Glutaredoxin Protects Cerebellar Granule Neurons from Dopamine-induced Apoptosis by Activating NF-kappa B via Ref-1*

Dvorah DailyDagger , Alexios Vlamis-Gardikas§, Daniel Offen, Leonid Mittelman||, Eldad Melamed, Arne Holmgren§**, and Ari BarzilaiDagger DaggerDagger

From the Dagger  Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel, the § Department of Biochemistry and Biophysics, Medical Nobel Institute for Biochemistry, Karolinska Institute, S-171 77 Stockholm, Sweden, the  Department of Neurology and Felsenstein Medical Research Institute, Rabin Medical Center and Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel, and the || Interdepartmental Core Facility, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel

Received for publication, September 6, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis. E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-kappa B activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although DA significantly reduced NF-kappa B binding activity, Grx2 could stimulate the binding of NF-kappa B to DNA by: (i) translocating NF-kappa B from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-kappa Balpha , and (ii) activating the binding of pre existing nuclear NF-kappa B. The DNA binding activity of NF-kappa B itself was essential for neuronal survival. Overexpression of I-kappa B dominant negative gene (I-kappa B-Delta N) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-kappa B binding activity. These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-kappa B.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The thiol-disulfide metabolism and balance contributes to the maintenance of the cellular redox (reduction/oxidation) state. Thiol redox control (1) can affect a protein's synthesis and folding, the assembly into multimeric complexes, the enzymatic activity or the binding activity of transcription factors (Refs. 2 and 3; for review, see Ref. 4). Intracellular redox-active molecules such as thioredoxin, glutaredoxin, and Ref-1, and low molecular weight thiols such as glutathione (GSH), distribute and maintain a reduced cytosolic environment in the normal cell.

Dopamine (DA),1 the endogenous neurotransmitter of the nigro-striatal pathway, is a powerful oxidant that exerts its toxic potential through its oxidative metabolites. Administration of DA into the rat striatum (5) caused pre- and postsynaptic damage. Intraventricular injection of DA into rats resulted in dose-dependent death of the animals (6). In vitro studies have shown that DA can cause cell death in mesencephalic, striatal, and cortical primary neuron cultures (7-11). Moreover, we have shown that DA-induced cell death in sympathetic, cerebellar granule neurons, PC-12 cells, and thymocytes has all the characteristic features of apoptotic cell death (12-14).

Glutaredoxins (Grx) are generally 10-kDa proteins, which catalyze GSH-disulfide oxidoreductions via two redox-active cysteine residues (15). The active site sequence (Cys-Pro-Tyr-Cys) is conserved in a variety of species (16-23). Glutaredoxins can reduce either intramolecular disulfides as in the case of ribonucleotide reductase (24-26), or mixed disulfides between a thiol-containing compound and GSH (25, 27). Escherichia coli contains three glutaredoxins, with Grx2 being the predominant in terms of concentration and catalytic activity (80% of total GSH-oxidoreductase activity in E. coli) (28). Currently, only one human glutaredoxin has been reported (23). The protein is generally considered intracellular but may also be found in human plasma (29). Apart from its activity with ribonucleotide reductase, human glutaredoxin can reduce the GSH-mixed disulfide of inactive oxidized nuclear factor-I and thus restore its DNA binding activity (30). Glutaredoxin has a role in reduction of mixed disulfides in cells exposed to oxidative stress (31).

The nuclear factor-kappa B NF-kappa B/Rel is one of the transcription factors whose DNA binding activity is regulated by a redox mechanism. Agents that modify free sulfhydryls such as N-ethylmaleimide and diamide inactivate the DNA binding of NF-kappa B, whereas reductants such as dithiothreitol (DTT), beta -mercaptoethanol, and thioredoxin enhance its DNA binding activity (for review, see Ref. 4; Ref. 32). NF-kappa B regulates the expression of various genes related to the immune response, stress, inflammation, and the inhibition of apoptosis (33, 34). Ordinarily, NF-kappa B proteins are expressed in an inactive form bound to an inhibitory protein referred to as I-kappa B. Following appropriate stimuli (UV radiation, inflammatory cytokines, phorbol esters, reactive oxygen species (ROS)), NF-kappa B is released from the cytoplasmic complex by phosphorylation and ubiquination-dependent degradation of I-kappa B. The I-kappa B-released NF-kappa B dimer translocates into the nucleus, where it binds to cognate DNA sequences and activates transcription of specific target genes (35-37). NF-kappa B is activated in a number of in vivo model systems of brain injury: brain trauma, focal ischemia, and kainate-induced seizure (38-41). Immunohistochemical analysis of brain sections from Alzheimer's disease patients revealed that NF-kappa B was activated in the most damaged areas of the brain (42). In Parkinson's disease patients, the number of neurons with NF-kappa B-stained nuclei was 70-fold higher than that of control subjects, suggesting that translocation of NF-kappa B to the nucleus was related to the pathophysiology of the disease (43).

Ref-1 regulates the activity of several transcription factors, including AP-1 elements (Jun-Fos dimers) and NF-kappa B. In addition, Ref-1 possesses apurinic/apyrimidinic endonuclease DNA repair activity against DNA damage caused by ROS, UV, and IR radiation (44-46). It is expressed in subpopulations of cells in the brain, including granule neurons of the cerebellum (47, 48). Ref-1 itself is subject to redox control and was shown to interact with thioredoxin (49).

