S-Nitrosoglutathione Reductase Plays Opposite Roles in SH-SY5Y Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis

Abstract

Oxidative and nitrosative stresses have been reported as detrimental phenomena concurring to the onset of several neurodegenerative diseases. Here we reported that the ectopic modulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) differently impinges on the phenotype of two SH-SY5Y-based in vitro models of neurodegeneration, namely, Parkinson's disease (PD) and familial amyotrophic lateral sclerosis (fALS). In particular, we provide evidence that GSNOR-knocking down protects SH-SY5Y against PD toxins, while, by contrast, its upregulation is required for G93A-SOD1 expressing cells resistance to NO-releasing drugs. Although completely opposite, both conditions are characterized by Nrf2 localization in the nuclear compartment: in the first case induced by GSNOR silencing, while in the second one underlying the antinitrosative response. Overall, our results demonstrate that GSNOR expression has different effect on neuronal viability in dependence on the stimulus applied and suggest that GSNOR could be a responsive gene downstream of Nrf2 activation.

Figure 1

Characterization of siGSNOR cells. (a) Western blotting of GSNOR in total cell lysates of SH-SY5Y cells transiently transfected for 18 h with siRNA against GSNOR (siGSNOR) or control scramble siRNA (siScr). (b) Analyses of GSNOR activity in siGSNOR and siScr cells. Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Tubulin was selected as loading control. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗∗ P < 0.01.

Figure 2

Cell death in siGSNOR cells. (a) Direct cell count upon Trypan blue staining of siScr and siGSNOR SH-SY5Y cells treated for 24 h with 0.1 to 10 mM MPP⁺. (b) Cytofluorimetric histogram of siScr and siGSNOR SH-SY5Y cells treated for 24 h with 1.25 or 2.5 mM MPP⁺ upon propidium iodide staining percentage of subG1 (apoptotic) population is shown. (c) Western blotting of PARP1 in total cell lysates of siScr and siGSNOR SH-SY5Y cells treated for 24 h with or without 2.5 mM MPP⁺. Densitometric analyses reporting the ratio cleaved: full length of PARP1 immunoreactive bands are shown on the top. (d) Direct cell count upon Trypan blue staining of siScr and siGSNOR SH-SY5Y cells treated for 24 h with 50 μM 6OHDA. Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Tubulin was selected as loading control. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 3

ROS production and protein carbonylation in GSNOR-downregulating systems. (a) Cytofluorimetric analyses of H₂O₂ in siScr and siGSNOR SH-SY5Y cells upon 2′,7′-dihydrodichlorofluorescein (DCF) staining. (b) Cytofluorimetric analyses of H₂O₂ and O₂ ^•− in GSNOR-KO and WT PCN upon 2′,7′-dihydrodichlorofluorescein (DCF) or (c) dihydroethidine (DHE) staining. (d) Western blot analyses of protein carbonyls from mesencephalon obtained from GSNOR-KO and WT brains. Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Actin was selected as loading control. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 4

DJ-1 and AKT in siGSNOR cells and GSNOR-KO brains. Western blot analyses of DJ-1, as well as basal and phosphoactive AKT in total extracts obtained from (a) siScr and siGSNOR SH-SY5Y cells or (b) GSNOR-KO and WT brains. Densitometric analyses of DJ-1 and phospho-AKT are shown on the top of the corresponding Western blot normalized to actin and basal AKT, respectively. (c) Biotin switch assay followed by pull-down with streptavidin and revealed with anti-DJ-1 antibody, in total extracts obtained from GSNOR-KO and WT brains. Western blot analysis indicates that DJ-1 was S-nitrosylated (present in the pull-down). Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Actin or GAPDH were selected as loading controls. (d) Direct cell count upon Trypan blue staining of siScr and siGSNOR SH-SY5Y cells transfected or not with siRNA against DJ-1 (siDJ-1) and treated for 24 h with 2.5 mM MPP⁺. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 5

Nuclear localization of Nrf2 in GSNOR-downregulating systems. (a) Fluorescence microscopy analyses of WT and GSNOR-KO PCN upon staining with anti-Nrf2 antibody (red), phalloidin (green), and Hoechst 33342 (blue), with the last two probes used to selectively highlight actin (cytoskeleton) and nuclei, respectively. Western blot analyses of Nrf2 in nuclear and cytosolic fractions obtained from (b) siScr and siGSNOR SH-SY5Y cells or (c) GSNOR-KO and WT brains. Western blots shown are representative of at least n = 3 independent experiments that gave similar results. SOD1 and histone 2B (H2B) were selected as loading and purity controls of cytosol and nuclei, respectively. (d) Direct cell count upon Trypan blue staining of siScr and siGSNOR SH-SY5Y cells treated for 24 h with 2.5 mM MPP⁺ in the presence or absence of 1 mM of the Nrf2 pharmacological inhibitor trigonelline. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 6

Characterization and sensitivity of G93A models to nitrosative stress. Biotin switch assay of total protein-SNOs (a) and analysis of GSNOR activity (b) performed on spinal cord lysates of WT and G93A-SOD1 expressing mice (G93A). (c) Cytofluorimetric analyses of apoptosis upon propidium iodide staining performed on parental SH-SY5Y cells (WT) and G93A-SOD1 mutants (G93A) transfected or not with siRNA against GSNOR (siGSNOR) and treated for 24 h with 400 μM DPTA. Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Tubulin was selected as loading control. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗ P < 0.05 and ^∗∗∗ P < 0.001; n.s.: not significant.

Figure 7

Characterization of the lowNO G93A resistant clone. (a) Western blotting of nNOS and GSNOR in total cell lysates of parental SH-SY5Y (WT) and the resistant lowNO G93A clone. (b) Fluorescence microscopy analyses of parental SH-SY5Y (WT) and the lowNO G93A clone. Anti-Grp75 (red) and Hoechst 33342 were used to visualize mitochondria and nuclei, respectively. (c) Fluorescence microscopy analyses of mitochondrial transmembrane potential (Δψ _m) of WT and the lowNO G93A clone by JC-1. Red fluorescence, high Δψ _m and green fluorescence, low Δψ _m. (d) Cytofluorimetric analyses of H₂O₂ and O₂ ^•− in SH-SY5Y (WT) and the lowNO G93A clone upon 2′,7′-dihydrodichlorofluorescein (DCF) or (c) dihydroethidine (DHE) staining. (e) Western blot analyses of protein carbonyls from total cell lysates obtained from WT and the lowNO G93A clone. (f) Cytofluorimetric analyses of apoptosis upon propidium iodide staining performed on parental SH-SY5Y cells (WT), sensitive G93A, and the lowNO G93A clone treated for 24 h with 400 μM DPTA. Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Actin or tubulin was selected as loading controls. Graphs shown represent the mean of data ± SD of n = 3 independent experiments, ^∗ P < 0.05 with respect to trigonelline untreated cells.

Figure 8

Role of Nrf2 in the lowNO G93A resistant clone. (a) Western blot analyses of Nrf2 in nuclear and cytosolic fractions obtained from parental SH-SY5Y (WT) and the lowNO G93A clone. (b) Cytofluorimetric analyses of apoptosis upon propidium iodide staining performed on parental SH-SY5Y cells (WT) and the lowNO G93A clone treated for 24 with DPTA in the presence or absence of the Nrf2 inhibitor trigonelline (0.5 μM). Western blots shown are representative of at least n = 3 independent experiments that gave similar results. Lactate dehydrogenase (LDH) and histone H2B were selected as loading and purity controls. Graphs shown represent the mean of data ± SD of n = 3 independent experiments.