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Glutathione transferase protects neuronal cultures against four hydroxynonenal toxicity
Free Radical Biology & Medicine, Vol. 25, No. 8, pp. 979 –988, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98/$–see front matter
PII S0891-5849(98)00186-5
Original Contribution GLUTATHIONE TRANSFERASE PROTECTS NEURONAL CULTURES AGAINST FOUR HYDROXYNONENAL TOXICITY CHENGSONG XIE,* MARK A. LOVELL,*†
and
WILLIAM R. MARKESBERY*‡
*Sanders-Brown Center on Aging, Departments of †Chemistry, ‡Neurology, and ‡Pathology, University of Kentucky, Lexington, KY, USA (Received 5 May 1998; Revised 29 June 1998; Accepted 10 July 1998)
Abstract—Peroxidation of polyunsaturated fatty acids (PUFA), particularly arachidonic acid, leads to the generation of reactive aldehydes, including 4-hydroxynonenal (HNE). Recent studies have demonstrated an increase in lipid peroxidation, a decline in PUFA, as well as an increase in HNE, and a decrease in glutathione transferase (GST) in the brain in Alzheimer’s disease. Four-hydroxynonenal is toxic to cultured neurons and to the brain of experimental animals. Although glutathione (GSH) has been shown to offer protection against HNE, no enzymatic system has been described which serves to detoxify these reactive species in neuronal cultures. Here, we describe the use of GST in the protection of neuronal cultures against HNE toxicity. Glutathione transferases are a superfamily of enzymes functioning to catalyze the nucleophilic attack of GSH on electrophilic groups on a second substrate. These enzymes function efficiently with 4-hydroxyalkenals, particularly HNE, as substrates. To investigate the protective effects of GST against HNE, primary hippocampal cultures were pretreated with GST before exposure to toxic doses of HNE which led to a statistically significant enhancement in cell survival. Pretreatment of cultures with equivalent levels of heat inactivated GST or antibody against GST did not offer protection against HNE. Control cultures pretreated with GST also demonstrated enhanced survival compared with control cells receiving no pretreatment. These data suggest that GST may be an important source of protection against the toxic effects of HNE. © 1998 Elsevier Science Inc. Keywords—Glutathione transferase (GST), 4-Hydroxynonenal (HNE), Neuronal culture, Protection, Alzheimer’s disease (AD)
a, b unsaturated aldehyde that is generated during the peroxidation of PUFA, particularly arachidonic acid [13]. This reactive by-product possibly plays a role in the pathogenesis of AD [14 –16]. Recently, we observed statistically significant increases in HNE levels in ventricular fluid [17] and in the brain in AD [18]. Glutathione transferases (GST) (E.C. 2.5.1.18) are a superfamily of enzymes that function to catalyze the nucleophilic attack of the sulfhur atom of glutathione (GSH) on electrophilic groups of a second substrate [19]. GST are present in many organs [20 –24] and function to inactivate toxic products of oxygen metabolism, such as 4-hydroxyalkenals. We recently observed a marked decline in GST activity in brain and ventricular fluid in AD subjects compared with normal control subjects [25]. Based on these observations, we initiated studies to evaluate the possible protective effects of GST against HNE in primary rat hippocampal cultures.
INTRODUCTION
One of the leading hypotheses regarding the pathogenesis of Alzheimer’s disease (AD) is the involvement of increased levels of oxidative stress in neuronal degeneration (reviewed in [1]). This increased oxidative stress in the brain in AD is characterized by increases in lipid peroxidation [2], protein oxidation [3,4], and DNA oxidation [5,6] and decreases in polyunsaturated fatty acids (PUFA) [7,8]. Additionally, markers of oxidative stress including nitrotyrosine, protein carbonyls and advanced Maillard reaction end products are present in neurofibrillary tangles (NFT) and/or senile plaques in AD [9 –12]. One product of oxidative stress that has received considerable attention is 4-hydroxynonenal (HNE). HNE is an Address correspondence to: Mark A. Lovell, 101 Sanders-Brown Center on Aging, University of Kentucky, 800 South Limestone St., Lexington, KY 40536-0230, USA; Tel: (606) 257-5566; Fax: (606) 323-2866. 979
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C. XIE et al. MATERIALS AND METHODS
Primary cortical and hippocampal cultures were established from gestation day 18 rat embryos as described by Mattson et al. [26] Cells were plated at a density of 50/mm2 in polyethyleneamine coated plastic 35 mm dishes. The cultures were maintained in Eagle’s minimum essential medium supplemented with 10% (v/v) qualified bovine serum (Gibco BRL, Life Technologies, Inc., Grand Island, NY) and containing 20 mM KCl, 1 mM pyruvate in an environment of 94% air/6% CO2 at 37°C. Experiments were carried out on cells 7– 8 days in culture. HNE (Cayman Chemical, Ann Arbor, MI) was used at 1 mM final concentration which has been shown to induce significant cell death after 24 h of treatment. The GST used was the mu isoform (Calbiochem, La Jolla, CA). For treatment, cultures were chosen at random and the culture medium switched to serum free Locke’s solution that consisted of 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 10 mM glucose, 5 mM HEPES pH 7.2 with 10 mg/l gentamicin sulfate. Cell survival was assessed by counting the number of undamaged neurons in premarked microscopic fields before and at each indicated time point (3, 6, 16 and 24 h). To ensure the validity of cell counts, cell viability was assessed using the MTT reduction assay in which MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] is converted to blue formazan crystals in cells as a result of mitochondrial electron transport chain reaction [27,28] and thus measures cell viability. For this assay, 10 ml MTT (1 mg/ml) was added to each dish after the appropriate treatment time and the dishes incubated for 45 min. The medium was removed and the dishes washed three times in PBS. The formazan product was solubilized in 1 ml dimethyl sulfoxide (DMSO) and the absorbance measured at 595 nm using a Bio-Tek UV-900C plate reader. Results of the assay were expressed as a percentage of control absorbance. For treatment, cultures were switched to Locke’s solution and GST added at varying concentrations for 6 h before addition of HNE. To assess whether function of the enzyme or simple chemical interaction with the protein afforded the protection, an equivalent concentration of GST that had been inactivated by heating at 100°C for 10 min was added to cultures. To verify that the protective effects observed were due to the presence of GST, cells were pretreated with a 1:100 dilution of a polyclonal rabbit antibody raised against recombinant human GST M1-1 (Oxford Biomedical Research, Oxford, MI) for 4 h before adding 100 nM GST. After incubation in 100 nM GST, 1 mM HNE was added and cell viability assessed as described above. GST activity was assayed according to the method of