The data presented here demonstrate that E. coli and human glutaredoxins could protect cerebellar granule neurons from DA-induced death by activating the DNA binding activity of NF-kappa B through Ref-1.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Postnatal day 8 BALB/c mice were obtained from Tel Aviv University Animal Care Facility (Glasberg Animal Research Tower). Culture media, sera, and trypsin (0.25% in 0.05% EDTA) were from Biological Industries Co. (Beit Hemeek, Israel). 32P was obtained from PerkinElmer Life Sciences, Sephadex G-25 from Amersham Pharmacia Biotech (Uppsala, Sweden), and NF-kappa B inhibitor (SN50) from Promega (Madison, WI). Rabbit antibodies against NF-kappa B p65 and Ref-1 were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). I-kappa B-Delta N plasmid was a gift from Prof. Yinon Ben-Neriah. E. coli Grx2 was a recombinant preparation purified to homogeneity as described (50). The single and double cysteine to serine mutants in the active site of Grx2 were prepared as described previously (50), but without His tags, and purified to homogeneity with the method for the wild type protein. Antibodies to Grx2 were raised in a rabbit by standard techniques. Recombinant human Grx was prepared as described by (51).

Primary Culture of Cerebellar Granule Neurons-- Cultures of highly enriched granule neurons were obtained from postnatal day 8 BALB/c mice (52). Cells were dissociated by trypsinization and plated in standard medium (basal Eagle's medium, 10% fetal calf serum, 25 mM KCl, 2 mM glutamine, 50 µg/ml gentamycin, and 250 ng/ml amphotericin B, supplemented with 1 mg/ml glucose (53) on dishes coated with poly-L-lysine (cell density, 7 × 106 cells/35-mm diameter dish, 1.5 × 106 cells/well for a 24-well plate, 2.5 × 105 cells/well for a 96-well plate). Cytosine-beta -arabinofuramoside (Ara-C) (10 µM) was added to the medium 18-22 h after plating to prevent replication of non neuronal cells (54).

Treatments-- DA (3-hydroxytyramine hydrochloride, CH8502, Sigma) was dissolved directly in the proper culture medium. Cerebellar granule neurons were maintained in standard medium for 6-7 days. The medium was then replaced with serum-free standard medium with DA, rhGrx, Grx2, or Grx2 mutants, for various periods of time. Control cultures were maintained in serum-free standard medium.

Prior to addition to the neurons, glutaredoxins were reduced with 2 mM DTT for 20 min at 37 °C. DTT was removed by two spin columns of Sephadex G-25 equilibrated with phosphate-buffered saline (PBS).

Analysis of Neuronal Viability-- The viability of the cultures (96-well plates) after treatment with DA and/or glutaredoxins was assessed either by Alamar Blue assay (AccuMed) or by 4,6-diaminodiphenyl-2-phenylindole (DAPI) staining. Alamar reagent (1:10) was added to the treated cells for 2-2.5 h at 37 °C. The viability was evaluated by subtracting the fluorescence of the medium alone (without cells) from the fluorescence of the cells at 530 nm excitation wavelength and 590 nm emission wavelength. Cells were fixated with 4% paraformaldehyde for 30 min, rinsed with PBS, and incubated with 5 µg/ml DAPI for 5 min. Nuclei were visualized under UV light. Each assay was carried out in triplicate.

Determination of the Apoptotic Commitment Point-- Neurons were treated with 600 µM DA. At different time points following DA exposure (1-24 h), the drug was washed out and replaced with fresh medium without DA. Similar sets of untreated cells served as control. Cell survival was monitored 24 h after DA administration, as mentioned above.

Protein Content-- Protein cell content was determined according to the method of Bradford (55) using bovine serum albumin as standard.

Immunocytochemistry and Nuclear Staining with DAPI-- Cerebellar granule neurons were grown on glass coverslips coated with poly-L-lysine hydrobromide (P2636, Sigma).Treated neurons were fixed for 30 min in ice-cold 4% paraformaldehyde/PBS, washed with PBS (pH 7.4), permeabilized, and blocked for 20 min with 0.2% Triton X-100 in 10% normal goat serum. Cells were then incubated with anti-p65 or anti-Ref-1 antibodies (1:50) in PBS containing 2% normal goat serum for 1 h at 37 °C, washed, and then incubated for 1 h with Cy3- or Cy2-conjugated secondary antibody (Jackson Laboratories, Bar Harbor, ME). To identify the cellular location of Ref-1 or p65, the DNA of the cells were stained with DAPI.

Protein Biotinylation-- Sulfosuccinimidyl-6-(biotinamido)hexanoate (catalog no. 21335) (biotin) was purchased from Pierce. Grx2 and C9S-C12S mutant were incubated with biotin (75 µg of biotin/mg of protein) in PBS buffer for 16 h at 4 °C. Residual biotin was removed using Sephadex G-25 column before being given to cultures. Biotinylation was confirmed by Western blot analysis using streptavidin-horseradish peroxidase.

Biotinylated Grx2 and C9S-C12S mutant were incubated for 30 min with the neurons. Thereafter, the slides were washed, fixed for 5 min in methanol (-20 °C) followed by a 2-min incubation in acetone (-20 °C), and blocked with 1% bovine serum albumin and goat gamma -globulin (Jackson Laboratories) (200 µg/ml) for 1 h. Next, the slides were washed and incubated with Cy3-conjugated streptavidin (0.5 µg/ml, Jackson Laboratories) for 30 min. After overnight washing in PBS, the slides were mounted.

To prevent bleaching of the fluorescence, moviol with n-propyl gallate was added to the slides before being excited with fluorescein and rhodamine filters.

Treatment Affecting Coated Pit Structure-- Hypertonic treatment, which blocks endocytosis via coated pits by dispersing the underlying clathrin lattices (55), was performed by incubating the culture for 15 min in PBS supplemented with 0.45 M sucrose. The cells were kept in hypertonic medium during incubation with Grx2.

