Journal of Alzheimer’s Disease 2 (2000) 123–131 123
Vitamin E Prevents Alzheimer’s Amyloid β-Peptide
(1-42)-Induced Neuronal Protein Oxidation and
Reactive Oxygen Species Production
Servet M. Yatina,c, Sridhar Varadarajan1 and oxidant vitamin E. To test the hypothesis that vitamin
D. Allan Butterfielda,b,* E’s protective effect may be due to inhibition of fibril
formation, electron microscopy studies were under-
taken. Vitamin E does not inhibit Aβ(1-42) fibril for-
Department of Chemistry and Center of mation, suggesting that the neuroprotection afforded
Membrane Sciences, University of Kentucky, by this molecule stems from other processes, most
Lexington, USA probably through the scavenging of Aβ-associated
Sanders-Brown Center on Aging, Univer- free radicals. These results may have implications on
the treatment of Alzheimer’s disease.
sity of Kentucky, Lexington, USA
Current address: ERPRC/Neurochemistry,
Harvard Medical School, Southborough, ABBREVIATIONS: Aβ, amyloid β-peptide; AD,
USA Alzheimer’s disease; APP, amyloid precursor protein;
DCF-DA, dicholorofluorescin diacetate; DNPH, 2,4-
dinitrophenylhydrazine; PBN, phenyl-α-tertbutyl-
Communicated by Mark S. Kindy nitrone; ROS, reactive oxygen species; SP, senile
ABSTRACT: Amyloid β-peptide (Aβ) is a 42-43
amino acid peptide known to accumulate in Alz-
heimer’s disease (AD) brain. We previously reported
that the neurotoxicity caused by Aβ is a result of its INTRODUCTION
associated free radicals, which can play an important
role in generating oxidative stress. Aβ(25-35)- Alzheimer’s disease (AD) is the most com-
associated oxidative stress-induced neuronal death in mon form of senile dementia, affecting more
vitro is well established by many laboratories, includ-
than 4 million people in the US alone. There are
ing ours. However, the oxidative stress-induced by
the full-length [Aβ(1-42)] peptide is not well investi-
360,000 new cases of AD annually in the United
gated. The protective effect of antioxidant vitamin E States (22). Unfortunately, there is yet no ef-
in full-length peptide-induced oxidative stress also has fective therapy available for this deadly disease.
not been reported. Here, we report that the increased The only two approved drugs available for AD
protein oxidation, reactive oxygen species (ROS) for- patients, both targeting the improvement of cog-
mation, and neurotoxicity induced by Aβ(1-42) in nitive function with modest effect, are cholin-
primary rat embryonic hippocampal neuronal culture esterase inhibitors. However, AD pathogenesis,
are prevented by the free radical scavenger and anti- although not very well known, involves more
than a deficiency of neurotransmitters. The
* Corresponding author: D. Allan Butterfield, De- presence of neurofibrillary tangles (32), extra-
partment of Chemistry, Center of Membrane Sciences cellular deposits of insoluble amyloid that is the
and Sanders-Brown Center on Aging, 121 Chemistry- main constituent of the senile plaques (SP) (36),
Physics Bldg., University of Kentucky, Lexington, and neuronal and synapse loss (11,12) are the
Kentucky 40506-0055, USA, Tel.: +1 606 257 3184, most pronounced pathological hallmarks of the
Fax: +1 606 257 5876, E-mail: email@example.com
1387-2877/00/$8.00 © 2000 – IOS Press. All rights reserved
124 S.M. Yatin et al. / Vitamin E
disease. Amyloid β-peptide (Aβ) is a 39-43 stress. In the present study we investigated the
amino acid peptide derived from the proteolytic role of the antioxidant and the free radical scav-
processing of β protein precursor (βPP). Evi- enger vitamin E in protection against Aβ(1-42)-
dence supporting an essential role of Aβ in AD associated free radical-induced protein oxidation,
includes: mutations in presenilin 1 and presenilin reactive oxygen species formation (ROS), and
2 or βPP, that lead to early onset AD, are associ- cell death in primary rat embryonic hippocampal
ated with excess Aβ deposition and oxidative neuronal culture. In addition, the effect of vita-
stress (33,37); Down’s syndrome patients, who min E on Aβ (1-42) fibril formation has been in-
carry three copies of chromosome 21, that en- vestigated.
