5 research outputs found
Processing of rRNA is altered in L166P overexpressing cells.
<p>(A) Schematic diagram showing pre-rRNA structure and processing steps. Positioning of A0, 1 and 4 cleavage sites on rRNA processing intermediates is shown. ETS, external transcribed spacer; ITS, internal transcribed spacer. (B) Analysis of steady-state levels of rRNA precursor and processing intermediates. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) and empty vector (c) were treated with Bars, 5 µM MG132 for 16 h, or left untreated. Total RNA was extracted and levels of pre-rRNA and processing intermediates were analyzed by qPCR with primers targeting A0 , 1 and 4 cleavage sites. Standard deviations are calculated on four replicas from two independent experiments. *, p<0.05. (C) Analysis of rRNA processing by metabolic labeling. L166P (L) and control (c) cells were treated as in A. After treatment, cells were pulse-labeled with <sup>32</sup>P-orthophosphate for 1 h and chased with cold medium for 3 h. RNA was extracted and equal quantities were separated on denaturing agarose gel. Nascent rRNA was visualized by autoradiography after gel drying (upper panel). rRNA processing intermediates and mature forms are indicated on the right. Loading was verified with ethidium bromide staining (lower panel).</p
Mutant DJ-1 L166P inhibits TTRAP nucleolar localization after proteasome inhibition.
<p>(A) TTRAP nucleolar localization is inhibited in L166P expressing cells. SH-SY5Y cells stably transfected with empty vector (c), FLAG-DJ-1 wt (W) or L166P (L) were treated with 5 µM MG132 for 16 h. Triple immunofluorescence was performed with anti-NPM (blue), anti-TTRAP (green) and anti-FLAG (red) antibodies. Images are representatives of three independent experiments. Data have been confirmed on two independent clones for each cell line. Bars, 10 µm. (B) Quantification of TTRAP nucleolar localization. Cells as in A. At least 200 cells from two independent experiments were counted and scored for TTRAP in the nucleolus (*, p<0.05). (C) Localization of TTRAP is unaffected in cells depleted of endogenous DJ-1. SH-SY5Y cells stably expressing an inducible short-hairpin RNA targeting DJ-1 (siDJ-1, clones A and B) or a scramble shRNA control (scramble, clones a and b) were treated with doxycycline for 10 days to induce silencing of DJ-1 expression. Then cells were treated with 5 µM MG132 for 16 h. Immunofluorescence was performed with anti-TTRAP (green) and anti-NCL (red) antibodies. Nuclei were visualized with DAPI (blue). Images are representatives of two independent experiments. Bars, 10 µm. (D) Quantification of TTRAP nucleolar localization in siDJ-1 cells. Data were collected as in B. (NS, not-significant). (E) DJ-1 is efficiently depleted in siDJ-1 cells. Total protein lysates were prepared from cells as in C. Levels of endogenous DJ-1 were measured by western blot with anti-DJ-1 antibody. Beta-actin was detected as loading control. (F) TTRAP nucleolar localization is altered by mutant huntingtin. SH-SY5Y cells were transfected with huntingtin N-terminal fragment fused to GFP with WT (Gln<sup>21</sup>, Q21) or mutated (Gln<sup>150</sup>, Q150) polyglutamine expansion. Endogenous TTRAP was visualized by indirect immunofluorescence (red). N-HTT was visible by GFP autofluorescence. Nuclei were visualized with DAPI (blue). Bars, 10 µm. (F) Quantification of TTRAP nucleolar localization in N-HTT expressing cells. Data were collected as in B on GFP-positive cells (*, p<0.05; NS, not-significant).</p
TRAF6 E3 ligase activity contributes to L166P-mediated exclusion of TTRAP from nucleolar cavities.
