title: Rapid cytotoxicity screening platform for amyloid inhibitors using a membrane-potential sensitive fluorescent probe.
abstract: The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid binding and the regulation of membrane potential are critical to the onset and progression of neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and prion diseases. Advancing the understanding of this field requires the application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-β(1-42) (Aβ(1-42)) to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the cytotoxicity of α-Syn119 and Aβ(1-42) as determined by conventional CC8 and ATP assays. Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We envisage that this work would accelerate the development of a rapid and cost-effective high-throughput screening platform in drug discovery for AD and PD.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, Koganei, Japan.
authors.forename: Jihoon
authors.initials: J
authors.lastname: Kim
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Wataru
authors.initials: W
authors.lastname: Yoshida
authors.forename: Natsuki
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Veloso
authors.forename: Kagan
authors.initials: K
authors.lastname: Kerman
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
authors.forename: Koji
authors.initials: K
authors.lastname: Sode
mesh_terms: D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000975:Antioxidants / Q000737:chemistry; D001463:Barbiturates / Q000737:chemistry; D015374:Biosensing Techniques; D002392:Catechin / Q000031:analogs & derivatives / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D044950:Flavanones / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry; D010446:Peptide Fragments / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000633:toxicity; D013858:Thiobarbiturates / Q000737:chemistry; D051844:alpha-Synuclein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D000975:Antioxidants; D001463:Barbiturates; D044950:Flavanones; D005456:Fluorescent Dyes; D007555:Isoxazoles; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013858:Thiobarbiturates; D051844:alpha-Synuclein; C075222:amyloid beta-protein (1-42); C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol; C006680:baicalein; D002392:Catechin; C045651:epigallocatechin gallate