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 Previous Article | Next Article 
The Journal of Neuroscience, June 15, 2002, 22(12):4833-4841
A Novel Function of Monomeric Amyloid  -Protein Serving as an
Antioxidant Molecule against Metal-Induced Oxidative Damage
Kun
Zou,
Jian-Sheng
Gong,
Katsuhiko
Yanagisawa, and
Makoto
Michikawa
Department of Dementia Research, National Institute for Longevity
Sciences, Obu, Aichi 474-8522, Japan
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ABSTRACT |
Aggregated and oligomeric amyloid  -protein (A) is known
to exhibit neurotoxicity. However, the action of A monomers on neurons is not fully understood. We have studied aggregation
state-dependent actions of A and found an oligomer-specific effect
of A on lipid metabolism in neurons (Michikawa et al., 2001 ). Here,
we show a novel function of monomeric A1-40, which is the major
species found in physiological fluid, as a natural antioxidant molecule that prevents neuronal death caused by transition metal-induced oxidative damage. Monomeric A1-40, which is demonstrated by
SDS-PAGE after treatment with glutaraldehyde, protects neurons cultured in a medium containing 1.5 µM Fe(II) without
antioxidant molecules. Metal ion chelators such as EDTA, CDTA
(trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid), and DTPA
(diethylenetriamine-N,N,N',N",N"-penta-acetic acid, an iron-binding protein, transferrin, and antioxidant
scavengers such as catalase, glutathione, and vitamin E also inhibit
neuronal death under the same conditions. Monomeric A1-40 inhibits
neuronal death caused by Cu(II), Fe(II), and Fe(III) but does not
protect neurons against H2O2-induced damage.
Monomeric A1-40 inhibits the reduction of Fe(III) induced by
vitamin C and the generation of superoxides and prevents lipid
peroxidation induced by Fe(II). A1-42 remaining as a monomer also
exhibits antioxidant and neuroprotective effects. In contrast,
oligomeric and aggregated A1-40 and A1-42 lose their
neuroprotective activity. These results indicate that monomeric A
protects neurons by quenching metal-inducible oxygen radical generation
and thereby inhibiting neurotoxicity. Because aggregated A is known
to be an oxygen radical generator, our results provide a novel concept
that the aggregation-dependent biological effects of A are
dualistic, being either an oxygen radical generator or its inhibitor.
Key words:
Alzheimer's disease; amyloid -protein; transition
metals; oxygen radicals; antioxidant; neuronal death
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INTRODUCTION |
One of the neuropathological
hallmarks of Alzheimer's disease (AD) is the formation of
extracellular amyloid deposits (Selkoe, 1994). The major component of
the amyloid deposits is the 39-42 amino acid peptide of the amyloid
-protein (A) (Glenner and Wong, 1984; Masters et al., 1985). One
of the A species, ending with a C terminus at residue 40 (A1-40), is the predominant soluble species in biological fluids
(Vigo-Pelfrey et al., 1993; Ida et al., 1996). The longer form of A,
ending at residue 42 (A1-42), accumulates initially and
predominantly in parenchymal plaques (Roher et al., 1993; Iwatsubo et
al., 1994). A1-42 is normally produced and secreted by cells in
much lower quantities than A1-40, which represents ~90% of the
total secreted A. It is believed that aggregated A exerts
neurotoxicity and initiates the progressive pathophysiology of AD
(Mattson et al., 1993; Pike et al., 1993; Lorenzo and Yankner, 1994;
Hartley et al., 1999). However, the function of monomeric A on
neurons is not yet fully understood.
It has been reported that the levels of metals such as zinc, iron, and
copper are significantly concentrated in senile plaques (Smith et al.,
1997; Lovell et al., 1998b). These observations followed original
reports showing that these metals promote A aggregation (Bush et
al., 1994a,b; Huang et al., 1997; Atwood et al., 1998), which is
reversed by treatment with chelators in vitro (Huang et al.,
1997) and in vivo (Cherny et al., 2001). In support of these
findings, a recent study has clearly demonstrated that zinc and copper
induce non--sheeted A aggregation but inhibit -sheeted
aggregation and fibril formation (Yoshiike et al., 2001). Other studies
have suggested that accumulated metals support the AD pathology as a
possible source of reactive oxygen radicals (Smith et al., 1997; Lovell
et al., 1998b; Sayre et al., 2000).
Recent studies showed that the surrounding regions of A deposits in
brains of patients with AD and Down's syndrome have no damage
(Nunomura et al., 2000, 2001) and that there is an inverse correlation
between A burden and the levels of oxidized nucleic acids in the AD
brain (Cuajungco et al., 2000b). Although aggregated A is reported
to generate free radicals (Hensley et al., 1994; Schubert and Chevion,
1995; Kay, 1997; Huang et al., 1999a; Monji et al., 2001b), these lines
of evidence imply a new function of A other than that of a radical
generator. A previous report has suggested its antioxidant activity for
lipoproteins (Kontush et al., 2001); however, no explanation has been
provided as to the mechanism behind the disparate results from
different laboratories regarding A-induced oxidative stress versus
others suggesting antioxidant properties.
In light of the above, we have studied the aggregation state-dependent
actions of A on neurons (Michikawa et al., 2001; Gong et al., 2002).
Here, we show that monomeric A1-40 and also A1-42 serve as
antioxidant molecules protecting neurons from oxygen radicals
generated in a metal-dependent manner, providing new insights into the
strategy for developing a therapy for patients with AD.
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MATERIALS AND METHODS |
Reagents and preparation. Synthetic human A1-40
was purchased from Peptide Institute Inc. (Osaka, Japan; lot numbers
510116 and 501001) and Bachem (Bubendorf, Switzerland; lot number
0538913). A40-1 (lot number D539530) was purchased from Bachem, and
A1-42 (lot number 510523), A1-16 (lot number 490704), and
A25-35 (lot number 500701) were purchased from Peptide Institute
Inc. A1-40, A1-42, and A25-35 were dissolved in DMSO at 2 mM and then diluted with distilled water to a
concentration of 200 µM. Although the solution
was clear, it is known that an A solution contains short fibrils
(Naiki et al., 1998). To remove short fibrils, A solutions were
centrifuged at 100,000 × g for 1 hr at 4°C, using a
Beckman Optima TLX table ultracentrifuge and a Beckman TLA-120.2
fixed-angle rotor. A1-16 was directly dissolved in water to a
concentration of 200 µM. Oligomeric A1-40
was prepared as described previously (Michikawa et al., 2001).
