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INTRODUCTION |
Most of the recent advances in Alzheimer's disease
(AD)1 stem from the study of
a 40-42-amino acid peptide called the amyloid
protein (A
) as
the essential pathologic marker of this disorder (1, 2). In brains
afflicted with AD, deposits of A
in the form amyloid fibrils are
widespread within senile plaques and in cerebral and meningeal blood
vessels (3, 4). Interestingly, A
is normally produced as a soluble
peptide (5-8), and whether this form of A
is the immediate
precursor of the amyloid deposits is still unknown. Synthetic peptides
homologous to A
1-40 and A
1-42, however, undergo spontaneous
rearrangements of their initial secondary structure, generating
oligomeric and polymeric species with higher content of
-sheets
(9-15). Such changes are either promoted or inhibited by numerous
factors (9, 14, 16-22).
The secondary structure determines several important properties of A
that may be relevant to the pathogenesis of AD. First, it has been
demonstrated that the amyloid peptide is neurotoxic (23-25) and that
this characteristic is associated with formation of
-sheets (15,
26-31) or amyloid fibrils (31). Second, the ability of A
to form
fibrils is directly correlated with the content of
-sheet structures
adopted by the peptide (32). In this regard, it has been proposed that
peptides with high contents of
-sheets can act as seeds for
nucleation and fibril formation (33, 34). Finally, A
peptides with
high contents of
-sheets become partially resistant to proteolytic
degradation, and this may be a crucial mechanism in amyloid deposition
(35). Such protease resistance and insolubility features, shared by all
known forms of amyloidoses, prevent amyloid removal from tissue
deposits. Thus, by preventing the formation of
-sheets one could not
only reduce neurotoxicity but also facilitate clearance of A
via
increased proteolytic degradation.
It has recently been found that melatonin has cytoprotective properties
against A
toxicity (36). In the process of investigating the
mechanisms of action of melatonin, new properties of this hormone were
uncovered. As determined by CD, electron microscopy, and 1H
NMR, melatonin interacted with A
and had a profound inhibitory effect on the formation of
-sheets and fibrils. Most interestingly, the observed changes in A
conformation appear to depend on specific structural characteristics of the hormone rather than on its recently established antioxidant properties (37).
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MATERIALS AND METHODS |
Circular Dichroism Spectroscopy--
Peptides A
1-40 and
A
1-42 were synthesized in the W. M. Keck Foundation (Yale
University, CT), and their purity was evaluated by amino acid sequence
and laser desorption mass spectrometry as described (32). Aliquots of
A
1-40 and A
1-42 at a concentration of 250 µM in 5 mM Tris-HC1, pH 7.4, were incubated at room temperature alone or with 100 µM of either melatonin or the melatonin
analog 5-hydroxy-N-acetyl-tryptamine (NAT) (Sigma) or
N-t-butyl-
-phenylnitrone (PBN) (Sigma), a powerful free
radical scavenger structurally unrelated to melatonin. Because of the
antioxidant properties of melatonin (37) and because oxidative
conditions may promote fibril formation (34, 38), NAT and PBN were
specifically selected both as controls for the method and to discount
for possible "nonspecific" antioxidant effects of melatonin in the
phenomenon described here.
Spectra in the far ultraviolet light (190-250 nm) were recorded at
various time intervals with a Jasco-720 spectropolarimeter as described
(32) using a cell path of 0.01 cm. Experiments with A
1-42
necessitated shorter incubation times due to the more fibrillogenic
properties and faster aggregation exhibited by the longer peptide. 40 scans/experimental condition were obtained at 0.2 nm intervals over the
wavelength range 190-250 nm. The data were analyzed by the Lincomb
algorythm (39) to obtain the percentages of the different secondary
structures motifs.
Electron Microscopy--
To determine whether melatonin
displayed inhibitory effects on amyloid fibril formation, transmission
electron microscopy was performed following a standard method
previously described (40) using a Phillips CM100 microscope and
Formvar-coated nickel grids. A
1-40 was incubated at the same
concentrations as noted for the CD and NMR experiments in the presence
or absence of melatonin, and fibril formation was monitored at 0, 12, 24, 36, and 48 h in three independent experiments. Additional
controls containing A
plus NAT and A
plus PBN were incubated in
parallel for 48 h. To determine the minimal inhibitory
concentration of melatonin on fibril formation, we performed
experiments in which several melatonin concentrations (1 nM, 10 nM, 1 µM, 10 µM, 100 µM, and 200 µM) were
added to tubes containing 250 µM A
1-40, incubated for
48 h, and then examined.
