?-Glutamylcysteine Ethyl Ester-Induced
Up-Regulation of Glutathione Protects
Neurons Against A?(1–42)-Mediated
Oxidative Stress and Neurotoxicity:
Implications for Alzheimer’s Disease
Debra Boyd-Kimball,1Rukhsana Sultana,1Hafiz Mohmmad Abdul,1and
D. Allan Butterfield1,2*
1Department of Chemistry, Center for Membrane Sciences, University of Kentucky, Lexington, Kentucky
2Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky
Glutathione (GSH) is an important endogenous antioxi-
dant found in millimolar concentrations in the brain. GSH
levels have been shown to decrease with aging. Alzhei-
mer’s disease (AD) is a neurodegenerative disorder as-
sociated with aging and oxidative stress. A?(1–42) has
been shown to induce oxidative stress and has been
proposed to play a central role in the oxidative damage
detected in AD brain. It has been shown that administra-
tion of ?-glutamylcysteine ethyl ester (GCEE) increases
cellular levels of GSH, circumventing the regulation of
GSH biosynthesis by providing the limiting substrate. In
this study, we evaluated the protective role of up-
regulation of GSH by GCEE against the oxidative and
neurotoxic effects of A?(1–42) in primary neuronal cul-
ture. Addition of GCEE to neurons led to an elevated
mean cellular GSH level compared with untreated con-
trol. Inhibition of ?-glutamylcysteine synthetase by buthi-
onine sulfoximine (BSO) led to a 98% decrease in total
cellular GSH compared with control, which was returned
to control levels by addition of GCEE. Taken together,
these results suggest that GCEE up-regulates cellular
GSH levels which, in turn, protects neurons against pro-
tein oxidation, loss of mitochondrial function, and DNA
fragmentation induced by A?(1–42). These results are
consistent with the notion that up-regulation of GSH by
GCEE may play a viable protective role in the oxidative
and neurotoxicity induced by A?(1–42) in AD brain.
© 2005 Wiley-Liss, Inc.
Key words: Alzheimer’s disease; glutathione; amyloid
A?(1–42) has been shown to induce oxidative stress
both in vitro and in vivo (Yatin et al., 1999a; Butterfield
and Lauderback, 2002; Drake et al., 2003a). Oxidative
stress is extensive in AD, a neurodegenerative disease
associated with cognitive decline and aging (Subbarao et
al., 1990; Hensley et al., 1995; Markesbery, 1997; Butter-
field et al., 2001, 2002a). Consequently, A?(1–42) has
been implicated as a causative agent in AD (Varadarajan et
al., 2000; Buttefield, 2002, 2003). The lipid-soluble anti-
oxidant vitamin E has been shown to inhibit the oxidative
damage induced by A?(1–42), suggesting that reactive
oxygen species (ROS) play a role (Yatin et al., 2000).
Glutathione (GSH) is a tripeptide (?-gluta-
mylcysteinylglycine) found in intracellular concentrations
of 1–3 mM in the brain (Cooper, 1997). GSH is located in
both the cytosol and the mitochondria within cells and acts
as a vital endogenous antioxidant to combat oxidative
stress. Cysteine is the limiting amino acid in GSH biosyn-
thesis, and, as a result, ?-glutamylcysteine is the limiting
substrate for GSH synthesis. ?-Glutamylcysteine syn-
thetase (?-GCS), the enzyme that catalyzes the formation
of the dipeptide ?-glutamylcysteine, is the rate-limiting
enzyme in GSH synthesis, and this enzyme is feedback
inhibited by GSH itself (Anderson and Luo, 1998). It has
been proposed that administration of the rate-limiting
substrate, ?-glutamylcysteine, will circumvent GSH-
mediated feedback inhibition in GSH biosynthesis (Drake
et al., 2002), because this dipeptide is a substrate for GSH
synthase (Cooper, 1997). Moreover, modification of the
substrate by esterification [?-glutamylcysteine ethyl ester
(GCEE)] has been shown to facilitate transport of the
compound across the plasma membrane, where it is dees-
terified and can be acted upon by GSH synthetase to
catalyze the formation of GSH (Anderson et al., 1985;
Anderson and Meister, 1989). Such up-regulation of GSH
*Correspondence to: Prof. D. Allan Butterfield, Department of Chemistry,
Center for Membrane Sciences, and Sanders-Brown Center on Aging, 121
Chemistry-Physics Building, University of Kentucky, Lexington, KY
40506-0055. E-mail: firstname.lastname@example.org
Received 18 July 2004; Revised 3 November 2004; Accepted 8 November
Published online 27 January 2005 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.20394
Journal of Neuroscience Research 79:700–706 (2005)
© 2005 Wiley-Liss, Inc.
