aggregates, and adjusted to 200 µM of α-Syn119. The equimolar inhibitors (baicalein,

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EGCG, and myricetin) and 400 µM PQQ were mixed with α-Syn119 solution. These samples

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were incubated in 1.2 mL of the same buffer at 37°C with shaking at 700 rpm in a 2 mL tube.

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Incubated α-Syn119 was sampled in every 6 h (0, 6, 12, 18, 24 h) for evaluation of cytotoxicity.

.

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The Aβ1-42 was prepared as described in the previous report30.The concentration of Aβ1-42 was

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adjusted to 50 µM in PBS (5% DMSO). For evaluation of inhibitors, the solution contained 40

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µM Aβ1-42 in the absence or presence of 50 µM inhibitors (with the exception of 100 µM PQQ).

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These samples were incubated at pH 7.3 at 37°C with no shaking in a 0.5 mL tube. Incubated

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Aβ1-42 was sampled at 0, 8, 18, 24 h for evaluation of cytotoxicity.

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Fibril formation was monitored by TfT fluorescence. Aliquots of 10 µL were removed from

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the incubated samples and added to 1.0 mL of 25 µM TfT in PBS buffer (pH 7.3). TfT


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fluorescence was recorded at 486 nm with excitation at 450 nm using an ARVO MX 1420 multi-

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label counter (PerkinElmer).

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Cell culture. The human osteosarcoma cell line obtained from Clontech U2-OS were routinely

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cultured in McCoy’s 5A medium containing 10% horse serum and maintained at 37°C in a

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humidified incubator with an atmosphere of 5% CO2. For cytotoxicity studies, the medium was

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removed and fresh medium was gently added before plating.

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Novel cytotoxicity evaluation system using membrane potential sensitive probe: DiBAC

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assay. U2-OS cells were plated at a density of 10,000 cells per well on 96-well plates in 100 µL

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of medium. After 24 h of incubation, medium was removed and cells were washed twice using

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100 µl of assay buffer (20 mM HEPES, 120 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2,

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and 5 mM Glucose [pH 7.4]) containing 5 µM DiBAC4(3). Assay buffer (90 µL) containing 5

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µM DiBAC4(3) was added to each well and incubated for 30 min at 37°C in a humidified

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incubator with an atmosphere of 5% CO2. Cells were then exposed to α-Syn119 (final

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concentration; 0, 2, 4, 7, 14, 20, 28 µM) and Aβ1-42 (f.c.; 1, 2.5, 5, 10, 20 µM) samples (10 µL),

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and DiBAC4(3) fluorescence intensity (A) was immediately recorded at 520 nm with excitation

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at 495 nm using an ARVO MX 1420 multi-label counter (PerkinElmer). The same volume of

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PBS was added to cells and the fluorescence intensity was measured (B) as the control. In our

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technique, incubated amyloidogenic proteins (α-Syn119 or Aβ1-42) was added to U2-OS cells in

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assay solution containing DiBAC4(3). Since the interaction between amyloidogenic protein and

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DiBAC4(3) might have affected the change of fluorescence intensity, in this study, we performed

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the control experiments using amyloidogenic protein and DiBAC4(3) as a blank. Actually in the


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absence of cells, a 10 µL sample of amyloidogenic proteins and PBS was added to 90 µL of

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assay buffer containing 5 µM DiBAC4(3) and the fluorescence intensity was measured as

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background value (C and D). The results shown were calculated as ∆ fluorescence intensity =

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(A-B) - (C-D). It confirmed that there is no obvious change of fluorescence intensity at 520 nm

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by addition of incubated α-Syn119 or Aβ1-42 in the absence of DiBAC4(3).

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Conventional cytotoxicity evaluation of amyloidogenic proteins. U2-OS cells were plated at

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a density of 10,000 cells per well on 96-well plates in 100 µL of medium. After 24 h of

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incubation, 10 µL of medium was removed and 10 µL of incubated amyloidogenic protein

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sample was added (α-Syn119 f.c.; 0, 2, 4, 7, 14, 20, 28 µM and Aβ1-42 f.c.; 1, 2.5, 5, 10, 20 µM)

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or PBS was added as a control. The cells were exposed for an additional 48-72 h at 37°C in a

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humidified incubator with an atmosphere of 5% CO2.

