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. 2013 Apr 25;8(4):e61874.
doi: 10.1371/journal.pone.0061874. Print 2013.

Effect of alanine replacement of l17 and f19 on the aggregation and neurotoxicity of arctic-type aβ40

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Effect of alanine replacement of l17 and f19 on the aggregation and neurotoxicity of arctic-type aβ40

Yi-Ru Chen et al. PLoS One. .

Abstract

Alzheimer's disease is the most common form of neurodegenerative disease. Beta-amyloid peptides (Aβ) are responsible for neuronal death both in vitro and in vivo. Previously, L17 and F19 residues were identified as playing key roles in the stabilization of the Aβ40 conformation and in the reduction of its neurotoxicity. In this study, the effects of L17A/F19A mutations on the neurotoxicity of Aβ genetic mutant Arctic-type Aβ40(E22G) were tested. The results showed that compared to Aβ40(E22G), Aβ40(L17A/F19A/E22G) reduced the rate of conformation conversion, aggregation, and cytotoxicity, suggesting that L17 and F19 are critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Aβ. Aβ40(L17A/F19A/E22G) also had decreased damage due to reactive oxygen species. The results are consistent with the discordant helix hypothesis, and confirm that residues 17-25 are in the discordant helix region. Compared to Aβ40(L17A/F19A), reduction in aggregation of Aβ40(L17A/F19A/E22G) was less significantly decreased. This observation provides an explanation based on the discordant helix hypothesis that the mutation of E22 to G22 of Aβ40(E22G) alters the propensity of the discordant helix. Arctic-type Aβ40(E22G) aggregates more severely than wild-type Aβ40, with a consequential increase in toxicity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Conformational changes of Aβ40 using far ultraviolet circular dichroism spectra.
(A) Aβ40, (B) Aβ40(L17A/F19A), (C) Aβ40 (E22G), (D) Aβ40(L17A/F19A/E22G) peptides at various incubated times. The concentration of Aβ peptides was 60 µM at pH 7.0 and 37°C.
Figure 2
Figure 2. Plot of molar ellipticity at 218 nm versus incubation time.
(A) Aβ40 (▪), (B) compare Aβ40 (▪) with Aβ40(L17A/F19A) (□), (C) Aβ40(E22G) (•), (D) compare Aβ40(E22G) with Aβ40(L17A/F19A/E22G) (○).
Figure 3
Figure 3. Kinetics of the aggregation process.
(A) Curve fitting with Aβ40 (▪), (B) Group Aβ40 (▪) versus Aβ40(L17A/F19A)(□), (C) Curve fitting with Aβ40(E22G) (•), (D) Group Aβ40(E22G) (•) versus Aβ40(L17A/F19A/E22G) (○). The aggregation assay was performed with 60 µM Aβ peptides (Aβ: Th-T = 2∶1) at 37°C.
Figure 4
Figure 4. Oligomerization and fibrillization of Aβ peptides.
(A) Representative western blots showing oligomeric and fibrillar Aβ peptides. Incubated Aβ peptides at 60 µM concentration at 37°C on day 0 (upper) and day 1, day 2 and day 3 Approximate molecular weights (in kD) determined using molecular weight markers are shown on the left-hand side. (B) FT-IR spectra of 0.2 mM Aβ peptides on day 3.
Figure 5
Figure 5. Negative stained TEM images of fibrils formed by Aβs.
Fibrils formed from 60 µM peptides in phosphate buffer, pH 7.0, 37°C on day 5. All TEM images are 200,000× magnification. The scale bar indicates 200 nm.
Figure 6
Figure 6. Cell viability determined by the MTT assay.
Survival percentages of SHSY5Y cells incubated with 30 µM Aβs for 72 hours. Wells containing SHSY5Y cells without Aβ peptides were used as a control group.*p≤0.05 versus control was considered statistically significant. **p≤0.01.
Figure 7
Figure 7. Formation of reactive oxygen species (ROS) by SHSY5Y cells.
(A) Flow cytometry after DCFH-DA staining to measure ROS production of cells treated with 30 µM Aβ peptides for 48 hours. (B) Total cells are 10000 events. P1 and P2 populations are compared with control cells without peptide (red). The histogram showing Aβ40 is blue, Aβ40(L17A/F19A) is orange, Aβ40(E22G) is green, and Aβ40(L17A/F19A/E22G) is olive.

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Grants and funding

This work was supported by grants from National Science Council of Taiwan, ROC (NSC96-2311-B-010-010-MY3 and NSC100-2113-M-010-001 to T.H.L. and NSC100-2627-M-715-001 and NSC101-2627-M-715-001 to Y.C.C.), Taipei Veterans General Hospital, Taiwan, Republic of China (V96C1-022, V96S4-002 to T.H.L.) and a grant from Ministry of Education, Aiming for the Top University Plan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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