Characterization of N-PrP-interacting proteins.

<p><b>A. Schematic representation of the experimental approach.</b> Parietal cortex samples of a non-AD and an AD brain were dissolved in lysis buffer (25% w/v) homogenized with 25 strikes in a glass douncer, and centrifuged (7,500 x g, 4°C). The supernatant was added to the PrP23-114 beads and incubated overnight at 4°C. As a control, lysates were incubated with cysteine-modified beads. To purify interaction partners, beads were washed extensively in lysis buffer. Finally, beads were pelleted by centrifugation and boiled in SDS buffer. The SDS-soluble fraction was separated by SDS-PAGE and proteins co-purified with the beads were identified by ESI-MS/MS after tryptic in-gel digestion. <b>B. Venn diagram of proteins interacting with PrP23-114 in non-AD and AD brain.</b> The overlap represents PrP23-114-interacting proteins identified in both brain samples.</p

Secretory proteins are enriched in the fraction of PrP23-114-interacting proteins derived from AD brain.

<p>A, B, C. Cellular localization of the N-PrP-interactors identified only in non-AD (A) or only in AD brain (B) or identified in both (C). D. Enrichment of N-PrP-interactors in AD or non-AD brain according to their cellular localization.</p

Brain samples used for the analysis.

<p>Brain samples used for the analysis.</p

Alterations in TRAP1 levels influence [A53T]α-Synuclein-induced sensitivity to oxidative stress and mitochondrial effects in HEK293 cells.

<p>HEK293 cells were transfected with plasmids promoting [A53T]α-Synuclein or TRAP1 expression. Empty vector transfection served as control. In addition, RNAi-mediated silencing of endogenous TRAP1 was induced (siTRAP1). Cells transfected with indicated plasmid combinations were treated with (A) hydrogen peroxide (100 µm) or (B) rotenone (200 µM) to induce oxidative stress. Cell numbers were analyzed to monitor survival. (C–E) HEK293 without oxidative stress treatment overexpressing the indicated proteins, or with RNAi-mediated silencing of TRAP1 were analyzed for (C) ATP production via Complex I, (D) total ATP content, and (E) mitochondrial membrane potential. Statistical analysis of displayed bar graphs was performed using ANOVA followed by Newman-Keuls Multiple Comparison Test. (A, B) Biologically relevant comparisons are indicated in bar graphs. (C) Differences compared to control are indicated. (A, B, C) A detailed summary of all comparisons is summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002488#pgen.1002488.s008" target="_blank">Figure S8</a>. (D, E) Only cells with [A53T]α-Synuclein expression and TRAP1 reduction displayed significant differences in statistical analysis as indicated in graph. All other comparisons were not significant. *p<0.05; **p<0.01; ***p<0.001; ns = not significant.</p

TRAP1 overexpression mitigates detrimental effects induced by neuronal [A53T]α-Synuclein expression.

<p>(A) Overexpression of [A53T]α-Synuclein under control of <i>ddc-GAL4</i> resulted in reduction of DA in fly heads at 4 weeks, which was potentiated by TRAP1 deficiency (<i>TRAP1[KG]/+;ddc>A53T/+</i>), but mitigated by TRAP1 overexpression (<i>ddc>A53T/hTRAP1</i>). (B) <i>ddc>A53T</i> flies display a reduction of TH-positive neurons, which was potentiated by TRAP1 deficiency, but rescued to control levels by TRAP1 overexpression. (C) In negative geotaxis assays <i>ddc>A53T</i> flies displayed a time-dependent decline in locomotion. Reduction of TRAP1 enhanced the inability to climb (although not significant), while overexpression of hTRAP1 provided a significant rescue effect (comparison of <i>ddc>A53T/+</i> vs <i>ddc>A53T/hTRAP1</i> at 4 weeks: p<0.05). Statistics in (A, B): ANOVA followed by Newman-Keuls Multiple Comparison Test; (C): 2-way ANOVA followed by Bonferroni post-hoc tests. Displayed are biologically relevant comparisons. *p<0.05; **p<0.01; ***p<0.001; ns = not significant. (D) Alterations in TRAP1 levels did not influence PolyQ-induced rough eye phenotypes. Light micrographs of external eye structures show that PolyQ-induced REP was suppressed by parallel expression of HSP70. In contrast, neither overexpression of hTRAP1 or silencing of endogenous TRAP1 by RNAi had an obvious impact on external eye structure. Expression of mitochondrial localized GFP (mito-GFP) served as control.</p