has been shown to protect both synaptosomes and mito-
chondria against peroxynitrite-mediated oxidative stress
(Drake et al., 2002, 2003b).
A?(1–42) has been shown to deplete GSH levels in
astrocytes (Abramov et al., 2003). Such glial cells play a
major role in supplying the metabolic substrates and pre-
cursors of GSH to neurons. Consequently, A?(1–42) may
lead to depletion of GSH as an available antioxidant in
neurons. Additionally, GSH levels decrease with age, leav-
ing neurons vulnerable to oxidative damage initiated by
A?(1–42) (Liu and Choi, 2000). Up-regulation of GSH
could play a therapeutic role in AD (Butterfield et al.,
In this study, we evaluated the role of GCEE up-
regulation of GSH in neurons as a protective therapeutic
mechanism against A?(1–42)-mediated oxidative stress
and neurotoxicity. We show that GCEE up-regulation of
GSH in neurons protects against A?(1–42)-induced pro-
tein oxidation, loss of mitochondria function, and DNA
MATERIALS AND METHODS
All chemicals were of the highest purity and were ob-
tained from Sigma (St. Louis, MO) unless otherwise noted.
A?(1–42) was purchased from Anaspec (San Jose, CA), with
HPLC and MS verification of purity. The peptide was stored in
the dry state at –20°C until use. The OxyBlot protein oxidation
detection kit was purchased from Chemicon International (Te-
mecula, CA). Anti-4-hydroxynonenal was purchased from Al-
pha Diagnostic International (San Antonio, TX).
Cell Culture Experiments
Neuronal cultures were prepared from 18-day-old
Sprauge-Dawley rat fetuses (Yatin et al., 1999b). A? peptides
were dissolved in sterile water that had been stirred over
Chelex-100 resin. The peptides were preincubated for 24 hr at
37°C prior to addition to cultures. The final concentration of
the peptides in the cell culture was 10 ?M, and the effects of A?
on the neurons were measured after 24 hr of exposure. GCEE
was added to cell cultures 1 hr prior to addition of A? peptide.
Mitochondrial function was evaluated by the 3-[4,5-
dimethylthiazol-2-yl)-2,5-diphenyl] tetrazolium bromide (MTT)
reduction assay. Briefly, MTT was added to each well at a final
concentration of 1.0 mg/ml and then incubated for 1 hr. The
dark blue formazan crystals formed in intact cells were extracted
with 250 ?l dimethyl sulfoxide (DMSO), and the absorbance
was read at 595 nm with a microtiter plate reader (Bio-Tek
Instruments, Winooski, VT).
Endogenous GSH was measured with a Glutathione Assay
Kit (Cayman Chemical, Ann Arbor, MI). Briefly, cells were
deproteinated by treatment with 10% (w/v) metaphosphoric
acid (Aldrich, Milwakee, WI) and centrifuged at 2,000g for
2 min. The supernatant was collected and treated with 4 M
triethanolamine. Standards of oxidized GSH (GSSG) were pre-
pared varying from 0 to 8.0 ?M. Fifty microliters of standard
was aliquoted per well to establish a standard curve ranging from
0 to 16.0 ?M GSH. Fifty microliters of sample was added per
well. One hundred fifty microliters of Assay Cocktail [consisting
of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, cofactor
mixture (NADP?and glucose-6-phosphate), enzyme mixture
(GSH reductase, and glucose-6-phosphate dehydrogenase] and
5,5?-dithiobis-2-nitrobenzoic acid (DTNB) were added to each
well, and the absorbance was followed at 405 nm for 30 min. All
measurements were made in triplicate. The average absorbance
at 25 min was calculated for each standard and sample. A plot of
the corrected absorbance vs. the concentration of the GSH
standards (in ?M) was utilized to calculate the average concen-
tration of GSH present in the samples.
