?-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.
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.
Abramov AY, Canevari L, Duchen MR. 2003. Changes in intracellular
calcium and glutathione in astrocytes as the primary mechanism of amy-
loid neurotoxicity. J Neurosci 23:5088–5095.
Anderson ME, Lou JL. 1998. Glutathione therapy: from prodrugs to genes.
Semin Liver Dis 18:415–424.
Anderson ME, Meister A. 1989. Glutathione monoesters. Anal Biochem
Anderson ME, Powrie F, Puri RN, Meister A. 1985. Glutathione mono-
ethyl ester: preparation, uptake by tissues, and conversion to glutathione.
Arch Biochem Biophys 239:538–548.
Behl C. 1999. Vitamin E and other antioxidants in neuroprotection. Int J
Vit Nutr Res 69:213–219.
Butterfield DA. 2002. Amyloid beta-peptide (1–42)-induced oxidative
stress and neurotoxicity: implications for neurodegeneration in Alzhei-
mer’s disease brain. Free Rad Res 36:1307–1313.
Butterfield DA. 2003. Amyloid beta-peptide [1–42]-associated free radical-
induced oxidative stress and neurodegeneration in Alzheimer’s disease
brain: mechanisms and consequences. Curr Med Chem 10:2651–2659.
Butterfield DA, Lauderback CM. 2002. Lipid peroxidation and protein
oxidation in Alzheimer’s disease brain: potential causes and consequences
involving amyloid beta-peptide-associated free radical oxidative stress.
Free Rad Biol Med 32:1050–1060.
Butterfield DA, Stadtman ER. 1997. Protein oxidation processes in aging
brain. Adv Cell Aging Gerontol 2:161–191.
Butterfield DA, Drake J, Pocernich C, Castegna A. 2001. Evidence of
oxidative damage in Alzheimer’s disease brain: central role for amyloid
beta-peptide. Trends Mol Med 7:548–554.
Butterfield DA, Castegna A, Lauderback CM, Drake J. 2002a. Evidence
that amyloid beta-peptide-induced lipid peroxidation and its sequelae in
Alzheimer’s disease brain contribute to neuronal death. Neurobiol Aging
Butterfield DA, Pocernich CB, Drake J. 2002b. Elevated glutathione as a
therapeutic strategy in Alzheimer’s disease. Drug Disc Res 56:328–437.
Cooper AJL. 1997. Glutathione in the brain: disorder of glutathione me-
tabolism. In: The molecular and genetic basis of neurological disease.
Newton, MA: Butterworth-Heinemann. p 1195–1230.
Darzynkiewicz Z, Li X, Gong J. 1994. Assays of cell viability: discrimina-
tion of cells dying by apoptosis. Methods Cell Biol 41:15–38.
Drake J, Kanski J, Varadarajan S, Tsoras M, Butterfield DA. 2002. Elevation
of brain glutathione by ?-glutamylcysteine ethyl ester protects against
peroxynitrite-induced oxidative stress. J Neurosci Res 68:776–784.
Drake J, Link CD, Butterfield DA. 2003a. Oxidative Stress precedes fibrillar
deposition of Alzheimer’s disease amyloid ?-peptide (1–42) in a trans-
genic Caenorhabditis elegans model. Neurobiol Aging 24:415-420.
Drake J, Sultana R, Aksenova M, Calabrese V, Butterfield DA. 2003b.
Elevation of mitochondrial glutathione by gamma-glutamylcysteine ethyl
ester protects mitochondria against peroxynitrite-induced oxidative stress.
J Neurosci Res 74:917–927.
Esterbauer H, Schaur RJ, Zollner H. 1991. Chemistry and biochemistry of
4-hydroxynonenal, malonaldehyde and related aldehydes. Free Rad Biol
Hensley K, Hall N, Subramaniam R, Cole P, Harris M, Aksenov M,
Aksenova M, Gabbita P, Wu JF, Carney JM, Lovell M, Markesbery WR,
Butterfield DA. 1995. Brain regional correspondence between Alzhei-
mer’s disease histopathology and biomarkers of protein oxidation. J Neu-
Liu R, Choi J. 2000. Age-associated decline in ?-glutamylcysteine syn-
thetase gene expression in rats. Free Rad Biol Med 28:566–574.
Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. 1997. A
role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in
disruption of ion homeostasis and neuronal death induced by amyloid
beta-peptide. J Neurochem 68:255–264.
Markesbery MR. 1997. Oxidative stress hypothesis in Alzheimer’s disease.
Free Rad Biol Med 23:134–147.
Medina S, Martinez M, Hernanz A. 2002. Antioxidants inhibit the human
cortical neuron apoptosis induced by hydrogen peroxide, tumor necrosis
factor alpha, dopamine, and beta-amyloid peptide 1–42. Free Rad Res
Selkoe DJ. 2001. Alzheimer’s disease results from the cerebral accumulation
and cytotoxicity of amyloid beta-protein. J Alzheimers Dis 3:75–80.
Subbarao KV, Richardson JS, Ang LC. 1990. Autopsy samples of Alzhei-
mer’s cortex show increased peroxidation in vitro. J Neurochem 55:342–
Uchida K. 2003. 4-Hydroxynonenal: a product and mediator of oxidative
stress. Prog Lipid Res 42:318–343.
Varadarajan S, Yatin S, Aksenova M, Butterfield DA. 2000. Review:
Alzheimer’s amyloid beta-peptide-associated free radical oxidative stress
and neurotoxicity. J Struct Biol 130:184–208.
Xie C, Lovell MA, Markesberry WR. 1998. Glutathione transferase pro-
tects neuronal cultures against four hydroxynonenal toxicity. Free Rad
Biol Med 25:979–988.
Yatin SM, Varadarajan S, Link CD, Butterfield DA. 1999a. In vitro and in
vivo oxidative stress associated with Alzheimer’s amyloid ?-peptide (1–
42). Neurobiol Aging 20:325–330.
Yatin SM, Yatin M, Aulick T, Ain KB, Butterfield DA. 1999b. Alzheimer’s
amyloid ?-peptide associated free radicals increase rat embryonic neuronal
polyamine uptake and ornithine decarboxylase activity: protective effect
of vitamin E. Neurosci Lett 263:17–20.
Yatin SM, Varadarajan S, Butterfield DA. 2000. Vitamin E prevents Alz-
heimer’s amyloid beta-peptide (1–42)-induced neuronal protein oxida-
tion and reactive oxygen species production. J Alzheimers Dis 2:123–131.
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