Is glutathione an important neuroprotective effector molecule against amyloid beta toxicity?

Cellular thiols are critical moieties in signal transduction, regulation of gene expression, and ultimately are determinants of specific protein activity. Whilst protein bound thiols are the critical effector molecules, low molecular weight thiols, such as glutathione, play a central role in cytoprotection through (1) direct consumption of oxidants, (2) regeneration of protein thiols and (3) export of glutathione containing mixed disulphides. The brain is particularly vulnerable to oxidative stress, as it consumes 20% of oxygen load, contains high concentrations of polyunsaturated fatty acids and iron in certain regions, and expresses low concentrations of enzymic antioxidants. There is substantial evidence for a role for oxidative stress in neurodegenerative disease, where excitotoxic, redox cycling and mitochondrial dysfunction have been postulated to contribute to the enhanced oxidative load. Others have suggested that loss of important trophic factors may underlie neurodegeneration. However, the two are not mutually exclusive; using cell based model systems, low molecular weight antioxidants have been shown to play an important neuroprotective role in vitro, where neurotrophic factors have been suggested to modulate glutathione levels. Glutathione levels are regulated by substrate availability, synthetic enzyme and metabolic enzyme activity, and by the presence of other antioxidants, which according to the redox potential, consume or regenerate GSH from its oxidised partner. Therefore we have investigated the hypothesis that amyloid beta neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels. Using PC12 cells as a model system, amyloid beta 25-35 caused a shift in DCF fluorescence after four hours in culture. This fluorescence shift was attenuated by both desferioxamine and NGF. After four hours, cellular glutathione levels were depleted by as much as 75%, however, 24 hours following oxidant exposure, glutathione concentration was restored to twice the concentration seen in controls. NGF prevented both the loss of viability seen after 24 hours amyloid beta treatment and also protected glutathione levels. NGF decreased the total cellular glutathione concentration but did not affect expression of GCS. In conclusion, loss of glutathione precedes cell death in PC12 cells. However, at sublethal doses the surviving fraction respond to oxidative stress by increasing glutathione levels, where this is achieved, at least in part, at the gene level through upregulation of GCS. Whilst NGF does protect against oxidative toxicity, this is not achieved through upregulation of GCS or glutathione. Introduction Increasing age is the most reliable and robust risk factor for susceptibility to neurodegenerative disease (Bains & Shaw, 1997). Lovell et al., (1995) showed support for the concept that the brain in Alzheimer’s disease (AD) is under increased oxidative stress (OS) demonstrating lipid peroxidation changes in areas where degenerative changes occur. Further evidence suggesting that the pathogenesis of AD is as a result of oxidative damage includes elevated levels of iron in AD brains and a colocalised reduction in antioxidant status (Good et al., 1996). Whilst the exact mechanisms underlying oxidative stress remain unclear, it has been proposed that the peptidergic fragment of amyloid beta (Aβ) that accumulates in AD may exert its toxicity through peroxide generation (Huang et al., 1999). Glutathione is arguably the most important AOX and free radical scavenger present in cells (Valencia et al., 2001), where glutathione-associated metabolism is a major mechanism for cellular protection against agents that generate OS. Glutathione participates in detoxification at several different levels, and may scavenge reactive oxygen species (ROS), reduce peroxides, or be conjugated with electrophilic compounds. Thus, glutathione provides the cell with multiple defences not only against ROS but also against their toxic products. Most importantly, many of the glutathione-dependent proteins are inducible and therefore represent a means whereby cells can adapt to OS (Hayes & McLellan, 1999). The GSH redox status is critical for various biological events that include