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- a 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 b 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 c 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 plaques 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: firstname.lastname@example.org 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. DISCUSSION 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 D C 180 DCF Fluo. (Ave. Pixel Intensity) 160 * 140 120 100 80 ** 60 40 20 0 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). 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