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

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenaseNOR3(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamideNOC181-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazenePBSphosphate-buffered salineWTwild typeAβamyloid βDOXdoxycyclineMETHmethamphetamine (2S)-N-methyl-1-phenyl-propan-2-amineTHtyrosine hydroxylaseTgtransgenicHPLChigh performance liquid chromatography. 2The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenaseNOR3(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamideNOC181-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazenePBSphosphate-buffered salineWTwild typeAβamyloid βDOXdoxycyclineMETHmethamphetamine (2S)-N-methyl-1-phenyl-propan-2-amineTHtyrosine hydroxylaseTgtransgenicHPLChigh performance liquid chromatography. 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 (IC50 = 202 μm). Low concentrations of dopamine (50–100 μm) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200–300 μm)-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 μm). 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. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenaseNOR3(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamideNOC181-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazenePBSphosphate-buffered salineWTwild typeAβamyloid βDOXdoxycyclineMETHmethamphetamine (2S)-N-methyl-1-phenyl-propan-2-amineTHtyrosine hydroxylaseTgtransgenicHPLChigh performance liquid chromatography. 2The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenaseNOR3(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamideNOC181-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazenePBSphosphate-buffered salineWTwild typeAβamyloid βDOXdoxycyclineMETHmethamphetamine (2S)-N-methyl-1-phenyl-propan-2-amineTHtyrosine hydroxylaseTgtransgenicHPLChigh performance liquid chromatography. 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 (IC50 = 202 μm). Low concentrations of dopamine (50–100 μm) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200–300 μm)-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 μm). 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. glyceraldehyde-3-phosphate dehydrogenase (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene phosphate-buffered saline wild type amyloid β doxycycline methamphetamine (2S)-N-methyl-1-phenyl-propan-2-amine tyrosine hydroxylase transgenic high performance liquid chromatography. glyceraldehyde-3-phosphate dehydrogenase (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene phosphate-buffered saline wild type amyloid β doxycycline methamphetamine (2S)-N-methyl-1-phenyl-propan-2-amine tyrosine hydroxylase transgenic high performance liquid chromatography.