Yatin 20et 20al 202000 20J 20Alzheimer s 20Disease 202 20123 131

Document Sample
Yatin 20et 20al 202000 20J 20Alzheimer s 20Disease 202 20123 131 Powered By Docstoc
					Journal of Alzheimer’s Disease 2 (2000) 123–131                                                                  123
IOS Press

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:

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

  A                                                                                                     B

  10 µm


                                                                     DCF Fluo. (Ave. Pixel Intensity)



                                                                                                         80                          **



                                                                                                              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).

                                                      Table 1
           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-
cals.                                                      ACKNOWLEDGEMENTS
   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
                                                                (1992) 944–950.
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.

G4j0t9rI G4j0t9rI