<p>(A) Expression of TRAF6 DN partially rescues TTRAP nucleolar localization in L166P cells. SH-SY5Y cells stably expressing mutant DJ-1 were transfected with GFP-tagged TRAF6 (wt or deleted of the N-terminus, DN), or with empty vector control, as indicated. After 24 h from transfection, cells were treated for 16 h with MG132. TTRAP localization was followed by indirect immunofluorescence coupled with GFP autofluorescence. Bars, 10 µm. (B) Quantification of TTRAP nucleolar localization. Cells were as in A. At least 100 transfected cells with comparable GFP levels from two independent experiments were counted and scored for TTRAP in the nucleolus (*, p<0.05). (C) Knock-down endogenous TRAF6 expression partially rescues TTRAP nucleolar localization. SH-SY5Y cells stably transfected with mutant DJ-1 L166Pwere transfected with siRNA oligonucleotides targeting endogenous TRAF6 (siTRAF6) or with a scramble control siRNA (Scr). After 72 h from transfection, cells were treated for 16 h with MG132. Immunofluorescence was performed with anti-TTRAP (green), anti-TRAF6 (red), anti-NPM (blue). Bars, 15 µm. (D) Quantification of TTRAP nucleolar localization. Cells were as in C. Analysis was performed as in B on cells with reduced TRAF6 expression (*, p<0.05). (E) Total cell lysates were prepared from SH-SY5Y cells transfected as in C. Cells were treated with MG132 for 16 h or left untreated. Expression of endogenous TRAF6 and TTRAP was measured with specific antibodies. Protein loading was controlled by beta-actin. Molecular weight markers (MWM) are indicated for each gel (kDa). Images are representative of two independent experiments.</p
TTRAP is present in cytoplasmic Lewy bodies and in the nucleolus in surviving dopaminergic neurons in PD <i>post-mortem</i> brains.
<p>(A) TTRAP localization in normal and PD dopaminergic neurons. Cryo-sections of post-mortem brain tissues were taken from Substantia Nigra of healthy individuals and PD patients, as indicated. Immunohistochemistry was performed with anti-TTRAP antibody (green). Nuclei were stained with DAPI (blue). Dopaminergic neurons were identified by neuromelanin (black) in transmitted light (TL). Overlays of fluorescence (merge) and fluorescence with TL (TL merge) are shown. Panels are representative images showing TTRAP nuclear, cytoplasmic diffused, cytoplasmic aggregated and nucleolar localization. The percentage of dopaminergic neurons with nuclear, cytoplasmic or nucleolar TTRAP staining is indicated on the right. Bars, 15 µM. (B) TTRAP-containing cytoplasmic aggregates are Lewy bodies. Immunohistochemistry of Substantia Nigra from PD brains was performed with anti-TTRAP (green) and anti-alpha-synuclein (red) antibodies. Images at low (1×) and high (10×) magnification are shown.</p
Mutant DJ-1 L166P does not inhibit nucleolar localization of PML and p53 upon proteotoxic stress.
<p>(A) Nucleolar localization of PML. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) or empty vector (c) were treated with 5 µM MG132 for 16 h. Endogenous TTRAP (green), PML (red) and NPM (blue) localization was analyzed by triple immunofluorescence. Images are representatives of two separate experiments performed on two independent clones for each cell line. (B) Quantification of PML nucleolar localization. Cells were treated as in A. 200 cells from two independent experiments were counted and scored for PML in the nucleolus (*, p<0.05). (C) Mutant DJ-1 L166P does not affect levels of endogenous PML. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) or empty vector (c) were treated with 5 µM MG132 for 16 h or left untreated, as indicated. The expression of endogenous PML and overexpressed FLAG-DJ-1 were measured with anti-PML and anti-FLAG antibodies, respectively. Beta-actin was detected as loading control. (D) Quantification of PML levels. Densitometric analysis of protein bands was performed on two independent experiments. Relative expression of endogenous PML was normalized to beta-actin. Data are expressed as the percentage of untreated condition in empty cells (c). (E) Nucleolar localization of p53. Cells were exactly as in A. Localization of endogenous p53 (green), NPM (blue) and FLAG-DJ-1 (red) were analyzed by triple immunofluorescence. Bars, 10 µm. (F) Quantification of p53 nucleolar localization. Analysis was performed as in B (NS, non significant).</p