Samples (5 ?l) were incubated for 20 min at room tem-
perature with 5 ?l of 12% sodium dodecyl sulfate (SDS) and
10 ?l of 2,4-dinitrophenylhydrazine (DNPH) that was diluted
10 times with water from a 200 mM stock. The samples were
neutralized with 7.5 ?l of neutralization solution (2 M Tris in
30% glycerol). The resulting sample (250 ng) was loaded per
well in the slot-blot apparatus. Samples were loaded onto a
nitrocellulose membrane under vacuum pressure. The mem-
brane was blocked with 3% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS) containing 0.01% (w/v) so-
dium azide and 0.2% (v/v) Tween 20 (wash blot) for 1 hr and
incubated with a 1:100 dilution of anti-DNP polyclonal anti-
body in wash blot for 1 hr. After completion of the primary
antibody incubation, the membranes were washed three times in
wash blot for 5 min each. An anti-rabbit IgG alkaline phospha-
tase secondary antibody was diluted 1:8,000 in wash blot and
added to the membrane for 1 hr. The membrane was washed in
wash blot three times for 5 min each and developed using
Sigmafast Tablets (BCIP/NBT substrate). Blots were dried,
scanned with Adobe Photoshop (San Jose, CA), and quantitated
with Scion Image.
Electron microscopy was used to assess the ability of the
A? peptides to form fibrils upon incubation in solution for
24 hr. Aliquots of 5 ?l of the peptide solutions that were used
for the cell culture experiments were placed on a copper mesh
formvar carbon-coated grid. After 1–1.5 min of incubation at
room temperature, excess liquid was drawn off, and samples
were counterstained with 2% uranyl acetate. Air-dried samples
were examined in a Philips Tecnai Biotwin 12 transmission
electron microscope (FEI, Eindhoven, The Netherlands) at
80 kV. Images were captured with a 2K ? 2K digital camera
(Advanced Microscopy Techniques).
Analysis of DNA Fragmentation
Cell death was measured by Hoescht 332584 (1 ?g/ml)
followed by propidium iodide (PI; 5 ?g/ml) staining and de-
tected by fluorescence microscopy (Darzynkiewicz et al., 1994).
Cortical neuronal cells were treated with A? (10 ?M) alone for
24 hr or pretreated with GCEE (for 1 hr) followed by A?
(10 ?M) and incubated for 24 hr. Cultures were rinsed three
times in PBS, fixed with 4% paraformaldehyde for 10 min at
37°C, rinsed, and stained with Hoechst 332584 or PI for 10 min
GCEE Protects Against A?(1–42) Oxidative Stress701
at room temperature. Images were obtained sequentially with
Hoechst 332584, then PI. Random areas (approximately seven)
in the cell culture dishes were selected, and the number of
apoptotic cells was counted in neurons from untreated control,
treated with A?(1–42) alone, or pretreated with GCEE fol-
lowed by A? (10 ?M) groups. The average of the apoptotic cells
in each of the respective groups was determined as a percentage
of the untreated control.
ANOVA followed by Student’s t-test was used to deter-
mine statistical significance. P ? 0.05 was considered significant.
GCEE Inhibited A?(1–42)-Induced Protein
Oxidation in a Concentration-Dependent Manner
A 60% increase in protein oxidation in neurons was
induced following 24 hr of treatment with A?(1–42)
(Fig. 1). Pretreatment of neurons with GCEE was shown
tration-dependent manner. Protein oxidation was signifi-
cantly decreased by 1 hr of pretreatment of neuronal
cultures with 750 ?M GCEE; however, protein carbonyl
values reached control levels following pretreatment with
1 mM GCEE for 1 hr prior to the addition of A?(1–42).