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The number of viable cells was monitored by the Cell Counting Kit-8 (CC8 assay; Dojindo

14

Molecular Technologies, Inc., Kumamoto, Japan) and cytotoxicity was evaluated. This assay is

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based on cellular respiration, which was measured by the cellular reduction of tetrazolium

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formazan, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium

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(WST-8). After replacement of fresh 90 µL medium, 10 µL of stock WST-8 was added and the

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incubation was continued for another 2-3 h. Absorbance values at 450 nm were determined with

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an ARVO MX 1420 multi-label counter (PerkinElmer). The absorbance of the cells treated with

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PBS was measured in the same way as a control (100%).

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Cell viability was also measured by CellTiter-Glo Luminescent Cell Viability Assay Kit

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obtained from Promega (ATP assay), according to the manufacturer’s protocol. This assay

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provided a homogeneous technique for determining the number of viable cells in culture based


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on quantitation of ATP, which indicated the presence of metabolically active cells31, 32. After

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replacement of 100 µL fresh medium, 100 µL of stock solution was added and the incubation

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was continued for 5 min. The luminescence values were determined with an ARVO MX 1420

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multi-label counter. The results are expressed as percentages of bioluminescence obtained in

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control cells treated with PBS instead of amyloidogenic proteins. The data are reported as

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cytotoxicity = whole cells (100%) - cell viability (%). The cell viability (%) was calculated by

7

the CC8 assay by monitoring cell respiration or the ATP assay by monitoring cellar content of

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ATP from cells incubated with the amyloidogenic protein samples relative to control cells

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exposed to PBS.

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Results and Discussion

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Development of a novel cytotoxicity evaluation system using membrane potential-

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sensitive fluorescent probe DiBAC4(3). During the incubation of amyloidogenic proteins, their

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quaternary structures change from a monomeric state to unstable oligomeric intermediates that

15

ultimately progress towards the formation of highly-ordered amyloid fibrils. However, the

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increase in amyloid-induced cytotoxicity over the course of self-assembly has been attributed

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specifically to the accumulation of toxic oligomers at the membrane. Herein, U2-OS cells were

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exposed to either α-Syn119 or Aβ1-42, which were prepared with incubations for various periods

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of time, and their cytotoxicities were subsequently evaluated using conventional CC8 and ATP

20

assays. Simultaneously, the fluorescence response of DiBAC4(3) was monitored during the

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exposure of U2-OS cells to α-Syn119 or Aβ1-42 samples. The fluorescence data was then

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compared with the cytotoxicity results obtained from the conventional assays.


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Initially, the time course analysis of α-Syn119 and Aβ1-42 aggregation was established (Figure

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2a and b) using a well-described fluorescent dye, Thioflavin T (TfT). The binding of TfT to the

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β-sheet-rich core structure of fibrils was accompanied by a characteristic increase in fluorescence

4

intensity at approximately 482 nm33. A rapid increase in fluorescence was observed after a lag

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phase of 10 h for α-Syn119 and 5 h for Aβ1-42. Lee et al. reported that several kinds of oligomeric

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species are formed during fibril formation34. We therefore exposed the cells to α-Syn119 and

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Aβ1-42 that had been sampled at selected time intervals of incubation.

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For the conventional assays, U2-OS cells were exposed to the α-Syn119 samples for 72 h. In

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the CC8 assay, the reduction of the water soluble tetrazolium salt, WST-8 (2-(2-methoxy-4-

10

nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) served as the indicator of

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cell respiratory activity (Figure 2c). The exposure of cells to α-Syn119 samples with no prior

12

incubation resulted in an 18% decrease in viable cell number, indicating obvious cytotoxicity of

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the sample. The cytotoxicity increased with longer periods of α-Syn119 incubation. The

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exposure of cells to α-Syn119 after 6- and 12-h incubation periods resulted in significant

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cytotoxicity, as indicated by a 29% and 35% decrease, respectively, in viable cell numbers.