transcriptional activation of specific genes, modulation of redox-regulated signal transduction, regulation of cell proliferation, apoptosis, and inflammation (Rahman & MacNee, 2000). In addition, it has been shown previously that GSH levels decrease following addition of cytotoxic agents, and at the time of onset of apoptosis (van den Dobbelsteen et al., 1996; Froissard & Duval, 1994). The intracellular synthesis of GSH is mainly regulated by gamma glutamyl cysteinyl synthetase (γ-GCS) (Richman & Meister, 1975). Differences or alterations in the levels of protein can occur by a number of different mechanisms including alterations in the level of gene transcription and alterations in the stability or translatability of the resulting RNA. The expression of γ-GCS is sensitive to OS, where the existence of the OS-response element, AP-1, on the γ-GCSh (g-GCS heavy subunit) promoter has been clarified (Mulcahy et al.,1997). Maintenance of a high intracellular (GSH)/(GSSG) ratio (>90%) minimises the accumulation of disulfides and provides a reducing environment within the cell. If there is a shift in the GSH/GSSG redox buffer, a variety of cellular signalling processes are influenced, such as activation and phosphorylation of stress kinases (JNK, p38, PI-3K) via sensitive cysteine-rich domains; activation of sphingomyelinase-ceramide pathway, and activation of the transcription factors AP-1 and NF-κB, eventually leading to increased gene transcription (Rahman & MacNee, 2000). Froissard et al., (1997) have reported that there is strong evidence to support the existence of a close relationship between the level of intracellular glutathione and cell survival in PC12 cells, where mitochondrial GSH is critical for the maintenance of mitochondrial function and cellular viability (Seyfried et al., (1999). However, whether a drop in GSH levels precede intracellular ROS production during apoptosis or vice versa is not certain. Hence any role of GSH depletion alone in triggering apoptosis is not clear (Hall, 1999). Evidence is accumulating for an important role for glutathione in detoxification processes in the brain. Depletion of glutathione in newborn rats using buthionine sulfoximine (BSO) leads to mitochondrial damage in the brain (Jain et al , 1991). Furthermore, application of beta amyloid peptide, glutamate receptor agonists and BSO cause GSH depletion in cultured neurones and lead to induction of apoptosis. H2O2 has previously been suggested to mediate Aβ cytotoxicity based on antioxidant inhibition of toxicity and demonstration of lipid peroxidation in treated cells (Behl et al., 1994b). The toxic effects of several other ROS generating compounds has been shown to be abrogated by neurotrophic factors such as BDNF, NGF, GDNF and bFGF (Cheng and Mattson, 1995; Chao and Lee). It has been postulated that this may be due to upregulation of the concentration of glutathione and/or the activities of antioxidant enzymes. Therefore we have investigated the hypothesis that Aβ neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels at the gene level. Herein, we demonstrate that Aβ toxicity is associated with alterations in cellular redox status, and whilst NGF affords protection against toxicity, this is independent of GCS expression. Methods Maintenance of PC12 cell line PC12 cells were routinely cultured in 75cm flasks in a water jacketed humidified 5% CO2 incubator with maintenance media. (RPMI 1640 media with Glutamax I; 10% (v/v) HS, 5% (v/v) FBS, penicillin (0.5U/ml) and streptomycin (0.5mg/ml). Every 2-3 days, spent PC12 cell media was removed and replaced with fresh maintenance media, which had been pre-warmed to 37°C. Cells were passaged once a week, as described in section 2.2.1.3, when the cells were at a density of approximately 5 x 10 per ml. For experimental purposes, cells were seeded at 2 x 10 cells per ml and allowed to rest for 4 hours prior to treatment for times and doses indicated. Determination of cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium (MTT) assay Cells (2 x 10 cells per ml, 100μl per well) were seeded into 96 well flat bottomed microtitre plates. Two hours prior to completion of the experiment, MTT solution (25μl of 5mg/ml in 0.01M PBS) was added to all wells including blanks. Plates were then incubated for a further 2 hours at 37°C, 5% CO2. Lysis buffer (100μl of 20% w/v SDS, in DMF (50%), dH2O(50%), pH 4.7 adjusted with 2.5% of 80% glacial acetic acid) was then added to each well and the plates incubated for a further 16 hours at 37°C, 5%CO2. The absorbance of the each well was then read at 570nm in a 96 well plate reader. Dichlorofluorescein diacetate (DCFDA) staining Cells (2 x 10 per ml, 2ml) were incubated with test agents in 35mm well plates. 