Effect of TRAP1 mutation on modification of [A53T]α-Synuclein toxicity.

<p>(A) Western blot analysis of HEK293 lysates transfected with indicated constructs showed similar expression levels of TRAP1[WT] and TRAP1[D158N] and a reduction of endogenous TRAP1 by siTRAP1. Blot was probed with TRAP1-specific antibody. ß-Actin served as loading control. (B, C) Effect of TRAP1[WT] and TRAP1[D158N] on [A53T]α-Synuclein-induced effects in HEK293 cells. (B) Effect of TRAP1[D158N] on [A53T]α-Synuclein-induced toxicity. Cell survival after rotenone (200 µM) treatment was monitored. Compared to TRAP1[WT], cells expressing TRAP1[D158N] displayed a significant reduction in survival (t-test, **p<0.01). (C) Assessment of ATP production via Complex I in unstressed cells with [A53T]α-Synuclein expression revealed a significant reduction of ATP levels in TRAP1[D158N] versus TRAP1[WT] expressing cells (t-test, ***p<0.001).</p

[A53T]α-Synuclein expression in fly heads results in age-dependent loss of DA.

<p>(A) Western blot showing abundance of human [A53T]α-Synuclein after aminergic neuron (<i>ddc-GAL4</i>)-specific expression in lysates of fly heads. While flies without [A53T]α-Synuclein transgene do not show any detectable signal for [A53T]α-Synuclein in Western blot, an increase of [A53T]α-Synuclein protein levels was observed by increasing the copy number of transgenes. Syntaxin served as a loading control and molecular weight markers are indicated. (B) Whereas <i>ddc>A53T/+</i> flies displayed no significant difference in longevity as compared to <i>ddc/+</i> flies, flies homozygous for <i>ddc>A53T</i> showed a significant decrease (p<0.001, Log rank test). (C) Compared to controls (<i>ddc/+</i>), <i>ddc>A53T</i>/+ flies showed a significant age-dependent loss of DA at 3 and 4 weeks post eclosion (*p<0.05 vs. control). (D) Only <i>ddc>A53T</i> flies showed a significant decrease (***p<0.001) in DA concentration in fly heads at 4 weeks. Comparisons of multiple controls were not significant (4 week values as per cent of 1 week values; ANOVA followed by Newman-Keuls Multiple Comparison Test).</p

TRAP1 overexpression protects rat cortical neurons from [A53T]α-Synuclein-induced sensitivity to rotenone.

<p>(A) Western blot analysis of cells infected with lentivirus promoting either GFP, TRAP1 or [A53T]α-Synuclein expression. For visualization, the blot was probed with either TRAP1- or α-Synuclein-specific antibodies, respectively. ß-Actin was used for normalization. (B) Rat cortical neurons infected with lentivirus promoting GFP expression were stained for neuronal marker NeuN (red) and DNA (Hoechst, blue). A high percentage of cells showed co-localization between GFP and the neuronal marker NeuN, indicative for high infection efficacy. Scale bar indicates 43 µm. (C) Quantification of cell survival after 16 h of rotenone treatment of cells infected with viruses mediating expression of indicated protein. Significant differences compared to control (GFP) are indicated. All other comparisons revealed highly significant differences (p<0.001) in statistical analysis (ANOVA followed by Newman-Keuls Multiple Comparison Test). **p<0.01; ***p<0.001; ns = not significant.</p