Transferrin, insulin, progesterone, putrescine, selenite, superoxide
dismutase (SOD), catalase, glutathione, vitamin E, and vitamin E
acetate were obtained from Sigma (St. Louis, MO). The B27 supplement
and B27 minus antioxidants (B27-AO) were purchased from Invitrogen
(Grand Island, NY). EDTA was purchased from Eastman Kodak Company
(Rochester, NY).
trans-1,2-diaminocyclohexane-N,N,N',N'-tetra-acetic acid (CDTA),
diethylenetriamine-N,N,N',N",N"-penta-acetic
acid (DTPA), iron sulfate heptahydrate, iron nitrate
nonahydrate, and copper sulfate pentahydrate were obtained from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). Monoclonal antibody,
namely, anti-4-hydroxy-2-nonenal (4-HNE) antibody, which recognizes
oxidized 4-HNE, was purchased from NOF Corporation (Tokyo, Japan).
Cell culture. All experiments were performed in compliance
with existing laws and the institutional guidelines. Cerebral cortical neuronal cultures were prepared from Sprague Dawley rats at embryonic day 17 as described previously (Michikawa and Yanagisawa, 1998). The
dissociated single cells were suspended in a feeding medium and plated
onto poly-D-lysine-coated 12-well plates at a
cell density of 5 × 10 5. The
feeding medium consisted of DMEM/F12 containing 0.1% bovine albumin
fraction V solution (Invitrogen) and N2 (Bottenstein and Sato, 1979),
B27, or B27-AO supplements.
Quantification of neuron survival. For assessment of cell
viability of cultured neurons, phase-contrast photomicrographs were taken before treatment and at various time points after treatment. The
number of viable neurons on each micrograph was determined in premarked
microscope fields (10× objective). Viable neurons were identified on
the basis of morphological criteria. Neurons with intact neurites with
uniform diameter and soma with a smooth round appearance were
considered viable, whereas neurons with fragmented neurites and
shrunken cell bodies were considered nonviable. In a pilot study, cell
viability was confirmed by testing cell membrane permeability using
propidium iodide (PI) or by staining with a viable cell-specific
marker, calcein AM, as described previously (Michikawa and Yanagisawa,
1998). Neurons were stained with Hoechst 33342 (bis-benzamide; 2.5 µg/ml), to visualize their nuclear morphology. Most neurons died
during culturing in DMEM/F12 medium supplemented with B27-AO (B27-AO
medium) or DMEM/F12 medium supplemented with N2 and
FeSO4, CuSO4, or
Fe(NO3)3. For each
determination of cell viability, 1000-1400 cells were counted.
Thioflavin-T binding assay for aggregated A.
Determination of the aggregated state of A in solution was performed
on the basis of a previously established method (LeVine, 1995;
LeVine, 1999). The conditioned media, in which the cultures were
incubated with A, were collected. Each well contained 50 µl of
each medium in 1 ml per well of 5 µM
thioflavin-T in 50 mM glycin-NaOH, pH 8.5. Steady-state fluorescence intensities for each sample were determined
in 48-well plates with a multiplate reader (Fluoroskan Ascent,
Labsystems Inc., Franklin, MA) (excitation 446 nm, emission 490 nm).
The culture media to which A was not added were used as the background.
Cross-linking of A with glutaraldehyde.
SDS-PAGE of cross-linked fA1-40 and iA1-40 was performed as
described previously (Levine, 1995). Briefly, 8 µg of each peptide in
a stock solution (200 µM) was diluted to 35 µl with H2O. One-tenth volume of glutaraldehyde (3.5 µl of 0.625% diluted from a 25% stock solution) was added to
each solution followed by the addition of an excess amount of
NaBH4 (10 µl of 0.175 M, 6.6 mg/ml, in 0.1 M
NaOH). After 10 min of incubation, 15 µl of the SDS-PAGE sample
buffer containing 100 mM dithiothreitol and 20%
sucrose was added. Boiling of the amyloid peptides in the sample buffer
was avoided, because SDS-resistant multimeric complexes are formed from
non-cross-linked peptides during heating in SDS (LeVine, 1995). Then,
20 µl of the mixture was subjected to SDS-PAGE using a 4-20%
gradient gel as described previously (Michikawa et al., 2001). The gel
was then visualized by silver staining. To compare the aggregation
status of A1-40 and A1-42 in 8 mM sodium
phosphate, pH 7.4, or DMEM/F12 medium, freshly dissolved A1-40 and
A1-42 were incubated at 20 µM for 3 hr at
37°C in each solution. The protein concentration of each solution of
A1-40 and A1-42 was determined, 2.8 µg of each peptide was
subjected to cross-linking, and the peptides were subjected to
electrophoresis and silver staining.
Iron reduction assays. Assays were performed according to a
previously reported method (Huang, 1999a). A1-40 (10 µM), A1-42 (10 µM),
DTPA (10 and 300 µM) or Fe(III) (25 µM), and
3-(2-pyridyl)-5,6-bis(4-sulfo-phenyl)-1,2,4-triazine (PDT)
(250 µM) were added to 1 ml of 8 mM sodium phosphate, pH 7.4, and rotated at
25°C for 6 hr. Vitamin C (10 µM) was then added, and the mixture was further incubated at 37°C for 14 hr. A
solution containing Fe(III) and A1-40 at the same concentrations in
the absence of the indicator PDT was used to determine the background
levels of this assay system. The absorbance at 562 nm indicates the
amount of reduced iron ion, Fe(II).
