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...

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Background: There is currently no strong evidence for a linkage between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer disease (AD). Results: GAPDH aggregates enhanced amyloid-␤ peptide (A␤) amyloidogenesis and augmented A␤40-induced neurotoxicity, both in vitro and in vivo, concomitant with mitochondrial dysfunction. Conclusion: GAPDH aggregates accelerate A␤ amyloidogenesis. Significance: A␤ amyloidogenesis associated with GAPDH aggregation might underlie AD pathogenesis.

Background:The link between poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear-translocated glyceradehyde-3-phosphate dehydrogenase (GAPDH) in neurons under oxidative/nitrosative stress remains unknown. Results: The N terminus of nuclear GAPDH binds with PARP-1, and this complex promotes PARP-1 overactivation both in vitro and in vivo. Conclusion: Nuclear GAPDH is a key PARP-1 regulator. Significance: GAPDH/PARP-1 signaling underlies oxidative/nitrosative stress-induced brain damage such as stroke.

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.

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The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+.