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, 284 (49), 34331-41

Glyceraldehyde-3-phosphate Dehydrogenase Aggregate Formation Participates in Oxidative Stress-Induced Cell Death

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Glyceraldehyde-3-phosphate Dehydrogenase Aggregate Formation Participates in Oxidative Stress-Induced Cell Death

Hidemitsu Nakajima et al. J Biol Chem.

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(2) is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC(50) = 202 microM). Low concentrations of dopamine (50-100 microM) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200-300 microM)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 microM). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage.

Figures

FIGURE 1.
FIGURE 1.
The active site cysteine (Cys-152) plays an essential role in oxidative stress-induced disulfide-bonded GAPDH aggregate formation. A, shown is a ribbon diagram of human GAPDH monomer (PDB code 1U8F). Side chains of three cysteines (152, 156, and 247) are indicated as yellow bonds. B, enzymatic activities of WT-, C152A-, C156S-, and C247A-GAPDH are indicated as a percentage of that of WT GAPDH. Data are indicated as the means ± S.D. of four samples (t test; **, p < 0.01). C, time course and concentration dependence of the increase in turbidity of GAPDH solutions in the presence of NOR3 are shown. Recombinant GAPDH (0.6 mg/ml) was treated with or without NOR3 (100 μm) at 37 °C for the indicated times (left panel) or treated with or without the indicated concentrations of NOR3 at 37 °C for 72 h (right panel). Data are presented as the means ± S.D. of three samples. D, NOR3-induced disulfide-bonded aggregates of GAPDH are shown. Samples without NOR3 were used as controls (left panel). The reaction mixtures treated with or without NOR3 (100 μm) at 37 °C for 72 h were centrifuged, generating supernatants (S) and pellets (P). The samples were subjected to either reduced or non-reduced 5–20% SDS-PAGE (right panels). E, semiquantification of the insolubility of GAPDH derived from D is shown as a pellet/(pellet + supernatant) ratio. The pellet or supernatant value is expressed as all of intensity of each lane, which was measured by Scion image software. Data are indicated as the means ± S.D. of three samples (t test; **, p < 0.01 versus WT).
FIGURE 2.
FIGURE 2.
Structural analysis of aggregated GAPDH. A, shown are far UV (upper panels) and near UV (lower panels) CD spectra of WT- or C152A-GAPDH-treated without (black lines) or with 100 μm NOR3 (red lines) at 37 °C for 1 h. The data are expressed as molar residue ellipticity (θ). B, thioflavin-S binding-dependent fluorescence of recombinant GAPDH treated with 100 μm NOR3 at 37 °C for 72 h is shown. Data are the means ± S.D. of four samples (t test; **, p < 0.01 versus WT). C, Congo Red birefringence of aggregated proteins is shown. WT GAPDH treated with 100 μm NOR3 for 72 h at 37 °C was shown under polarized lights. Aggregated Aβ1–42 (20 μm) was prepared by established methods (22). The magnification is ×20 (upper panels) or ×100 (lower panels). A.U., arbitrary units.
FIGURE 3.
FIGURE 3.
Dopamine-induced cell death, GAPDH aggregation, and nuclear translocation in SH-SY5Y cells. A, concentration-dependent effects of dopamine on the viability of SH-SY5Y cells are shown. The cell viabilities were measured at 48 h after dopamine treatment. Data are the means ± S.D. of four samples (Dunnet's test; **, p < 0.01 versus no dopamine). The IC50 value (202 μm) is calculated from the concentration dependence of the inhibition curves using nonlinear regression analyses assisted by a GraphPad Prizm 4 software. B, dopamine-induced disulfide-bonded aggregates of insoluble GAPDH in SH-SY5Y cells are shown. The particulate fractions treated without or with dopamine (50–300 μm) were subjected to non-reduced 5–20% SDS-PAGE. WB, Western blot. C, immunofluorescence of GAPDH (red) in SH-SY5Y cells treated without or with dopamine (50–300 μm) for 48 h is shown. Nuclei were stained by 4′,6-diamidino-2-phenylindole (blue). Arrows or arrowheads indicate cells with GAPDH aggregates or nuclear translocation, respectively. The scale bar = 20 μm. IHC, immunohistochemistry; endo, endogenous. D, semiquantifications of the number of the cells with aggregates are shown. Data are the means ± S.D. of three samples (Dunnet's test; **, p < 0.01 versus no dopamine). E, semiquantification of the number of the cells with nuclear translocation are shown. Data are the means ± S.D. of three samples (Dunnet's test; **, p < 0.01 versus no dopamine).