Measurement of superoxide levels. The levels of
intracellular superoxide anion radicals were measured using
hydroethidium (HE), which is oxidized to a fluorescent ethidium cation
by superoxides, using methods similar to those described previously
(Bindokas et al., 1996). In brief, cells were incubated for 30 min in
the presence of 5 µM HE (Molecular Probes,
Eugene, OR) at 37°C in 5% CO2 atmosphere, and
confocal images of cell-associated HE fluorescence were acquired
(excitation = 488 nm and emission >560 nm).
Western blot analysis for determination of lipid
peroxidation. Cerebral cortices of Sprague Dawley rats at
embryonic day 17 were isolated and minced with a cutter and incubated
in PBS in the presence of 3 or 5 µM Fe(II) with
or without fA1-40 at 10 µM for 4 hr at
37°C. The fragments were then homogenized in RIPA buffer (150 mM NaCl, 10 mM Tris-HCl, pH
7.5, 1% Nonidet P-40, 0.1% SDS, and 0.25% sodium deoxycholate) and
centrifuged at 10,000 × g for 10 min at 4°C, and the
supernatants were recovered. Protein concentrations of the supernatants
were determined by the BCA method (Pierce, Rockford, IL). Western blot
analysis was performed according to the methods described previously
(Michikawa et al., 2001). In brief, 24 µg of each protein was
separated by 4-20% gradient SDS-PAGE and electrotransferred onto
nitrocellulose membranes. The membranes were incubated with monoclonal
primary antibody, anti-4-HNE antibody, at 2 µg/ml overnight at 4°C.
The membranes were then washed in PBS containing 0.05% Tween 20 (PBS-T) three times, followed by incubation with HRP-conjugated goat
anti-mouse IgG (1:5000 dilution) for 1 hr at room temperature. The
membranes were washed four times in PBS-T and visualized with an ECL
kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).
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RESULTS |
Freshly dissolved A1-40 protects neurons against death induced
by antioxidant-depleted medium
We studied the effect of A1-40 on neuronal viability. The
medium used was DMEM nutrient mixture (DMEM/F12, 50:50)
containing B27-AO. When neurons were incubated in the B27-AO medium,
cultured neurons appeared healthy 40 hr after plating; however,
neuronal death was induced 48 hr after plating, and most of the cells
were dead 64 hr after plating (Fig.
1a-c). In
contrast, neuronal death was inhibited in the presence of freshly
dissolved A1-40 (fA1-40) at a concentration of 5 µM 64 hr after plating (Fig. 1d).
Addition of DMSO at a final concentration of 1% DMSO to the B27-AO
medium did not prevent or accelerate neuronal death (Fig.
1e). Figure 1e shows the time-dependent curves of
neuronal viability of the cultures treated with fA1-40 at various
concentrations. The neuronal death induced by incubation in the B27-AO
medium was inhibited by fA1-40 in a dose-dependent manner. Neuronal
viability was maintained at the initial levels when the cultures were
treated with fA1-40 at concentrations of 10 and 20 µM until 4 d after the commencement of the
treatment (Fig. 1e). The neurons at each time point were
stained with Hoechst 33342 and PI. The viable neurons at culture
day 2 had larger swollen cell bodies (Fig. 1b), and the
nuclei of dead neurons were shrunken as demonstrated by Hoechst 22336 and PI staining (Figs. 1f,h). The effect of
freshly prepared A1-42 on neuronal viability was also examined.
fA1-42 could not inhibit neurotoxicity but rather promoted neuronal
death (Fig. 1e).
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Figure 1.
fA1-40 inhibits neuronal death induced in an
antioxidant-depleted medium. Rat cortical neurons were cultured in the
B27-AO medium without A1-40 treatment: a, 40 hr
culture; b, 48 hr culture;
c, 64 hr culture; d, with fA1-40 (5 µM) treatment 4 hr after plating, 64 hr culture.
a-c are the same microscope field.
e, Cell viability in the cultures treated with A:
none ( ); DMSO vehicle ( ); A1-40 at 2 µM ( ),
10 µM ( ), and 20 µM ( ); and A1-42
at 10 µM ( ). Data represent means ± SE;
n = 6 each. Six independent experiments showed
similar results. Representative Hoechst staining showing the nuclear
morphology of cortical neurons with or without A1-40 treatment is
shown in f, g. Cell membrane permeability
is indicated by PI staining in h,
i.
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Monomeric A1-40, but not oligomeric A1-40, has an ability
to protect neurons in the B27-AO medium
We examined the effect of incubated A1-40 (iA1-40), which
was filtered and the protein concentration of which was determined before addition, on neuronal viability cultured in the B27-AO medium.
As shown in Figure 2a,
neuronal death occurred 72 hr after the commencement of the treatment,
which was completely inhibited by fA1-40, but not by iA1-40, at
a concentration of 5 µM. Results of the
quantitative analysis of neuronal viability within 72 hr of incubation
are shown in Figure 1b, showing that fA1-40 at concentrations of 5 and 10 µM completely
inhibited neuronal death, whereas iA1-40 inhibited neuronal death
at 10 µM but not at 5 µM. To determine the oligomeric state of A,
the reaction of the conditioned medium of each culture with
thioflavin-T was determined. As shown in Figure 2c, the
value of the conditioned medium of the cultures treated with iA1-40
was significantly higher than that treated with fA1-40, indicating
that iA1-40 contains highly oligomerized A. To determine more
directly that the amount of monomeric A was decreased and that of
oligomeric A was increased, a cross-linking study of each A
sample was performed. As shown in Figure 2d, fA1-40
contains mostly monomers, whereas iA1-40 contains many oligomers,
including dimers, trimers, and tetramers, in addition to decreased
levels of monomers. These results indicate that A monomers have a
neuroprotective activity and that the lack of neuroprotective activity
of iA1-40 at 5 µM is not caused by its
toxic effect on neurons but rather by the low levels of monomers.