Ricci et al. [29] which follows the conjugation of 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole (NBD-Cl) with GSH as catalyzed by GST. The NBD-Cl complex is a yellow compound that is stable and strongly absorbs at 419 nm with a molar absorption coefficient of 14.5 mM21cm21. For the assay, 2 ml of 0.1 M sodium acetate buffer (pH 5.0) was mixed with 200 ml 0.2 mM NBD-Cl and 100 ml 0.5 mM reduced GSH. The reaction was initiated by addition of 200 ml of concentrated protein isolated from culture medium. All reagents were used at room temperature with the exception of the protein which was stored on ice until use. The assay is extremely sensitive to temperature and may be greatly affected by reagents that are below room temperature. For Western blot analysis, the protein from the medium was concentrated by centrifugation through a 10 kDa molecular weight cut off filter. The cells were rinsed three times in PBS, scraped and lysed in 200 ml of a lysis buffer consisting of 20 mM imidazole (pH 7.4) containing 0.6 mM EGTA and 0.1 mM PMSF. The membrane fraction was isolated by centrifugation at 100,000 3 g for 45 min and 4°C. The resulting supernatant was decanted and the cell membrane pellet resuspended in 60 ml PBS. Protein content of the membrane and cytosolic fractions was determined using the Pierce BCA method. Forty micrograms of protein was subjected to separation on a 15% SDS gel. The proteins were transferred to nitrocellulose and the blot incubated overnight in 5% dry milk in 0.5% Tween-20/Tris buffered (TTBS) saline, to block non-specific binding of the primary antibody. The blot was incubated for 3 h in a 1/1000 dilution of GST antibody in 5% dry milk/TTBS. The GST antibody is the polyclonal rabbit antibody described above. The antibody is specific for the GST monomer at 25 kDa. After incubation in primary antibody, the blot was rinsed three times in TTBS over 30 min and incubated in a 1/300 dilution of alkaline phosphatase conjugated anti-rabbit IgG for 1 h. The blot was washed three times in TTBS and protein bands visualized by incubation in BCIP-NBT (Sigma, St. Louis, MO). Experimental levels of oxidized and reduced GSH, glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R), were measured in cultured cortical neurons. For GSH and GSH-Px and GSSR-R activity assays, cortical cell cultures were treated with HNE (1 mM) and GST (100 nM) for 16 h as described above. Medium was removed and the cells washed three times in PBS. Cells from 4 dishes/treatment group were scraped into 1 ml of a homogenization buffer containing HEPES buffer (pH 7.4) with 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4 and 0.6 mM MgSO4 and homogenized 10 strokes using a chilled Dounce homogenizer. The resulting homogenate was centrifuged at 100,000 3 g for 1 h at 4°C. Total GSH concentrations were determined using the
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Fig. 1. Survival of primary hippocampal cells in response to treatment with four-hydroxynonenal (HNE). There is a time and concentration dependent decrease in cell survival with HNE treatment.
method of Tietze [30] as modified by Eyer and Podhradsky [31]. Oxidized GSH was determined using the method of Griffith [32]. To determine total GSH levels, 200 ml of homogenate was mixed with 10 ml 4 M triethanolamine (Sigma, St. Louis, MO) and vortexed. The samples were microfuged at 14,000 rpm for 2 min to pellet any remaining protein. One hundred microliters of the supernatant was removed for oxidized GSH measurements and mixed with 1 ml 2-vinylpyridine (Neat, Sigma, St. Louis, MO) to block reduced GSH. The samples were vortexed and incubated at room temperature for 1 h. After incubation, 50 ml of each solution was mixed with 120 ml MES buffer (4-morpholineethane sulfonic acid, pH 6.0) containing 2 mM sodium EDTA in a 96 well plate. Ten microliters 1 mM 5-59-dithiobis (2 nitrobenzoic acid) (DTNB) in 0.5% NaHCO3 and 10 ml 5 mM NADPH in 0.5% NaHCO3 was added and the reaction initiated by addition of 10 ml 0.025 mg/ml GSSG-R in 50 mM sodium phosphate buffer containing 2 mM EDTA (pH 7.0). The plates were incubated 25 min at room temperature on an orbital shaker. Absorbances
were measured at 405 nm using a Bio-Tek UV-900C plate reader. Concentrations of total and oxidized GSH were determined based on a calibration curve of standard oxidized GSH levels. Results are an average of three repeats and are expressed as pmol/mg protein. Reduced GSH results represent the difference between total GSH and oxidized GSH levels. Protein content was determined using the Pierce BCA method (Sigma, St. Louis, MO). GSH-Px activity was assayed according to the method of Paglia and Valentine [33] as modified by Mizuno and Ohta [34]. For the assay, 200 ml of the supernatant were mixed with 1.6 ml 68 mM KH2PO4 buffer (pH 7.2) containing 1.35 mM EDTA, 10 ml 66 units/ml GSSG-R, 100 ml 1.8 mM NADPH, 10 ml 200 mM NaN3 and 50 ml 50 mM reduced GSH. The reaction was initiated by the addition of 100 ml 15 mM H2O2. The absorbance was measured every min for three min at 340 nm using a Gynesys 5 UV-Vis spectrophotometer. Each sample was analyzed in duplicate with results for three repeats expressed as units/mg protein where 1 unit 5 1 nmol NADPH oxidized/min.
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Fig. 2. Primary hippocampal cells before and after 16 h treatments. (A) Control cells in serum free Locke’s medium alone; (B) Cells treated with 1 mM HNE. Cell death is indicated by vacuolation of cell bodies and extensive neurite fragmentation; (C) Cells treated with 1 mM HNE and 100 nM GST. No significant degenerative changes are present in treated cells.
GSSG-R activity was assayed using the method of Mizuno and Ohta [34] Two hundred microliters of supernatant was mixed with 1.6 ml 68 mM KH2PO4 buffer (pH 7.0) containing 1.35 mM EDTA along with 100 ml 1.8 mM NADPH. The reaction was initiated by addition of 220 ml 13 mM oxidized GSH. Absorbance was followed for 3 min at 340 nm as described above. Analyses were carried out in duplicate with results expressed as the mean 6 SEM units/mg protein with 1 unit 5 1 nmol NADPH oxidized/min. RESULTS
As shown in Fig. 1, HNE leads to a time and concentration dependent decrease in neuron survival as evidenced by neurite fragmentation and cell body vacuola-
tion (Fig. 2B). However, pretreatment of cultures with GST led to protection against a 1 mm toxic dose of HNE (Fig. 3). Assessment of cell survival using the MTT assay yielded similar results to counting of undamaged cells. Plots of cell survival assessed by the MTT assay versus cell counts yielding correlation coefficients of 0.9 – 0.998. As shown in Fig. 3, GST at concentrations greater than 2 nM, led to a statistically significant ( p , .05) increase in cell survival at all time points including 24 h. GST at 0.2 nM offered only slight protection against HNE. At 24 h, 2 nM GST offered 36.2% protection, 20 nM GST 38.9% protection, 100 nM 44.4%, and 200 nM 66% protection which was greater than survival in control cells. One hundred nM GST alone led to a 25% increase in survival compared with control cells. Cells pretreated with a 1:100 dilution of antibody against GST
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Fig. 3. Glutathione transferase offers (statistically significant, p , .05) protection against HNE at concentrations greater than 0.2 nM in a concentration dependent manner. Heat inactivated enzyme offered no protection against HNE. Antibody against GST, inhibited the protective effects of the enzyme.