Confocal Microscopy-- Fluorescently-stained cells were analyzed using Zeiss confocal laser scanning microscope. Zeiss LSM 410 inverted (Oberkochen, Germany) is equipped with a 25-milliwatt krypton-argon laser (488 and 568 maximum lines) and 10-milliwatt helium-neon laser (633 maximum lines). A 40× numeric aperture/1.2 C Apochromat water-immersion lens (Axiovert 135M, Zeiss) was used for all imaging.

Western Blot Analysis-- Western blot analysis was performed as described by Harlow and Lane (57), using 12.5% polyacrylamide gels. Each lane was loaded with an equal amount of protein extracts (30 or 60 µg), which, following electrophoresis, was transferred to an Immobilon polyvinylidene difluoride membrane for 1.5 h. Blots were stained with Ponceau to verify equal loading and transfer of proteins. Membranes were then probed with anti-Ref1 (Santa Cruz Biotechnology), anti-Ikappa B alpha  or anti-phospho-Ikappa B alpha  (Ser32) antibodies (New England Biolabs) (1:1000). Intensity of the signal was determined by ECL-Plus detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).

Nuclear and Cytoplasmic Extracts-- Neurons (7 × 106 cells) were washed with phosphate-buffered saline, scraped off, pelleted, and resuspended in 30 µl of hypotonic buffer A (10 mM Tris-HCl (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and protease inhibitors). After 15 min on ice, Nonidet P-40 was added (0.6%) and the lysates were spun down at 14,000 rpm at 4 °C. The supernatant was removed (cytoplasmic extract), and the nuclear pellet was resuspended in 20 µl of buffer B (20 mM Tris-HCl (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and protease inhibitors) with frequent vortexing for 30 min at 4 °C. Finally, the nuclear extract was spun at 14,000 rpm for 10 min and the supernatant was used for electrophoretic mobility shift assays (EMSA).

EMSA-- The binding reaction mixture containing 10 mM Tris-HCl (pH 7.9), 60 mM KCl, 0.4 mM dithiothreitol, 10% glycerol, 2 µg of bovine serum albumin, 1 µg of poly(dI-dC), 15,000 cpm of 32P-labeled kappa B oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, WI) was incubated for 30 min with 5 µg of nuclear extract. For specificity control, a 50-fold excess of unlabeled probe was applied. In the antibody supershift assay, the reaction mixture minus probe was incubated with 2 µg of p50 and/or p65 antibodies (Santa Cruz Biotechnology) for 15 min at room temperature. Products were analyzed on a 5% acrylamide gel made up in 1× TGE (50 mM Tris, 400 mM glycine, 2 mM EDTA). Dried gels were exposed to x-ray film or to phosphor screen (Molecular Dynamics). Quantitative data were obtained using PhosphorImager analysis (Molecular Dynamics).

Antisense Oligonucleotide Treatment-- Ref-1 antisense oligonucleotide and the complementary sense oligonucleotide were synthesized and high performance liquid chromatography-purified by Sigma Genosys Ltd. The oligonucleotides were labeled with fluorescein at the 5' end and phosphorotioated at the 3' end (at the positions marked by *) to confer nuclease resistance. The sequence of the Ref-1 antisense probe was 5'-TTCCCCGCTTTGGCAT*C*G*C-3' and the sense 5'-GCGATCCCAAAGCGGGGAA-3' (nucleotides -3 to 16). The probe did not show homology to other known genes according to the GenBankTM data base.

The oligonucleotides were dissolved (10 mM stock) in distilled water prior to use and diluted to a final concentration of 5 and 10 µM in serum-free medium. Cerebellar granule neuons were treated with oligonucleotides 6 days after plating.

Cotransfection of Cultured Cerebellar Granule Neurons-- Cerebellar granule neurons (1.5 × 106) were transfected 6 days after plating using the calcium phosphate method (58). Cells were cotransfected with a plasmid encoding green fluorescent protein (GFP) and with pl I-kappa B-Delta N gene (pRC-CMV-I-kappa B-Delta N), or with the GFP plasmid and the pRC-CMV empty vector. Cotransfections were carried out using a ratio of 1:1. 24 h after transfection, the medium was replaced by serum-free medium with or without DA and/or Grx2, and neuronal viability was analyzed by counting GFP positive cells under microscope (×20). For each treatment, four different fields from four different wells were analyzed.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Glutaredoxin Activity Protects Neuronal Cells from DA- induced Deaths (Fig. 1)-- To determine whether glutaredoxin could protect neuronal cells from DA-induced death, cerebellar granule neurons were exposed to 600 µM DA for 5 h, in the presence of various concentrations (2-13.8 µM) of reduced E. coli Grx2, Grx2 mutants C12S (exhibits 70% of the wild type activity) and C9S-C12S (inactive) and recombinant human glutaredoxin (rhGrx) (Fig. 1). In the absence of Grx2, only 20% of the exposed neurons survived. Increasing concentrations of wild type Grx2 rescued exposed neurons from apoptotic death (84%) (Fig. 1A). The protective effect was lower for the Grx2 mutant C12S (52%), whereas no protection was conferred by the double thiol mutant (Grx2 C9S-C12S) (Fig. 1A). rhGrx showed an almost identical protection profile as E. coli Grx2 (Fig. 1B). Counting condensed and apoptotic nuclei using DAPI staining revealed that Grx2 (4-13 µM) treatment conferred full protection against DA toxicity (Fig. 1C). These results show that glutaredoxins (E. coli and human) protected neurons from DA-induced death. The redox activity of Grx2 was essential for its protective effect.



