(Received for publication, April 17, 1995; and in revised form, August 17, 1995)
From the
-Amyloid protein, which assembles into pathological
aggregates deposited in Alzheimer's disease brain tissue,
exhibits N-terminal heterogeneity both in vitro and in
vivo. To investigate the effects of this N-terminal heterogeneity
on the assembly characteristics and biophysical properties of
-amyloid, we synthesized a series of peptides with progressively
shortened N termini (initial residues at positions
1,
4,
8,
12, and
17) and C termini extending to residue
40 or
42. We report that peptides with N-terminal deletions
exhibit enhanced peptide aggregation relative to full-length species,
as quantitatively assessed by sedimentation analyses. Overall,
sedimentation levels were greater for peptides terminating at residue
42 than for those terminating at residue
40. To determine if
established biophysical features of the full-length protein were
maintained in the truncated peptides, structural and bioactive
properties of these peptides were examined and compared. Full-length
and truncated peptides exhibiting aggregation showed circular dichroism
spectra consistent with predominant
-sheet conformation, fibrillar
morphology under transmission electron microscopy, and significant
toxicity in cultures of rat hippocampal neurons. These data demonstrate
that N-terminal deletions enhance aggregation of
-amyloid into
neurotoxic,
-sheet fibrils and suggest that such peptides may
initiate and/or nucleate the pathological deposition of
-amyloid.
-Amyloid (A
) (
)is a normal, soluble protein
40-42 amino acid residues in length that, in neuropathological
conditions such as Alzheimer's disease (AD), self-assembles into
insoluble fibrils, forming characteristic extracellular deposits termed
senile plaques(1) . Since a variety of histological, molecular
genetic, and in vitro and in vivo studies provide
evidence consistent with the possibility that A
significantly
contributes to the initiation and/or progression of neurodegenerative
changes in AD(2, 3, 4) , investigation of the
production, assembly, and bioactivity of A
is crucial for the
successful understanding of and therapeutic intervention in AD and
related disorders.
A is derived from proteolytic processing of
its precursor, A
precursor protein (A
PP), by least two
distinct and incompletely defined pathways (see (5) for
review). In the secretory pathway, transmembrane A
PP is cleaved by
-secretase between
16 and
17(6) , thus
precluding the formation of full-length A
(
1-40/42) but
generating a 3-kDa
17-40/42
fragment(7, 8, 9, 10) . In the
endosomal/lysosomal pathway, A
PP is degraded into several
C-terminal fragments containing the complete A
sequence(11, 12) that require additional cleavage at
the A
amino (
-secretase) and carboxyl (
-secretase)
termini to generate A
. Although the predominant form of A
contains the
1-40 sequence, significant N- and C-terminal
heterogeneity has been reported recently in studies of both cell
culture (7, 9, 13, 14, 15) and
human fluids and
tissues(13, 16, 17, 18, 19, 20, 21, 22, 23) ,
suggesting multiple forms of A
that vary in primary structure by a
few to several amino acids.
Previous studies suggest that C-terminal
heterogeneity of A has profound effects upon the initiation and
progression of AD. Specifically, increased length of the hydrophobic C
terminus both enhances in vitro aggregation of A
(24, 25, 26) and appears to promote early
deposition of plaque A
in AD brains(27) . In addition, an
increased relative production of longer C-terminal forms of A
,
demonstrated both in vitro(14) and in AD brain (28) , appears to underlie the pathologic action of
A
PP
mutations linked to early onset familial AD.
The effects of N-terminal heterogeneity of A on its assembly
and bioactivity characteristics are not well defined. The significance
of this issue is underscored by recent data suggesting that early stage
plaques may be composed primarily of A
peptides with truncated N
termini(23, 29) . The possibility of enhanced
amyloidogenicity of A
peptides with truncated N termini is
consistent with a previous study that reported decreased in vitro solubilities of synthetic A
peptides
8-,
9-, and
10-43 relative to
1-,
2-, and
4-43(30) .
In order to examine
the effects of A N-terminal heterogeneity on both peptide assembly
and bioactivity, we have synthesized two series of A
peptides with
progressive N-terminal deletions (beginning at positions
1,
4,
8,
12, and
17), one of which terminates at
residue
40 (
n-40 series) and the other at
42 (
n-42 series). These A
peptides have
been examined for their rates and levels of peptide assembly, fibrillar
ultrastructures under electron microscope, secondary structures by
circular dichroism, and bioactivities with cultured neurons.
Given the N- and C-terminal heterogeneity of A isolated
from AD brain tissue(16, 17, 18, 19, 20, 21, 22, 23) as
well as the demonstrated profound influence of C-terminal heterogeneity
on A
assembly in vitro(24, 25, 26) and apparently in vivo(27) , the
potential influence of N-terminal heterogeneity on A
assembly is
an intriguing possibility that may significantly contribute to the
pathological deposition of A
. In this study, we sought to
determine (i) how the length of the N terminus influences the overall
levels and kinetics of A
aggregation and (ii) whether aggregates
of N-terminal truncated A
retain the biophysical properties of
full-length A
.