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Figure 2.
fA1-40 but not iA1-40 protects neurons in
the B27-AO medium. Neurons were treated with fA1-40 or iA1-40
24 hr after plating. Phase-contrast photomicrographs were taken
(a), and the cell viability was determined
(b) 48 hr after the commencement of each
treatment. a, p < 0.0001 versus
fA1-40 (5 µM), iA1-40 (10 µM), and
fA1-40 (10 µM). c, Thioflavin-T
fluorescence with the conditioned medium of each cultured neuron
treated with fA1-40 or iA1-40 for 3 d. b,
p < 0.01 versus iA1-40. d,
Detection of oligomeric A in fA and iA samples by
cross-linking with glutaraldehyde. fA1-40 or iA1-40 (2.5 µg)
was cross-linked with glutaraldehyde and subjected to a 4-20%
SDS-PAGE. The gel was then visualized by silver staining.
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Metal-binding protein and metal chelators inhibit neuronal death in
the B27-AO medium
Because neurotoxicity was induced in the media deficient of
antioxidant reagents, it is reasonable to assume that the A-mediated neuronal protection may possibly be explained by an antioxidant action
of A. Antioxidant actions include a direct antioxidant effect, and
the indirect actions of fA1-40 include quenching of metal ions to
inhibit secondary generation of free radicals. Thus, we examined the
effect of molecules that have antioxidant activities. As shown in
Figure 3a, catalase,
glutathione, vitamin E acetate, and vitamin E inhibited neuronal death
at culture day 2. Catalase and vitamin E acetate and vitamin E
partially and completely inhibited cell death at culture day 4, respectively; however, SOD did not show any neuroprotective activity.
Because we have observed that the N2 supplements (Bottenstein and Sato, 1979) inhibited neuronal death induced by incubation in the B27-AO medium, we examined the inhibitory effect of each component of N2
supplements. Figure 3b shows that among the components
examined, only transferrin successfully inhibited neuronal death.
Because transferrin is known to bind iron, inhibiting cell death by
quenching the iron-dependent generation of reactive oxygen radicals
(Halliwell and Gutteridge, 1989), it is reasonable to postulate that
iron in DMEM/F12 plays a critical role in neuronal death in the B27-AO medium. Thus, we next examined the effect of various iron chelators on
neuronal death under these conditions. EDTA (400 µM), CDTA (40 µM), and
DTPA (8 µM) protected neurons against toxicity
induced in the B27-AO medium at culture day 4 (Table
1).
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Figure 3.
Transferrin and antioxidant scavengers inhibit
neuronal death that occurred in the B27-AO medium. a,
SOD (1500 U/ml), catalase (21,600 U/ml), glutathione (450 µg/ml),
vitamin E acetate (1 µg/ml), or vitamin E (1 µg/ml) was added to
neuronal cultures maintained in the B27-AO medium 4 hr after plating.
b, N2 supplements or each component of N2 supplements,
transferrin (100 µg/ml), insulin (5 µg/ml), progesterone (0.0063 µg/ml), putrescine (16.11 µg/ml), and selenite (0.0052 µg/ml) was
added to neuronal cultures maintained in the B27-AO medium 4 hr after
plating. Neuronal viability was determined as described in Materials
and Methods at culture days 1, 2, and 4 (a) or 1, 2, and 7 (b).
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Monomeric A1-40 protects neurons against iron- and
copper-mediated neuronal toxicity
Because the culture medium, DMEM/F12 supplemented with B27-AO,
contains 1.5 µM Fe (II), 124 nM Fe(III), and
5.2 nM Cu(II), our findings that neuronal death induced in
the B27-AO medium is prevented by antioxidant scavengers, metal
chelators, and transferrin indicate that neurotoxicity is induced by
oxygen radicals generated in an Fe(II)-mediated manner. Thus, we next
determined whether transition metal ions, such as iron and copper ions,
and H2O2 exhibit
neurotoxicity on cultured neurons, and whether fA1-40 has the
ability to prevent this toxicity. Twenty-four hours after plating,
neuronal cultures were treated with 1.5 µM
CuSO4, 3.0 µM
FeSO4, 25 µM
Fe(NO3)3, and 30 µM H2O2 in
the presence or absence of 5 µM fA1-40 in the N2
medium. After 24 hr incubation, photographs were taken, and the
neuronal viability was determined. As shown in Figure
4, Cu(II), Fe(II), Fe(III), and
H2O2 caused cell death (a, c, e, and g,
respectively). fA1-40 at a concentration of 5 µM inhibited cell death caused by these metals
(Fig. 4b,d,f) but did not
inhibit cell death caused by
H2O2 (Fig. 4h),
indicating that protection of neurons by fA1-40 is not caused by a
direct antioxidant activity but by an indirect one via interaction with metal ions. The quantitative analysis of these experiments is shown in
Figure 4i. We performed additional experiments to determine the effect of transferrin on neuronal toxicity induced by these metals.
We found that 3.8 µM transferrin inhibited 3.0 µM Fe(II)- and 25 µM
Fe(III)-mediated neurotoxicity, but even 13 µM
transferrin did not inhibit 1.5 µM
Cu(II)-mediated neurotoxicity (data not shown), supporting the idea
that Fe(II) but not Cu(II) is responsible for the generation of oxygen
radicals and induces toxicity. This is supported by the fact that the
B27-AO medium contains 1.5 µM Fe(II), which is
sufficiently high to induce cell toxicity, whereas it contains much
lower concentrations of Cu(II) and Fe(III).
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Figure 4.
f A1-40 inhibits neuronal death induced by
transition-metal ions. Neurons were cultured in N2 medium for 24 hr,
followed by treatment with 1.5 µM CuSO4
(a, b), 3.0 µM Fe
SO4 (c, d), 25 µM Fe(NO3)3,
(e, f), and 30 µM
H2O2 (g,
h), and incubated for another 24 hr; photographs were
taken to determine cell viability. a, c,
e, and g represent control cultures, and
b, d, f, and
h represent neurons treated with fA1-40 (5 µM) in addition to metal ions. i,
Quantitative analysis of these treatments 24 hr after the commencement
of the metal treatment. Open and closed
bars indicate cell viability in the cultures in the absence or
presence, respectively, of fA1-40 (5 µM).