codes βPP, show AD pathology; transgenic ani-
mals overexpressing mutant βPP show increased
Aβ content in the brain and also exhibit oxidative MATERIALS AND METHODS
stress (reviewed in (4)). AD brain is under ex-
tensive oxidative stress manifested by lipid per- Materials and Experimental Treatment of
oxidation, increased protein oxidation in the SP Cultures
rich region, hippocampus and frontal cortex,
compared to SP poor region, cerebellum, in AD Synthetic Aβ(1-42) (AnaSpec, San Jose, CA,
brain (38,19), DNA oxidation, mitochondrial Lot # 5811) was dissolved in double-distilled
dysfunction and widespread peroxynitrite dam- water immediately before use at a concentration
age (reviewed in (27,8)). of 1 mg/ml. This stock solution was incubated
Involvement of oxidative stress in the mecha- for 24 h for the formation of Aβ aggregates and
nism of Aβ-induced neurotoxicity is supported by fibrils (confirmed by electron microscopy, see
reports from several laboratories (2-4,7,8,16,18, below), then added to cultures to produce a final
28,35,40,44-47). An Aβ-associated free radical concentration of 10 µM. Pretreatment with
oxidative stress model for neuronal death in AD 50 µM vitamin E (pure α-tocopherol, Sigma) and
brain was proposed by our laboratory (7,8). In treatment with Aβ(1-42) were performed in
this model, Aβ inserts into the plasma membrane growth medium.
of neuronal or glial cells and oxygen-dependent,
reactive free radicals are formed. Those free Neuronal Culture
radicals can attack the neuronal plasma mem-
brane, induce Ca2+ influx, disturb cell membrane Hippocampal neuronal cultures were prepared
functions, increase protein oxidation, and induce from SD E18 as described previously (44,45).
lipid peroxidation (reviewed in (8)). In agree- Briefly, rat hippocampi were dissected and incu-
ment with our hypothesis Aβ(1-42) is reported to bated for 15 min in a solution of 2 mg/mL trypsin
reduce Cu2+ to Cu+, i.e., an electron is derived in Ca2+- and Mg2+- free Hanks’ balanced salt so-
from the peptide to reduce the metal ion forming lution (HBSS) buffered with 10 mM HEPES
a peptide radical (20). Aβ neurotoxicity is re- (Gibco). The tissue was then exposed for 2 min
ported to be attenuated by a number of antioxi- to soybean trypsin inhibitor (1 mg/mL in HBSS)
dants and free radical scavengers, such as phenyl- and rinsed 3 times in HBSS. Cells were dissoci-
α-tert-butylnitrone (PBN), EUK-8, estrogen, vi- ated by trituration and plated at a density of 75-
tamin E and, idebenone (2-4,15,39,42,45,46). 100 cells/mm2. At the time of plating, the culture
The majority of these studies were performed dishes contained 2 mL of Eagle’s minimum es-
using either Aβ(25-35) or Aβ(1-40), yet, Aβ(1- sential medium (MEM;GIBCO) that also con-
42) is the major form of Aβ deposited early in tained (final concentrations) 100 mL/L fetal bo-
AD brain (48). Thus it would be important to vine serum (Sigma), 1 mM L-glutamine, 20 mM
establish whether Aβ(1-42) induces oxidative KCl, 1 mM pyruvate, and 40 mM glucose. After
stress. If so, a chain-breaking antioxidant such as a 5 h period to allow cell attachment, the original
vitamin E would be predicted to ameliorate this medium was removed and replaced with 1.6 mL
S.M. Yatin et al. / Vitamin E 125
of fresh medium of the same composition. After with the membrane for 1 hour at 37 ºC. Mem-
a 24 h period, 1.2 mL MEM was replaced with branes were washed after every step in washing
1.0 mL of Neurobasal medium (Gibco) contain- buffer (PBS with 0.01% sodium azide and 0.2%
ing (final concentrations) 2% v/v B-27 or N-2 Tween 20) for 10 min at room temperature.