demonstrated a statistically significant decrease in cell survival at all time points ( p , .05, Fig. 3) with decreases in survival in excess of those observed for cells treated with HNE alone. This increase in toxicity may be due to the inactivation of endogenous native GST by the antibody. Figure 2C shows the decrease in cellular damage in cultures pretreated with GST at 100 nM. Control cultures pretreated with GST also exhibited increased survival compared with control cells receiving no enzyme (Fig. 3). To determine if the protective effect of GST was merely from a chemical interaction with HNE, cultures were treated with an equivalent concentration of enzyme that had been heat inactivated at 100°C for 10 min. The heat inactivated enzyme did not offer any protection against HNE, indicating that the enzyme must be functionally active for protection (Fig. 3). To determine the effectiveness of HNE as a substrate for GST activity, varying concentrations of HNE were added in place of GSH in an activity assay which measures the conjugation of an electrophilic compound
(NBD-Cl) with GSH to form a complex that absorbs at 419 nm. HNE functions in a dose dependent manner as a substrate for action of GST (Fig. 4) in the absence of exogenous GSH. In fact, HNE at an equivalent concentration of GSH functions more effectively as a substrate for conjugation of NBD-Cl. To determine if the conjugation of HNE with GST inactivated the enzyme, GST was incubated with HNE for 5 min at 37°C and the excess HNE removed via filtration through a 5 kDa molecular weight cut off filter. The isolated GST was used in the activity assay as described above. Results of the assay indicate that nonenzymatic conjugation of HNE to GST functionally inactivated the enzyme. Western blot analysis of GST protein (Fig. 5), concentrated from culture medium and from cell homogenate (cytosolic and membrane fractions), indicated that the exogenously added GST remained and functioned in the medium. However, analysis of cell homogenate indicated the presence of low levels of native GST (mw 50 kDa) in both fractions (data not shown).
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Fig. 4. Four-hydroxynonenal (HNE) functions effectively as a substrate for GST mediated conjugation to NBD-CL in the absence of exogenous glutathione. HNE substituted for GSH functioned more effectively as a substrate for conjugation with NBD-CL.
To determine the effect of GST alone and in combination with HNE on levels of oxidized and reduced GSH, as well as the activities of GSH-Px and GSSG-R, enzyme
activities and concentrations were determined in cortical cultures. Total GSH levels were significantly depleted ( p , .05) in cells treated for 16 h with HNE alone
Fig. 5. Western blot analysis of (GST) protein isolated from Locke’s medium. Lane 1—molecular weight markers; Lane 2—purified GST protein; Lane 3—protein isolated from medium from control cells; Lane 4 —GST isolated from a dish treated with 2 nM GST; Lane 5—GST isolated from a dish treated with 20 nM GST; Lane 6 —GST isolated from a dish treated with 100 nM GST. The blot indicates that the protein functions in the medium.
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Table 1. Total, reduced and oxidized glutathione, glutathione peroxidase and glutathione reductase in cortical neurons treated with HNE alone and HNE 1 GST
Control 1 mM HNE 1 mM HNE 1 100 nM GST
Total GSH (pmol mg protein)
Oxidized GSH (pmol/mg protein)
Reduced GSH (pmol/mg protein)
GSH-Px (U/mg protein)
GSSG-R (U/mg protein)
9.32 6 2.1 2.93 6 0.37* 9.37 6 3.71
0.386 6 0.04 0.39 6 0.34 060
8.93 6 2.1 2.54 6 0.50 9.37 6 3.71
0.0011 6 0.00006 0.00128 6 0.000014 0.0013 6 0.0001*
0.0057 6 0.002 0.0047 6 0.006 0.0157 6 0.004**
*p , .05, compared to control cells. **p , .05 compared to both control and HNE treated cells.
(2.93 6 0.37 pmol/mg protein) compared with control cells (9.32 6 2.1 pmol/mg protein) and were marginally significantly increased in cells treated with HNE in conjunction with GST (9.37 6 3.71 pmol/mg protein ( p , .08). Oxidized GSH levels were not significantly altered in cells treated with HNE for 16 h (0.39 6 0.34 pmol/mg protein) compared with control cells (0.386 6 0.04 pmol/mg protein), whereas cells treated with HNE in the presence of GST demonstrated no observable oxidized GSH (Table 1). Reduced GSH levels were significantly depleted in HNE treated cells as reflected in the decrease in total GSH, whereas the level of reduced GSH in GST treated cells was slightly higher than control cells. Analysis of levels of GSH-Px activity indicated no significant differences between control (0.0011 6 0.00006 U/mg protein) and HNE treated cells (0.00128 6 0.00014 U/mg protein), whereas GST treated cells demonstrated a slight but statistically significant increase in enzyme activity (0.0013 6 0.0001 U/mg protein) (Table 1). In addition, GSSG-R activities were not significantly altered in HNE treated cells (0.0047 6 0.0006 U/mg protein) compared with control cells (0.0057 6 0.002 U/mg protein). However, GST treated cells demonstrated statistically significant ( p , .05) increases in GSSG-R activity (0.0157 6 0.004) compared with both control and HNE treated cells (Table 1). DISCUSSION
HNE is the most toxic aldehydic product of lipid peroxidation. It inhibits DNA, RNA and protein synthesis, and interferes with enzyme activity [13]. It is extremely reactive and cross-links with proteins through interaction with lysine, histidine, serine and cysteine residues [35,36]. Although the protein interaction may serve to diffuse the toxicity of HNE, no defense mechanism against HNE has been described in neurons. Our previous studies in short postmortem interval brains revealed a significant increase in HNE in multiple brain regions in AD compared with normal control brains [18]. These increases reached statistical significance in AD amygdala, and hippocampus and parahippocampal gyrus, regions showing the most pronounced