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Fig. 1.   Neuroprotective activity of Grx2, Grx2 mutants, and rhGrx against DA-induced death. Cerebellar granule neurons were incubated with 600 µM DA for 5 h in the presence or absence of various concentrations of Grx2 (), C9S mutant (down-triangle), or C9S-C12S mutant (black-square) (A) or rhGrx (B). Cell viability was determined by Alamar blue assay or by counting condensed and apoptotic nuclei as shown by DAPI staining (C). ***, p < 0.001; **, p < 0.025; *, p < 0.05. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test (n = 4).

Grx2 Penetrates into the Granule Neurons (Fig. 2)-- To examine whether Grx2 could penetrate neurons, neurons were incubated with 13 µM Grx2 for 30 min, fixed, and exposed to anti Grx2 antibodies. No cross-reacting material was detected (data not shown). After incubating neurons with biotinylated Grx2, cross-reacting material was observed throughout 100% of the cells (cytoplasm and nucleus, Fig. 2C). No signal was obtained from samples in which biotinylated Grx2 was omitted (Fig. 2A). To exclude the possibility that the streptavidin binds to residual levels of free biotin and not to biotinylated Grx2, the neurons were exposed to biotin eluted from Sephadex column (similar to biotinylated proteins), fixed, and then reacted with labeled streptavidin. No signal was obtained under these conditions (data not shown). These results demonstrate that small amounts of wild type Grx2 could penetrate into neurons. Since the inactive double thiol mutant C9S-C12S could also be detected intracellularly (Fig. 2E), it is most likely that the penetration of Grx2 is independent of the redox activity of the protein.



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Fig. 2.   Biotinylated Grx2 and C9S-C12S mutant penetrate into the granule neurons. Cerebellar granule neurons were incubated for 30 min with 13 µM biotinylated Grx2 or C9S-C12S mutant, washed, fixed, reacted with Cy3-conjugated streptavidin, and analyzed by confocal microscopy. A, cells reacted with Cy3-conjugated streptavidin only (without biotinylated proteins). C and E, detection of intracellular Grx2 and C9S-C12S mutant, respectively. B, D, and F, Nomarski imaging of the cells presented in A, C, and E, respectively. Bar indicates 10 µm.

To examine whether coated pit vesicles were involved in Grx2 endocytosis, neurons were exposed to biotinylated Grx2 in the presence of 0.45 M sucrose, which disperses coated pit structures. An indistinguishable pattern of Grx2 permeabilization was observed in the presence or absence of sucrose, suggesting that Grx2 penetrates neurons via mechanisms other than those of coated pit vesicles (data not shown).

DA Decreases whereas Grx2 Increases NF-kappa B Binding Activity in Cerebellar Granule Neurons (Figs. 3 and 4)-- To determine the duration of exposure to DA required to trigger the apoptotic process, neurons were exposed to DA for various incubation times, followed by removal of DA and re-addition of serum-free medium. Neuronal viability was assessed after 24 h. Cells recovered well after exposure to DA for 1 h. 50% of the neurons did not recover after 1.5-2 h of exposure (data not shown), which was therefore set as the commitment time for apoptosis.



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Fig. 3.   NF-kappa B binding activity in cerebellar granule neurons following exposure to DA and/or Grx2. A, EMSA showing NF-kappa B binding activity in nuclear extracts from cerebellar granule neurons treated with 600 µM DA, or with 8.3 µM Grx2 or cotreated with both DA and Grx2 for 1 and 2 h. Sister cultures were incubated in serum-free medium and served as control. To analyze the specificity of the shifted bands, nuclear proteins were pre-exposed to 50-fold excess of unlabeled NF-kappa B consensus sequence, followed by incubation with radiolabeled probe (Competition). The arrows indicate the positions of the NF-kappa B-DNA complexes. B, quantitative analysis of NF-kappa B induction is represented as percentage of untreated cells (n = 5). ***, p < 0.001; **, p < 0.025; *, p < 0.05. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test.



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Fig. 4.   NF-kappa B binding activity in cerebellar granule neurons following treatment with wild type Grx2 and mutants. A, EMSA showing NF-kappa B binding activity in nuclear extracts from cerebellar granule neurons treated with 8.3 µM Grx2, or with C12S monothiol mutant (partially active), or with C9S-C12S double thiol mutant (inactive). The first lane shows the NF-kappa B complexes isolated from untreated neurons. The arrows indicate the positions of NF-kappa B-DNA complexes. B, quantitative analysis of NF-kappa B induction is represented as percentage of untreated cells (n = 4). **, p < 0.025; *, p < 0.05. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test.

NF-kappa B is a transcription factor known to respond to redox signals and could thus be a potential signal molecule to mediate Grx2 activity. We initially examined the DNA binding activity of NF-kappa B in neurons exposed to DA and/or Grx2 for 1 and 2 h (2 h being the commitment time). EMSA revealed three distinct bands (Fig. 3). To examine whether these bands were specific to NF-kappa B, nuclear extracts were exposed to radiolabeled probes in the presence of a 50-fold excess of unlabeled sequences. All three bands were significantly reduced in the presence of the unlabeled oligonucleotides, indicating that the three distinct bands were NF-kappa B-specific. Supershift analysis revealed that the upper shifted band corresponds to the p50/p65 heterodimer, the middle band to the p50/p50 homodimer and the lower band did not contain either p50 or p65 subunits.

Exposure of cerebellar granule neurons to DA for 1 h did not affect the binding activity of NF-kappa B. Further exposure led to a significant decrease in the intracellular levels of DNA-bound NF-kappa B. On the other hand, incubation of the neurons with Grx2 significantly augmented the DNA binding activity of NF-kappa B in a time-dependent manner. Rescuing concentrations of Grx2 could abrogate the DA effect on NF-kappa B binding.