30 minutes before the end of incubation, DCFDA solution (20μl of 7.5mM diluted stock in PBS) was added to the plates. After exactly 30 minutes cells were immediately harvested with a Gilson pipette and a rubber policeman and transferred to a flow cytometry tube. Samples were then immediately run through a Coulter EPICS flow cytometer with the intensity of light scatter and the FL1 fluorescence emitted between 505 and 535nm following excitation at 488nm (argon laser) was recorded for individual nucleoids. The median X was calculated for 10,000 nucleoid events. Glutathione (GSH) Assay Glutathione (reduced and oxidised) levels were determined using modified microtitre plate method (Punchard et al., 1994). Actual glutathione levels were calculated by equating the changes in absorbance over time alongside standards of known GSH concentrations included on each plate. Glutathione (GSH), reduced standards were freshly prepared for each experiment from a stock (100mM in sterile distilled water). Cells (2x10) were then harvested and PBS (1ml) added to wash the pellet. Tubes were recentrifuged at 6600 x g for 1.5 minutes and the PBS was then removed, making sure to leave the pellet dry. SSA (3.33μl of 1% made up in distilled water) was then added to precipitate the protein and the tubes were immediately centrifuged at 13000 x g for 1.5 minutes. Stock buffer (96.6μl of 125mM sodium phosphate, 6.3mM disodium EDTA, pH 7.5 autoclaved) was then added to each tube. Standards and samples (25μl) were then aliquoted into a flat bottomed plates (96 well) in triplicate, the remaining aliquot was immediately frozen at -70°C for later use in the GSSG assay. Daily buffer (150μl of 0.3mg NADPH/ml stock buffer) and DTNB solution (50μl of 6mM DTNB in daily buffer) was then added to all wells. The plate was then loaded onto the carriage of a 96 well plate reader and glutathione reductase solution (25μl of 20U/ml) was then added as quickly as possible to initiate the reaction. The OD was then recorded every minute for 5 minutes at 410nm. Glutathione (GSSG) Assay Oxidised glutathione (GSSG) standards were freshly prepared for each experiment from an oxidised glutathione stock (1.5μM in sterile distilled water). The remaining 25μl aliquot from the GSH assay was employed for this assay. To the frozen aliquot and standards (25μl), 2-vinylpyridine (2-VP), triethanolamine (0.5μl) were added. Samples and standards were then vortexed and centrifuged for 30 seconds at 13000 x g. Aliquots of standards and samples (10μl) were then plated out into a microtitre plate (96 well plate). Daily buffer (75μl of 0.3mg NADPH/ml stock buffer) and DTNB solution (25μl of 6mM DTNB in daily buffer) were added to the wells. The plate was then loaded onto the carriage of a 96 well plate reader and glutathione reductase solution (12.5μl of 20U/ml) was then added as quickly as possible to initiate the reaction. The OD was then recorded every minute for 5 minutes at 410nm. Estimation of Protein Concentration Protein concentrations were assessed according to a modified version of Sigma Procedure No. TPRO-562, based on the method of Smith et al, (1985). Protein standards of six concentrations were prepared from the protein standard solution (bovine serum albumin, 1mg/ml) diluted in coating buffer (15mM sodium carbonate, 35mM sodium hydrogen carbonate, pH 9.2). Each standard (10μl) was plated out in triplicate into a 96 well flat bottomed microtitre plate. Unknown samples (10μl) were also plated out in triplicate. Bicinchoninic acid solution (200μl) containing copper (II) sulphate pentahydrate 4% solution (50:1) was then added to all wells (samples and standards), the plate was then incubated at 37°C for 30 minutes. The absorbance of the plate was then measured at 570nm in a 96 well plate reader. Unknowns were calculated from a standard curve prepared using