Fig. 1. Protein oxidation as indexed by protein carbonyls. Relative to
untreated control, increased protein carbonyls were observed for 24 hr
of treatment of neuronal cell cultures with 10 ?M A?(1–42). Lower
levels of protein carbonyls were observed with 10 ?M A?(1–42) and
pretreatment with 500 ?M GCEE or 10 ?M A?(1–42) and pretreat-
ment with 750 ?M GCEE. Complete inhibition of A?(1–42)-induced
protein oxidation was found with treatment of cultures with 1 mM
GCEE 1 hr prior to addition of 10 ?M A?(1–42). Results are given as
the mean ? SEM of four independent treatments. *P ? 0.003, **P ?
0.001; n ? 4. The statistical comparison was performed between
untreated control and A? treatment sets.
Fig. 2. Neurotoxicity A?(1–42) and protection by GCEE as measured
by MTT reduction. The results are shown as mean ? SEM of three
independent measurements. Relative to untreated neuons, treatment of
neurons with 10 ?M A?(1–42) showed a significant decrease in MTT
reduction. Likewise, 1 hr of pretreatment of cell cultures with 500 ?M
or 750 ?M GCEE resulted in a significant decrease in A?(1–42)-
mediated MTT reduction. Such changes were not observed for 1 hr of
pretreatment with 1 mM GCEE, followed by 10 ?M A?(1–42). *P ?
0.05, **P ? 0.0001; n ? 3. The statistical comparison was performed
between control and A? treatment data sets.
Fig. 3. GSH assay. Cell cultures were pretreated with 500 ?M buthi-
onine sulfoximine (BSO) for 6 hr to inhibit ?-glutamylcysteine syn-
thetase, and 1 mM GCEE was then added. Cells were collected after
24 hr of treatment with GCEE and assayed for total GSH. The results
are shown as mean ? SEM of four independent measurements. Treat-
ment of neurons with BSO led to a significant loss of GSH. No
significant difference was noted in GSH levels between control,
GCEE-, and BSO- plus GCEE-treated neurons. *P ? 0.0005; n ? 4.
The statistical comparison was performed between control and each
treatment data set.
702 Boyd-Kimball et al.
This 1 mM level is well within the level of GSH in the
brain (Cooper, 1997).
GCEE Protects Mitochondria From A?(1–42)-
Mediated Oxidative Stress in a Concentration-
A?(1–42) was shown to decrease MTT reduction by
45% compared with untreated control (Fig. 2). Pretreat-
ment of neurons with 500 ?M GCEE for 1 hr followed by
A?(1–42) did not show any change in MTT reduction
compared with A?(1–42) alone. Pretreatment of neurons
with 750 ?M GCEE and A?(1–42) did show some pro-
tection of mitochondrial function compared with that
induced by A?(1–42) alone but still showed a significant
decrease in MTT reduction compared with untreated
control. However, addition of 1 mM GCEE 1 hr prior to
the addition of A?(1–42) showed no significant loss in
MTT reduction compared with control, which is consis-
tent with the concentration of GCEE that protected neu-
rons against A?(1–42)-mediated protein oxidation (Fig.
1). Because 1 mM GCEE protected neurons against pro-
tein oxidation and prevented loss of mitochondrial func-
tion, this concentration was used for all subsequent exper-
GCEE Increases Endogenous GSH Levels
To determine whether GCEE protected neuons by
acting as a GSH mimetic or by serving as a substrate for
GSH synthase for the up-regulation of GSH, an assay of
total GSH was conducted (Fig. 3). Treatment of cells with
1 mM GCEE led to a mean 20% increase in GSH com-
pared with control, but this increase was not statistically
different from the GSH level of untreated controls. Inhi-
bition of ?-glutamylcysteine synthetase by 500 ?M bu-
thionine sulfoximine (BSO) led to a significant reduction
in cellular GSH levels (2% of control); however, inhibition
of ?-glutamylcysteine synthetase followed by addition of
1 mM GCEE raised total GSH levels to 88% of control
and was not found to vary significantly from control. That
is, GCEE apparently led to elevated GSH levels by having
the deesterified dipeptide serve as a substrate for GSH
synthase, in that de novo synthesis of GSH via
?-glutamylcysteine synthetase was not possible in the pres-
ence of BSO.