The Grx2 C12S monothiol mutant activated slightly the DNA binding activity of NF-kappa B (3-fold activation) compared with wild type Grx2 (10-fold activation). The levels of DNA-bound NF-kappa B remained unchanged when neurons were incubated with the redox inactive C9S-C12S Grx2 mutant. These results demonstrate that only redox-active Grx2 could increase the DNA binding activity of NF-kappa B.

Grx2 Affects Both the Translocation of NF-kappa B and Its DNA Binding Activity (Figs. 5 and 6)-- At least two separate mechanisms can account for alterations in the binding activity of NF-kappa B: the redox state of NF-kappa B and its translocation from the cytoplasm to the nucleus. To investigate whether Grx2 was affecting the redox state of NF-kappa B, isolated nuclei were exposed to 8.3 µM Grx2 for 30 min at 37 °C. Significant stimulation of NF-kappa B binding activity to DNA was observed (Fig. 5). Treatment of nuclear extracts with 1 mM GSH had almost no effect on NF-kappa B binding activity. Exposure of isolated nuclei to DA resulted in reduced levels of NF-kappa B binding, as observed also in whole cell experiments (Fig. 3). NF-kappa B binding activity decreased also after exposure of cerebellar granule neurons or their nucleic extracts to hydrogen peroxide (30 µM), suggesting that ROS lead to decreased NF-kappa B DNA binding activity in those cells (data not shown). DA-induced loss of NF-kappa B binding activity was restored by GSH or Grx2. However, Grx2 (8.3 or 16.6 µM) was more efficient than 1 mM GSH. These results suggest that NF-kappa B proteins inactivated by oxidative compounds could be reactivated by reducing agents (glutaredoxin, GSH). In addition, a portion of the nuclear NF-kappa B proteins is not bound to DNA. Upon the appropriate conditions, this portion can be readily activated by Grx2 and bind to DNA.



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Fig. 5.   Modulation of NF-kappa B binding activity by oxidizing and reducing agents in isolated nuclear extracts. A, nuclear proteins were isolated from neurons grown in serum-containing medium. The binding activity of untreated nuclear extracts was used as reference (Control). Isolated nuclear extracts were treated for 30 min at 37 °C with 8.3 µM Grx2, or 1 mM GSH or 600 µM DA, in the presence or absence of 1 mM GSH, 8.3 or 16.6 µM Grx2 as indicated (n = 4). B, **, p < 0.025; *, p < 0.05; ··, p < 0.025, between DA-treated neurons and DA+GSH- or DA+Grx-treated neurons. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test.



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Fig. 6.   Grx2 affects the translocation of NF-kappa B and its binding to DNA. A, EMSA showing NF-kappa B DNA binding activity in nuclear extracts isolated from cerebellar granule neurons treated with 600 µM DA, 8.3 µM Grx2, cotreated with 600 µM DA + 8.3 µM Grx2, or left untreated. The second and fourth lanes show NF-kappa B activity following addition of 8.3 µM Grx2 to isolated nuclear extracts isolated from control cells or DA-treated cells, respectively. Note that nuclear levels of NF-kappa B were significantly higher in nuclear extracts isolated from Grx2-treated neurons compared with the maximum NF-kappa B binding following addition of Grx2 to the isolated nuclear extract. The same level of NF-kappa B activation was found after addition of 8.3 µM or 16.6 µM Grx2 for 30, 60, or 90 min at 37 °C, and was therefore considered "maximum binding." B, immunolocalization of p65 following treatment with Grx2 and DA. Cerebellar granule neurons were exposed to Grx2 or to 600 µM DA for 2 h, fixed, and analyzed by indirect immunofluorescence using polyclonal anti-p65 antibodies. Note that NF-kappa B levels in condensed and apoptotic nuclei are very low. Bar indicates 10 µm. C, Grx2 increases I-kappa Balpha phosphorylation and degradation. Cerebellar granule neurons were exposed to Grx2 for different time points as indicated. Cells were harvested and total proteins isolated from Grx2-treated and control untreated cells were separated on 12.5% polyacrylamide gel and blotted onto polyvinylidene difluoride membrane. The membrane was stained with Ponceau to verify that equal amounts of protein were transferred, after which the blot was reacted with anti-I-kappa Balpha and anti-phosphorylated I-kappa Balpha antibodies (1:1000, New England Biolabs, Beverly, MA). Secondary antibody was conjugated goat anti-rabbit horseradish peroxidase. The blots were developed using the ECL system, and a representative Western blot is shown (n = 4).

Next we examined whether Grx2 was affecting the translocation of NF-kappa B (Fig. 6). Administration of Grx2 (8.3 µM) to neuronal cells caused a 10-fold stimulation (10.6 ± 2.6) in NF-kappa B binding activity (Fig. 6A). In comparison, treatment of isolated nuclei with Grx2 resulted in a maximum 2-fold stimulation (Fig. 6A, Nuc + Grx2). The differences in the extent of activation of NF-kappa B between whole cells and isolated nuclei suggested that Grx2 was having an additional cytoplasmic effect. This effect repeated itself upon DA treatment. Exposure of neuronal cells to DA reduced the DNA-bound NF-kappa B by 25 ± 2.2% (Fig. 6A). When Grx2 was administered to isolated nuclear extracts from DA-treated cells, a maximum 2-fold increase in NF-kappa B binding was measured (Fig. 6A, DA + Nuc + Grx2). On the other hand, administration of DA and Grx2 to whole neurons resulted in 4-fold increase in NF-kappa B binding activity (3.75 ± 0.78) (Fig. 6A). Confocal microscopy clearly revealed that Grx2 caused nuclear accumulation of p65 whereas DA reduced nuclear levels of p65 (Fig. 6B). To further analyze the mechanism by which Grx2 affected NF-kappa B binding activity, we examined whether Grx2 was affecting the levels of I-kappa Balpha . As shown in Fig. 6C, administration of Grx2 to neurons led to a time-dependent increase in I-kappa Balpha phosphorylation, which was followed by I-kappa Balpha degradation. In conclusion, these results show that the major mechanism for NF-kappa B activation following Grx2 treatment is NF-kappa B translocation from the cytosol to the nucleus. In addition, nuclear Grx2 could affect the binding of nuclear NF-kappa B directly or indirectly, presumably by reducing the NF-kappa B proteins.