For the more amyloidogenic A
1-42, experiments were preformed at the
same peptide concentration (250 µM) in the presence or absence of melatonin at various concentrations (100 nM, 1 µM, 10 µM, 100 µM, and 200 µM). The formation of amyloid fibrils was monitored at 0, 2, and 6 h.
Nuclear Magnetic Resonance Spectroscopy--
To further explore
structural changes of A
by melatonin we performed one-dimensional
1H NMR spectroscopic studies on A
1-40. The NMR approach
has the distinct advantage of being able to specifically locate the
amino acid side chains that bind to a particular ligand (41). The solution conditions for the NMR and CD studies were similar, except that deuterated water (D20) in phosphate buffer was used in
the NMR study. All 1H NMR spectra were obtained at 600 MHz
using a Varian UnityPlus-600 spectrometer, and the data were processed
using the FELIX program (version 95.0, Biosym, Inc.). The NMR solutions
were prepared in D20 (0.6 ml) with sodium phosphate buffer
(5 mM, pH 7.5), perdeuterated Na2EDTA (0.5 mM), NaN3 (0.05 mM), and
3-(trimethylsilyl) propionate-2,2,3,3-d4 (0.05 mM), the last of which serves as an internal chemical shift reference at 0 ppm. The NMR measurements were performed at 10 °C and
the residual protium absorption of D20 was suppressed by
low power irradiation during the recycle delay. For all spectra, 128 scans were required with a total recycle delay of 4.2 s, which included an acquisition time and recycle delay of 2.2 and 2.0 s,
respectively. The digital resolution of the acquired data was 0.24 Hz/pt, which was reduced to 0.12 Hz/pt by zero-filling the data once
before processing. To further improve the resolution, before Fourier
transformation spectra were multiplied by a Lorentzian-to-Gaussian weighting factor. This experiment was duplicated on two different days.
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RESULTS |
Circular Dichroism Studies--
As expected, the content in
-sheet conformation of A
1-40 incubated alone increased over time
from 52% at time 0 to 66% after 24 h at 37 °C (Fig.
1A). These results are in
qualitative agreement with previous work (42). The relative proportion
of the structures was dramatically changed by addition of melatonin to
sister tubes. At time 0, there was an immediate increase of the random
conformation, whereas the original
-sheet content markedly
diminished (Fig. 1A, left panel). This effect was
not observed with NAT or PBN. The amount of
-sheet structures for
A
1-40 plus melatonin decreased over time, reaching 24% after
24 h of incubation (Fig. 1A, right panel).
At 24 h, no structural changes were again detected in control
experiments with the melatonin analog NAT, and only small effects were
observed with PBN. Experiments with the more amyloidogenic A
1-42
showed qualitatively similar results (Fig. 1B). Melatonin caused an immediate reduction in the amount of
-sheet structures at
time 0 from 89 to 65% (Fig. 1B, left panel).
This percentage continued to decrease to 59% after 4 h of
incubation (Fig. 1B, right panel). As observed
with A
1-40, such striking structural changes were not elicited in
parallel control preparations containing A
1-42 plus NAT or PBN.

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Fig. 1.
Circular dichroism studies of A 1-40
(A) and A 1-42 (B) alone or in the presence
of melatonin. Spectral tracings were generated as described under
"Materials and Methods" and expressed in terms of mean residue
ellipticity in units of deg cm2 dmol 1 after
substraction of buffer base-line spectra (including melatonin, NAT, or
PBN when indicated) and smoothed by a computer assisted algorythm
provided by Jasco Co. The curves designate the spectra of
A alone (solid line), A plus NAT (short
dashes), A plus PBN (long dashes), or A plus
melatonin (short and long dashes). Left
panels indicate results obtained at time 0; right
panels show the values obtained at 24 h for A 1-40 and
4 h for A 1-42. The corresponding percentages of the different
secondary structure motifs are shown in the tables below each
respective tracing. An average of 40 scans/independent experimental
condition was obtained. An independent experiment yielded qualitatively
similar data.