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Cytotoxicity reached a maximum after 18 h of α-Syn119 incubation, resulting in approximately

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55% cell death.

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In the ATP assay, the total ATP content, related to the number of metabolically active cells,

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was measured (Figure 2d). The exposure of U2-OS cells to the initial α-Syn119 sample with no

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prior incubation resulted in a 24% decrease in the viable cell number compared to the control.

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Exposure to α-Syn119 samples after 6- and 12-h incubations resulted in significant cytotoxicity,

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as indicated by a 32% and 46% decrease, respectively, in viable cell numbers. The cytotoxicity

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reached a maximum after 24 h of α-Syn119 incubation, resulting in approximately 54% cell


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death. Therefore, the results of ATP assay suggested that the increased incubation time resulted

2

in increased α-Syn119 cytotoxicity, similar to the cytotoxicity results obtained from the CC8

3

assay.

4

For the evaluation of our novel biosensing system, the same α-Syn119 samples that were

5

evaluated by the CC8 and ATP assays, were added to U2-OS cells in an assay buffer containing

6

DiBAC4(3). The fluorescence emission of DiBAC4(3) at 520 nm was immediately measured.

7

The background signal of DiBAC4(3) was approximately 3000 (Figure S-1). Figure 2e shows the

8

observed increase in the fluorescence intensity by the addition of various α-Syn119 samples. The

9

fluorescence intensity increased for up to 12 h of incubation, and showed a greater than 7-fold

10

increase compared with the initial α-Syn119 sample. These results indicated that the time-scale

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of cytotoxic effects was in good correlation with the data obtained using the conventional assays.

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Figure 2. Cytotoxicity evaluation of incubated amyloidogenic proteins using novel biosensing

3

system

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200 µM of α-Syn119 (a) and 50 µM of Aβ1-42 (b) were incubated in PBS buffer, pH 7.4. The time

5

course of α-Syn119 and Aβ1-42 aggregation was obtained from the TfT fluorescence assay

6

analysis. (c-e) Pre-incubated α-Syn119 (final concentration 20 µM) was added to U2-OS cells.

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After 72 h of exposure, the cytotoxicity upon exposure to amyloidogenic proteins was measured

8

by CC8 reduction (c) and ATP-dependent luminescence (d). The change of fluorescence

9

intensity of DiBAC4(3) was measured instantly (e). (f-h) Pre-incubated Aβ1-42 (final

10

concentration 5 µM) was added to U2-OS cells. After 72 h of exposure, the cytotoxicity was

11

determined using CC8 assay (f) and ATP assay (g). The change of fluorescence intensity of

12

DiBAC4(3) was measured instantly (h). Error bars represent the standard deviation of triplicate

13

measurements (n=3).


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We then investigated the correlation between the fluorescence response of DiBAC4(3) and the

2

α-Syn119 sample concentration. U2-OS cells were exposed to various concentrations (0, 2, 7, 14,

3

and 28 µM) of α-Syn119 for 24 h. Initially, the cytotoxicity of the α-Syn119 samples was

4

evaluated using CC8 and ATP assays after 48 h of exposure to cells. In the CC8 assay, the

5

exposure of cells to 1.4, 7, and 28 µM of α-Syn119 resulted in 14%, 22%, and 44% decrease,

6

respectively, in cell viability (Figure 3a). In the ATP assay, the exposure of 3.5, 7, and 28 µM of

7

α-Syn119 resulted in 8%, 14%, and 36% decrease, respectively, in cell viability (Figure 3b).

8

These results indicated that cytotoxicity of incubated α-Syn119 increased in a concentration-

9

dependent manner.