(Gibco) depending on the experiment, 2 mM L- Washed membranes were developed using
glutamine (Gibco), 0.5% w/v D-(+) glucose. On BCIP-NBT solution (SigmaFast tablets, Sigma).
the fifth day two-thirds of the Neurobasal me- Western blots were analyzed using computer-
dium was replaced with freshly prepared Neuro- assisted imaging (MCID/M4 software supplied
basal medium of the same composition. Cultures by Imaging Research (St. Catharines, Ontario,
were maintained at 37 ºC in a 5% CO2/95% room Canada)).
air-humidified incubator at all times. Cultures
were aged between 9–11 days before use in the ROS Measurement
experiments described. In B27/Neurobasal, glial
growth is reported to be less than 0.5% of the Intracellular reactive oxygen species were
nearly pure neuronal population (5). detected by the dicholorofluorescin diacetate
(DCF-DA) assay as described previously
Protein Carbonyl Measurement (44,45). Briefly, cells were loaded with DCFH-
DA (Molecular Probes, Inc.) by incubating in
To determine the level of protein oxidation an the non-CO2 incubator for 50 min, then were
Oxidized Protein Detection Kit (Oxyblot, washed three times with warm MSF buffer.
ONCOR Cat # S7150-Kit) was used as de- Fluorescence visualization was performed using
scribed previously (44,45). This kit is based on a confocal laser scanning microscope (Molecu-
immunochemical detection of protein carbonyl lar Dynamics, Sarastro 2000) coupled to an in-
groups derivatized with 2,4-dinitrophenyl- verted microscope (Nikon). Fluorescence was
hydrazine (DNPH) (23). excited at 488 nm and emission filtered using a
Briefly, the samples were treated with 20 mM 510 nm barrier filter. Cells scanned were cho-
2,4-dinitrophenylhydrazine (DNPH) in 10% tri- sen randomly.
fluoroacetic acid and derivatization-control so-
lution and incubated for 15–30 min. After de- Cell Toxicity Studies
rivatization and neutralization with 2M tris/30%
glycerol and 19% 2-mercaptoethanol, cell pro- Cell death was estimated by counting the
teins were separated by SDS-PAGE. Following number of neurons that internalized Trypan blue
electrophoresis, proteins were transferred on dye. After 48 h of incubation with or without
nitrocellulose for further immunoblotting analy- vitamin E (50 µM final concentration) and Aβ
sis. Western blotting was performed according (1-42) (10 µM final concentration), cells were
to the procedure adapted from Glenney (14). rinsed three times with 1 mL PBS (pH, 7.4).
The transfer of proteins on nitrocellulose after Trypan blue (Sigma) (0.4%) was added to cells
SDS-PAGE was completed in two hours. The together with 300 µL PBS and incubated for 10
transfer buffer was Tris-Glycine pH 8.5 with min. Sixteen different microscopic areas were
20% methanol. After the transfer, membranes counted for Trypan blue uptake, which indexes
were blocked in 3% BSA (in PBS with sodium cell death. Data are given as percentages of cor-
azide 0.01% and Tween-20 0.2%) for 1 hour at responding vehicle-treated control values.
room temperature. Rabbit anti-DNP antibody
from ONCOR Oxyblot kit (1:150 working dilu- Aβ Fibril Formation Analysis
tion) was used as a primary antibody. Secon-
dary antibodies (anti-Rabbit IgG conjugated Fibril formation was assayed as previously de-
with alkaline phosphatase, Sigma) were diluted scribed (8). Briefly, aliquots of 5 µL of the pep-
in the blocking solution 1:15,000 and incubated tide solution incubated for 24 h were placed on a
126 S.M. Yatin et al. / Vitamin E
copper formvar carbon-coated grid. After 1–5 guishable between Aβ(1-42) and Aβ(1-42) in the
min of incubation at room temperature, excess presence of vitamin E (Fig. 3), suggesting that
liquid was drawn off, and samples were counter- vitamin E does not inhibit Aβ(1-42) fibril forma-
stained with 2% uranyl acetate. Air-dried sam- tion.
ples were examined in a Hitachi 7000 transmis-
sion electron microscope at 75 kV.
