Nuclear Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)

Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard.

Deprenyl Enhances the Teratogenicity of Hydroxyurea in Organogenesis Stage Mouse Embryos

Hydroxyurea, an antineoplastic drug, is a model teratogen. The administration of hydroxyurea to CD1 mice on gestation day 9 induces oxidative stress, increasing the formation of 4-hydroxy-2-nonenal adducts to redox-sensitive proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the caudal region of the embryo. GAPDH catalytic activity is reduced, and its translocation into the nucleus is increased. Because the nuclear translocation of GAPDH is associated with oxidative stress–induced cell death, we hypothesized that this translocation plays a role in mediating the teratogenicity of hydroxyurea. Deprenyl (also known as selegiline), a drug used as a neuroprotectant in Parkinson’s disease, inhibits the nuclear translocation of GAPDH. Hence, timed pregnant CD1 mice were treated with deprenyl (10mg/kg) on gestation day 9 followed by the administration of hydroxyurea (400 or 600mg/kg). Deprenyl treatment significantly decreased the hydroxyurea-induced nuclear translocation of GAPDH in the caudal lumbosacral somites. Deprenyl enhanced hydroxyurea-mediated caudal malformations, inducing specifically limb reduction, digit anomalies, tail defects, and lumbosacral vertebral abnormalities. Deprenyl did not augment the hydroxyurea-induced inhibition of glycolysis or alter the ratio of oxidized to reduced glutathione. However, it did dramatically increase cleaved caspase-3 in embryos. These data suggest that nuclear GAPDH plays an important, region-specific, role in teratogen-exposed embryos. Deprenyl exacerbated the developmental outcome of hydroxyurea exposure by a mechanism that is independent of oxidative stress. Although the administration of deprenyl alone did not affect pregnancy outcome, this drug may have adverse consequences when combined with exposures that increase the risk of malformations.

Overexpression and nuclear translocation of glyceraldehyde‐3‐phosphate dehydrogenase in acetaminophen acutely intoxicated rats

The mechanism of acetaminophen (AAP) hepatotoxicity involves glutathione depletion, oxidative stress and mitochondrial dysfunction leading to necrosis. Recently, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was highlighted as a mediator of cell death.AimTo evaluate GAPDH expression and subcellular distribution in liver of AAP acute intoxicated rats.M&MMale Wistar rats were injected i.p. with 1 g/kg b.w. of AAP (AAP group) or vehicle (C group) (n=4). After 24 h, liver GAPDH expression (mRNA and protein) was studied by RT‐PCR and Western‐Blot (WB). Nuclear, mitochondrial, and cytosolic GAPDH content was evaluated in hepatic subcellular fractions by WB. Results were expressed as mean±SD and statistically analized with Student‐t‐test (* p<0.05 vs C).ResultsGAPDH mRNA (AAP:408±221*; C:100±26) and protein expression (AAP:292±127*; C:100±7) were significantly increased in treated rats. Subcellular distribution of GAPDH changed for nucleus (AAP:232±95*; C:100±49) but not for mitochondrial and cytosolic fractions.ConclusionAAP acute intoxication increased GAPDH expression due to a transcriptional mechanism, which questions its use as a housekeeping gene for either RT‐PCR or WB studies. Under AAP overdose, the GAPDH increase its content in the nucleus sugesting a role in AAP toxicity.

Nuclear GAPDH: changing the fate of Müller cells in diabetes

Müller cells, the primary glial cells are a crucial component of the retinal tissue performing a wide range of functions including maintaining the blood-retinal barrier. Several studies suggest that diabetes leads to Müller cell dysfunction and loss. The pathophysiology of hyperglycemia-induced cellular injury of Müller cells remains only poorly understood. Recently, the concept that translocation of the predominantly cytosolic glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the nucleus and its accumulation in this cellular compartment alters transcriptional events associated with cell death induction has gained major interest. High glucose conditions induce nuclear translocation and accumulation of GAPDH in the nucleus of Müller cells in vivo and in vitro. With regards to Müller cell dysfunction, the effects of nuclear accumulation of GAPDH are multifaceted. Considering the functional versatility of GAPDH including gene regulation, DNA repair, telomere protection, etc., it is of immense importance to explore possible GAPDH actions to unravel the mysteries around the role of GAPDH in hyperglycemia-induced cellular changes in order to develop novel therapeutic strategies. Therefore, this review focuses on the molecular events associated with the nuclear translocation of GAPDH and how it affects the fate of Müller cells in diabetes.

