26 research outputs found
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
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
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
[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
ASYN biochemical state.
<p><b>A. Native Gels.</b> Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. <b>B. STED microscopy.</b> Selected mutants were imaged in order to characterize the fine structure of the inclusions. <b>C. Thioflavin S staining.</b> H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. <b>D-E</b>. <b>Triton X-100 solubility assay and quantification.</b> H4 cells show that all mutants form detergent insoluble species. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.</p
Selected genes investigated by qPCR in PD patients-derived samples.
<p>-ΔCts plotted for 10 genes chosen for qPCR validation in cDNA obtained from 16 patients with either slow or rapid progression of the disease. Data is expressed as means ± SD of triplicates. T-test was used for statistical analysis with significance level of p<0.05. *p<0.05; **p<0.01; ***p<0.0005.</p
ASYN secretion is inversely correlated with toxicity.
<p><b>A. Secretion of ASYN. B. Toxicity measurements.</b> Medium from H4 cells were collected to determine the secretion and the percentage cytotoxicity for each mutant. To measure the release of ASYN, an ELISA assay was performed. Using the same media we also measured the release of lactate dehydrogenase as a measure of cytotoxicity. We observed that these values were inversely correlated with those obtained in the release/secretion experiments. A decrease trend particularly for TP and Y125F detected in terms of secretion, was higher in toxicity. n = 3. <b>C. Correlation between Secretion and Toxicity.</b> The graph shows the inverse trend in secretion and toxicity.</p
ASYN partially co-localizes with endosomes/lysosomes.
<p><b>A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants.</b> Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. <b>B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1.</b> We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.</p
Correlation between the effects of ASYN mutations on oligomerization and inclusion formation.
<p>The graph depicts how mutations affect oligomerization and inclusion formation, enabling the selection of mutants with different effects. Values were attributed to ASYN mutations according to the results from the two models (oligomerization and inclusion formation) using WT ASYN as reference (center of the graph).</p