13 research outputs found
New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease
International audienceThe causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neu-roblastoma cell model of PD induced by MPP + , the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway , regulating cathD activity and affecting two main biological processes, α-synuclein ag-gregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology
Interplay between CathD, glycosaminoglycans (heparan sulfate (HS), chondroitin sulfate (CS)) and α-synuclein on MPP<sup>+</sup>-stressed cells.
<p>See details in the text.</p
Glycosaminoglycans modulation of α-synuclein aggregation and degradation by cathepsin D <i>in vitro</i>.
<p>a) <i>In vitro</i> α-synuclein aggregation in the presence of different GAGs. Commercial α-synuclein (25 μg/mL) was incubated 24 h at 37°C in the presence of 1, 10 or 100 μg/mL of commercial Hep, CS or HS. Monomeric, dimeric and oligomeric forms of α-synuclein were detected by western blot and quantified with ImageJ software. b)Inhibition of cathD degradation of α-synuclein by different commercial GAGs. α-synuclein was incubated in the presence of cathD (50 mU/mL) and pepstatin A, Hep, HS or CS (100 μg/mL) during 30 min at 37°C. Residual α-synuclein was detected by western blot and quantified with ImageJ software. Results are presented as the mean ± S.E.M. *** p<0.01 compared to control.</p
Characterization of MPP<sup>+</sup>-induced apoptosis.
<p>Total, mitochondrial and cytosolic extracts were obtained from normal and MPP<sup>+</sup>-stressed SHSY5Y cells (0.5 mM, 6 h).a) CathD activity in cytosolic extracts. CathD activity was normalized by cell number in each sample and expressed as percentage of the activity in normal cells. Results are expressed in percentage of normal cell extract levels and presented as mean ± S.E.M. of four independent experiments in triplicate. *** p<0.01 compared to control cells. b) Immunofluorescence detection of total cathD in cells. Left: unstressed control cells (Ctrl), right: MPP<sup>+</sup>-stressed cells (6 h). Cells were observed with a confocal microscope Zeiss Axio Observer Z.1. Scale bar represents 10 μm. Negative control omitting the first antibody but in the presence of the second one (anti-goat Fluo 546) did not show any signal. c) Analysis by western blot of Bax relocation in mitochondrial membranes at 6 h, just after the end of stress. The amount of protein was normalized by the number of cells to avoid taking into account the increase of protein amount induced by the MPP<sup>+</sup> stress. Succinate dehydrogenase-A (SDHA) was used as the loading control. This immunoblot is representative of three independent experiments in duplicate. d) Assessment of respiratory chain function 24 h after MPP<sup>+</sup> treatment. The respiratory control index (RCI) i.e. ratio [state 3 rate] / [state 4 rate] was calculated. Results are expressed as percentage of unstressed control group and represent three independent experiments in triplicate. Results are presented as the mean ± S.E.M. *** p<0.01 compared to control cells.</p
Regulation of cathepsin D activity by glycosaminoglycans.
<p>a) SH-SY5Y cells were treated with sodium chlorate (75 mM) during 24 h and sample were extracted at different times after sodium chlorate removal. GAGs amounts were measured in cells after 0, 1, 3 and 6 h after the end of the chlorate treatment. Results are presented as mean ± S.E.M. of three independent experiments in triplicate. *** p<0.001 compared to control. b) Total cathD activity was measured at different times after sodium chlorate removal (75 mM, 24h). Results are expressed in percentage of cathD in normal cell extract and presented as mean ± S.E.M. of three independent experiments in triplicate. *** p<0.001 compared to control cells. c) GAGs were extracted from control, MPP<sup>+</sup>-stressed cells, chlorate treated cells 3 and 6 h after chlorate removalas described in Material and Methods. GAG amounts were measured by DMMB assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116641#pone.0116641.ref027" target="_blank">27</a>]. The activity of cathD was measured in the presence of various concentrations of these extracted GAGs (white bars: 1μg/mL; light grey2μg/mL, medium grey:5 μg/mL and dark grey: 7 μg/mL) and expressed as the percentage of the specific CathD activity. Results are mean ± S.E.M. of three independent experiments in duplicate. ** p<0.005; *** p<0.001 compared to control cathD activity, ψ p<0.01; ψψ p<0.005; ψψψ p<0.001 compared to results obtained with 1 μg/mL of GAGs, ΦΦ p<0.005 comparison between 3 and 6 h extracted GAGs. d) Total cathD activity was measured after different times (0, 1, 3 and 6 h) of treatment with MPP<sup>+</sup> (0.5 mM) with or without pre-treatement of cell with chlorate (75 mM, 24h). CathD activity was normalized by cell number in each sample and expressed as percentage of the activity in normal cells. ** p<0.005; *** p<0.001 compared to control. e) CathD activity in cells subjected or not to MPP<sup>+</sup> stress (6 h) and followed by commercial GAGs treatment (Hep, HS and CS, 1 μg/mL). After 24 h, GAGs effects were compared to the complete inhibition of cathepsin activity by pepstatin A (peps), a specific inhibitor of this enzyme and expressed as percentage of cathD activity in control cells.</p
Characterization of α-synuclein aggregation in cells after MPP<sup>+</sup> stress.