NF-kappa B Activation Is Essential for Neuronal Viability (Figs. 7 and 8)-- To explore whether NF-kappa B activation was important for neuronal survival, neurons were exposed to SN50, a peptide that inhibits NF-kappa B translocation (59). Even at low concentrations (9 µM), SN50 was very toxic (data not shown), suggesting that inhibition of NF-kappa B translocation rendered neurons more vulnerable. However, the peptide could have had other nonspecific effects and was thus be toxic to neurons through other mechanisms. To specifically inhibit NF-kappa B translocation, neurons were cotransfected with different amounts of I-kappa B dominant negative gene (60) (I-kappa B-Delta N) and green fluorescent protein (GFP). I-kappa B-Delta N cannot be phosphorylated and thus will not release NF-kappa B proteins. Around 0.1% of neurons were cotransfected. To assess whether overexpression of I-kappa B-Delta N inhibited NF-kappa B translocation in GFP-positive neurons, cells were cultured on coverslips, fixed, and reacted with p65 antibodies. Confocal microscopy revealed that the immunoreactivity of nuclear and cytoplasmic p65 levels was reduced in the cotransfected neurons (green cell in Fig. 7), confirming that I-kappa B-Delta N inhibited the translocation of NF-kappa B to the nucleus.



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Fig. 7.   Overexpression of I-kappa B-Delta N reduced p65 immunoreactivity. Cerebellar granule neurons were cotransfected with I-kappa B-Delta N and GFP, fixed, and reacted with anti-p65 antibodies. A shows fluorescently labeled neuron (GFP-positive cell), and B demonstrates reduction in p65 immunoreactivity of this cell (white arrow) compared with nontransfected surrounding cells. Transfected cells with GFP alone did not show reduced immunoreactivity of p65 (data not shown). Bar indicates 10 µm.



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Fig. 8.   Overexpression of I-kappa B-Delta N caused neuronal death. Cerebellar granule neurons were cotransfected with I-kappa B-Delta N and GFP for 24 h, after which the amount of fluorescently-labeled neurons was monitored as a function of time in the presence or absence of DA (A). Treating the cotransfected neurons with Grx2 did not affect their viability (B). ***, p < 0.001; **, p < 0.025; *, p < 0.05; ··, p < 0.025 between Grx2+DA-treated cells and Grx2+DA-cotransfected cells. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test.

We next examined whether inhibition of NF-kappa B translocation affected neuronal viability. Twenty-four hours after transfection, the number of cotransfected green fluorescent neurons was identical to control GFP-transfected cells. Thereafter, the medium was replaced and neuronal viability was determined (Fig. 8). In the presence of 1 µg/0.2 ml I-kappa B-Delta N, neuronal viability was decreased as a function of time and reached a steady level of 65 ± 3.3% S.D. after 5 h. Increasing I-kappa B-Delta N to 5 µg/0.2 ml resulted in a similar rate of neuronal death (59 ± 8.5% S.D.) (Fig. 8A). Cotransfection of the neurons with pRC/CMV plasmid and GFP had no effect on neuronal viability, suggesting that cotransfection per se did not alter the viability. Administration of 600 µM for 5 h resulted in 54% neuronal death. Cotransfected neurons were slightly but not significantly more sensitive to DA (52.6% and 45% neuronal death with 1 and 5 µg of I-kappa B-Delta N, respectively) (Fig. 8A). These results are in agreement with our previous experiments (EMSA of NF-kappa B and immunolocalization of p65) showing reduced NF-kappa B activity and accumulation of p65 in the cytoplasm after DA treatment. Moreover, Grx2 was incapable of rescuing cotransfected neurons (Fig. 8B).



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Fig. 9.   Ref-1 expression after treatment with Grx2 and/or DA. Cerebellar granule neurons were treated for 2 h with 8.3 µM Grx2, 600 µM DA and cotreated with Grx2 and DA. The neurons were fixed, reacted with anti-Ref-1 antibodies and Cy2-conjugated goat anti-rabbit antibodies, and analyzed by confocal microscopy. The left panels show immunoreactivity of anti-Ref-1 antibody, the middle panels show nuclear staining with DAPI, and the right panels show superposition of DAPI and anti-Ref-1 immunoreactivity. Bar indicates 10 µm.

Grx2 Regulates NF-kappa B Binding Activity through Ref-1 (Figs. 9-11)-- In untreated cells, Ref-1 was preferentially expressed in the nucleus (Fig. 9, top panel). Two hours of incubation with DA caused slight nuclear condensation and a dramatic reduction in Ref-1 immunoreactivity (Fig. 9, second panel) (in accordance with Western blot analysis, not shown). Grx2 significantly stimulated the expression of both nuclear and cytoplasmic Ref-1 (Fig. 9, third panel). Treating the neurons with Grx2 mutant C9S-C12S did not alter Ref-1 expression (data not shown), suggesting that the latter is dependent on a redox-active glutaredoxin. Grx2 could offset the suppression of Ref-1 expression by DA. In the presence of both DA and Grx2, Ref-1 levels were even higher than those of untreated control neurons (Fig. 9, bottom panel).