Siah-1 Protein Is Necessary for High Glucose-induced Glyceraldehyde-3-phosphate Dehydrogenase Nuclear Accumulation and Cell Death in Müller Cells

The translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the nucleus has closely been associated with cell death induction. However, the mechanism of this process has not been completely understood. The E3 ubiquitin ligase siah-1 (seven in absentia homolog 1) has recently been identified as a potential shuttle protein to transport GAPDH from the cytosol to the nucleus. Previously, we have demonstrated that elevated glucose levels induce GAPDH nuclear accumulation in retinal Müller cells. Therefore, this study investigated the role of siah-1 in high glucose-induced GAPDH nuclear translocation and subsequent cell death in retinal Müller cells. High glucose significantly increased siah-1 expression within 12 h. Under hyperglycemic conditions, siah-1 formed a complex with GAPDH and was predominantly localized in the nucleus of Müller cells. siah-1 knockdown using 50 nm siah-1 small interfering RNA significantly decreased high glucose-induced GAPDH nuclear accumulation at 24 h by 43.8 +/- 4.0%. Further, knockdown of siah-1 prevented high glucose-induced cell death of Müller cells potentially by inhibiting p53 phosphorylation consistent with previous observations, indicating that nuclear GAPDH induces cell death via p53 activation. Therefore, inhibition of GAPDH nuclear translocation and accumulation by targeting siah-1 promotes Müller cell survival under hyperglycemic conditions.

A Novel Role for Glyceraldehyde-3-Phosphate Dehydrogenase and Monoamine Oxidase B Cascade in Ethanol-Induced Cellular Damage

Alcoholism is a major psychiatric condition at least partly associated with ethanol (EtOH)-induced cell damage. Although brain cell loss has been reported in subjects with alcoholism, the molecular mechanism is unclear. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monoamine oxidase B (MAO B) reportedly play a role in cellular dysfunction under stressful conditions and might contribute to EtOH-induced cell damage. Expression of GAPDH and MAO B protein was studied in human glioblastoma and neuroblastoma cell lines exposed to physiological concentrations of EtOH. Expression of these proteins was also examined in the prefrontal cortex from human subjects with alcohol dependence and in rats fed with an EtOH diet. Coimmunoprecipitation, subcellular fractionation, and luciferase assay were used to address nuclear GAPDH-mediated MAO B activation. To test the effects of inactivation, RNA interference and pharmacological intervention were used, and cell damage was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) and hydrogen peroxide measurements. Ethanol significantly increases levels of GAPDH, especially nuclear GAPDH, and MAO B in neuronal cells as well as in human and rat brains. Nuclear GAPDH interacts with the transcriptional activator, transforming growth factor-beta-inducible early gene 2 (TIEG2), and augments TIEG2-mediated MAO B transactivation, which results in cell damage in neuronal cells exposed to EtOH. Knockdown expression of GAPDH or treatment with MAO B inhibitors selegiline (deprenyl) and rasagiline (Azilect) can block this cascade. Ethanol-elicited nuclear GAPDH augments TIEG2-mediated MAO B, which might play a role in brain damage in subjects with alcoholism. Compounds that block this cascade are potential candidates for therapeutic strategies.

Proteomic Analysis of the Differential Protein Expression Reveals Nuclear GAPDH in Activated T Lymphocytes

Despite the important role of T cell activation in the adaptive immunity, very little is known about the functions of proteins that are differentially expressed in the activated T cells. In this study, we have employed proteomic approach to study the differentially expressed proteins in activated T cells. A total of 25 proteins was characterized that displayed a decreased expression, while a total of 20 proteins was characterized that displayed an increased expression in the activated T cells. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified unexpectedly as one of the up-regulated proteins. Western blot analysis of proteins separated by 2-dimensional gel electrophoresis had identified several modified GAPDHs which were detectable only in the activated T cells, but not in resting T cells. These modified GAPDHs had higher molecular mass and more basic PI, and were present in the nucleus of activated T cells. Promoter occupancy studies by chromatin immunoprecipitation assay revealed that nuclear GAPDH could be detected in the promoter of genes that were up-regulated during T cell activation, but not in the promoter of genes that were not unaffected or down-regulated. Our results suggest that nuclear GAPDH may function as transcriptional regulator in activated T cells.

