kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2009.13.4.273 kjpp-13-273 Original Article Inhibition of β-amyloid1-40 Peptide Aggregation and Neurotoxicity by Citrate ParkYong Hoon 1 ∗ KimYoung-Jin 2 SonIl Hong 1 2 YangHyun Duk 1 2 ∗ Inam Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Gunpo 435-040, Korea Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Gunpo 435-040, Korea Corresponding to: Hyun Duk Yang, Inam Neuroscience Research Center, Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, 1142, Sanbon-dong, Gunpo 435-040, Korea. (Tel) 82-31-390-2422, (Fax) 82-31-390-2422, (E-mail) hyundyang@gmail.com

Yong Hoon Park and Young-Jin Kim contributed equally to this work.

 082009 17082009 13 4 273279 20052009 31072009 17082009 Copyright © 2009 Korean J Physiol Pharmacol 2009 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The accumulation of β-amyloid (Aβ) aggregates is a characteristic of Alzheimer's disease (AD). Furthermore, these aggregates have neurotoxic effects on cells, and thus, molecules that inhibit Aβ aggregate formation could be valuable therapeutics for AD. It is well known that aggregation of Aβ depends on its hydrophobicity, and thus, in order to increase the hydrophilicity of Aβ, we considered using citrate, an anionic surfactant with three carboxylic acid groups. We hypothesized that citrate could reduce hydrophobicity and increase hydrophilicity of Aβ1-40 molecules via hydrophilic/electrostatic interactions. We found that citrate significantly inhibited Aβ1-40 aggregation and significantly protected SH-SY5Y cell line against Aβ1-40 aggregates-induced neurotoxicity. In details, we examined the effects of citrate on Aβ1-40 aggregation and on Aβ1-40 aggregates-induced cytotoxicity, cell viability, and apoptosis. Th-T assays showed that citrate significantly inhibited Aβ1-40 aggregation in a concentration-dependent manner (Th-T intensity: from 91.3% in 0.01 mM citrate to 82.1% in 1.0 mM citrate vs. 100.0% in Aβ1-40 alone). In cytotoxicity and viability assays, citrate reduced the toxicity of Aβ1-40 in a concentration-dependent manner, in which the cytotoxicity decreased from 107.5 to 102.3% as compared with Aβ1-40 aggregates alone treated cells (127.3%) and the cell viability increased from 84.6 to 93.8% as compared with the Aβ1-40 aggregates alone treated cells (65.3%). Furthermore, Hoechst 33342 staining showed that citrate (1.0 mM) suppressed Aβ1-40 aggregates-induced apoptosis in the cells. This study suggests that citrate can inhibit Aβ1-40 aggregation and protect neurons from the apoptotic effects of Aβ1-40 aggregates. Accordingly, our findings suggest that citrate administration should be viewed as a novel neuroprotective strategy for AD.

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The effect of citrate on aggregate formation of Aβ1-40 was determined by Th-T fluorometric assays. Aβ1-40 at 20 μM was incubated with citrate at 0.01, 0.1, or 1.0 mM at 37°C for 6 days. The fluorescence intensities of Aβ1-40 aggregate formation were measured using a spectrofluorometer. Results are means±SDs of three experiments. The asterisk (∗) indicates a significant (p<0.05) difference between treatments with Aβ aggregates alone and Aβ plus citrate.

The effect of citrate on the viability of SH-SY5Y cell line. The cells were treated for 3 days with the solution of 20 μM Aβ1-40 with/without citrate preincubated at 37°C for 6 days. Results are means±SDs of three experiments. The asterisk (∗) indicates a significant (p<0.05) difference between treatment with Aβ alone and treatment with Aβ plus citrate.

The protective effect of citrate on Aβ1-40 aggregates-induced neuronal damage of SH-SY5Y cell line. The cells were treated for 3 days with the solution of 20 μM Aβ1-40 with/without citrate preincubated at 37°C for 6 days. Results are means±SDs of three experiments. The asterisk (∗) indicates a significant (p<0.05) difference between treatment with Aβ alone and treatment with Aβ plus citrate.

Inhibitory effect of citrate on Aβ1-40-induced apoptosis. Apoptotic body formation was observed under a fluorescent microscope after Hoechst 33342 staining. Hoechst labeling shows (A) control (untreated cells), (B) increased nuclear condensation and fragmentation in 20 μM Aβ1-40 treated cells and (C) decreased nuclear condensation and fragmentation in cells treated with 20 μM Aβ1-40 plus 1.0 mM citrate preincubated for 6 days. The cells were labeled with 10 μg/ml Hoechst 33342 for 1 h after exposure to Aβ1-40 treatments for 3 days. Original magnification: ×1,000.