Figure 1.
Glycosaminoglycans modulation of α-synuclein aggregation and degradation by cathepsin D in vitro.
a) In vitro 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.
Figure 2.
Characterization of MPP+-induced apoptosis.
Total, mitochondrial and cytosolic extracts were obtained from normal and MPP+-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+-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+ 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+ 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.
Figure 3.
Characterization of α-synuclein aggregation in cells after MPP+ stress.
a) Typical immunoblot of α-synuclein in stressed (MPP+, 6h)) and unstressed (Ctrl) cells. b) Total and c) oligomeric forms of α-synuclein measured on stressed (MPP+) 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+-stressed cells. Cells were observed with a confocal microscope Zeiss Axio Observer Z.1.
Table 1.
Glycosaminoglycans in control and MPP+-stressed cells.
Figure 4.
GAGs metabolism in normal and MPP+-stressed cells.
Analysis of enzymes involved in GAGs metabolism by Real Time PCR. Real time PCR of Hs2st, Hs3st1, Hs3st2, Hs3st3, Hs3st3A1, Hs3st3B1, Hs3st4, Hs3st5, Hs3st6, Hs6st1, Hs6st2vL, Hs6st2vS, Hs6st3, NDTS1, NDST2, NDST3, NDST4, Chst8 v1 and v2, Chst8 v3, Chst10, Chst11, Chst12, Chst14, epimerase and heparanase were performed with RNA extracts from MPP+-stressed (24 h) and normal cells. Values were normalized using geNorm software, an accurate normalisation of gene expression with multiple references genes [30]. The following reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutamine synthetase and alpha-tubulin were used. Each measure is the mean +/- S.E.M. from measurements performed on 3 independent cultures. * p<0.01 compared to control cells. Light grey bars: HS biosynthesis enzymes; dark grey bars: CS biosynthesis enzymes; white bar: HS degradation enzyme.
Figure 5.
Regulation of cathepsin D activity by glycosaminoglycans.
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+-stressed cells, chlorate treated cells 3 and 6 h after chlorate removalas described in Material and Methods. GAG amounts were measured by DMMB assay [27]. 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+ (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+ 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.
Figure 6.
Effect of glycosaminoglycans sulfation on α-synuclein accumulation.
a) Total α-synuclein accumulation was detected after 6 h in normal (Ctrl) or MPP+-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+ 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+ 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.
Figure 7.
Co-localization of cathepsin D, glycosaminoglycans and α-synuclein in MPP+-stressed cells.
a) Immunofluorescence detection of CathD (green) and α-synuclein (red) in unstressed (Ctrl) and MPP+-stressed cells (6 h). b) Immunofluorescence co-labeling of CS (green) and α-synuclein (red) in normal (Ctrl) and MPP+-stressed cells (6 h). c) Immunofluorescence co-labeling of HS (green) and α-synuclein (red) in normal (Ctrl) and MPP+-stressed cells (6 h). Observations were done with a confocal microscope Zeiss Axio Observer Z.1. Stars and arrows indicate areas where co-localization is observed.
Figure 8.
Interplay between CathD, glycosaminoglycans (heparan sulfate (HS), chondroitin sulfate (CS)) and α-synuclein on MPP+-stressed cells.
See details in the text.