The Aβ-mediated decrease in estradiol levels is prevented by AG18051.

<p>(A) Scheme of pre- and co-incubation treatment. (B) Pre-treatment of cells with AG18051 for 24 hours prior to adding Aβ42 maintains estradiol levels compared to the vehicle control, (C) as does co-treatment. **, <i>P</i><0.01.</p

Effects of the ABAD inhibitor AG18051 on cell viability and estradiol levels.

<p>(A) LDH assay of SH-SY5Y human neuroblastoma cells incubated with increasing concentrations of AG18051 (normalized to 1 for control) shows that the ABAD inhibitor is not toxic at concentrations of 0.1 µM and below. (B) Treatment of SH-SY5Y cells with increasing concentrations of AG18051 causes reduced levels of estradiol. <b>*</b>, <i>P</i><0.05.</p

Aβ42 binds to and impairs ABAD activity, while HA (human amylin) does not.

<p>(A) Treatment of SH-SY5Y human neuroblastoma cells with Aβ42 causes decreased levels of estradiol, indicative of an impairment of ABAD activity, while HA does not. Results are means ± SE, (n = 5 to 6 per group), <b>**</b>, <i>P</i><0.01 (B) Pull-down of ABAD from SH-SY5Y cells shows that different from HA, Aβ42 can bind to ABAD <i>in vitro</i>. (C) Structure of the ABAD inhibitor, AG18051 (adapted from Kissinger et al., JMB 2004).</p

The ABAD inhibitor AG18051 partially prevents the toxicity of HA, but not its metabolic impairment.

<p>(A) Co-incubation of HA with AG18051 partially maintains levels of LDH release in SH-SY5Y cells suggesting that the toxicity of HA is partially mediated by ABAD. (B) Treatment with 0.5 µM HA significantly decreases metabolic activity as shown with the MTT assay, which is not prevented with co-incubation with AG18051. <b>*</b>, <i>P</i><0.05; <b>**</b>, <i>P</i><0.01.</p

The ABAD inhibitor AG18051 prevents the toxicity and metabolic impairment caused by Aβ.

<p>(A) Co-incubation of AG18051 and Aβ42 maintains the Aβ42-induced change in LDH levels at baseline levels. (B) The metabolic impairment as determined with the MTT assay is also prevented by co-incubation of Aβ42 with AG18051. (C) Pre-incubation of the cells with AG18051 for 24 hours prior to adding Aβ42 is similarly protective to Aβ's toxicity as measured with the MTT assay. <b>*</b>, <i>P</i><0.05; <b>**</b>, <i>P</i><0.01; <b>***</b>, <i>P</i><0.001.</p

High-resolution respirometry revealed a reduction of oxygen consumption in Aβ42-treated cells that was restored after pre-treatment with AG18051.

<p>O₂ flux and consumption by vital cells was measured after addition of different agents: pyruvate/glutamate, ADP, FCCP, rotenone. Two-way ANOVA revealed a significant difference between the cellular respiration of the cells treated either with vehicle, Aβ42 or AG18051 alone, or AG18051 plus Aβ42 (p<0.0001) (see scheme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028887#pone-0028887-g003" target="_blank">Fig. 3A</a>). The respiratory rates of mitochondria were significantly reduced in Aβ42-treated cells compared to control (vehicle treated) cells and cells pre-treated with AG18051 (24 h) before exposure to Aβ42. Values represent the means ± S.E. from n = 3–5 independent measurements. Post-hoc Bonferroni's Multiple Comparison Test analysis for single experimental respiratory conditions: *, p<0.05; **, p<0.01; ***, p<0.001.</p

AG18051 pre-treatment prevents ROS generation also induced by HA.

<p>As for Aβ, HA causes reduced cellular (A) as well as mitochondrial ROS (B), e.g. reduced mitochondrial superoxide anion radicals (C). Levels are restored to vehicle upon pre-treatment with AG18051. <b>*</b>, <i>P</i><0.05; <b>***</b>, <i>P</i><0.001.</p

Pull-down of ABAD from SH-SY5Y cells shows that different from HA, Aβ42 can bind to ABAD <i>in vitro</i>.

<p>The presence of AG18051 significantly decreases the amount of ABAD pulled down by biotinylated Aβ42 (BAβ). *, p<0.05.</p