FIGURE 4.
FIGURE 4.
Correlation between GAPDH aggregation and dopamine-induced cell death. These plots indicate all of a datum of both the number of cells with GAPDH aggregates (%) and its viability (%) in single sample. The closed circles indicate as above the IC50 value. The correlation value (r2 = 0.96) is calculated using linear regression analyses assisted by GraphPad Prizm 4 software.
FIGURE 5.
FIGURE 5.
Dopamine-induced GAPDH aggregation, nuclear translocation, and cytotoxicity in DOX-inducible, GAPDH-expressing SH-SY5Y cells. A, effects of dopamine on the viability of WT GAPDH expressing cells with (+) or without (−) DOX treatment are shown. Data are the means ± S.D. of four samples (t test; **, p < 0.01 versus DOX (−) control cells). B, shown are the effects of dopamine on the viability of C152A-GAPDH expressing cells with (+) or without (−) DOX treatment are shown. Data are the means ± S.D. of four samples. C, immunofluorescence of Myc-tagged WT (upper panels) or C152A (lower panels)-GAPDH (green) in DOX-inducible cells treated without or with dopamine (50–300 μm) for 48 h is shown. Nuclei were stained by 4′,6-diamidino-2-phenylindole (red). Arrows or arrowheads indicate cells with GAPDH aggregates or the nuclear translocation, respectively. The scale bar = 20 μm. D, immunofluorescence of total GAPDH (red) including both endogenous and Myc-tagged induced WT- and C152A-GAPDH in DOX-inducible cells treated without or with dopamine (300 μm) for 48 h are shown (upper panels). Mock cells in the presence of DOX indicate a control. The lower panels show the imaged particles, namely total GAPDH aggregates. IHC, immunohistochemistry. E, quantifications of the number of particles per cell are shown. Data are the means ± S.D. of 20 samples (t test; **, p < 0.01 versus mock control cell).
FIGURE 6.
FIGURE 6.
Effects of deprenyl (DEP) on dopamine-induced GAPDH-aggregation, nuclear translocation, and cytotoxicity. A, no effects of DEP on the NOR3-induced increase of the turbidity of solution of WT GAPDH are shown. Deprenyl at the indicated concentrations were preincubated for 30 min at 4 °C before the NOR3 treatment. Data are the means ± S.D. of four samples. B, no effects of deprenyl on the dopamine-elicited aggregation of endogenous GAPDH in SH-SY5Y cells are shown. Deprenyl at 10 nm was preincubated for 30 min at 37 °C before the dopamine treatment. Data are the means ± S.D. of three samples. C, significant effects of deprenyl on the dopamine-elicited nuclear translocation of endogenous GAPDH in SH-SY5Y cells are shown. Data are the means ± S.D. of three samples (t test; **, p < 0.05 versus control cells). D, partial effectiveness of deprenyl on dopamine-elicited cytotoxity is shown. Deprenyl significantly prevented cytotoxicity only at 100 μm dopamine treatment. Data are the means ± S.D. of four samples (t test; **, p < 0.01 versus control).
FIGURE 7.
FIGURE 7.
METH-treated GAPDH transgenic mice show robust GAPDH aggregate formation and a marked reduction in dopaminergic neurons in the nigra-striatum. A, effects of METH treatment on oxidative stress levels in the striatum of WT mice are shown. DNP, dinitrophenol. WB, Western blot. B, effects of METH treatment on dopamine levels in the striatum of WT mice are shown. C, Western blotting analysis of GAPDH aggregate formation in the striatum of METH-treated WT mice are shown. D, double immunofluorescence staining of TH and GAPDH in the substantia nigra of METH-treated WT mice is shown. TH-positive dopaminergic neurons (green) and GAPDH-positive plaques (red) are shown. E, Western blotting analysis of GAPDH aggregate formation in the striatum of METH-treated WT or GAPDH-Tg mice is shown. F, dopamine levels in the striatum of METH-treated GAPDH-Tg mice are shown. G, semiquantification of TH levels in the midbrain of METH-treated GAPDH-Tg mice is shown. All of data in these graphs are the means ± S.E. of WT (littermate controls) (n = 15), WT METH-treated (n = 12), GAPDH-Tg control (n = 8), or GAPDH-Tg METH-treated (n = 8) animals (t test; *, p < 0.05; **, p < 0.01 versus WT).

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