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Monomeric A1-40 inhibits vitamin C-mediated reduction
of Fe(III)
Because reduced metal ions are known to generate oxygen radicals
that initiate subsequent reactions of radical productions (Halliwell
and Gutteridge, 1984), we determined whether fA1-40 has any effect
on Fe(III) reduction. To examine the inhibitory effect of fA1-40 on
Fe(III) reduction, a vitamin C-mediated metal reduction system was
used. As shown in Figure 5a,
fA1-40 inhibited Fe(III) reduction mediated by vitamin C. In
addition to fA1-40, fA1-42, A1-16, A25-35, and a metal
ion chelator, DTPA, also inhibited Fe(III) reduction. Because the
action of A is known to depend on the state of aggregation of the
peptides, we next determined the aggregation states of A used in
this study by cross-linking of peptides with glutaraldehyde and
subsequent silver staining. As shown in Figure 5b, most of
both A1-40 and A1-42 incubated in 8 mM
sodium phosphate buffer and A1-40 incubated in DMEM/F12 for 3 hr
were found to be monomers, whereas fA1-42 incubated in DMEM/F12 for
3 hr was found to form aggregation (Fig. 5b, *), and the
amount of monomeric A1-42 was significantly decreased (Fig.
5b).
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Figure 5.
Inhibitory effect of A peptides on vitamin
C-mediated Fe(III) reduction and the aggregation state of A peptides
in PB and DMEM/F12. a, A peptides were incubated with
Fe(III) (25 µM) and PDT (250 µM), followed
by the addition of vitamin C (10 µM) and subsequent
incubation for 14 hr at 37°C. The effects of freshly dissolved A
peptides (10 µM) (fA1-40, fA1-42, fA1-16, and
fA25-35, all of which were dissolved in distilled water to make a
stock solution at 200 µM) and DTPA (10 and 300 µM) on the reduction of Fe(III) were determined. The
amount of reduced iron ions was determined by measuring the absorbance
at 562 nm. Data represent means ± SE; n = 5 replicate wells. p < 0.001 (a) and p < 0.0001 (b) versus control. b, Freshly
dissolved A1-40 and A1-42 peptides at 20 µM were
incubated for 3 hr at 37°C in 8 mM sodium phosphate
buffer, pH 7.4 (lanes 1, 3), or in
DMEM/F12 medium (lanes 2, 4). The
aggregation state of A1-40 (lanes 1,
2) and A1-42 (lane 3,
4) was visualized by SDS-PAGE and silver staining
after cross-linking as described in Materials and Methods. Note that
A1-42 aggregated immediately in DMEM/F12 (*), but the majority of
both peptides, A1-40 in both solutions and A1-42 in PB,
remained as a monomer.
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Generation of superoxides in the B27-AO medium and Fe(II)-induced
lipid peroxidation are inhibited by monomeric A1-40
Using HE dye, we examined whether the generation of oxygen
radicals is enhanced in the B27-AO medium and whether this enhancement is inhibited by fA1-40. As shown in Figure
6a, strong signals of oxidized
ethidium dye were observed in some viable neurons with swollen cell
bodies (Fig. 6a, arrows) and dead neurons with shrunken cell bodies (Fig. 6c), whereas the signals with
ethidium dye in the cultures treated with fA1-40 (5 µM) were not detected (Fig. 6b). We
then examined the effect of fA1-40 on Fe(II)-induced lipid
peroxidation in rat brain cortices by investigating the production of
4-HNE-modified proteins, a product of lipid peroxidation in rat brains,
using a monoclonal antibody against 4-HNE-modified proteins. As shown
in Figure 6e (*), the amount of 4-HNE-modified proteins
increased in brains incubated in PBS in the presence of 3 and 5 µM Fe(II), whereas treatment with 10 µM fA1-40 attenuated this increase. This
result indicates that fA1-40 prevented lipid peroxidation of the
brain tissues induced by oxygen radicals generated by the Fenton
reaction.
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Figure 6.
fA1-40 inhibits the generation of superoxide
and lipid peroxidation. Neurons were treated with (a,
c) or without (b, d) 5 µM fA1-40 4 hr after plating and were cultured for 48 hr in the B27-AO medium. The cultures were then incubated with 5 µM HE fluorescence (a, b)
for 30 min, and transmissive light micrographs of these cultures were
taken. Note that the increased signal of superoxides was observed in
swollen neurons (arrows) as well as in shrunken neurons
in cultures without fA (a). e,
The effect of A on the production of lipid peroxidation in rat
cerebral cortices in the presence of Fe(II). Cerebral cortices were
isolated, minced with a cutter, and incubated in PBS in the presence of
3 and 5 µM Fe(II) with or without fA1-40 at 10 µM for 4 hr at 37°C. The fragments were then
homogenized in RIPA buffer and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant of the homogenate
was subjected to Western blot analysis using anti-4-HNE antibody as the
primary antibody.
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Comparison of effects of various kinds of A species on
neuronal protection
We examined the neuroprotective effect of A1-42, A1-16,
and A25-35, in addition to A1-40, on cultured cells incubated in the B27-AO medium. Treatment of A1-40 at a concentration of 5 µM inhibited neuronal death at a percentage of 95 ± 5, whereas A1-42 at concentrations of 0.01, 0.1, 1, 2, 5, 10, and
20 µM, A1-16 at concentrations of 1, 2, 5, 10, 20, and 40, or A25-35 at concentrations of 1, 2, 5, 10, 20, and 40 did
not prevent neuronal death 48 hr after the commencement of incubation
(Table 2). In the case of A1-42, the
thioflavin-T value of the culture medium at the end of treatment
significantly increased compared with that of the culture medium
treated with A1-40, A1-16, or A25-35, indicating that
A1-42 becomes highly aggregated in a culture medium. However,
because A1-40, when it remains as a monomer, has an antioxidant
effect in the in vitro assay system (Fig. 5a), we
next performed an experiment to determine whether monomeric A1-42
at a concentration of 5 µM has a
neuroprotective effect on neurons. Because Congo red is known to
inhibit oligomerization of A by stabilizing A monomer (Podlisny
et al., 1995, 1998), we used Congo red to maintain A1-42 as a
monomer. As shown in Table 2, concurrent treatment of 100 µM Congo red with 5 µM
A1-42 inhibited neuronal death, whereas treatment with 100 µM Congo red alone did not. The thioflavin-T
value of these conditioned media was not determined, because Congo red
affects the thioflavin-T assay system. These data indicate that
monomeric A, regardless of its species, A1-40 or A1-42,
rescues neurons.