RESULTS There are several therapeutic approaches that
target different alterations seen in AD. These
A prediction of our model for neurotoxicity in include acetylcholinesterase inhibition to prevent
AD brain (8) is that Aβ(1-42)-induced neurotoxic- the loss of the neurotransmitter acetylcholine,
ity is a consequence of oxidative stress. If so, estrogen therapy, anti-inflammatory drugs, Aβ
Aβ(1-42) is predicted to lead to ROS formation aggregation inhibitors, and antioxidants. Most of
and to oxidation of neuronal proteins. Figure 1A these approaches are in development and still
represents a field of untreated neurons showing need extensive study in in vitro and in vivo sys-
low levels of ROS. Incubation of the neuronal tems before clinical trials begin. A recent report
culture with 10 µM of Aβ(1-42) for 48 h signifi- suggests that reduction in chain-breaking antioxi-
cantly increases ROS levels (Fig. 1B). Treatment dants in patients with dementia may reflect an
of the rat embryonic hippocampal neuronal culture increased free radical activity (13), supporting the
with 50 µM vitamin E 1 hour prior to 10 µM free radical hypothesis, for AD (8,27). We pre-
Aβ(1-42) administration prevented neurons from viously showed that the antioxidant vitamin E is
increased ROS formation (Fig. 1C). Quantifica- effective in preventing neuronal cells from
tion of the ROS data is shown Fig. 1D, which in- Aβ(25-35)-associated free radical-induced oxida-
dicates that Aβ(1-42) increases ROS formation tive stress in cell culture (45,46). Here we report
four-fold compared to control (*p < 0.001), and that Aβ(1-42)-associated free radicals increase
that Aβ(1-42)-induced ROS formation is signifi- neuronal ROS, protein oxidation and cell
cantly reduced by the free radical scavenger vita- death. Each detrimental effect is inhibited by the
min E [**p < 0.005 vs. Aβ(1-42)] (Fig. 1D). free radical scavenger vitamin E, supporting the
Protein carbonyl levels, a measure of protein free radical oxidative stress hypothesis of Aβ
oxidation (9), in neuronal culture treated with with this full length peptide that many research-
Aβ(1-42) were found to be 168% those of controls ers agree may be central to the pathogenesis of
(*p < 0.001 vs. control, Fig. 2). This increased AD.
protein oxidation was suppressed to control levels Consistent with our results and hypothesis, a
by pretreatment of cultures with vitamin E. recent report demonstrated that AD patients with
Cell survival was assessed by the Trypan blue moderately severe impairment respond favorably
exclusion assay. Table 1 shows that 10 µM Aβ (1- to α-tocopherol, as evidenced by slowing of the
42) significantly increased neuronal death progression of the disease (34). Another study
(p < 0.05). Consistent with the hypothesis that this employing 633 persons over 65 years and older
Aβ (1-42)-induced cell death results from peptide- suggests that use of the high dose vitamin E and
induced oxidative stress, pretreatment of neuronal vitamin C supplements may lower the risk of AD
cultures with 50 µM vitamin E prevented cell (30). Recently, a study reported by Brusco et al.
death (Table I). (6) employing monozygotic twins with the diag-
One possibility for protection by vitamin E nosis of AD for 8 years showed that one of the
against Aβ (1-42)-induced cell death is that this twins treated with the antioxidant melatonin had
antioxidant prevents Aβ fibril formation. Elec- a milder impairment of memory function. These
tron microscopy was used to demonstrate that reports not only suggest the involvement of oxi-
fibrils are equally obvious and not readily distin- dative stress in AD indirectly but also point out
S.M. Yatin et al. / Vitamin E 127
DCF Fluo. (Ave. Pixel Intensity)
Control Aβ(1-42) Vit E+Aβ(1-42)
Fig. 1. Inhibition of ROS formation by vitamin E, detected by the conversion of 2’,7’- dichlorofluorescin to 2’,7’-
dichlorofluorescein. Color images were taken by using a fluorescence confocal microscope. (A) A field of control
neurons showing low levels of background fluorescence. Color bar shows the fluorescence color code; moving up in
the color code indicates increase in ROS. (B) Neurons treated with 10 µM Aβ(1-42) for 48 h showing increase in
ROS. (C) Neurons treated with 50 µM vitamin E 1 h prior to 10 µM Aβ(1-42) addition. (D) Quantification of the
ROS formation indicates that vitamin E significantly prevents Aβ(1-42)-induced ROS formation. Error bars repre-
sent SEM values. *p < 0.001 vs. control, **p < 0.005 vs. Aβ(1-42) (n=3; each n is the average of 8-11 neurons).