histopathological alterations in AD. Montine et al. [37] demonstrated that an HNE protein adduct, the pyrrole adduct, is present in hippocampal NFT in AD. Borohydride reducible HNE adducts are present in pyramidal neurons in the hippocampus, entorhinal cortex and temporal neocortex in AD [38]. Sayre et al. [39] demonstrated the presence of lysine derived pyrrole adducts of HNE in approximately 50% of NFT-bearing neurons as well as in NFT-free neurons in AD hippocampus. In addition, we found a statistically significant increase in free HNE in AD ventricular fluid compared with normal control subjects [17]. The toxicity of HNE to neurons and the mechanisms of toxicity is defined clearly in neuron culture systems [14 –16]. In cultured hippocampal neurons, Mark et al. [40] showed that the addition of the amyloid beta peptide leads to the generation of HNE which acts to impair ion motive ATPase activity [16], thus leading to a disruption of calcium homeostasis and neuronal death. HNE mediates impaired glucose and glutamate transport in cultured hippocampal neurons [40] and in synaptosomal preparations [41]. HNE is capable of inducing apoptosis in PC12 cells and hippocampal neurons, suggesting that it is a mediator of oxidative stress induced apoptosis [15]. We observed that HNE causes a significant decrease in choline acetyltransferase activity, but not acetylcholinesterase activity, in CHP-134 neuroblastoma cell cultures [42]. Overall, the above studies coupled with those showing an increase in lipid peroxidation [2] and decrease in PUFA [7] in AD strongly suggest that HNE may play an important role in the pathogenesis of neuron degeneration in AD. Mammalian GST are divided into five classes, four of which are cytosolic, soluble proteins termed alpha, mu, pi, and theta [43], with the fifth being a membrane-bound protein [44]. The proteins are dimeric but split with an apparent molecular weight of 25 kDa in SDS-PAGE analysis. These enzymes have no known natural substrate for function [19], although peroxidized DNA [45], arachidonic oxides [46], cholesterol epoxides [47,48], and 4-hydroxyalkenals [49 –53], function quite effectively as substrates. GST and GSH function efficiently in
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the conjugation of by-products of oxidative stress, including HNE [45–53]. Although GSH can function alone in the conjugation of HNE via the formation of a thiolether linkage, the GST-catalyzed reaction occurs 300 – 600 times faster [13]. Zimniak et al. [54] showed that HepG2 cells overexpressing GST (form A4) offered protection against HNE toxicity. Our recently submitted study revealed a marked reduction of GST activity and protein levels in multiple brain regions in AD patients compared with normal control subjects [25]. These decreases reached statistical significance in the amygdala, hippocampus and parahippocampal gyrus, and inferior parietal lobule, regions showing the most significant elevations in HNE [18] and severe morphological changes. A statistically significant reduction in GST activity and protein also was found in ventricular CSF in AD compared with normal control subjects [25]. The present study demonstrates that GST enhances neuron survival against toxic levels of HNE. Although the observed protection is not markedly different between the concentrations of GST used in the study, there is a statistically significant positive correlation (r 5 10.99) between concentration of GST used and percentage enhanced survival. Our results indicate GST remains and is active in the culture medium and thus functions in the detoxification of HNE in the absence of exogenous GSH. Blockage of the protective effects observed with an antibody against GST coupled with an increase in toxicity of HNE, possibly through the blockage of endogenous GST, suggests that GST is responsible for the protective effects observed. In addition, we observed that HNE alone can function as efficiently as GSH as a substrate for GST conjugation. The protection provided by the mu form of the enzyme is dependent on functionally active GST because the heat inactivated enzyme offered no protection. GST prevents HNE from inducing GSH loss in cells which again is consistent with our observation of the action of GST in the culture medium. The decreased levels of oxidized GSH in GST treated cells are consistent with our observation of statistically significant increased levels of GSSG-R, the enzyme responsible for regenerating GSH. The GSH-Px and GSSG-R studies suggest that HNE does not directly impact their activities, although pretreatment of cells with GST led to significant enhancement of enzyme activities compared with control cells. This observation is consistent with the observed increase in survival in cells treated with GST. The data in the present study suggest that the loss of a protective enzyme could make neurons more vulnerable to the toxic effect of HNE. Coupled with our finding of diminished GST [25] and increased HNE in the brain in AD [18], this study suggests the lack of protection
against HNE by GST could lead to neuron degeneration in AD. It also suggests that enhancement of GST and GSH might have therapeutic benefits in AD. Acknowledgements — This research was supported by NIH Grants 1P01-AG05119 and 5P50-AG05144, and a grant from the Abercrombie Foundation. The authors are grateful for the editorial assistance of Paula Thomason and technical assistance of Jane Meara.
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GST protects against HNE [16] Mark, R. J.; Lovell, M. A.; Markesbery, W. R.; Uchida, K.; Mattson, M. P. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation in disruption of ion homeostasis and neuronal death induced by amyloid b-peptide. J. Neurochem. 68:255–264; 1997. [17] Lovell, M. A.; Ehmann, W. D.; Mattson, M. P.; Markesbery, W. R. Elevated 4-hydroxynonenal in ventricular fluid in Alzheimer’s disease. Neurobiol. Aging 18:457– 461; 1997. [18] Markesbery, W. R.; Lovell, M. A. Four-hydroxynonenal, a product of lipid peroxidation, is increased in the brain in Alzheimer’s disease. Neurobiol. Aging 19:33–36; 1998. [19] Mannervik, B.; Danielson, U. H. Glutathione transferases—Structure and catalytic activity. CRC Crit. Rev. Biochem. 