Glyceraldehyde-3-Phosphate Dehydrogenase–Monoamine Oxidase B-Mediated Cell Death-Induced by Ethanol is Prevented by Rasagiline and 1-R-Aminoindan

The inhibitors of monoamine oxidase B (MAO B) are effectively used as therapeutic drugs for neuropsychiatric and neurodegenerative diseases. However, their mechanism of action is not clear, since the neuroprotective effect of MAO B inhibitors is associated with the blockage of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-death cascade, rather than the inhibition of MAO B. Here, we provide evidence that GAPDH potentiates the ethanol-induced activity of MAO B and brain cell toxicity. The levels of nuclear GAPDH and MAO B activity are significantly increased in brain-derived cell lines upon 75 mM ethanol-induced cell death. Over-expression of GAPDH in cells enhances ethanol-induced cell death, and also increases the ethanol-induced activation of MAO B. In contrast, the MAO B inhibitors rasagiline and selegiline (0.25 nM) and the rasagiline metabolite, 1-R-aminoindan (1 muM) decreases the ethanol-induced MAO B, prevents nuclear translocation of GAPDH and reduces cell death. In addition, GAPDH interacts with transforming growth factor-beta-inducible early gene (TIEG2), a transcriptional activator for MAO B, and this interaction is increased in the nucleus by ethanol but reduced by MAO B inhibitors and 1-R-aminoindan. Furthermore, silencing TIEG2 using RNAi significantly reduces GAPDH-induced MAO B upregulation and neurotoxicity. In summary, ethanol-induced cell death, attenuated by MAO B inhibitors, may result from disrupting the movement of GAPDH with the transcriptional activator into the nucleus and secondly inhibit MAO B gene expression. Thus, the neuroprotective effects of rasagiline or 1-R-aminoindan on ethanol-induced cell death mediated by a novel GAPDH-MAO B pathway may provide a new insight in the treatment of neurobiological diseases including alcohol-use disorders.

Nuclear GAPDH Triggers Apoptosis

In addition to its functions in cellular metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is nitrosylated and translocated to the nucleus in response to stimuli that activate inducible nitric oxide synthase (iNOS). Sen et al . show that when nitrosylated GAPDH is translocated to the nucleus, it interacts with and is acetylated by the nuclear protein acetyltransferase p300 or its related homolog CBP (collectively p300/CBP). Lys 160 was the acetylated residue on GAPDH, and a K160R mutant was not acetylated and acted as a dominant-negative protein preventing acetylation of wild-type protein, but not inhibiting S -nitrosylation or nuclear translocation. When a nuclear localized version of GAPDH (NLS-GAPDH) was expressed in wild-type or iNOS -null macrophages, NLS-GAPDH bound to p300/CBP and was acetylated in response to lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treatment, which stimulates iNOS. A nuclear-localized GAPDH that had a mutation at the nitrosylation site also interacted with p300/CBP and was acetylated. Thus, nitrosylation does not appear to be required for the interaction with p300/CBP; instead, it seems to promote the nuclear localization of GAPDH. When p300 and CBP are autoacetylated, their acetylating activities are increased. LPS/IFN-γ stimulated p300 acetylation, and this was blocked by inhibition of iNOS or if GAPDH was depleted from the cells by RNA interference (RNAi). In vitro GAPDH also increased the acetylation of p300, and this did not require nitrosylation of GAPDH and was not enhanced by nitrosylation of GAPDH. To gain insight into how the interaction between GAPDH and p300/CBP may contribute to apoptosis, Sen et al . showed that NO stimulated the acetylation of p53 in human embryonic kidney (HEK) 293 cells, and this was prevented if the cells were expressing the K160R mutant, iNOS was inhibited, or GAPDH was depleted by RNAi. Chromatin immunoprecipitation experiments revealed that p53, GAPDH, and p300 were all present at the promoter of PUMA , which encodes a p53-regulated protein implicated in apoptosis. When peritoneal macrophages or the neuroblastoma cell line SH-SY5Y were transfected with GAPDH, cell death induced by LPS/IFN-γ or an NO donor, respectively, was increased, and when the cells were transfected with the K160R, mutant cell death was prevented. Thus, GAPDH appears to contribute to cell death triggered by an NO cascade by functioning in the nucleus to stimulate the acetyltransferase activity of p300/CBP, leading to the activation of p53 and proapoptotic gene expression. N. Sen, M. R. Hara, M. D. Kornberg, M. B. Cascio, B.-I. Bae, N. Shahani, B. Thomas, T. M. Dawson, V. L. Dawson, S. H. Snyder, A. Sawa, Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis. Nat. Cell Biol. 10 , 866-873 (2008). [PubMed]

Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis

Besides its role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) initiates a cell death cascade. Diverse apoptotic stimuli activate inducible nitric oxide synthase (iNOS) or neuronal NOS (nNOS), with the generated nitric oxide (NO) S-nitrosylating GAPDH, abolishing its catalytic activity and conferring on it the ability to bind to Siah1, an E3-ubiquitin-ligase with a nuclear localization signal (NLS). The GAPDH-Siah1 protein complex, in turn, translocates to the nucleus and mediates cell death; these processes are blocked by procedures that interfere with GAPDH-Siah1 binding. Nuclear events induced by GAPDH to kill cells have been obscure. Here we show that nuclear GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB binding protein (CBP) through direct protein interaction, which in turn stimulates the acetylation and catalytic activity of p300/CBP. Consequently, downstream targets of p300/CBP, such as p53 (Refs 10,11,12,13,14,15), are activated and cause cell death. A dominant-negative mutant GAPDH with the substitution of Lys 160 to Arg (GAPDH-K160R) prevents activation of p300/CBP, blocks induction of apoptotic genes and decreases cell death. Our findings reveal a pathway in which NO-induced nuclear GAPDH mediates cell death through p300/CBP.

New nuclear functions of the glycolytic protein, glyceraldehyde‐3‐phosphate dehydrogenase, in mammalian cells

Recent studies establish that the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is not simply a classical metabolic protein involved in energy production. Instead, it is a multifunctional protein with defined functions in numerous subcellular processes. New investigations establish a primary role for GAPDH in a variety of critical nuclear pathways apart from its already recognized role in apoptosis. These new roles include its requirement for transcriptional control of histone gene expression, its essential function in nuclear membrane fusion, its necessity for the recognition of fraudulently incorporated nucleotides in DNA, and its mandatory participation in the maintenance of telomere structure. Each of these new functions requires GAPDH association into a series of multienzyme complexes. Although other proteins in those complexes are variable, GAPDH remains the single constant protein in each structure. To undertake these new functions, GAPDH is recruited to the nucleus in S phase or its intracellular distribution is regulated as a function of drug exposure. Other investigations relate a substantial role for nuclear GAPDH in hyperglycemic stress and the development of metabolic syndrome. Considerations of future directions as well as the role of GAPDH post-translational modification as a basis for its multifunctional activities is suggested.

Aging of human glyceraldehyde-3-phosphate dehydrogenase is dependent on its subcellular localization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.

Rapid Shortening of Telomere Length in Response to Ceramide Involves the Inhibition of Telomere Binding Activity of Nuclear Glyceraldehyde-3-phosphate Dehydrogenase

Ceramide has been demonstrated as one of the upstream regulators of telomerase activity. However, the role for ceramide in the control of telomere length remains unknown. It is shown here that treatment of the A549 human lung adenocarcinoma cells with C(6)-ceramide results in rapid shortening of telomere length. During the examination of ceramide-regulated telomere-binding proteins, nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified to associate with both single- and double-stranded telomeric DNA with high specificity in vitro. The association of nuclear GAPDH with telomeres in interphase nuclei was also demonstrated by co-fluorescence in situ hybridization and chromatin immunoprecipitation analysis. Further data demonstrated that the nuclear localization of GAPDH is regulated by ceramide in a cell cycle-dependent manner parallel with the inhibition of its telomere binding activity in response to ceramide. In addition, the results revealed that nuclear GAPDH is distinct from its cytoplasmic isoform and that telomere binding function of nuclear GAPDH is strikingly higher than the cytoplasmic isoform. More importantly, the functional role for nuclear GAPDH in the maintenance and/or protection of telomeric DNA was identified by partial inhibition of the expression of GAPDH using small interfering RNA, which resulted in rapid shortening of telomeres. In contrast, overexpression of nuclear GAPDH resulted in the protection of telomeric DNA in response to exogenous ceramide as well as in response to anticancer drugs, which have been shown to induce endogenous ceramide levels. Therefore, these results demonstrate a novel function for nuclear GAPDH in the maintenance and/or protection of telomeres and also show that mechanisms of the rapid degradation of telomeres in response to ceramide involve the inhibition of the telomere binding activity of nuclear GAPDH.