<p>a) Typical immunoblot of α-synuclein in stressed (MPP<sup>+</sup>, 6h)) and unstressed (Ctrl) cells. b) Total and c) oligomeric forms of α-synuclein measured on stressed (MPP<sup>+</sup>) and unstressed (Ctrl) cells by Western Blot and quantified by ImageJ software. This immunoblot represents three independent experiments and is expressed as the mean ± S.E.M. *** p<0.01 compared to control cells. GAPDH was used as loading control. d) α-synuclein aggregation immunostaining with α-synuclein antibody in unstressed cells (Ctrl) and MPP<sup>+</sup>-stressed cells. Cells were observed with a confocal microscope Zeiss Axio Observer Z.1.</p
Effect of glycosaminoglycans sulfation on α-synuclein accumulation.
<p>a) Total α-synuclein accumulation was detected after 6 h in normal (Ctrl) or MPP<sup>+</sup>-treated cells and after chlorate treatment (75 mM, 24h). α-synuclein aggregation was visualized by immunostaining. Cells were observed with a confocal microscope Zeiss Axio Observer Z.1. b) Western blot analysis of total α-synuclein in lysate of cells subjected or not to MPP<sup>+</sup> stress (6 h) after treatment with sodium chlorate (75 mM, 24h) or the specific inhibitor of cathD, pepstatin A (Peps) (100 μM, 24h). Intensities of the bands obtained by western blot were quantified by ImageJ software and represent three independent experiments and are expressed as the mean ± S.E.M. *** p<0.001 compared to control cells. GAPDH was used as the loading control. c) Amounts of total α-synuclein in cells subjected or not to MPP<sup>+</sup> stress (6 h) and followed by commercial GAGs treatment (Hep, HS and CS, 1 μg/mL). After 24 h, α-synuclein was detected by western blot and quantified with ImageJ software.</p
Glycosaminoglycans in control and MPP<sup>+</sup>-stressed cells.
<p>*p<0.01,</p><p>**p<0.05 compared to control cells. Results are the mean ± S.E.M. of three independent experiments in duplicate.</p><p>Glycosaminoglycans in control and MPP<sup>+</sup>-stressed cells.</p
Final results of the PERSEE experiment
International audienceThe PERSEE breadboard, developed by a consortium including CNES, IAS, LESIA, OCA, ONERA and TAS since 2005, is a nulling demonstrator that couples an infrared nulling interferometer with a formation flying simulator able to introduce realistic disturbances in the set-up. The general idea is to prove that an adequate optical design can considerably relax the constraints applying at the spacecrafts level of a future interferometric space mission like Darwin/TPF or one of its precursors. The breadboard is now fully operational and the measurements sequences are managed from a remote control room using automatic procedures. A set of excellent results were obtained in 2011. The measured polychromatic nulling depth with non polarized light is 8.8 10-6 stabilized at 9 10-8 in the 1.65-2.45 mum spectral band (37 % bandwidth) during 100 s. This result was extended to a 7h duration thanks to an automatic calibration process. The various contributors are identified and the nulling budget is now well mastered. We also proved that harmonic disturbances in the 1-100 Hz up to several ten's of nm rms can be very efficiently corrected by a Linear Quadratic Control (LQG) if a sufficient flux is available. These results are important contributions to the feasibility of a future space based nulling interferometer