GCEE Protects Against A?(1–42) Loss of
Neuronal Network and Neuronal Death
Phase-contrast microscopy was used to examine
the morphological changes in the neurons following
treatment (Fig. 4). Neurons that were exposed to
10 ?M A?(1–42) for 24 hr (Fig. 4B) showed loss of
neuronal network and vacuoles in the perikarya, indi-
cating dying cells. Conversely, neurons pretreated with
1 mM GCEE 1 hr prior to addition of A?(1–42) (Fig.
4C) showed intact networks and cell bodies similar to
those of control neurons (Fig. 4A). The apoptotic (later
stage) and necrotic cells are positive for Hoechst stain-
ing and PI staining, whereas early apoptotic stages are
negative for PI staining. We used Hoechst staining and
PI for DNA fragmentation to distinguish between late-
stage apoptosis and necrosis vs. early-stage apoptosis
(Fig. 5). Neurons treated with A?(1–42) (Fig. 5B)
showed extensive DNA fragmentation by both stains,
from which we conclude that late apoptotic and ne-
crotic cells are found under the conditions of this ex-
periment. In contrast, pretreatment of neurons with
1 mM GCEE followed by addition of A?(1–42) (Fig.
5C) resulted in a significant reduction in DNA frag-
mentation and apoptotic cells similar to those in un-
treated control cells (Fig. 5A). The averages of late
apoptotic cells and necrotic cells were calculated and are
reflected in the bar graph (Fig. 5D). The results suggest
that there is significant (*P ? 0.05) DNA fragmentation
as a measure of cells undergoing late-stage apoptosis and
necrosis in the cells treated with A? (10 ?M).
GCEE Does Not Interfere With A?(1–42) Fibrils
To determine whether GCEE interacted with
A?(1–42) in such a way as to disturb the fibril morphol-
Fig. 4. Phase-contrast provides morphological insight. A: Control cells show extensive intact neu-
ronal network and cell bodies. B: Neuronal culture treated with 10 ?M A?(1–42) shows atrophied
neurons and loss of neuronal connections. C: Neuronal culture treated with 1 mM GCEE 1 hr prior
to treatment with 10 ?M A?(1–42) shows intact neuronal connections.
GCEE Protects Against A?(1–42) Oxidative Stress703
ogy of the peptide, EM studies were conducted (Fig. 6).
No significant difference was observed in the fibril mor-
phology between A?(1–42) (Fig. 6A) and A?(1–42) in
the presence of 1 mM GCEE (Fig. 6B).
Oxidative stress can lead to a variety of cellular
consequences, including altered protein conformation,
loss of protein function, increased protein aggregation,
Fig. 5. Hoechst and propidium iodide staining for DNA fragmenta-
tion. Control neurons (A) and neurons treated for 1 hr with 1 mM
GCEE followed by 10 ?M A?(1–42) (C) show little evidence of
apoptotic neurons. Conversely, 10 ?M A?(1–42) treatment of neurons
shows evidence of late-stage apoptosis and necrosis (arrows; B). See
text. D: The averages of late-stage apoptotic cells and necrotic cells
were calculated and are reflected in this bar graph. The results presented
are the mean ? SEM expressed as percentage of control values. Each
experiment was repeated three times with three independent samples.
*P ? 0.05 compared with the untreated control.
704 Boyd-Kimball et al.
decreased protein turnover, altered cellular redox poten-
tial, altered Ca2?homeostasis, and ultimately cell death.
A?(1–42) has been shown to induce oxidative stress and
neurotoxicity in vitro in a manner inhibited by the chain-
breaking antioxidant vitamin E, suggesting that ROS play
a pivotal role (Yatin et al., 1999a, 2000; Behl, 1999).
A?(1–42) has been shown to deplete GSH levels in as-
trocytes, leading to a significant loss of neurons in vitro
(Abramov et al., 2003). A?(1–42) has been implicated as
a causative agent in AD (Selkoe, 2001a), which is associ-
ated with oxidative stress and aging (Butterfield and Laud-
erback, 2002). GSH levels have been shown to decrease
with aging, leaving neurons vulnerable to ROS attack and
subsequent damage (Lui and Choi, 2000). Additionally,
exogenous GSH has been shown to prevent A?(1–42)-
induced apoptosis in human cortical neurons (Medina et
al., 2002). In this study, we tested the hypothesis that
up-regulation of GSH by GCEE would protect primary
neuronal cell cultures against A?(1–42)-mediated oxida-
tive stress and neurotoxicity.