Percent Survival Relative to Controls Following Aβ(1-42) Addition to Hippocampal Neuronal
Cultures With or Without Added Vitamin E*
Aβ(1-42) Aβ(1-42) Plus Vit. E
MEAN +/- SEM 76.1 +/- 4.56 (n = 6) 99 +/- 4.0 (n = 2)
P-Value < 0.05 N.S.
*Each n represents four replicates.
128 S.M. Yatin et al. / Vitamin E
Fig.2. The relative changes of protein carbonyl content in rat embryonic hippocampal culture treated with 10 µM
Aβ(1-42) and vitamin E + Aβ(1-42) for 48 hours. Error bars represent SEM values. Significance at *p < 0.001
(n = 4).
Aβ(1-42) Aβ(1-42) + Vit E
Fig. 3. Fibril formation assessed by electron microscopy. Aβ (1-42) at 1 mg/mL was dissolved in 1.7 µL DMSO,
with or without vitamin E, all of which was dispersed in a final volume of 500 µL deionized water. The final vita-
min E concentration was 50 µM. The mixture was incubated for 48 h at 37°C and EM images obtained as de-
scribed in Methods.
S.M. Yatin et al. / Vitamin E 129
the potential beneficial effects of free radical other antioxidants modulate oxidative stress as-
scavengers and antioxidants in the treatment of sociated with Aβ.
AD. The results shown in the current study, ob-
Inhibition of fibril formation has led to abro- tained by using Aβ(1-42)-treated neuronal cul-
gation of Aβ-induced neurotoxicity (25), al- tures, one of the in vitro systems with which
though others have challenged the proposed re- potentially useful therapeutics for AD can be
quirement for fibrillization as a prerequisite for studied, suggest that oxidative stress plays a
neurotoxicity (10). Recent reports suggest that major role in Aβ(1-42)-induced cell death and
aggregated, soluble Aβ peptides, so-called proto- can be prevented by the free radical scavenger
fibrils, are toxic (41), as are mixtures of soluble vitamin E. The present study in an in vitro sys-
aggregated Aβ in the presence of certain proteins tem is consistent with the in vivo findings of
but in the absence of fibrils (1,31). In the current protein oxidation in AD brain (19,33), and in
study, vitamin E, although an inhibitor of Aβ(1- transgenic C. elegans expressing Aβ(1-42) (47).
42)-induced neurotoxicity, protein oxidation, and Additionally, this study shows that antioxidant
ROS formation, did not inhibit fibril formation. treatment ameliorates Aβ(1-42)-induced oxida-
This result suggests that the neuroprotective ac- tive stress, which may have relevance to AD
tion of vitamin E occurs through a different treatment.
mechanism, the most obvious of which presuma-
bly is its scavenging of Aβ-associated free radi-
Vitamin E previously had been reported to
block Aβ-induced lipid peroxidation (21,26) and This work was supported in part by grants
neurotoxicity (17,29,46). Here, it is shown that NIH (AG-10836; AG-05119; AG-12423). The
vitamin E blocked Aβ(1-42)-induced neuronal authors gratefully acknowledge Dr. Mark
protein oxidation, ROS formation, and cell death Mattson and Dr. Jeffrey Keller for the use of the
confirming previous findings with the shorter confocal laser scanning microscope.
fragment, Aβ(25-35) (39,45). Others showed
that vitamin E protects against Aβ(1-42)-induced
learning and memory deficits in rats (42). In REFERENCES
contrast to numerous reports of the effectiveness
of vitamin E in preventing oxidative damage in 1. Aksenov MY, Aksenova MV, Butterfield DA,
and death to neurons, some reports suggest that Hensley K, Vigo-Pelfrey C. Carney JM, Gluta-
vitamin E is not totally protective against Aβ (25- mine synthetase-induced neurotoxicity accom-
35) (24,43). In one study, the authors describe an panied by abrogation of fibril formation and
Aβ-induced lipid peroxidation and ROS forma- amyloid β-peptide fragmentation, J Neurochem
tion that was inhibited by vitamin E, but this an- 66 (1996) 2050–2056.