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Electron Microscopy Studies--
Results of the ultrastructural
studies reflected the conformational changes and supported the
hypothesis that formation of
-sheet structures precede
fibrillogenesis (32, 33). In three independent experiments, fibrils
were abundant in the tubes containing A
1-40 alone incubated for
36 h. In contrast, no fibrils were detected for solutions of
A
1-40 plus melatonin up to 48 h (Fig. 2). Notably, fibrils were abundant and
easily identifiable in the tubes incubated for 48 h containing
A
alone, A
plus NAT, or A
plus PBN but not in the tubes
containing A
plus melatonin. Such a contrasting finding suggests
that the methoxy group at position 5 of the indolamine nucleus of
melatonin confers relative structural specificity to the observed
phenomena.

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Fig. 2.
A 1-40 fibril formation in the presence or
the absence of melatonin. A 1-40 incubated for 48 h either
alone (A) or with NAT (B), PBN (C), or
melatonin (D). Bar, 200 nm. Well formed amyloid
fibrils are easily recognized in A, B, and
C. Fibrils were not formed in D. EM grids were
extensively and carefully examined, and a negative result was only
documented when fibrils were totally absent from the grids.
These results were reproduced in three independent experiments.
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Fibril formation was inhibited in all tubes containing A
1-40 plus
melatonin at concentrations above 10 µM. To substantiate the negative results obtained with PBN, three different concentrations of this scavenger (10, 100, and 200 µM) were added to
tubes containing A
1-40, incubated for 48 h, and then examined.
Fibrils were abundant in all these tubes. In contrast, only amorphous
material was again identified in control sister tubes containing 250 µM A
1-40 plus 100 µM melatonin.
In the experiments with A
1-42, fibrils were identified in the tubes
containing the peptide alone incubated after 2 and 6 h (no fibrils
were seen at time 0). In contrast, only amorphous material was
identified in the tubes containing A
1-42 plus melatonin at these
time points (Fig. 3). All the
concentrations of melatonin used in the experiments with AB1-42 (see
"Materials and Methods") were effective to inhibit fibril
formation.

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Fig. 3.
A 1-42 fibril formation in the presence or
the absence of melatonin. A 1-42 was either incubated alone
(A) or with melatonin (B) as described under
"Materials and Methods." Fibrils were only found in the tubes
containing A 1-42 alone after 2 h of incubation (not shown) and
after 6 h of incubation (A). Only amorphous material
was seen in the tubes containing A 1-42 alone immediately after
dissolution (time 0, not shown) or containing A 1-42 plus melatonin
at the indicated time points and with a range of melatonin
concentrations. In (B), amorphous material as seen at one of
the concentrations of melatonin used is representatively illustrated
(in this picture, melatonin concentration was 100 µM and
incubation time was 6 h). Bar, 200 nm.
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Nuclear Magnetic Resonance Spectroscopy--
Shown in Fig.
4 are the downfield spectral regions for
the A
1-40 peptide, melatonin (Fig. 4E) and the A
1-40
with 0.4, 0.8, and 1.2 molar equivalents of melatonin (Fig. 4,
B-D). The three well resolved His-2H and His-4H signals are
consistent with the A
1-40 peptide being partly folded into an
ordered structure, which according to the CD studies should be
-sheet and random coil. If only random coil structure were present,
then degenerate signals should be present for His6,
His13, and His14 (43).

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Fig. 4.
Downfield 1H NMR spectral region
(600 MHz) of 0.25 mM A 1-40 peptide (A) and
5.0 mM melatonin (E), with the chemical
structure of melatonin provided above the upper plot.
The spectra in B, C, and D contain
0.25 mM A 1-40 plus 0.1, 0.2, and 0.3 mM
melatonin, respectively. Assignments for the aromatic signals of
melatonin and the A 1-40 peptide are shown, and those resonances
exhibiting changes in shifts are connected by dotted lines.
A duplicate independent experiment showed virtually identical
results.