the software package GraphPad PRISM. mRNA EXTRACTION Cells (2x10) were harvested into a RT grade centrifuge tube (1.5ml). Cells were centrifuged for 1.5minutes at 13000 x g in an Eppendorf Centrifuge 5415D. Supernatants were removed and PBS (1ml) added, cells were centrifuged for another 1.5minutes at 13000 x g and the PBS removed. The extraction was carried out using the Dynal mRNA direct kit protocol. Pellets were resuspended into 200μl Dynal lysis/binding buffer (100mM Tris-HCl, pH 7.5, 500mM LiCl, 10mM EDTA, pH8.0, 1% LiDS and 5mM DTT) and aspirated twenty times to fully resuspend each pellet with fresh tips being used for each tube. The solution was then aspirated five times using 1ml sterile syringes and sterile 21 gauge needles to shear the DNA. This was then repeated using 1ml sterile syringes and sterile 25 gauge needles. Tubes were then centrifuged for 1 minute at 13000 x g in an Eppendorf Centrifuge 5415D. Resuspended Dynal Oligo (dT)25 beads (30μl) were washed in Dynal lysis/binding buffer twice before being added to each tube of lysed cells. The tubes were then mixed on a Dynal sample mixer for 5 minutes at 22-25°C. Tubes were then magnetised on the Dynal MPC-E magnet (Magnetic particle concentrator for microtubes of Eppendorf type (1.5ml)) and the colourless solution removed. Dynal wash buffer A (200μl of 10mM Tris-HCl, pH 7.5, 0.15M LiCl, 1mM EDTA, 0.1% LiDS) was then added to the pellet and the cells resuspended in it. The tubes were then remagnetised the colourless solution removed and Dynal wash buffer A added (200μl) and the cells resuspended. The process was then repeated using Dynal wash buffer B (200μl of 10mM Tris-HCl, pH 7.5, 0.15M LiCl, 1mM EDTA). After two washes with wash buffer B the cell pellet was resuspended in DEPC treated water (30μl of 0.1%). This was then transferred to a fresh tube (1.5ml), and master mix was added (39μl of 10mM DTT (7.5μl of 100mM), 1mM dNTPs (7.5μl dNTP mix), 25U RNAsin (1.8μl), 1U (3μl) RQ1 RNase-free DNase, 0.1% DEPC treated water (4.2μl) and Expand buffer (15μl)). Each tube was aspirated twice and incubated at 37°C for 60 minutes. DNase was heat inactivated at 70°C for 10 minutes in a preheated dry heating block. Samples were then centrifuged at 13000 x g for 1 minute in an Eppendorf centrifuge 5415D. Reverse transcription of mRNA Two RT grade PCR tubes were labelled for each sample, one positive and one negative. RNAsin 15U (1μl) was added to the negative tubes and 30U (2μl) to the positive tubes, mRNA extraction product (46μl) was added to the positive tubes and mRNA extraction product (23μl) was added to the negative tubes. Expand RT 50U, (1μl) was then added to the positive tubes. For reverse transcription mRNA was incubated at 37°C for 1 hour in Expand RT buffer containing 10mM DTT, 1mM dNTPs, 25U RNAsin, 1U RQ1 RNase-free DNase. DNase was heat inactivated at 70°C for 10 minutes. The samples were then incubated at 42°C for 1 hour before being stored in the short term at 0 4°C, and at -20°C in the long term. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) and GCS polymerase chain reaction (PCR) The primers (50nmol of each) as stated in table 2.4 were synthesised by Gibco/BRL, Life Technologies, Paisley, Scotland. Each primer was diluted to 100 pmol/μl in sterile 1 x TE buffer (10mM Tris, 1mM EDTA, pH 8.0), and then further diluted tenfold in 1 x TE buffer and stored as 10μl aliquots at 20°C. Primer Sequences Primer Forward primer sequence (5’ – 3’) Reverse primer sequence (5’-3’) Reference GAPDH AGA ACA TCA TCC CTG CCT C GCC AAA TTC GTT GTC ATA CC Hall et al., 1998 GCS CCT TCT GGC ACA GCA CGT TG TAA GAC GGC ATC TCG CTC CT El Mouatassium et al., 2000 PCR was performed in PCR buffer with sterile water, 200mM dNTPs and 10pmol of each primer. All reactions were covered in a drop of mineral oil and hot start conditions were used, full details of each PCR as stated in table 2.5. Reactions were initiated with 2.5U (0.5μl) Taq DNA Polymerase. All liquid handling was conducted using filter tips. Optimal PCR Conditions PRIMER CONDITIONS CYCLES GAPDH 98°C 3 minutes + Taq 60°C 2 minutes 72°C 2 minutes ....................................... 94°C 30 seconds 60°C 30 seconds 72°C 30 seconds ....................................... 94°C 30 seconds 60°C 30 seconds 72°C 4 minutes 1 cycle