tioxidant did not prevent modification of mito- 2. Behl C, Davis J, Cole GM, Schubert D, Vitamin
chondrial proteins by a lipid peroxidation prod- E protects nerve cells from amyloid β protein
uct, 4-hydroxynonenal (43). In another study, no toxicity, Biochem Biophys Res Commun 186
antioxidant, among numerous used, was reported
3. Behl C, Davis J, Lesley R, Schubert D, Hydro-
to prevent Aβ-induced damage (24). The former
gen peroxide mediates amyloid β protein toxic-
report confirms some of the findings of numerous ity, Cell 77 (1994) 817–827.
laboratories, and the latter is in total disagree- 4. Behl C, Alzheimer’s disease and oxidative stress:
ment with a large preponderance of evidence implications for novel therapeutic approaches,
from numerous laboratories suggesting that Aβ Prog Neurobiol 57 (1999) 301–323.
is associated with oxidative stress (reviewed in 5. Brewer GJ, Torricelli JR, Evege EK, Price PJ,
(8)). It is our opinion that a fair summation of Optimized survival of hippocampal neurons in
the literature would suggest that vitamin E or B27-supplemented Neurobasal, a new serum-free
130 S.M. Yatin et al. / Vitamin E
medium combination, J Neurosci Res 35 (1993) 18. Harris M, Hensley K, Butterfield DA, Leedle RA,
567–576. Carney JM, Direct evidence of oxidative injury
6. Brusco LI, Marquez M, Cardinali DP, Monozy- produced by the Alzheimer’s Aβ(1-40) in cul-
gotic twins with Alzheimer’s disease treated with tured hippocampal neurons, Exp Neurol 131
melatonin: Case report, J Pineal Res 25 (1998) (1995) 193–202.
260–263. 19. Hensley K, Hall N, Subramaniam R, Cole GM,
7. Butterfield DA, Hensley K, Harris M, Mattson Harris M, Aksenov MY, Aksenova MV, Gabbita
MP, Carney JM, β-Amyloid peptide free radical P, Wu JF, Carney JM, Lovell MA, Markesbery
fragments initiate synaptosomal lipoperoxidation WR, Butterfield DA, Brain regional correspon-
in a sequence specific fashion: implications to dence between Alzheimer’s disease histopathol-
Alzheimer’s disease, Biochem Biophys Res ogy and biomarkers of protein oxidation, J Neu-
Commun 200 (1994) 710–715. rochem 65 (1995) 2146–2156.
8. Butterfield DA, β-Amyloid associated free radical 20. Huang X, Atwood CS, Hartshorn MA, Multhaup
oxidative stress and neurotoxicity: implications G, Goldstein LE, Scarpam RC, Cuajungcom MP,
for Alzheimer’s disease, Chem Res Toxicol 10 Graym DN, Lim J, Moir RD, Tanzi RE, Bush AI,
(1997) 495–506. The Aβ peptide of Alzheimer’s disease directly
9. Butterfield DA, Stadtman ER, Protein oxidation produces hydrogen peroxide through metal ion
processes in aging brain, Adv Cell Aging Geron- reduction, Biochemistry 38 (1999) 7609–7616.
tol 2 (1997) 161–191. 21. Koppal T, Subramaniam R, Drake J, Prasad MR,
10. Davis JN, Chisholm JC, The ‘amyloid cascade Butterfield DA, Vitamin E protects against amy-
hypothesis’ of AD: decoy or real McCoy?, Trends loid peptide (25-35)-induced changes in neocorti-
Neurosci 20 (1997) 154–159. cal synaptosomal membrane lipid structure and
11. Davies CA, Mann DMA, Sumpter PQ, Yates PO, composition, Brain Res 786 (1998) 270–273.
A quantitative morphometric analysis of the neu- 22. Kowall NW, Alzheimer disease 1999: A status
ronal and synaptic content of the frontal and tem- report, Alzheimer Dis Assoc Disord 13 (1999)
poral cortex in patients with AD, J. Neurol. Sci. Suppl 1:S11–16.