10

The fluorescence intensity of DiBAC4(3) in the presence of 28 µM α-Syn119 sample increased

11

more than 11- and 2-fold compared with the 2 µM and 7 µM ones, respectively (Figure 3c). We

12

have observed that the fluorescence intensity of DiBAC4(3) also increased with the addition of

13

pre-incubated α-Syn119 in a concentration-dependent manner. Plotting the cytotoxicity

14

determined by the CC8 and ATP assays after 48 h of exposure versus the fluorescence intensity

15

of DiBAC4(3) (Figure 3d), showed a high degree of linear correlation between our technique and

16

the conventional ones (R2 = 0.8964 and 0.8177 for CC8 and ATP assays, respectively).

17 18 19


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2 3

Figure 3. The correlation between novel biosensing system and conventional assays

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Samples of 24-h incubated α-Syn119 and 18-h incubated Aβ1-42 were added to U2-OS cells in

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several concentrations (f.c. 0, 2, 7, 14, 28 µM of α-Syn119 and 0, 1, 2.5, 5, 10, 20 µM of Aβ1-42).

6

After 48-h of exposure, the cytotoxicity was determined using CC8 assay (a, e) and ATP assay (b,

7

f). The fluorescence response of DiBAC4(3) was measured instantly (c, g). The calibration curve

8

shows the change in DiBAC4(3) fluorescence intensity determined immediately after addition of

9

incubated α-Syn119 (d) and Aβ1-42 (h) versus the cytotoxicity determined by CC8 (black square

10

and black dot-line) or ATP (white square and gray dot-line) assays. ). Error bars represent the

11

standard deviation of triplicate measurements (n=3).

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The cytotoxicity of Aβ1-42 was similarly evaluated with CC8 (Figure 2f) and ATP assays

2

(Figure 2g) after 72 h of exposure. In both cases, the cytotoxicity of Aβ1-42 increased with

3

incubation time, as was observed with α-Syn119 samples. Then, the correlation between the

4

fluorescence signals of DiBAC4(3) and the cytotoxicity of Aβ1-42 was studied using the CC8 and

5

ATP assays. The sample that was incubated for 8 h showed an almost 15-fold greater

6

fluorescence response than the one measured prior to the incubation (Figure 2h). These results

7

indicated that the observed increase in the fluorescence intensity of DiBAC4(3) in U2-OS cells

8

was also in good correlation with the data obtained using the conventional assays. When the

9

concentration of Aβ1-42 samples was ranged (0-20 µM), all three techniques showed good linear

10

correlation in a concentration-dependent manner (Figure 3e-g) using Aβ1-42 samples incubated

11

for 18 h (R2 = 0.9552 and 0.9107 by CC8 or ATP assays, respectively) (Figure 3h).

12

These results indicated that the observed fluorescence change of DiBAC4(3) after exposure to

13

the amyloidogenic proteins was in good correlation with their cytotoxicity, which was also

14

confirmed with the conventional assays. Therefore, the utilization of a membrane potential-

15

sensitive fluorescent probe, DiBAC4(3), was demonstrated here as a rapid and cost-effective

16

biosensing system for the cytotoxicity evaluation of amyloidogenic proteins at the early stages of

17

aggregation.

18

All data obtained with the newly developed biosensing system were validated using routinely

19

implemented assays. However, the major discrepancy between the conventional assays and the

20

novel technique was the difference in the cytotoxicity observed in samples after 18 h of

21

incubation. While the CC8 and ATP assays showed an increase in cytotoxicity with the sample

22

incubated more than 18 h, the DiBAC4(3) evaluation revealed decreased cytotoxicity with the

23

sample incubated more than 18 h (Figure 2e and h). These discrepancies between the DiBAC4(3)


16

Page 17 of 32

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1

technique and the conventional methods were attributed to the difference in the exposure time

2

and probably the composition of biomolecules showing cytotoxicity. In the conventional assays,

3

cytotoxicity was evaluated with a total amount of toxic molecules formed during the incubation

4

and also those formed during the exposure period (additional 24 – 48h). In contrast, the principle

5

of cytotoxicity evaluation with DiBAC4(3) fluorescence was based on the amount of toxic

6

molecules formed only during the sample incubation, as the fluorescence was analyzed

7

immediately after the exposure of cell to the sample. In addition, the novel method using

8

DiBAC4(3) is based entirely on the ion-channel theory. The amyloidogenic protein-induced

9

cytotoxicity resulting from other potential mechanisms, as described in the introduction, which

10

may result from other toxic molecular status, would not be estimated in this novel assay. In the

11

other words, our proposed technique would have a significant advantage in evaluating

12

cytotoxicity based on ion-channel theory at early stages.