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Fig. 10.   Reduction in Ref-1 expression after treatment with Ref-1 antisense. A, cerebellar granule neurons exposed for 2 h to 8.3 µM Grx2 in the presence of Ref-1 antisense (5 or 10 µM) or Ref-1 sense (5 or 10 µM) and were reacted with anti-Ref-1 antibodies. Green staining indicates the presence of antisense/sense oligonucleotides. Left panel shows Ref-1 immunoreactivity and antisense/sense staining, whereas the right panel shows only the Ref-1 immunoreactivity. Ref-1 immunoreactivity in untreated cells and in cells treated with Grx2 is shown in lowest right and left panel, respectively. Bar represents 10 µm. B, Western blot analysis of Ref-1 levels following treatment with Grx2 and antisense (AS, 5 and 10 µM) or sense (S, 5 and 10 µM). C, quantitative analysis of Ref-1 alterations is represented as percentage of untreated cells. **, p < 0.025; ··, p < 0.025; ·, p < 0.05 between Grx2-treated neurons and Grx2-treated neurons that were exposed to Ref-1 antisense. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test (n = 5).



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Fig. 11.   NF-kappa B binding activity in cerebellar granule neurons following treatment with Ref-1 antisense. A, EMSA showing NF-kappa B DNA binding activity in nuclear extracts isolated from cerebellar granule neurons treated with 8.3 µM Grx2 or pre-exposed to 5 and 10 µM antisense and 10 µM sense and then treated with Grx2. The first lane shows NF-kappa B binding activity from untreated granule neurons. B, quantitative analysis of NF-kappa B induction is presented as percentage of untreated cells (n = 4). **, p < 0.025; *, p < 0.05; ··, p < 0.025 between Grx2-treated neurons and Ref-1 antisense-treated neurons. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's t test.

Since the levels of Ref-1 and NF-kappa B DNA binding activity were elevated at the same time, it could be that the induction of Ref-1 was important for the activation of NF-kappa B. Exposure of neurons to Ref-1 antisense oligonucleotide reduced the expression levels of Ref-1, whereas the sense sequence had no effect (Fig. 10A). Similar results were obtained with Western blot analysis (Fig. 10, B and C). Grx2 could not activate NF-kappa B in the presence of Ref-1 antisense oligonucleotide in a concentration-dependent manner (Fig. 11). However, Grx2 enhanced the DNA binding activity of NF-kappa B in the absence of Ref-1 antisense, or in the presence of the sense oligonucleotide (Fig. 11). These results demonstrate that Grx2 stimulates the DNA binding activity of NF-kappa B through Ref-1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DA, the endogenous neurotransmitter of the nigro-striatal pathway, is a powerful oxidant that exerts its toxic potential through its oxidative metabolites. Reducing thiols such as DTT, NAC, and GSH are capable of neutralizing DA oxidative metabolites and thereby conferring protection against DA-induced apoptosis (14). In the search of more physiological thiol antioxidant, we found that E. coli Grx2 and human glutaredoxin could protect neuronal cells against DA-induced death by activating the NF-kappa B-dependent signal transduction pathway through Ref-1. Grx2 penetrated the neurons whether or not it was redox-active (Fig. 2). Our results are consistent with several studies that demonstrated that human Trx and its double thiol mutant were taken up similarly by 3T3 and MCF7 cells (2, 3). The mechanism by which Grx2 penetrates into the neurons is not clear. It does not involve a coated pit-dependent mechanism, perhaps by fluid phase, for example (61). Grx2 internalization per se is not sufficient to initiate signal transduction; it must catalyze GSH-disulfide oxidoreduction in its active form. Similarly, Gasdaska et al. (3) have shown that the redox activity of Trx is essential for its effects on cell proliferation. Our study does not rule out the possibility that Grx2 exerts its effects through specific interaction with cell surface receptors.

Next, we looked for the intracellular targets of Grx2. Transcription factors whose DNA binding activity is redox-dependent were good candidates (reviewed in Ref. 4). Such transcription factors are the NF-kappa B Rel family of proteins, which have conserved cysteine residues in their DNA binding domains (62, 63). Modification of the cysteines with diamide reduced their activity, whereas treatment with reducing agents enhanced DNA binding. In this work, redox-active Grx2 increased the DNA binding activity of NF-kappa B. The effect was observed in isolated nuclei, suggesting a direct effect on NF-kappa B (e.g. the reduction of a mixed disulfide between a conserved cysteine and GSH). However, Grx2 also stimulates NF-kappa B translocation, through a process that involves I-kappa Balpha phosphorylation and degradation. Several reports indicated that accumulation of ROS can induce I-kappa B phosphorylation and degradation (64-66). Our results show that a reductive agent such as Grx2 can induce I-kappa Balpha phosphorylation and/or ubiquitin-dependent degradation. This notion is consistent with the findings that glutathione peroxidase is involved in I-kappa Balpha phosphorylation (64). Furthermore, ubiquitin-conjugating enzymes have been shown to be redox-regulated by the ratio between GSH and GSSG (67, 68). Our results are partially consistent with the recent findings obtained by Hirota et al. (69), who showed that hGrx reduced the ability of tumor necrosis factor alpha  to phosphorylate I-kappa B but on the other hand enhanced NF-kappa B activation in HEK293 cells.

The fact that Grx2 stimulates NF-kappa B DNA activity through Ref-1 activation (Fig. 11) demonstrates a multiple signaling role for Grx2. It has been shown that AP-1 transcriptional activity is regulated by a direct association between Trx and Ref-1 (49). Our data show that Ref-1 expression levels are redox regulated. In the presence of DA which causes oxidative stress, Ref-1 levels were dramatically decreased (Fig. 10). Furthermore, no induction in Ref-1 levels was observed in the presence of the double thiol mutant. Ref-1 down-regulation was observed in certain cases of apoptosis, such as the myeloid leukemia cells line HL-60 (70) or following ischemia (71, 72). It is known that, after ischemia and reperfusion, mitochondrial production of superoxide radicals is increased. Thus, the reduced expression of Ref-1 after ischemia or DA treatment may be due to oxidative damage.