78 (1987) 151–164. 23. Levine RL, Williams JA, Stadtman ER, Shacter
12. DeKosky ST, Scheff SW, Synapse loss in frontal E, Carbonyl assays for determination of oxida-
cortex biopsies in AD: correlation with cognitive tively modified proteins, Methods Enzymol 233
severity, Ann Neurol 27 (1990) 457–464. (1994) 346–357.
13. Foy CJ, Passmore AP, Vahidassr MD, Young IS, 24. Lockhart BP, Benicourt C, Junien JL, Privat A,
Lawson JT, plasma chain-breaking antioxidants in Inhibitors of free radical formation fail to attenu-
Alzheimer’s disease, vascular dementia and ate direct beta- amyloid25-35 peptide-mediated
Parkinson’s disease, Q J Med 92 (1999) 39–45. neurotoxicity in rat hippocampal cultures, J Neu-
14. Glenney JR, Antibody probing on Western blots rosci Res 39 (1994) 494–505.
have been stained with India ink, Anal Biochem 25. Lorenzo, Yankner BA, Beta-amyloid neurotoxic-
156 (1986) 315–318. ity requires fibril formation and is inhibited by
15. Goodman Y, Mattson MP, Secreted forms of β- congo red, Proc Natl Acad Sci USA 6 (1994)
amyloid precursor protein protect hippocampal 12243–12247.
neurons against amyloid β-peptide toxicity and 26. Mark RJ, Fuson KS, May PC, Characterization of
oxidative injury, Exp Neurol 128 (1994) 175–182. 8-epiprostaglandin F2alpha as a marker of amy-
16. Gridley GR, Green PS, Simpkins JW, Low con- loid beta-peptide-induced oxidative damage, J
centrations of estradiol reduce β-amyloid (25-35)- Neurochem 72 (1999) 1146–1153.
induced toxicity, peroxidation and glucose utili- 27. Markesbery WR, Oxidative stress hypothesis in
zation in human SK-N-SH neuroblastoma, Brain Alzheimer’s disease, Free Radic Biol Med 23
Res 78 (1997) 158–165. (1997) 134–147.
17. Harkany T, Hortobagyi T, Sasvari M, Konya C, 28. Mattson MP, Barger SW, Cheng B, Lieberburg I,
Penke B, Luiten PG, Nyakas C, Neuroprotective Smith-Swintosky VL, Rydel RE, β-amyloid pre-
approaches in experimental models of beta- cursor protein metabolites and loss of neuronal
amyloid neurotoxicity: relevance to Alzheimer’s Ca2+ homeostasis Alzheimer’s disease, Trends
disease, Prog Neuropsychopharmacol Biol Psy- Neurosci 16 (1993) 409–414.
chiatry 23 (1999) 963–1008. 29. Mattson MP, Goodman Y, Different amyloido-
genic peptides share a similar mechanism of neu-
S.M. Yatin et al. / Vitamin E 131
rotoxicity involving reactive oxygen species and 40. Varadarajan S, Yatin S, Kanski J, Jahanshahi F,
calcium, Brain Res 676 (1995) 219–224. Butterfield DA, Methionine residue 35 is im-
30. Morris MC, Beckett LA, Scherr PA, Hebert LE, portant in amyloid β-peptide-associated free
Bennet DA, Field TS, Evans D, Vitamin E and radical oxidative stress, Brain Res Bull 50
vitamin C supplement use and risk of incident (1999) 133–141.
Alzheimer disease, Alzheimer Dis Assoc Disord 41. Walsh DM, Hartley DM, Kusumoto Y, Fezoui
12 (1998) 121–126. Y, Condron MM, Lomakin A, Benedek GB,
31. Oda T, Wals P, Osterbung H, Johnson S, Pasinetti Selkoe DJ, Teplow DB, Amyloid beta-protein
G, Morgan T, Rozovosky I, Stein WB, Synder S, fibrillogenesis. Structure and biological activity
Holzman T, Krafft G, Finch C, Clusterin (apo J) of protofibrillar intermediates, J Biol Chem 274
alters the aggregation of amyloid β peptide (Aβ 1- (1999) 25945–25952.
