Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-β (Aβ) deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out

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

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

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

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

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

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