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

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 amyloidlike 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 M). Low concentrations of dopamine (50 -100 M) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200 -300 M)-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 M). 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 wildtype 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) is a classic glycolytic enzyme that is also involved in cell death and neuropsychiatric conditions (1, 2). GAPDH mediates cell death under oxidative stress conditions at least in part through nuclear translocation together with Siah (3). In the nucleus, GAPDH activates p300/CBP and regulates gene transcription (4). The pathway can be blocked by deprenyl (Selegiline), a neuroprotective compound (5). Although nuclear translocation of GAPDH is known to cause cell death, other mechanisms of GAPDH-associated cell death may also exist.Several neurodegenerative diseases are characterized by the accumulation of misfolded proteins, resulting in intracellular and extracellular protein aggregates (6, 7). For instance, conformational changes in ␤-amyloid (A␤) in Alzheimer disease and ␣-synuclein in Parkinson disease lead to the formation of abnormal oligomers and amyloid fibrils (8). Similar to A␤ and ␣-synuclein, GAPDH is also amyloidogenic (9 -14). We previously reported the molecular mechanism underlying oxidative stressinduced amyloid-like aggregation of GAPDH using the purified rabbit GAPDH and demonstrated the critical role of the act...

Oxidation of 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopaminergic Metabolite, to a Semiquinone Radical and an ortho-Quinone

“…A high increase (22%) in protein reactivity was evident with the radical-quinone mixture, indicating that redox cycling and ROS production may contribute to the observed results. Furthermore, GAPDH is known to be sensitive to ROS modification, and GAPDH aggregation is involved in dopaminergic models of oxidative stress-induced cell death (45). This makes the cross-linking observed here potentially relevant as a specific mechanism of toxicity for DOPAL.…”

“…Because of its role as a redox-sensitive enzyme involved in glucose metabolism and multiple external thiol residues (12) and its cellular proximity to DA metabolism, GAPDH serves as an appropriate target for modification by DOPAL. Also, DA-induced aggregation of GAPDH results in increased oxidative stress and cytotoxicity to dopaminergic cell models (44,45). Although both the DOPAL radical and quinone increased protein cross-linking, this effect was more pronounced for the radical (29%) than for the quinone (6%).…”

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

“…In vivo studies show that GAPDH converts from its native soluble state into a non-native high molecular weight insoluble state during disease (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). For instance, insoluble aggregates of GAPDH have been observed in the affected tissues of patients with Alzheimer disease (13) and alcoholic liver cirrhosis (12).…”

“…Interestingly, GAPDH is also a susceptibility locus for late onset Alzheimer disease (21). Furthermore, robust aggregation of GAPDH has been detected in rodent models of motor neuron disease (17) and methamphetamine abuse (11). However, whether GAPDH aggregation is a causative factor of disease remains to be determined.…”

“…Current hypotheses of GAPDH aggregation stipulate an essential role for the oxidation and disulfide cross-linking of its cysteine residues (11,13,22,23). Indeed, GAPDH aggregates formed in vivo (11)(12)(13)18) and in vitro (10,11,18,22,23) have been shown to be disulfide-cross-linked.…”