A novel role for glyceraldehyde-3-phosphate dehydrogenase and monoamine oxidase B cascade in ethanol-induced cellular damage

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Effects of ethanol on expression of GAPDH and MAO B. The human glioblastoma U-118 MG and neuroblastoma SH-SY5Y were treated with 75 mM ethanol for 48 h for mRNA assay or for 72 h for MAO B catalytic activity assay. (A) GAPDH mRNA levels and (B) nuclear GAPDH protein levels were determined. Histone H4 was used as a loading control for nuclear proteins. (C) Immunofluorescence microscopy was performed with anti-GAPDH antibody. U-118 MG cells were plated on a four-well chamber slide and treated with or without ethanol for 48 h. Then the cells were immunostained by mouse anti-GAPDH antibody, followed by fluorescein-conjugated secondary antibody (red). Stained slides were mounted in the presence of DAPI for nuclear staining (blue). The GAPDH (red) and nucleus (blue) and the merge of both GAPDH and the nucleus are indicated at the top. (D) MAO B mRNA levels and MAO B catalytic activities were determined. Data represent the mean ± S.D. of four independent experiments. *, p < 0.0001 and #, p < 0.005 compared with respective controls (ethanol 0 mM).

Fig. 2

Protein expression of GAPDH and MAO B in the human prefrontal cortex in alcohol-dependent subjects. (A) Western Blot analysis of brain GAPDH and MAO B. A representative blot of protein expression from 4 normal controls and 4 alcohol-dependent subjects is shown. (B) Quantitative analysis of Western blotting. Each protein was analyzed separately. Graphs of the average optical density of GAPDH and MAO B (normalized to the density of actin) are shown for the control group and alcohol-dependent subjects. The relative intensity (relative optical density × pixel area) of autoradiographic bands from two independent preparations was evaluated. Graphs of the average optical density of GAPDH/actin and MAO B/actin for the individual subjects and mean values (horizontal lines) are shown with 20 subjects (n = 20) in both the control group (circles) and alcohol dependent group (diamonds or squares). Expression of GAPDH (p < 0.05) and MAO B (p < 0.05) is significantly increased in alcohol dependent subjects as compared to the normal control subjects.

Fig. 3

Protein expression of GAPDH and MAO B in the prefrontal cortex of rats fed with ethanol. Rats were fed with an ethanol diet or control diet for 28 days, and the protein levels of GAPDH and MAO B in the prefrontal cortex were examined by Western blotting. (A) Representative Western blots showing the immunolabelling of GAPDH or MAO B in the prefrontal cortex of 6 untreated controls and 6 ethanol-treated rats. The anti-actin antibody was used as the loading controls. (B) Quantitative analysis of Western blot results. Each GAPDH or MAO B protein’s autoradiographic band was evaluated from two independent preparations by its relative intensity (relative optical density × pixel area) and normalized to the density of actin. Graphs of the average optical density of GAPDH/actin and MAO B/actin for the individual subjects and mean values (horizontal lines) are shown with 10 rats (n = 10) for both the control group (circles) and ethanol-feeding group (diamonds or triangles). Expression of GAPDH (p < 0.02) and MAO B (p < 0.05) is significantly increased in the ethanol-fed group as compared to that of the unfed control group.

Fig. 4

Effect of ethanol-induced nuclear accumulation of GAPDH or TIEG2 (an MAO B transcriptional activator) and the presence of the GAPDH/TIEG2 complex in the nucleus. (A) Western blotting of nuclear TIEG2 or GAPDH. Nuclear proteins were isolated from cells that were treated with or without ethanol for 72 h and analyzed with anti-TIEG2 or with anti-GAPDH. Quantitative analysis of optical density of TIEG2 or GAPDH (normalized to the density of histone H4) is shown at the bottom. (B) Nuclear TIEG2 co-immunoprecipitation with GAPDH. Nuclear proteins were isolated from cells that were treated with or without ethanol for 72 h, immunoprecipitated by incubation with anti-TIEG2 antibody, and analyzed by Western blotting with anti-GAPDH antibody. Quantitative analysis is shown at the bottom. Data represent the mean ± S.D. of four independent experiments. *p < 0.001 compared with respective control group (without ethanol) which was taken as 1. (C) Transient transfection and luciferase assay for the interaction among GAPDH, TIEG2, and MAO B promoter in U-118 MG cells. MAO B 2 kb promoter-luciferase reporter gene was co-transfected with pcDNA3.1 (control), GAPDH (wild type)-, GAPDH mutant (K160R)- or GAPDH mutant (Cys150)-expression vector, TIEG2-expression vector or both GAPDH- and TIEG2-expression vectors into cells. After 24 h, cells were treated with ethanol (75 mM) daily for another 2 days. All measurements were performed in triplicate in three independent experiments. *, p < 0.01, **, p < 0.001 and #, p < 0.0001 compared with control (transfected with pcDNA3.1; lane 1).

Fig. 5

Effects of GAPDH-knockdown on MAO B mRNA level and DNA damage in U-118 MG cells. (A) Western blot analysis of GAPDH-knockdown mediated by siRNA. Cells were transfected with control-siRNA or GAPDH-siRNA for 3 days. Equal amounts of total protein from each supernatant solution were resolved by SDS/PAGE and blotted by anti-GAPDH antibody. (B) Effects of GAPDH-knockdown on MAO B mRNA level. Control-siRNA-transfected cells or GAPDH-knockdown cells were treated with ethanol (75 mM) for 2 days, and then the MAO B mRNA level was determined. *, p < 0.0005 compared with control-siRNA transfected cells in ethanol-treated group. (C) Fluorescence showing TUNEL(+) cells and TUNEL(−) cells after treatment with ethanol in control-siRNA transfected cells or in GAPDH-knockdown cells. (a) Photomicrographs show representative cells from each treatment group (ethanol treatment was 2 days); arrows indicate apoptotic cells. (b) Percentage of cells that contain damaged DNA (green fluorescence) induced by ethanol (0, 2 or 3 days) as revealed by the TUNEL assay. Experiments were done in duplicate in three independent evaluations. The average counted cell numbers are 775, 694, 758, 983, 716 and 695 from lane 1 to lane 6, respectively. *, p < 0.001 and ** p < 0.0001 compared with control-siRNA-transfected cells in the ethanol-treated groups.

Fig. 6

Effects of MAO B inhibitor (selegiline) on ethanol-induced GAPDH nuclear translocation, MAO B mRNA level and the generation of toxic H₂O₂. Cells were treated with 75 mM ethanol without or with selegiline (0.25 nM) for 48 h (for immunofluorescence and mRNA level) or 72 h (for Western blotting and measurement of H₂O₂ generation). GAPDH nuclear accumulation was determined by (A) immunofluorescence microscopy and (B) Western blotting. Nuclear proteins were isolated from cells that were treated with ethanol or with both ethanol and selegiline for 72 h and were analyzed with anti-GAPDH antibody as indicated. Quantitative analysis of optical density of GAPDH (normalized to the density of histone H4) is shown at the bottom. *, p < 0.0001 compared with ethanol-treated group (without selegiline). (C) MAO B mRNA levels and (D) generation of H₂O₂ were also determined. Controls were cells treated with ethanol alone which were taken as 100%. *, p < 0.001 and #, p < 0.005. Data represent the mean ± S.D. of three independent experiments.