22:283–337; 1988. [20] Warholm, M.; Guthenberg, C.; Mannervik, B.; von Bahr, C.; Glaumann, H. Identification of a new glutathione S-transferase in human liver. Acta Chem. Scand. B 34:607– 610; 1980. [21] Warholm, M.; Guthenberg, C.; Mannervik, B.; von Hahr, C. Purification of a new glutathione S-transferase (transferase mu) from human liver having high activity with benzo (a) pyrene-4,5oxide. Biochem. Biophys. Res. Commun. 98:512–519; 1981. [22] Mannervik, B.; Guthenberg, C.; Jensson, H.; Warholm, M.; Alin, P. Isozymes of gluatathione s-transferase in rat and human tissues. In: Larsson, A.; Orrenius, S.; Holmgren, A.; Mannervik, B., eds. Functions of glutathione: Biochemical, pharmacological, toxicological and clinical aspects, New York: Raven Press; 1983:75– 88. [23] Marcus, C. J.; Habig, W. H.; Jacoby, W. B. Glutathione transferase from human erythrocytes, nonidentity with the enzymes from liver. Arch. Biochem. Biophys. 188:287–293; 1978. [24] Guthenberg, C.; Akerfeldt, K.; Mannervik, B. Purification of glutathione S-transferase from human placenta. Acta Chem. Scand. B33:595–596; 1979. [25] Lovell, M. A.; Xie, C. S.; Markesbery, W. R. Glutathione transferase activity is decreased in brain and ventricular fluid in Alzheimer’s disease. Neurology. in press. [26] Mattson, M. P.; Barger, S. W.; Begley, J. G.; Mark, R. J. Calcium, free radicals and excitotoxic neuronal death in primary cell culture. Methods Cell Biol. 46:187–216; 1995. [27] Mosmann, T. Rapid colorimetric assay for cellular growth and survival. J. Immunol. Methods. 65:55– 63; 1983. [28] Musser, D. A.; Oseroff, A. R. The use of tetrazolium salts to determine sites of damage to the mitochondrial electron transport chain in intact cells following in vitro photodynamic therapy with Photofrin II. Photochem Photobiol. 59:621– 626; 1994. [29] Ricci, G.; Cacurri, A. M.; Lo Bello, M.; Pastore, A.; Piemonte, F.; Federici, G. Colorimetric and fluorometric assays of glutathione transferase based on 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Anal. Biochem. 218:463– 465; 1994. [30] Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Applications to mammalian blood and other tissues. Anal. Biochem. 27:502– 522; 1969. [31] Eyer, P.; Podhradsky, D. Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman’s reagent. Anal. Biochem. 153:57– 66; 1986. [32] Griffith, O. W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106:207–212; 1980. [33] Paglia, D. E.; Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab Clin. Med. 70:158 –169; 1967. [34] Mizuno, Y.; Ohta, K. Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of oxygen free radicals, and some of the related enzymes in adult and aged rat brains. J. Neurochem. 46:1344 –1352; 1986. [35] Jurgens, G.; Lang, J.; Esterbauer, H. Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim. Biophys. Acta 875:103–114; 1986. [36] Uchida, K.; Stadtman, E. R. Modification of histidine residues in
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C. XIE et al. pressing glutathione s-transferase mGSTA4-4. Toxicol. Appl. Pharmacol. 143:221–229; 1997. ABBREVIATIONS
AD—Alzheimer’s disease GSH— glutathione
GSH-Px— glutathione peroxidase GSSG-R— glutathione reductase GST— glutathione transferase HNE—Four-hydroxynonenal NBD-Cl—7-chloro-4-nitrobenzo-2-oxa-1,3 diazole PUFA—polyunsaturated fatty acids
PII S0891-5849(98)00186-5
Original Contribution GLUTATHIONE TRANSFERASE PROTECTS NEURONAL CULTURES AGAINST FOUR HYDROXYNONENAL TOXICITY CHENGSONG XIE,* MARK A. LOVELL,*†
and
WILLIAM R. MARKESBERY*‡
*Sanders-Brown Center on Aging, Departments of †Chemistry, ‡Neurology, and ‡Pathology, University of Kentucky, Lexington, KY, USA (Received 5 May 1998; Revised 29 June 1998; Accepted 10 July 1998)
Abstract—Peroxidation of polyunsaturated fatty acids (PUFA), particularly arachidonic acid, leads to the generation of reactive aldehydes, including 4-hydroxynonenal (HNE). Recent studies have demonstrated an increase in lipid peroxidation, a decline in PUFA, as well as an increase in HNE, and a decrease in glutathione transferase (GST) in the brain in Alzheimer’s disease. Four-hydroxynonenal is toxic to cultured neurons and to the brain of experimental animals. Although glutathione (GSH) has been shown to offer protection against HNE, no enzymatic system has been described which serves to detoxify these reactive species in neuronal cultures. Here, we describe the use of GST in the protection of neuronal cultures against HNE toxicity. Glutathione transferases are a superfamily of enzymes functioning to catalyze the nucleophilic attack of GSH on electrophilic groups on a second substrate. These enzymes function efficiently with 4-hydroxyalkenals, particularly HNE, as substrates. To investigate the protective effects of GST against HNE, primary hippocampal cultures were pretreated with GST before exposure to toxic doses of HNE which led to a statistically significant enhancement in cell survival. Pretreatment of cultures with equivalent levels of heat inactivated GST or antibody against GST did not offer protection against HNE. Control cultures pretreated with GST also demonstrated enhanced survival compared with control cells receiving no pretreatment. These data suggest that GST may be an important source of protection against the toxic effects of HNE. © 1998 Elsevier Science Inc. Keywords—Glutathione transferase (GST), 4-Hydroxynonenal (HNE), Neuronal culture, Protection, Alzheimer’s disease (AD)
a, b unsaturated aldehyde that is generated during the peroxidation of PUFA, particularly arachidonic acid [13]. This reactive by-product possibly plays a role in the pathogenesis of AD [14 –16]. Recently, we observed statistically significant increases in HNE levels in ventricular fluid [17] and in the brain in AD [18]. Glutathione transferases (GST) (E.C. 2.5.1.18) are a superfamily of enzymes that function to catalyze the nucleophilic attack of the sulfhur atom of glutathione (GSH) on electrophilic groups of a second substrate [19]. GST are present in many organs [20 –24] and function to inactivate toxic products of oxygen metabolism, such as 4-hydroxyalkenals. We recently observed a marked decline in GST activity in brain and ventricular fluid in AD subjects compared with normal control subjects [25]. Based on these observations, we initiated studies to evaluate the possible protective effects of GST against HNE in primary rat hippocampal cultures.