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The NMR spectra of the mixtures of melatonin and A
1-40 show changes
in chemical shifts indicative of binding and local conformational changes. The His-2H and His-4H signals shift downfield 0.05 and 0.02 ppm, respectively, whereas the aromatic peaks of melatonin also shift
downfield (Fig. 4). In addition, careful analysis of the upfield
spectral region (spectra not shown) revealed downfield shifts for the
Asp
CH2 groups (Table I).
Control NMR experiments with NAT showed only minor chemical shift
perturbations (± 0.01 ppm), suggesting a specificity for the
interaction of melatonin with A
. The lack of any line broadening or
separate peaks for the bound and free states indicates that the binding
is in the fast exchange limit (41). The downfield shifts can be
interpreted in terms of ring-current shift contributions, with the
shifted hydrogens becoming located in the planes of the melatonin and His aromatic ring (44). A remarkable feature is the identical shift
seen for each of the three His residues. This result together with the
observed non-linearity of the shifts with varied melatonin concentrations suggests that the binding is not localized to a particular His site on A
. Instead, the chemical shift changes are
consistent with a residue-specific interaction between melatonin and
any of the three His and Asp residues of A
.
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Table I
Titration studies of A (1-40) peptide with melatonin
All solutions contained 5 mM sodium phosphate buffer in
D2O, pH 7.5, at 10 °C with the chemical shifts referenced to
internal 3-(trimethylsilyl) propionate-2,2,3,3-d4.
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DISCUSSION |
Melatonin has a proposed role in the aging process (45, 46).
Decreased secretion of this hormone during aging is well documented
(47, 48), and more profound reductions are reported in populations with
dementia (49, 50). The reported lack of toxicity of melatonin and the
ease and rapidity with which this molecule crosses the blood-brain
barrier following oral administration (51) makes it a prime candidate
for experimental testing in humans. This hormone has been administered
to human subjects at very high doses (i.e. 1 g/day) without
any clinically significant toxicity (52). Our data clearly indicate
that under the conditions tested, melatonin modifies the secondary
structure of the A
peptide and inhibits the formation of amyloid
fibrils.
These newly found anti-amyloidogenic properties of melatonin are very
rare for endogenous substances. Because of the relationship between
oxidative stress and AD and the recently established antioxidant properties of this hormone, it was initially thought that the neuroprotective actions of melatonin were mostly due to its
intracellular antioxidant effects (36). However, the results presented
here suggest that the anti-amyloidogenic properties are dependant on structural interactions of the hormone with A
rather than on antioxidant properties exclusively. The His and Asp residues play important roles in
-amyloid fibril production and stability. Many
physiological constituents such as transthyretin and zinc can prevent
or promote aggregation by their affinities for the His residues of A
(53, 54). Additionally, imidazole-carboxylate salt bridges between the
side chains of the His+ and the Asp
residues
are critical to the formation of the amyloid
-sheet structures (12,
55-57). More significantly, disruption of these salt bridges promotes
fibril dissolution (58). One possibility is that melatonin promotes the
-sheet
random coil conversion by disruption of the
His+-Asp
salt bridges. Alternatively, the
described effects may result from a unique combination of structural
and antioxidant features of this molecule. More experiments are
necessary to clarify this interpretation and dissect the relationship
between cytoprotection, changes in peptide structure, and antioxidant
characteristics. The antioxidant properties of melatonin may provide
additional cytoprotection at the intracellular level (36).
The ratio melatonin:A
used in these studies is within physiologic
range, because the concentration of both substances in the brain are
normally around 1:1 during youth (both substances are at comparable
picomolar concentrations in brain tissue during the dark phase of the
cycle (59, 60)). However, limitations of the methods employed required
concentrations of melatonin and A
that deviate from actual
physiological conditions.
At this time, no information is available about the possible
therapeutic or preventive values of melatonin or of its potential efficacy at physiologic or pharmacologic dosages. It would be premature
to conclude that a subgroup of AD is caused by an age-related deficiency of this hormone, although such a possibility is nonetheless intriguing. The results reported here suggest that melatonin can provide a combination of antioxidant and anti-amyloidogenic features that can be explored either as a preventive or therapeutic treatment for AD or as a model for development of anti-amyloidogenic indole analogs.