Effect of tachykinin neuropeptides on monomeric
A1-40-mediated neuroprotection
Because a previous study has demonstrated the neurotrophic effects
of A1-40, which can be reversed by tachykinin neuropeptides (Yankner et al., 1990), we further examined whether the neuroprotective effect of monomeric A1-40 is inhibited by tachykinin neuropeptides. In our culture system, takykinine neuropeptides such as substance P,
physalaemin, eledoisin, neurokinin A, and neurokinin B at 1, 2, 5, 10, and 20 µM did not inhibit neuronal death. Moreover, substance P and physalaemin did not inhibit the neuroprotective effect
of A1-40 (Table 3). These results
indicate that the mechanism underlying the neurotrophic effects of
A1-40 is different from that underlying the antioxidant functions
of monomeric A.
View this table:
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Table 3.
Effect of substance P and physalaemin on the
neuroprotective effect of monomeric A1-40 in the B27-AO medium
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DISCUSSION |
In this study, we demonstrated a novel function of monomeric
A1-40 as an antioxidant molecule on cultured neurons. Monomeric A1-40 exhibits a neuroprotective effect on neurons by quenching transition metal-mediated oxygen radical generation; however, oligomeric A1-40 loses its neuroprotective activity. Monomeric A1-42 also exhibits a neuroprotective effect; however, when
monomeric A1-42 is incubated in the culture medium, it rapidly
aggregates and exhibits neurotoxicity, whereas monomeric A1-40
remains as a monomer under the same conditions and protects
neurons. These findings indicate a novel concept that the
biological action of A is dualistic. A, as a monomer, functions
as an antioxidant molecule, preventing the generation of oxygen
radicals, whereas oligomerized or aggregated A not only loses its
antioxidant activity but also contributes to the generation of oxygen
radicals (Kay, 1997; Monji et al., 2001a,b), disrupts lipid homeostasis
(Michikawa et al., 2001; Gong et al., 2002), and eventually exhibits
neurotoxicity (Mattson et al., 1993; Pike et al., 1993; Lorenzo and
Yankner, 1994).
Neuronal death induced in the B27-AO medium is inhibited by the
addition of radical scavengers, indicating that neuronal toxicity is
caused by oxygen radicals. The B27-AO medium contains 1.5 µM Fe(II), 124 nM Fe(III), and 5.2 nM Cu(II), and redox-active transition metals such as iron
and copper are known to stimulate oxygen radical chain reactions
(Halliwell and Gutteridge, 1984). Because 1.5 µM or
higher concentrations of Fe(II) and Cu(II) induce neuronal death in
culture [our unpublished data and previous reports (White et al.,
1999; Wang and Cynader, 2001)], Fe(II) is most likely responsible for
inducing neurotoxicity by generating oxygen radicals in the B27-AO
medium. Furthermore, the facts that transferrin successively protects
neurons in the B27-AO medium (Fig. 3b) and inhibits
Fe(II)-mediated neuronal death in N2 medium, whereas it does not
prevent Cu(II)-induced neuronal death (data not shown), strongly
support this notion. Thus, it is possible that fA1-40 protects
neurons from oxygen radicals generated in an Fe(II)-mediated manner.
Antioxidant actions include a direct antioxidant action such as that of
scavengers and indirect actions including the quenching metal ions to
inhibit secondary generation of free radicals. The neuroprotective
activity of monomeric A1-40 includes an inhibitory effect on the
generation of superoxides in cultured neurons and lipid peroxidation in
brains (Fig. 6). Furthermore, the direct inhibitory effects of
monomeric A1-40 on metal reduction induced by vitamin C are also
demonstrated (Fig. 5). These findings, together with the result showing
that monomeric A1-40 does not serve as a radical scavenger (Fig.
4), indicate that the neuroprotective activity of A1-40 is not
caused by a direct antioxidant effect but rather by an indirect effect
of this peptide, probably the sequestration of metal ions leading to
the quenching of the secondary generation of oxygen radicals as other
metal-binding proteins do (Halliwell and Gutteridge, 1989).
Free-radical involvement in AD pathogenesis is a well established
hypothesis (Lovell et al., 1998a; Markesbery and Lovell, 1998). A is
widely believed to serve as a neurotoxic molecule by producing oxygen
radicals leading to cell dysfunction and death (Behl et al., 1994;
Hensley et al., 1994). The oxygen radicals generated by the interaction
of A with redox-active metal ions are suggested to be the possible
source of A neurotoxicity, which is suppressed by the redox-inactive
form of zinc or metal ion chelators (Huang et al., 1999a,b; Cuajungco
et al., 2000a). These lines of evidence seem to contradict our present
results that monomeric A1-40 is a potent antioxidant molecule. This
discrepancy can be explained by the notion that the action of A is
aggregation state-dependent. We show that monomeric A1-40 protects
neurons from metal-induced neurotoxicity, whereas iA1-40 contains
fewer A monomers but more oligomers (Fig. 2d), which
could be the reason for the loss of its neuroprotective ability. This
is also the case for A1-42, because we have found that A1-42,
remaining as a monomer in PB, inhibits the reduction of Fe(III) caused
by vitamin C as does A1-40 (Fig. 5a), indicating that
monomeric A1-42 also functions as an antioxidant molecule. In
addition, the finding that A1-42, when it is maintained as a
monomer by coincubation with Congo red in DMEM/F12 medium, exhibits
neuroprotective activity (Table 2) strongly supports this notion.