INTRODUCTION
One of the leading hypotheses regarding the pathogenesis of Alzheimer’s disease (AD) is the involvement of increased levels of oxidative stress in neuronal degeneration (reviewed in [1]). This increased oxidative stress in the brain in AD is characterized by increases in lipid peroxidation [2], protein oxidation [3,4], and DNA oxidation [5,6] and decreases in polyunsaturated fatty acids (PUFA) [7,8]. Additionally, markers of oxidative stress including nitrotyrosine, protein carbonyls and advanced Maillard reaction end products are present in neurofibrillary tangles (NFT) and/or senile plaques in AD [9 –12]. One product of oxidative stress that has received considerable attention is 4-hydroxynonenal (HNE). HNE is an Address correspondence to: Mark A. Lovell, 101 Sanders-Brown Center on Aging, University of Kentucky, 800 South Limestone St., Lexington, KY 40536-0230, USA; Tel: (606) 257-5566; Fax: (606) 323-2866. 979
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C. XIE et al. MATERIALS AND METHODS
Primary cortical and hippocampal cultures were established from gestation day 18 rat embryos as described by Mattson et al. [26] Cells were plated at a density of 50/mm2 in polyethyleneamine coated plastic 35 mm dishes. The cultures were maintained in Eagle’s minimum essential medium supplemented with 10% (v/v) qualified bovine serum (Gibco BRL, Life Technologies, Inc., Grand Island, NY) and containing 20 mM KCl, 1 mM pyruvate in an environment of 94% air/6% CO2 at 37°C. Experiments were carried out on cells 7– 8 days in culture. HNE (Cayman Chemical, Ann Arbor, MI) was used at 1 mM final concentration which has been shown to induce significant cell death after 24 h of treatment. The GST used was the mu isoform (Calbiochem, La Jolla, CA). For treatment, cultures were chosen at random and the culture medium switched to serum free Locke’s solution that consisted of 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 10 mM glucose, 5 mM HEPES pH 7.2 with 10 mg/l gentamicin sulfate. Cell survival was assessed by counting the number of undamaged neurons in premarked microscopic fields before and at each indicated time point (3, 6, 16 and 24 h). To ensure the validity of cell counts, cell viability was assessed using the MTT reduction assay in which MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] is converted to blue formazan crystals in cells as a result of mitochondrial electron transport chain reaction [27,28] and thus measures cell viability. For this assay, 10 ml MTT (1 mg/ml) was added to each dish after the appropriate treatment time and the dishes incubated for 45 min. The medium was removed and the dishes washed three times in PBS. The formazan product was solubilized in 1 ml dimethyl sulfoxide (DMSO) and the absorbance measured at 595 nm using a Bio-Tek UV-900C plate reader. Results of the assay were expressed as a percentage of control absorbance. For treatment, cultures were switched to Locke’s solution and GST added at varying concentrations for 6 h before addition of HNE. To assess whether function of the enzyme or simple chemical interaction with the protein afforded the protection, an equivalent concentration of GST that had been inactivated by heating at 100°C for 10 min was added to cultures. To verify that the protective effects observed were due to the presence of GST, cells were pretreated with a 1:100 dilution of a polyclonal rabbit antibody raised against recombinant human GST M1-1 (Oxford Biomedical Research, Oxford, MI) for 4 h before adding 100 nM GST. After incubation in 100 nM GST, 1 mM HNE was added and cell viability assessed as described above. GST activity was assayed according to the method of
Ricci et al. [29] which follows the conjugation of 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole (NBD-Cl) with GSH as catalyzed by GST. The NBD-Cl complex is a yellow compound that is stable and strongly absorbs at 419 nm with a molar absorption coefficient of 14.5 mM21cm21. For the assay, 2 ml of 0.1 M sodium acetate buffer (pH 5.0) was mixed with 200 ml 0.2 mM NBD-Cl and 100 ml 0.5 mM reduced GSH. The reaction was initiated by addition of 200 ml of concentrated protein isolated from culture medium. All reagents were used at room temperature with the exception of the protein which was stored on ice until use. The assay is extremely sensitive to temperature and may be greatly affected by reagents that are below room temperature. For Western blot analysis, the protein from the medium was concentrated by centrifugation through a 10 kDa molecular weight cut off filter. The cells were rinsed three times in PBS, scraped and lysed in 200 ml of a lysis buffer consisting of 20 mM imidazole (pH 7.4) containing 0.6 mM EGTA and 0.1 mM PMSF. The membrane fraction was isolated by centrifugation at 100,000 3 g for 45 min and 4°C. The resulting supernatant was decanted and the cell membrane pellet resuspended in 60 ml PBS. Protein content of the membrane and cytosolic fractions was determined using the Pierce BCA method. Forty micrograms of protein was subjected to separation on a 15% SDS gel. The proteins were transferred to nitrocellulose and the blot incubated overnight in 5% dry milk in 0.5% Tween-20/Tris buffered (TTBS) saline, to block non-specific binding of the primary antibody. The blot was incubated for 3 h in a 1/1000 dilution of GST antibody in 5% dry milk/TTBS. The GST antibody is the polyclonal rabbit antibody described above. The antibody is specific for the GST monomer at 25 kDa. After incubation in primary antibody, the blot was rinsed three times in TTBS over 30 min and incubated in a 1/300 dilution of alkaline phosphatase conjugated anti-rabbit IgG for 1 h. The blot was washed three times in TTBS and protein bands visualized by incubation in BCIP-NBT (Sigma, St. Louis, MO). Experimental levels of oxidized and reduced GSH, glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R), were measured in cultured cortical neurons. For GSH and GSH-Px and GSSR-R activity assays, cortical cell cultures were treated with HNE (1 mM) and GST (100 nM) for 16 h as described above. Medium was removed and the cells washed three times in PBS. Cells from 4 dishes/treatment group were scraped into 1 ml of a homogenization buffer containing HEPES buffer (pH 7.4) with 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4 and 0.6 mM MgSO4 and homogenized 10 strokes using a chilled Dounce homogenizer. The resulting homogenate was centrifuged at 100,000 3 g for 1 h at 4°C. Total GSH concentrations were determined using the
GST protects against HNE
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Fig. 1. Survival of primary hippocampal cells in response to treatment with four-hydroxynonenal (HNE). There is a time and concentration dependent decrease in cell survival with HNE treatment.
method of Tietze [30] as modified by Eyer and Podhradsky [31]. Oxidized GSH was determined using the method of Griffith [32]. To determine total GSH levels, 200 ml of homogenate was mixed with 10 ml 4 M triethanolamine (Sigma, St. Louis, MO) and vortexed. The samples were microfuged at 14,000 rpm for 2 min to pellet any remaining protein. One hundred microliters of the supernatant was removed for oxidized GSH measurements and mixed with 1 ml 2-vinylpyridine (Neat, Sigma, St. Louis, MO) to block reduced GSH. The samples were vortexed and incubated at room temperature for 1 h. After incubation, 50 ml of each solution was mixed with 120 ml MES buffer (4-morpholineethane sulfonic acid, pH 6.0) containing 2 mM sodium EDTA in a 96 well plate. Ten microliters 1 mM 5-59-dithiobis (2 nitrobenzoic acid) (DTNB) in 0.5% NaHCO3 and 10 ml 5 mM NADPH in 0.5% NaHCO3 was added and the reaction initiated by addition of 10 ml 0.025 mg/ml GSSG-R in 50 mM sodium phosphate buffer containing 2 mM EDTA (pH 7.0). The plates were incubated 25 min at room temperature on an orbital shaker. Absorbances
were measured at 405 nm using a Bio-Tek UV-900C plate reader. Concentrations of total and oxidized GSH were determined based on a calibration curve of standard oxidized GSH levels. Results are an average of three repeats and are expressed as pmol/mg protein. Reduced GSH results represent the difference between total GSH and oxidized GSH levels. Protein content was determined using the Pierce BCA method (Sigma, St. Louis, MO). GSH-Px activity was assayed according to the method of Paglia and Valentine [33] as modified by Mizuno and Ohta [34]. For the assay, 200 ml of the supernatant were mixed with 1.6 ml 68 mM KH2PO4 buffer (pH 7.2) containing 1.35 mM EDTA, 10 ml 66 units/ml GSSG-R, 100 ml 1.8 mM NADPH, 10 ml 200 mM NaN3 and 50 ml 50 mM reduced GSH. The reaction was initiated by the addition of 100 ml 15 mM H2O2. The absorbance was measured every min for three min at 340 nm using a Gynesys 5 UV-Vis spectrophotometer. Each sample was analyzed in duplicate with results for three repeats expressed as units/mg protein where 1 unit 5 1 nmol NADPH oxidized/min.