42) and forms slowly sedimenting Aβ complexes 42. Yamada K, Tanaka T, Han D, Senzaki K,
that cause oxidative stress, Exp Neurol 136 Kameyama T, Nabeshima T, Protective effects
(1995) 22–31. of idebenone and α-tocopherol on Aβ(1-42)-
32. Pearson RC, Esiri MM, Hiorns RW, Wilcock GK, induced learning and memory deficits in rats:
Powell TP, Anatomical correlates of the distribu- implication of oxidative stress in β-amyloid-
tion of the pathological changes in the neocortex induced neurotoxicity in vivo, Eur J Neurosci 11
in Alzheimer disease, Proc Natl Acad Sci USA 82 (1999) 83–90.
(1985) 4531–4534. 43. Yao ZX, Drieu K, Szweda LI, Papadopoulos V,
33. Perry G, Smith MA, Is oxidative damage central Free radicals and lipid peroxidation do not me-
to pathogenesis of AD?, Acta Neurol Belgium 98 diate beta-amyloid- induced neuronal cell death,
(1998) 175–179. Brain Res 847 (1999) 203–210
34. Sano M, Ernesto C, Thomas RG, Klauber MR, 44. Yatin SM, Aksenova MV, Aksenov MY, Mark-
Schafer K, Grundman M, Woodbury P, Growdon esbery WR, Aulick T, Butterfield DA, Temporal
J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ, relations among amyloid β-peptide-induced free-
A controlled trial of selegiline, α-tocopherol, or radical oxidative stress, neuronal toxicity and
both as treatment for Alzheimer’s disease, The neuronal defensive responses, J Molec Neurosci
New England J Medicine 336 (1997) 1216–1223. 11 (1998) 183–197.
35. Schubert D, Behl C, Lesley R, Brack A, Dargusch 45. Yatin SM, Aksenov MY, Butterfield DA, The
R, Sagara Y, Kimura H, Amyloid peptides are antioxidant vitamin E modulates amyloid β-
toxic via a common oxidative mechanism, Proc peptide induced creatine kinease activity inhibi-
Natl Acad Sci USA 92 (1995) 1989–1993. tion and increased protein oxidation: Implica-
36. Selkoe DJ, The deposition of amyloid proteins in tions for the free radical hypothesis of Alz-
the aging mammalian brain: implications for Alz- heimer’s disease, Neurochem Res 24 (1999)
heimer’s disease, Ann Med 21 (1989) 73–76. 427–435.
37. Selkoe DJ, Amyloid β-protein and genetics of 46. Yatin SM, Yatin M, Aulick T, Ain KB, Butter-
Alzheimer’s disease, J Biol Chem 271 (1996) field DA, Alzheimer’s amyloid β-peptide asso-
18295–18298. ciated free radicals increase rat embryonic neu-
38. Subbarao KV, Richardson JS, Ang LC, Autopsy ronal polyamine uptake and ornithine decarbox-
samples of Alzheimer’s cortex show increased ylase activity: protective effect of vitamin E,
peroxidation in vitro, J Neurochem 55 (1990) Neurosience Lett 263 (1999) 17–20.
342–345. 47. Yatin SM, Varadarajan S, Link CD, Butterfield
39. Subramaniam R, Koppal T, Green M, Yatin S, DA, In vitro and in vivo oxidative stress associ-
Jordan B, Drake J, Butterfield DA, The free radi- ated with Alzheimer’s amyloid β-peptide (1-42),
cal antioxidant vitamin E protects cortical syn- Neurobiol. Aging 20 (1999) 325–330.
aptosomal membranes from Aβ(25-35) toxicity 48. Younkin SG The role of Aβ(1-42) in Alz-
but not from hydroxynonenal toxicity: relevance heimer’s disease, J Physiol Paris 92 (1998) 289–
to the free radical hypothesis of Alzheimer’s 292.
disease, Neurochem Res 23 (1998) 1403–1410.