However, when fA1-42 is incubated in DMEM/F12 medium that contains
salt, it aggregates rapidly (Fig. 5b, Table 2) and exhibits
neurotoxicity (Fig. 1e, Table 2), whereas fA1-40
remaining as a monomer under the same conditions protects neurons
(Figs. 1e, 5b). Thus, under physiological
conditions, A1-42, a highly amyloidogenic peptide, rapidly
aggregates, loses its neuroprotective activity, generates free
radicals, and subsequently exhibits neurotoxicity (Pike et al., 1993;
Lorenzo and Yankner, 1994; Roher et al., 1996; Kay, 1997; Huang et al.,
1999b; Cuajungco et al., 2000b; Monji et al., 2001a,b). These lines of
evidence suggest that it may not the differences in A species,
A1-40 or A1-42, but those in the state of aggregation,
monomers, or other states of aggregation such as oligomers or fibrils,
that determine whether the action of A is either neuroprotective or neurotoxic.
Another possible explanation for the discrepancy between the effect of
A1-40 and that of A1-42 on neuronal survival may be that
at low iron/A binding ratios, iron is captured by A and
sequestered from inducing oxygen radical generation, but at higher
iron/A ratios, the interaction of A and iron promotes oxygen
radical generation (Huang et al., 1999a). Because A1-42 is
suggested to bind iron with greater affinity than A1-40 (Atwood et al., 2000), it may be possible to postulate that
A1-42 may acquire gain of adverse action at lower concentrations
than A1-40.
One may say that because a previous study has demonstrated that
aggregated A does not lose the stoichiometry of copper binding (Atwood et al., 2000), an increased amount of oligomerized A may
undergo oxidization, reduce metal ions, and serve as an oxygen radical
generator (Huang et al., 1999a), leading to neuronal death. Actually,
at present we have no evidence indicating that oligomeric A has
lesser binding affinity to iron than monomeric A. This may be the
case for A1-42, because A1-42 that rapidly aggregates in the
culture medium not only loses its neuroprotective activity but also
exhibits neurotoxicity (Fig. 1e); however, this may not be
the case for A1-40. Our findings that 5 µM
iA1-40 loses its neuroprotective effect on neurons, whereas 10 µM iA1-40 protects neurons (Fig.
2b), do not favor the idea that the loss of neuroprotective function is caused by oxidized oligomeric A but favor the notion that monomeric but not oligomeric A1-40 can serve as an antioxidant molecule.
The last question to be addressed is that the neuroprotective effects
of monomeric A1-40 shown in our present study are the same as the
previously reported neurotrophic effects of A1-40, which can be
reversed by tachykinin neuropeptides (Yankner et al., 1990). However,
monomeric A1-40 has a neuroprotective effect even on mature neurons
at high concentrations, whereas takykinin neuropeptides including
substance P and physalaemin at 10 and 20 µM did not
inhibit neuronal death in our culture system. Moreover, substance P and
physalaemin did not reverse the neuroprotective effect of A1-40
(Table 3), indicating that the mechanism underlying the neurotrophic
effects of A1-40 is different from that underlying the antioxidant
functions of monomeric A.
The notion that monomeric A1-40 functions as an antioxidant is
supported by previous studies showing that the surrounding regions of
A deposits in the brains of patients with AD and Down's syndrome
have no damage (Nunomura et al., 2000, 2001) and that the inverse
correlation is found between A burden and levels of oxidized nucleic
acids in AD brain (Cuajungco et al., 2000b). Interestingly, a recent
report suggests that brain oxidative damage occurs before A
accumulation in the brains of a model mouse of AD amyloidosis
(Pratico et al., 2001). Previous reports have shown that A1-42
accumulates with aging, whereas A1-40 does not but accumulates in
AD brains (Funato et al., 1998), and that oxidative stress promotes
amyloidogenesis (Misonou et al., 2000). These lines of evidence may
allow us to assume that oxygen radicals generated in an age-dependent
manner enhance generation of A, which may protect neurons from
oxygen radical toxicity generated by metal-dependent chain reactions.
However, with the increasing amount of A serving as an antioxidant,
A aggregates with longer incubation periods in extracellular local
fluid and, in turn, exhibits neurotoxicity.
On the basis of our findings, we envisage that A may serve dual
actions both by being involved in mechanisms attempting to quench
oxidative stress and neurotoxicity probably by sequestrating metal ions
when A is in a monomeric state and by exhibiting neurotoxicity when
A is highly oligomerized and aggregated by generating oxygen radicals in a metal-mediated manner. Hence, although the toxic actions
of A have been exaggerated to date, our observations may provide a
new insight into the strategies for development of AD therapy that not
only reduction of the amount of A but also inhibition of A
aggregation could be the pivotal target for AD therapy.
| |
FOOTNOTES |
Received Dec. 3, 2001; revised March 19, 2002; accepted March 22, 2002.
This study was supported by a research grant for Longevity Sciences
(H11-001), Research on Brain Science from the Ministry of Health and
Welfare, and by Core Research for Evolutional Sciences and Technology, Japan.
Correspondence should be addressed to Dr. Makoto Michikawa, Department
of Dementia Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi, 474-8522, Japan. E-mail: michi{at}nils.go.jp.
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REFERENCES |
-
Atwood CS,
Moir RD,
Huang X,
Scarpa RC,
Bacarra NM,
Romano DM,
Hartshorn MA,
Tanzi RE,
Bush AI
(1998)
Dramatic aggregation of Alzheimer A by Cu(II) is induced by conditions representing physiological acidosis.
J Biol Chem
273:12817-12826[Abstract/Free Full Text].
-
Atwood CS,
Scarpa RC,
Huang X,
Moir RD,
Jones WD,
Fairlie DP,
Tanzi RE,
Bush AI
(2000)
Characterization of copper interactions with alzheimer amyloid peptides: identification of an attomolar-affinity copper binding site on amyloid 1-42.
J Neurochem
75:1219-1233[ISI][Medline].
-
Behl C,
Davis JB,
Lesley R,
Schubert D
(1994)
Hydrogen peroxide mediates amyloid -protein toxicity.
Cell
77:817-827[ISI][Medline].
-
Bindokas VP,
Jordan J,
Lee CC,
Miller RJ
(1996)
Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine.