Human and E. coli Grx2 differ significantly in their catalytic activity using the mixed disulfide between the beta -hydroxyethyl disulfide and GSH as a substrate (51). However, their protective profiles against DA-induced apoptosis were almost identical (Fig. 1). This may simply reflect that the two proteins have similar catalytic properties to their nonidentified in vivo substrate.

Monothiol Grx2 could protect neurons from DA-induced apoptosis, but to a lesser extent than the dithiol wild type glutaredoxin. Known glutaredoxins can reduce a disulfide substrate through a monothiol or a dithiol mechanism (73). For the reduction of protein disulfides, both cysteine residues (dithiol mechanism) are required. A single amino-terminal cysteine can specifically reduce protein-GSH mixed disulfides (monothiol mechanism; Refs. 26 and 74). In the latter case, GSH forms a mixed disulfide with glutaredoxin (GrxS-SG), while the protein component is released in its reduced form. The GrxS-SG can be reduced by GSH to give reduced glutaredoxin and oxidized glutathione (GSSG). The ability of monothiol Grx2 to confer increased DNA binding of NF-kappa B shows that the DNA binding of NF-kappa B is regulated by a GSH mixed disulfide mechanism.

The contribution of NF-kappa B to cell death and survival pathways is an intriguing and controversial issue. Our results show that NF-kappa B activation is important for the viability of cerebellar granule neurons and that Grx2 exerts its protective effects through the activation of NF-kappa B signaling pathways. First, inhibition of NF-kappa B translocation and binding by I-kappa B-Delta N overexpression causes neuronal death (Fig. 8). Second, Grx2 could not rescue the neurons overexpressing I-kappa B-Delta N (Fig. 8). Third, DA-induced neuronal death is correlated with a significant reduction in NF-kappa B DNA binding activity (Fig. 3) and a decrease in p65 levels in condensed and apoptotic nuclei (Fig. 6B). Our findings are in agreement with several recent reports suggesting a role for NF-kappa B in protection from apoptosis. Abrogation of NF-kappa B, either by deletion of RelA (p65) or by overexpression of I-kappa B-Delta N, sensitizes immune cells to apoptosis in response to tumor necrosis factor and DNA damaging agents (33, 34, 75, 76). NF-kappa B activation protected hippocampal neurons against oxidative stress-induced apoptosis (77). Furthermore, nerve growth factor protects sympathetic neurons by the activation of NF-kappa B (78). Increased NF-kappa B activity was found in surviving endothelial cells, whereas apoptotic cells show caspase-mediated cleavage of p65 (79). It was further shown that uncleavable caspase-resistant p65 protected the cells from apoptosis (79). In contrast, NF-kappa B has been implicated in the death of neurons induced by glutamate and beta -amyloid protein (80, 81), by ceramide-induced apoptosis in mesencephalic dopaminergic neurons (43), and in leukemia cells (82). It is possible that the opposing roles of NF-kappa B in cell survival and death stem from the diversity in subunit expression and subcellular compartmentation. In different cell types, the relative expression of the different members of the NF-kappa B family may influence the composition of the dimers and, as a consequence, the affinity of the activated complex to specific DNA sequence motifs located in the regulatory region of different genes. Moreover, activation of distinct patterns of genes by the simultaneous activation of other regulatory transcription factors may contribute to opposite cell fates in different cell types as well as within the same cell type. Tamatani et al. (83) showed that tumor necrosis factor protects hippocampal neurons by the induction of bcl-2 and bcl-x through the activation of NF-kappa B. These observations strengthen the notion that NF-kappa B can inhibit apoptosis by inducing the expression of antiapoptotic genes.

Recently, Nakamura et al. (29) showed that Grx and Trx are found in human plasma, and suggested that they may be part of plasma defense mechanisms against oxidative stress. We propose that, besides being part of the plasma defense mechanism, redox-active glutaredoxins function as a signal molecule that penetrates the cells and activates signal pathways by affecting redox regulation.

In summary, our results demonstrate that Grx2 protects cerebellar granule neurons from DA-induced apoptosis by activating NF-kappa B signaling pathways through Ref-1. Inhibition of NF-kappa B activity by overexpression of I-kappa B-Delta N causes neuronal death, indicating that the activation of a NF-kappa B signaling cascade is necessary to granule neuron survival. Studies will be needed to identify Grx- and NF-kappa B-induced genes that promote neuronal survival, and to clarify the precise role of redox regulation by glutaredoxin in signal transduction and neuronal protection.


    FOOTNOTES

* This work was supported in part by grants from the Israeli Academy of Sciences and Humanities and the Israeli Ministry of Health, by Swedish Cancer Society Grant 961, and by Swedish Medical Research Council Grant 03XS-13005-01A (to A. H. and A. V.-G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Supported by a Sackler fellowship at Tel Aviv University.

Dagger Dagger To whom correspondence should be addressed: Dept. of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-6409782; Fax: 972-3-6407643; E-mail: barzilai@post.tau.ac.il.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008121200


    ABBREVIATIONS

The abbreviations used are: DA, dopamine; Grx, glutaredoxin; DTT, dithiothreitol; ROS, reactive oxygen species; EMSA, electrophoretic mobility shift assay; DAPI, 4,6-diaminodiphenyl-2-phenylindole; GFP, green fluorescent protein; PBS, phosphate-buffered saline.


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DISCUSSION
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