A Novel CRM1-mediated Nuclear Export Signal Governs Nuclear Accumulation of Glyceraldehyde-3-phosphate Dehydrogenase following Genotoxic Stress

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

Alteration of nuclear glyceraldehyde-3-phosphate dehydrogenase structure in Huntington’s disease fibroblasts

The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be involved in neuronal disease and in programmed cell death. Recent investigations indicate an in vitro physical association between GAPDH and huntingtin, the mutated protein in Huntington’s disease (HD). Previous studies reveal the functional diversity of GAPDH as a membrane, cytoplasmic and nuclear protein. These activities are independent of its classical glycolytic function. Thus, huntingtin–GAPDH interactions could affect not only energy production but also result in pleiotropic effects involving various biochemical pathways in HD cells. We now report the identification of a nuclear high molecular weight (HMW) GAPDH species in Huntington’s disease cells. In contrast, nuclei from age-matched control normal human cells did not contain the HMW GAPDH species. Further, this GAPDH structure was not observed in HD whole cell sonicates which are characterized by normal GAPDH activity. The disruption of intracellular structure is implicit in the preparation of whole cell sonicates. Therefore, these results suggest that the dissociation of the GAPDH protein from its high molecular weight structure results in the recovery of its function. These findings reveal a singular, new subcellular phenotype in HD cells. As such, they indicate an interrelationship between nuclear GAPDH function and huntingtin localization in this CAG expansion neuronal disease.

Induction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in rat brain after focal ischemia/reperfusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, has been recently identified to be involved in the initiation of neuronal apoptosis. To investigate the serial changes and cellular localization of GAPDH expression, and its role in ischemia/reperfusion-induced neuronal apoptosis, the authors analyzed immunohistochemically brain areas of rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Nuclear overexpression of GAPDH was noted in the ischemic core area after 2 hours of MCAO without reperfusion. During the subsequent reperfusion, nuclear accumulation of GAPDH in this area decreased in a time-dependent manner. However, cytoplasmic and nuclear GAPDH immunoreactivity was detected in neurons of the penumbra area of the parietal cortex, in rats subjected to 2-hour MCAO followed by 3-hour reperfusion. The increase of nuclear GAPDH immunoreactivity was persistently noted up to 48 hours of reperfusion, whereas cytoplasmic immunoreactivity correlated inversely with the duration of reperfusion. Moreover, double staining revealed colocalization of nuclear GAPDH and TUNEL in the penumbra area. The authors' study demonstrated that overexpression of GAPDH and nuclear translocation occurred in both the ischemic core and penumbra area soon after focal ischemia. These processes could be viewed as an early marker of ischemia/reperfusion-induced apoptotic neuronal death. The results suggest that GAPDH may play a critical role in the progression and spread of ischemic neuronal damage.

Nuclear translocation of GAPDH-GFP fusion protein during apoptosis.

Increased expression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are early, critical events in several forms of apoptosis. In order to investigate the subcellular trafficking of GAPDH in vivo, the localization of a GAPDH-green fluorescent protein (GFP) fusion was studied in PC12, HEK 293 and COS-1 cells. In control cells, fusion protein autofluorescence was largely restricted to the cytoplasm, rather than the nuclear concentration favored by GFP alone. In contrast, as early as 30 min after an insult, nuclear fluorescence increased in all cell lines studied. The fusion protein redistribution paralleled the dynamics of endogenous GAPDH. These data suggest that some nuclear GAPDH observed during apoptosis represents protein previously resident in the cytosol. This construct provides an in vivo monitor for an early change in apoptosis.

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis.

Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.

Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.