J Neurosci
16:1324-1336[Abstract/Free Full Text].
-
Bottenstein JE,
Sato GH
(1979)
Growth of a rat neuroblastoma cell line in serum-free supplemented medium.
Proc Natl Acad Sci USA
76:514-517[Abstract/Free Full Text].
-
Bush AI,
Pettingell Jr WH,
Paradis MD,
Tanzi RE
(1994a)
Modulation of A adhesiveness and secretase site cleavage by zinc.
J Biol Chem
269:12152-12158[Abstract/Free Full Text].
-
Bush AI,
Pettingell WH,
Multhaup G,
Paradis MD,
Vonsattel JP,
Gusella JF,
Beyreuther K,
Masters CL,
Tanzi RE
(1994b)
Rapid induction of Alzheimer A amyloid formation by zinc.
Science
265:1464-1467[Abstract/Free Full Text].
-
Cherny RA,
Atwood CS,
Xilinas ME,
Gray DN,
Jones WD,
McLean CA,
Barnham KJ,
Volitakis I,
Fraser FW,
Kim Y,
Huang X,
Goldstein LE,
Moir RD,
Lim JT,
Beyreuther K,
Zheng H,
Tanzi RE,
Masters CL,
Bush AI
(2001)
Treatment with a copper-zinc chelator markedly and rapidly inhibits -amyloid accumulation in Alzheimer's disease transgenic mice.
Neuron
30:665-676[ISI][Medline].
-
Cuajungco MP,
Faget KY,
Huang X,
Tanzi RE,
Bush AI
(2000a)
Metal chelation as a potential therapy for Alzheimer's disease.
Ann NY Acad Sci
920:292-304[Abstract/Free Full Text].
-
Cuajungco MP,
Goldstein LE,
Nunomura A,
Smith MA,
Lim JT,
Atwood CS,
Huang X,
Farrag YW,
Perry G,
Bush AI
(2000b)
Evidence that the -amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of A by zinc.
J Biol Chem
275:19439-19442[Abstract/Free Full Text].
-
Funato H,
Yoshimura M,
Kusui K,
Tamaoka A,
Ishikawa K,
Ohkoshi N,
Namekata K,
Okeda R,
Ihara Y
(1998)
Quantitation of amyloid -protein (A) in the cortex during aging and in Alzheimer's disease.
Am J Pathol
152:1633-1640[Abstract].
-
Glenner GG,
Wong CW
(1984)
Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.
Biochem Biophys Res Commun
120:885-890[ISI][Medline].
-
Gong JS, Sawamura N, Zou K, Sakai J, Yanagisawa K, Michikawa
M (2002) Amyloid -protein affects cholesterol metabolism
in cultured neurons: implications for pivotal role of cholesterol in
the amyloid cascade. J Neurosci Res, in press.
-
Halliwell B,
Gutteridge JM
(1984)
Oxygen toxicity, oxygen radicals, transition metals and disease.
Biochem J
219:1-14[ISI][Medline].
-
Halliwell B,
Gutteridge JMC
(1989)
Protection by sequestration of metal ions.
In: Free radicals in biology and medicine, Ed 2, pp 131-133 New York: Oxford UP.
-
Hartley DM,
Walsh DM,
Ye CP,
Diehl T,
Vasquez S,
Vassilev PM,
Teplow DB,
Selkoe DJ
(1999)
Protofibrillar intermediates of amyloid -protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons.
J Neurosci
19:8876-8884[Abstract/Free Full Text].
-
Hensley K,
Carney JM,
Mattson MP,
Aksenova M,
Harris M,
Wu JF,
Floyd RA,
Butterfield DA
(1994)
A model for -amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease.
Proc Natl Acad Sci USA
91:3270-3274[Abstract/Free Full Text].
-
Huang X,
Atwood CS,
Moir RD,
Hartshorn MA,
Vonsattel JP,
Tanzi RE,
Bush AI
(1997)
Zinc-induced Alzheimer's A1-40 aggregation is mediated by conformational factors.
J Biol Chem
272:26464-26470[Abstract/Free Full Text].
-
Huang X,
Atwood CS,
Hartshorn MA,
Multhaup G,
Goldstein LE,
Scarpa RC,
Cuajungco MP,
Gray DN,
Lim J,
Moir RD,
Tanzi RE,
Bush AI
(1999a)
The A peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.
Biochemistry
38:7609-7616[Medline].
-
Huang X,
Cuajungco MP,
Atwood CS,
Hartshorn MA,
Tyndall JD,
Hanson GR,
Stokes KC,
Leopold M,
Multhaup G,
Goldstein LE,
Scarpa RC,
Saunders AJ,
Lim J,
Moir RD,
Glabe C,
Bowden EF,
Masters CL,
Fairlie DP,
Tanzi RE,
Bush AI
(1999b)
Cu(II) potentiation of Alzheimer A neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.
J Biol Chem
274:37111-37116[Abstract/Free Full Text].
-
Ida N,
Hartmann T,
Pantel J,
Schroder J,
Zerfass R,
Forstl H,
Sandbrink R,
Masters CL,
Beyreuther K
(1996)
Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay.
J Biol Chem
271:22908-22914[Abstract/Free Full Text].
-
Iwatsubo T,
Odaka A,
Suzuki N,
Mizusawa H,
Nukina N,
Ihara Y
(1994)
Visualization of A42(43) and A40 in senile plaques with end-specific A monoclonals: evidence that an initially deposited species is A42(43).
Neuron
13:45-53[ISI][Medline].
-
Kay CJ
(1997)
Mechanochemical mechanism for peptidyl free radical generation by amyloid fibrils.
FEBS Lett
403:230-235[Medline].
-
Kontush A,
Berndt C,
Weber W,
Akopyan V,
Arlt S,
Schippling S,
Beisiegel U
(2001)
Amyloid- is an antioxidant for lipoproteins in cerebrospinal fluid and plasma.
Free Radic Biol Med
30:119-128
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TOP
ABSTRACT
INTRODUCTION