Treatment of neurons with 1 mM GCEE was shown
to elevate GSH levels 24 hr after administration, suggest-
ing that GCEE was able to cross the plasma membrane of
the neuron and provide a substrate for the synthesis of
GSH. Moreover, BSO was used to inhibit ?-gluta-
mylcysteine synthetase, which prevented de novo synthe-
sis of GSH and significantly depleted the cellular GSH
level. Upon addition of GCEE to BSO-treated neurons,
GSH levels returned to control, which is consistent with
the notion that ?-glutamylcysteine was a substrate for
glutathione synthase, an enzyme that catalyzes the addition
of C-terminal glycine to this dipeptide to form GSH.
Additionally, pretreatment of neurons with GCEE pro-
tected these cells against A?(1–42)-mediated loss of mi-
tochondrial function, protein oxidation, loss of neuronal
network, and apoptotic cells. Taken together, these results
suggest GCEE induces up-regulation of GSH, which, in
turn, protects neurons against protein oxidation and neu-
GCEE was unable to protect neurons against lipid
peroxidation (data not shown). This finding is particularly
important for two reasons. First, GSH is located in the
cytosol and is not lipid soluble. Consequently, GSH would
not play a primary role in protection of the lipid bilayer
from free radical attack. Second, and more importantly,
A?(1–42) may exert its cytotoxic effect by inserting into
the lipid bilayer as small, soluble aggregates, where it
initiates a lipid peroxidation cascade, followed by protein
oxidation (Varadarajan et al., 2000; Butterfield, 2002; But-
terfield and Lauderback, 2002). GCEE-mediated up-
regulation of GSH does not interfere with A?(1–42)
aggregation (Fig. 6) or insertion into the lipid bilayer,
because lipid peroxidation was still present (data not
shown). However, GCEE up-regulation of GSH was able
to protect the cell from protein oxidation. This is partic-
ularly important in that the index of lipid peroxidation
used was 4-hydroxy-2-trans-nonenal (HNE). HNE is an
?,?-unsaturated alkenal and a strong electrophilic product
of lipid peroxidation that can readily react by Michael
addition with cysteine, lysine, and histidine residues in
proteins to introduce a carbonyl functionality (Esterbauer
et al., 1991; Butterfield and Stadtman, 1997; Mark et al.,
1997; Uchida, 2003). As noted, in this study, the index of
protein oxidation was protein carbonyls. Protein carbony-
lation in neurons pretreated with GCEE followed by
A?(1–42) was not found to be significantly different from
that in control. These results suggest that, although GCEE
up-regulation of GSH was unable to prevent lipid peroxi-
dation, it was able to protect against damage mediated by
the lipid peroxidation product HNE. GSH has been
shown to react directly with electrophiles, such as HNE,
or indirectly in a reaction catalyzed by glutathione
S-transferase (Esterbauer et al., 1991; Xie et al., 1998).
Moreover, HNE can diffuse from its site of origin to react
with cytosolic proteins (Butterfield and Stadtman, 1997).
In summary, we have shown that GCEE can cross
the plasma membrane of neurons in culture and increase
cellular GSH levels. Moveover, this increase in GSH levels
Fig. 6. EM studies of fibril morphology. The extensive fibril formation of A?(1–42) (A) is unaffected
in the presence of 1 mM GCEE (B).
GCEE Protects Against A?(1–42) Oxidative Stress705
can protect neurons from alterations in mitochondrial
function, increased protein oxidation, loss of neuronal
network, and apoptosis induced by A?(1–42). This find-
ing is of importance in that A?(1–42) may play a central
role in the pathogenesis of AD, and GSH is an vital
endogenous antioxidant found in millimolar concentra-
tions in the brain (Cooper, 1997), although GSH levels
decrease with age (Liu and Choi, 2000) . Thus, agents such
as GCEE, which may react directly with ROS or by
increasing the availability of GSH in the brain, may pro-
vide therapeutic benefit in oxidative stress-associated neu-
rodegenerative diseases such as AD (Butterfield et al.,
This work was supported in part by NIH grants
AG-05119 and AG-10836 to D.A.B.
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