13 14

Development of a novel biosensing system for screening the potential amyloidogenesis

15

inhibitors. The key role of oxidative stress in the mechanism of amyloid-induced cytotoxicity

16

has been supported by protective properties observed in cells exposed to antioxidants such as

17

tocopherol, lipoic acid, or reduced glutathione35,

18

antioxidant compounds (pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-

19

gallate (EGCG), and myricetin) that interact with α-Syn119 and Aβ1-42. These natural

20

polyphenolic compounds have drawn much attention due to their ability to prevent fibril

21

formation, which has potential therapeutic implications39, 40, 41, 42, 43, 44. We investigated whether

22

the inhibitory effect of these natural polyphenolic compounds on α-Syn119 and Aβ1-42

23

cytotoxicity could be detected by our biosensing system.

36, 37, 38

. We studied well-characterized


17


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Page 18 of 32

1

Initially, the formation of amyloid fibrils was monitored using TfT fluorescence. An aliquot of

2

α-Syn119 (200 µM) was incubated for 18 h in the absence or presence of an equimolar quantity

3

of inhibitors (with the exception of 400 µM PQQ). As reported, all inhibitors predominantly

4

slowed down α-Syn119 fibril formation (Figure 4a). These samples containing both α-Syn119

5

and inhibitors were then added to U2-OS cells, and their cytotoxicity was evaluated by our novel

6

biosensing system. We confirmed that PQQ, baicalein, EGCG and myricetin did not affect

7

fluorescence intensity of DiBAC4(3) in our biosensing system (Figure S-2). The samples

8

containing PQQ, EGCG, and myricetin exhibited a 33%, 45%, and 48% decrease, respectively,

9

in fluorescence intensity compared to the fluorescence data obtained from the incubation of α-

10

Syn119 alone (Figure 4b), indicating a lowered cytotoxicity resulting from the oligomeric

11

species. Significantly, baicalein decreased in fluorescence intensity by 70% compared to the

12

response recorded with α-Syn119 sample alone. Recent reports that baicalein inhibits the

13

fibrillation of α-Syn and is capable of disaggregating existing fibrils further supports the high

14

activity of baicalein observed here43, 45.

15

We also evaluated the interactions of these inhibitors with Aβ1-42. The samples of 40 µM Aβ1-

16

42 were

incubated in the presence or absence of 50 µM inhibitors (with the exception of 100 µM

17

PQQ). After 12 h of incubation, the addition of inhibitors to Aβ1-42 resulted in a 50% decrease of

18

fibril formation compared to fibril formation measured in the absence of inhibitors (Figure 4c).

19

These samples containing Aβ1-42 and inhibitors were then exposed to U2-OS cells, and

20

cytotoxicity was evaluated by measuring the change of DiBAC4(3) fluorescence. As a result, all

21

samples containing the inhibitors showed 20–30% decrease in the fluorescence intensity

22

compared with the data obtained from Aβ1-42 alone (Figure 4d). Our results indicated that all the

23

tested inhibitors successfully decreased the cytotoxic effects of Aβ1-42. In agreement with our


18

Page 19 of 32

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1

results, it has been well-documented that PQQ and EGCG had an inhibitory effect on Aβ1-42

2

cytotoxicity,39, 46 while myricetin has been observed to decrease cytotoxicity caused by Aβ1-4041.

3

The novel biosensing system clearly showed that the reduction of α-Syn119 and Aβ1-42

4

cytotoxicity by various inhibitors could be determined in a cost-effective and rapid format.