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Fig. 2. Primary hippocampal cells before and after 16 h treatments. (A) Control cells in serum free Locke’s medium alone; (B) Cells treated with 1 mM HNE. Cell death is indicated by vacuolation of cell bodies and extensive neurite fragmentation; (C) Cells treated with 1 mM HNE and 100 nM GST. No significant degenerative changes are present in treated cells.
GSSG-R activity was assayed using the method of Mizuno and Ohta [34] Two hundred microliters of supernatant was mixed with 1.6 ml 68 mM KH2PO4 buffer (pH 7.0) containing 1.35 mM EDTA along with 100 ml 1.8 mM NADPH. The reaction was initiated by addition of 220 ml 13 mM oxidized GSH. Absorbance was followed for 3 min at 340 nm as described above. Analyses were carried out in duplicate with results expressed as the mean 6 SEM units/mg protein with 1 unit 5 1 nmol NADPH oxidized/min. RESULTS
As shown in Fig. 1, HNE leads to a time and concentration dependent decrease in neuron survival as evidenced by neurite fragmentation and cell body vacuola-
tion (Fig. 2B). However, pretreatment of cultures with GST led to protection against a 1 mm toxic dose of HNE (Fig. 3). Assessment of cell survival using the MTT assay yielded similar results to counting of undamaged cells. Plots of cell survival assessed by the MTT assay versus cell counts yielding correlation coefficients of 0.9 – 0.998. As shown in Fig. 3, GST at concentrations greater than 2 nM, led to a statistically significant ( p , .05) increase in cell survival at all time points including 24 h. GST at 0.2 nM offered only slight protection against HNE. At 24 h, 2 nM GST offered 36.2% protection, 20 nM GST 38.9% protection, 100 nM 44.4%, and 200 nM 66% protection which was greater than survival in control cells. One hundred nM GST alone led to a 25% increase in survival compared with control cells. Cells pretreated with a 1:100 dilution of antibody against GST
GST protects against HNE
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Fig. 3. Glutathione transferase offers (statistically significant, p , .05) protection against HNE at concentrations greater than 0.2 nM in a concentration dependent manner. Heat inactivated enzyme offered no protection against HNE. Antibody against GST, inhibited the protective effects of the enzyme.
demonstrated a statistically significant decrease in cell survival at all time points ( p , .05, Fig. 3) with decreases in survival in excess of those observed for cells treated with HNE alone. This increase in toxicity may be due to the inactivation of endogenous native GST by the antibody. Figure 2C shows the decrease in cellular damage in cultures pretreated with GST at 100 nM. Control cultures pretreated with GST also exhibited increased survival compared with control cells receiving no enzyme (Fig. 3). To determine if the protective effect of GST was merely from a chemical interaction with HNE, cultures were treated with an equivalent concentration of enzyme that had been heat inactivated at 100°C for 10 min. The heat inactivated enzyme did not offer any protection against HNE, indicating that the enzyme must be functionally active for protection (Fig. 3). To determine the effectiveness of HNE as a substrate for GST activity, varying concentrations of HNE were added in place of GSH in an activity assay which measures the conjugation of an electrophilic compound
(NBD-Cl) with GSH to form a complex that absorbs at 419 nm. HNE functions in a dose dependent manner as a substrate for action of GST (Fig. 4) in the absence of exogenous GSH. In fact, HNE at an equivalent concentration of GSH functions more effectively as a substrate for conjugation of NBD-Cl. To determine if the conjugation of HNE with GST inactivated the enzyme, GST was incubated with HNE for 5 min at 37°C and the excess HNE removed via filtration through a 5 kDa molecular weight cut off filter. The isolated GST was used in the activity assay as described above. Results of the assay indicate that nonenzymatic conjugation of HNE to GST functionally inactivated the enzyme. Western blot analysis of GST protein (Fig. 5), concentrated from culture medium and from cell homogenate (cytosolic and membrane fractions), indicated that the exogenously added GST remained and functioned in the medium. However, analysis of cell homogenate indicated the presence of low levels of native GST (mw 50 kDa) in both fractions (data not shown).
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Fig. 4. Four-hydroxynonenal (HNE) functions effectively as a substrate for GST mediated conjugation to NBD-CL in the absence of exogenous glutathione. HNE substituted for GSH functioned more effectively as a substrate for conjugation with NBD-CL.
To determine the effect of GST alone and in combination with HNE on levels of oxidized and reduced GSH, as well as the activities of GSH-Px and GSSG-R, enzyme
activities and concentrations were determined in cortical cultures. Total GSH levels were significantly depleted ( p , .05) in cells treated for 16 h with HNE alone
Fig. 5. Western blot analysis of (GST) protein isolated from Locke’s medium. Lane 1—molecular weight markers; Lane 2—purified GST protein; Lane 3—protein isolated from medium from control cells; Lane 4 —GST isolated from a dish treated with 2 nM GST; Lane 5—GST isolated from a dish treated with 20 nM GST; Lane 6 —GST isolated from a dish treated with 100 nM GST. The blot indicates that the protein functions in the medium.
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Table 1. Total, reduced and oxidized glutathione, glutathione peroxidase and glutathione reductase in cortical neurons treated with HNE alone and HNE 1 GST
Control 1 mM HNE 1 mM HNE 1 100 nM GST
Total GSH (pmol mg protein)
Oxidized GSH (pmol/mg protein)
Reduced GSH (pmol/mg protein)
GSH-Px (U/mg protein)
GSSG-R (U/mg protein)
9.32 6 2.1 2.93 6 0.37* 9.37 6 3.71
0.386 6 0.04 0.39 6 0.34 060
8.93 6 2.1 2.54 6 0.50 9.37 6 3.71
0.0011 6 0.00006 0.00128 6 0.000014 0.0013 6 0.0001*
0.0057 6 0.002 0.0047 6 0.006 0.0157 6 0.004**
*p , .05, compared to control cells. **p , .05 compared to both control and HNE treated cells.
