第718回 千葉医学会例会・第二内科例会 14.

No correlation between pThr514 CRMP2 and pSer396 tau in LBD parietal cortex. a Bar graph of mean (± SEM) pSer396 tau to total tau ratios. Scatter plots of pThr514 CRMP2 with pSer396 tau : total tau in total homogenate fractions of b LBD (DLB + PDD), c DLB and d PDD parietal cortex, with insets indicating rho and p values. Available N for control = 19; PDD = 19 and DLB = 20. No significant differences (p > 0.05) were found for multiple pair-wise comparisons of pSer396 : total tau between groups (Kruskal-Wallis H tests), or for pThr514 CRMP2 correlations with pSer396 tau : total tau ratios (Spearman). (PDF 106 kb

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

Additional file 6: Figure S6. of Increased phosphorylation of collapsin response mediator protein-2 at Thr514 correlates with β-amyloid burden and synaptic deficits in Lewy body dementias

No correlation between pThr514 CRMP2 and insoluble pSer129 α-synuclein immunoreactivity in LBD parietal cortex. a Bar graphs of pSer129 α-synuclein normalized to α-synuclein immunoreactivities in the insoluble fraction (mean ± SEM in arbitrary units) with representative immunoblots. Scatter plots of pThr514 CRMP2 with pSer129 α-synuclein immunoreactivity of b LBD (DLB + PDD), c DLB and d PDD parietal cortex, with insets indicating rho and p values. Available N for control (C) = 19; PDD (P) = 19 and DLB (D) = 19. One DLB sample was excluded as an outlier due to pSer129 α-synuclein value > 8, the inclusion of which did not alter the significance of the results (data not shown). *p < 0.05, **p < 0.01, significant differences for multiple pair-wise comparisons (Kruskal-Wallis H with Dunn’s post-hoc tests). No significant differences (p > 0.05) were found for pThr514 CRMP2 correlations with pSer129 α-synuclein (Spearman). (PDF 86 kb

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

Additional file 1: Figure S1. of Increased phosphorylation of collapsin response mediator protein-2 at Thr514 correlates with β-amyloid burden and synaptic deficits in Lewy body dementias

CRMP2 is concentrated in cytosol-enriched fractions of postmortem human neocortex. Representative CRMP2 immunoblots of total and cytosol-enriched (“Cytosolic”) fractions of postmortem human neocortex (5 μg protein loaded per lane). β-actin was used as a control to show concentration of soluble cytoskeletal proteins, including CRMP2, in the cytosol-enriched fractions using the fractionation procedure described in Methods. (PDF 116 kb

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

Additional file 5: Figure S5. of Increased phosphorylation of collapsin response mediator protein-2 at Thr514 correlates with β-amyloid burden and synaptic deficits in Lewy body dementias

No correlation between pThr514 CRMP2 and α-synuclein immunoreactivity in LBD parietal cortex. a Bar graph of α-synuclein immunoreactivity (mean ± SEM in arbitrary units) with representative immunoblots, with GAPDH as loading control. Scatter plots of pThr514 CRMP2 with α-synuclein immunoreactivity in total homogenate fractions of b LBD (DLB + PDD), c DLB and d PDD parietal cortex, with insets indicating rho and p values. Available N for control (C) = 19; PDD (P) = 19 and DLB (D) = 20. No significant differences (p > 0.05) were found for multiple pair-wise comparisons of α-synuclein between groups (one-way ANOVA with Bonferroni’s post-hoc tests), or for pThr514 CRMP2 correlations with α-synuclein (Spearman). (PDF 195 kb

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