5

Demuro et al. reported that soluble amyloid oligomers rapidly elevated intracellular Ca2+

6

concentration of neuroblastoma cell

7

applied to neuroblastoma cell. Because Aβ1-40 has also been reported to form ion-channel-like

8

structures in the membrane48, we believe that our biosensing system could also be used to

9

measure Aβ1-40 cytotoxicity. A compound that inhibits oligomerization but has a similar

10

fluorescence profile as DiBAC4(3) might not be identified by a high throughput screen based on

11

our system. However, false positives, such as compounds that interact with DiBAC4(3) or the

12

cell membrane, may be easily eliminated via secondary assays. For example, one possible assay

13

may be to add the test compound at the end of the incubation period, immediately prior to

14

exposure with the cells and DiBAC4(3). This would help distinguish the desired compounds that

15

prevent oligomerization from those that do not, but which produce similar fluorescence results.

16

The described system can be readily miniaturized in a microfluidic high-throughput format for

17

screening large libraries of candidate drug molecules.

47

, suggesting that our biosensing system could also be

18 19


19


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Page 20 of 32

1 2

Figure 4. Evaluation of potential inhibitors for amyloidogenic proteins using novel biosensing

3

systems

4

(a) The formation of amyloid fibrils was monitored by the increase in TfT fluorescence after α-

5

Syn119 (200 µM) was incubated for 18 h in the absence or presence of an equimolar quantity of

6

inhibitors (with the exception of 400 µM PQQ). (c) The samples of 40 µM Aβ1-42 were also

7

incubated and monitored in the absence or presence of 50 µM compounds (with the exception of

8

100 µM PQQ). These samples containing α-Syn119 (b) or Aβ1-42 (d) with inhibitors were then

9

added to U2-OS cells and their cytotoxicity was evaluated by our novel biosensing system. Error

10

bars represent standard deviation of triplicate measurements (n=3).

11 12


20

Page 21 of 32

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1

Increasing evidence points to a central role of non-specific membrane permeabilization by

2

toxic oligomers leading to oxidative stress with the early alterations of the intracellular redox

3

state and free Ca2+ levels. Ca2+ / ROS dyshomeostasis would activate the glutamate-gated

4

calcium channels leading to a chain of events including mitochondrial damage and apoptosis49, 50,

5

51

6

elucidate some of the factors affecting protein misfolding and aggregation in AD and PD. In this

7

report, a novel fluorescence-based technique was developed to investigate the growth of toxic

8

oligomers, resulting in membrane perturbation and cell damage.

. Understanding the molecular basis of the membrane-oligomer interactions may help to

9

Although the conventional cytotoxicity assays require over 24 h to perform reliable analysis,

10

the DiBAC4(3)-based biosensing system provides a significant amount of information within the

11

first 35 min. The data of this novel biosensing system showed only average 2.26% error, in

12

contrast, conventional techniques exhibited average of 4.98% error between cell samples (data

13

not shown). This difference was mostly attributed to the variance in exposure times after the

14

addition of amyloidogenic proteins. In conventional assays, during a long exposure periods,

15

some cells enter the growth phase, and a slight difference of exposure conditions may affect the

16

cellular status, and eventually alter the vulnerability of cells against the cytotoxicity of

17

amyloidogenic proteins. It is also possible that the conformations of added protein samples could

18

have changed during the exposure periods. By contrast, this novel biosensing system required no

19

exposure time for evaluation of cytotoxicity, thus the fluctuations caused by undesired cell

20

culture conditions and transitions between aggregation states of proteins are significantly limited.

21 22

Conclusions


21


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Page 22 of 32

1

Our biosensing system provided a rapid and cost-effective platform for high-throughput

2

screening of compounds capable of mitigating cytotoxicity caused by amyloidogenic proteins

3

related to neurodegenerative diseases. Future design of therapeutic interventions should be

4

primarily targeted at avoiding the appearance of early aggregates; such a goal can be

5

accomplished by reducing the load of misfolded proteins populated at equilibrium.

6


22

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1


Supporting Information Available

2


23


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Page 24 of 32

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