(2.93 6 0.37 pmol/mg protein) compared with control cells (9.32 6 2.1 pmol/mg protein) and were marginally significantly increased in cells treated with HNE in conjunction with GST (9.37 6 3.71 pmol/mg protein ( p , .08). Oxidized GSH levels were not significantly altered in cells treated with HNE for 16 h (0.39 6 0.34 pmol/mg protein) compared with control cells (0.386 6 0.04 pmol/mg protein), whereas cells treated with HNE in the presence of GST demonstrated no observable oxidized GSH (Table 1). Reduced GSH levels were significantly depleted in HNE treated cells as reflected in the decrease in total GSH, whereas the level of reduced GSH in GST treated cells was slightly higher than control cells. Analysis of levels of GSH-Px activity indicated no significant differences between control (0.0011 6 0.00006 U/mg protein) and HNE treated cells (0.00128 6 0.00014 U/mg protein), whereas GST treated cells demonstrated a slight but statistically significant increase in enzyme activity (0.0013 6 0.0001 U/mg protein) (Table 1). In addition, GSSG-R activities were not significantly altered in HNE treated cells (0.0047 6 0.0006 U/mg protein) compared with control cells (0.0057 6 0.002 U/mg protein). However, GST treated cells demonstrated statistically significant ( p , .05) increases in GSSG-R activity (0.0157 6 0.004) compared with both control and HNE treated cells (Table 1). DISCUSSION
HNE is the most toxic aldehydic product of lipid peroxidation. It inhibits DNA, RNA and protein synthesis, and interferes with enzyme activity [13]. It is extremely reactive and cross-links with proteins through interaction with lysine, histidine, serine and cysteine residues [35,36]. Although the protein interaction may serve to diffuse the toxicity of HNE, no defense mechanism against HNE has been described in neurons. Our previous studies in short postmortem interval brains revealed a significant increase in HNE in multiple brain regions in AD compared with normal control brains [18]. These increases reached statistical significance in AD amygdala, and hippocampus and parahippocampal gyrus, regions showing the most pronounced
histopathological alterations in AD. Montine et al. [37] demonstrated that an HNE protein adduct, the pyrrole adduct, is present in hippocampal NFT in AD. Borohydride reducible HNE adducts are present in pyramidal neurons in the hippocampus, entorhinal cortex and temporal neocortex in AD [38]. Sayre et al. [39] demonstrated the presence of lysine derived pyrrole adducts of HNE in approximately 50% of NFT-bearing neurons as well as in NFT-free neurons in AD hippocampus. In addition, we found a statistically significant increase in free HNE in AD ventricular fluid compared with normal control subjects [17]. The toxicity of HNE to neurons and the mechanisms of toxicity is defined clearly in neuron culture systems [14 –16]. In cultured hippocampal neurons, Mark et al. [40] showed that the addition of the amyloid beta peptide leads to the generation of HNE which acts to impair ion motive ATPase activity [16], thus leading to a disruption of calcium homeostasis and neuronal death. HNE mediates impaired glucose and glutamate transport in cultured hippocampal neurons [40] and in synaptosomal preparations [41]. HNE is capable of inducing apoptosis in PC12 cells and hippocampal neurons, suggesting that it is a mediator of oxidative stress induced apoptosis [15]. We observed that HNE causes a significant decrease in choline acetyltransferase activity, but not acetylcholinesterase activity, in CHP-134 neuroblastoma cell cultures [42]. Overall, the above studies coupled with those showing an increase in lipid peroxidation [2] and decrease in PUFA [7] in AD strongly suggest that HNE may play an important role in the pathogenesis of neuron degeneration in AD. Mammalian GST are divided into five classes, four of which are cytosolic, soluble proteins termed alpha, mu, pi, and theta [43], with the fifth being a membrane-bound protein [44]. The proteins are dimeric but split with an apparent molecular weight of 25 kDa in SDS-PAGE analysis. These enzymes have no known natural substrate for function [19], although peroxidized DNA [45], arachidonic oxides [46], cholesterol epoxides [47,48], and 4-hydroxyalkenals [49 –53], function quite effectively as substrates. GST and GSH function efficiently in
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the conjugation of by-products of oxidative stress, including HNE [45–53]. Although GSH can function alone in the conjugation of HNE via the formation of a thiolether linkage, the GST-catalyzed reaction occurs 300 – 600 times faster [13]. Zimniak et al. [54] showed that HepG2 cells overexpressing GST (form A4) offered protection against HNE toxicity. Our recently submitted study revealed a marked reduction of GST activity and protein levels in multiple brain regions in AD patients compared with normal control subjects [25]. These decreases reached statistical significance in the amygdala, hippocampus and parahippocampal gyrus, and inferior parietal lobule, regions showing the most significant elevations in HNE [18] and severe morphological changes. A statistically significant reduction in GST activity and protein also was found in ventricular CSF in AD compared with normal control subjects [25]. The present study demonstrates that GST enhances neuron survival against toxic levels of HNE. Although the observed protection is not markedly different between the concentrations of GST used in the study, there is a statistically significant positive correlation (r 5 10.99) between concentration of GST used and percentage enhanced survival. Our results indicate GST remains and is active in the culture medium and thus functions in the detoxification of HNE in the absence of exogenous GSH. Blockage of the protective effects observed with an antibody against GST coupled with an increase in toxicity of HNE, possibly through the blockage of endogenous GST, suggests that GST is responsible for the protective effects observed. In addition, we observed that HNE alone can function as efficiently as GSH as a substrate for GST conjugation. The protection provided by the mu form of the enzyme is dependent on functionally active GST because the heat inactivated enzyme offered no protection. GST prevents HNE from inducing GSH loss in cells which again is consistent with our observation of the action of GST in the culture medium. The decreased levels of oxidized GSH in GST treated cells are consistent with our observation of statistically significant increased levels of GSSG-R, the enzyme responsible for regenerating GSH. The GSH-Px and GSSG-R studies suggest that HNE does not directly impact their activities, although pretreatment of cells with GST led to significant enhancement of enzyme activities compared with control cells. This observation is consistent with the observed increase in survival in cells treated with GST. The data in the present study suggest that the loss of a protective enzyme could make neurons more vulnerable to the toxic effect of HNE. Coupled with our finding of diminished GST [25] and increased HNE in the brain in AD [18], this study suggests the lack of protection
against HNE by GST could lead to neuron degeneration in AD. It also suggests that enhancement of GST and GSH might have therapeutic benefits in AD. Acknowledgements — This research was supported by NIH Grants 1P01-AG05119 and 5P50-AG05144, and a grant from the Abercrombie Foundation. The authors are grateful for the editorial assistance of Paula Thomason and technical assistance of Jane Meara.
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C. XIE et al. pressing glutathione s-transferase mGSTA4-4. Toxicol. Appl. Pharmacol. 143:221–229; 1997. ABBREVIATIONS
AD—Alzheimer’s disease GSH— glutathione
GSH-Px— glutathione peroxidase GSSG-R— glutathione reductase GST— glutathione transferase HNE—Four-hydroxynonenal NBD-Cl—7-chloro-4-nitrobenzo-2-oxa-1,3 diazole PUFA—polyunsaturated fatty acids