Correlation between serum BDNF levels and scores on the <i>Perceived Stress Scale</i> (PSS) by insomnia severity groups according to the <i>Insomnia Severity Index</i> (ISI).

<p>Analyses showed a significant correlation (partial correlation controlled for smoking) between BDNF and stress only in subjects with no insomnia (<i>r_p</i> = −0.511, p = 0.013) compared to subjects with sub threshold (<i>r_p</i> = 0.069, <i>p</i> = 0.814) or clinical insomnia (<i>r_p</i> = 0.199, <i>p</i> = 0.608). White squares represent subjects with no insomnia, black circles represent subjects with sub threshold and black triangles represent subjects with clinical insomnia. (Inset) Mean serum BDNF levels of the insomnia severity groups. Plotted means and standard errors estimated by ANCOVA with serum BDNF as dependent variable, insomnia severity group as independent variable and smoking as covariate. For all three insomnia severity groups the overall effect on serum BDNF was not significant (<i>F</i>(2) = 2.67; <i>p</i> = 0.080). Contrasts showed that serum BDNF levels in the group with no insomnia were significantly higher compared to the groups reporting sub threshold and clinical insomnia (<i>F</i>(1) = 5.33; <i>p</i> = 0.026); (no insomnia n = 24; sub threshold insomnia n = 16, clinical insomnia n = 10). * Denotes statistical significance at <i>p</i><0.05</p

Descriptive characteristics of study participants.

<p>A total sample size of N = 50 was included in the analysis. Descriptive data are presented in means (M) and standard deviations (SD). Absolute numbers of participants are given (N) and expressed as percentage (%).</p><p><b>Abbreviations:</b> means (<i>M</i>); standard deviation (<i>SD</i>), brain-derived neurotrophic factor (BDNF); Insomnia Severity Index (ISI); Perceived Stress Scale (PSS); restless legs syndrome (RLS), periodic limb movement (PLM).</p

Stress experience in subjects suffering from current insomnia.

<p>Correlation between insomnia severity score (indicated by the <i>Insomnia Severity Index</i> (ISI)) and stress perception (indicated by the <i>Perceived Stress Scale</i> (PSS)). Analysis showed a significant correlation between scores on the ISI and the PSS across the whole sample (<i>r_p</i> = 0.548, <i>p</i><0.001). * Denotes statistical significance at <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

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

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

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

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