To investigate the influence of N-terminal
deletions on A assembly properties, we solubilized the
n-40 and
n-42 peptides and
quantitatively determined the levels of peptide aggregates at various
time points by sedimentation assay. As illustrated in Fig. 1A, the
n-40 peptides
exhibited initially low levels of sedimentation, which gradually
increased over 7 days. This progressive increase in peptide aggregation
is consistent with previous observations of the time-dependent nature
of A
aggregation(25, 26, 31) .
Figure 1:
A peptides with N-terminal
deletions exhibit enhanced sedimentation. A,
n-40 peptides show negligible levels of sedimentation at
day 0 that gradually increase by day 7. B,
n-42 peptides exhibit relatively high sedimentation
levels, which reach maintained, maximal values by day 2. Means
(±S.E.) of triplicate values are shown. * denotes significant (p < 0.05) peptide sedimentation relative to
non-centrifuged peptide samples.
In
comparison to the n-40 series, the
n-42 peptides exhibited more rapid and extensive
assembly as evidenced by high initial sedimentation and early
attainment of maintained peak values; maximal levels were reached
within 2 days for
1-,
4-, and
8-42 and
by the initial time point (i.e. within minutes of
solubilization) for
12- and
17-42 (Fig. 1B). The significantly greater amounts of
sedimentable aggregates and more rapid kinetics observed in the
n-42 relative to the
n-40
peptides are consistent with the conclusions of previous studies that
the length of the C terminus is a critical variable in A
assembly(24, 25, 26, 31) . More
importantly, we observed a general trend in the
n-42 series of increasing levels of sedimentation
with decreasing length of N terminus, with the exception of increased
sedimentation levels for
8-42 at the 2- and 7-day time
points; a similar relationship was also apparent in the
n-40 peptides. Note that in both series of
peptides, the greatest levels of sedimentation were observed in those
peptides beginning at either residue
8 or
17.
The
increased relative aggregation of A peptides with N-terminal
deletions shown by the above data suggests that these and/or related
peptides may be the initial A
species deposited in senile plaques;
recent observations in AD brain support this
possibility(27, 29) . Interestingly, A
deposited
in early stage or diffuse plaques generally lacks the
-sheet
fibrillar structure and associated degenerating neurites characteristic
of classic senile plaques. Thus, we sought to determine whether
N-terminal deletions might affect previously defined bioactive and
structural properties of full-length A
, including neurotoxicity,
-sheet conformation, and fibrillar structure.
Our previous
studies have demonstrated that the presence of peptide aggregates in
solutions of full-length A accurately predicts significant
neurotoxic activity(31, 35, 36) . To
determine whether this assembly/toxicity relationship also
characterizes A
peptides with N-terminal deletions, we examined
potential neurotoxic activities of the
n-40 and
n-42 peptides in cultured hippocampal neurons.
Within the
n-40 peptide series, neurotoxicity
induced by the truncated peptides was greater than that induced by
1-40 (Fig. 2A). Neurotoxicity generally
increased within the peptide series between the 0- and 7-day time
points, a trend that parallels the sedimentation data.
Figure 2:
A peptides with N-terminal deletions
induce significant neurotoxicity in cultures of hippocampal neurons. A,
n-40 peptides induce the greatest cell
loss at day 7, the time point associated with maximal sedimentation (Fig. 1A). Note the enhanced toxicity of
n-40 deleted peptides relative to
1-40. B,
n-42 peptides cause the greatest cell
loss at day 0, a time point associated with maximal or near maximal
sedimentation values (Fig. 1B). At day 7, all
n-42 peptides induce comparable but diminished
levels of toxicity. * denotes significant (p < 0.05)
differences in cell viability relative to untreated controls;** denotes
significant (p < 0.05) differences relative to
1-42 at the day 0 time point.
Like the
n-40 peptides, the
n-42
peptides also exhibited significant neurotoxicity (Fig. 2B). The greater toxic activities of the
n-42 series may reflect in part their higher
sedimentation levels. Toxicity of
n-42 peptides was
generally greatest at day 0 and showed a significant decrease in
intensity with decreasing length of the peptide N termini. At the day 7
time point, neurotoxicity was comparable between the different
n-42 peptides but generally reduced within
individual peptides relative to the initial time point. The decreased
neurotoxicities, both across the peptide series at day 0 and within
individual peptides over the two time points, may reflect higher order
assembly (i.e. aggregation of A
oligomers) of these
A
peptide aggregates resulting in a corresponding decrease in
aggregate-cell interactions, in agreement with previous
observations(37) . Thus, although the property of neurotoxicity
for individual A
peptides is predicted well by their tendency to
form sedimentable peptide aggregates, the relationship between these
two factors does not necessarily exhibit a strict quantitative
correlation(31) .
In addition to neurotoxicity, aggregating
A peptides are also characterized by specific structural features,
including
-sheet secondary structure (24, 37) and
fibrillar
morphology(25, 38, 39, 40) ; like
aggregation measures, these factors have been demonstrated to be
associated with A
neurotoxicity(32, 37, 41) . The following
structural studies focused on the
n-42 series,
since these peptides not only exhibited significantly higher levels of
sedimentation and neurotoxicity than the
n-40
series but also have been demonstrated to be the initial A
species
deposited within AD plaques(27, 29) .
Examination
of CD spectra was used to provide qualitative information regarding the
secondary structure of n-42 peptides in solution.
Predominant
-sheet structure is recognized by a single negative
peak near 218 nm, as opposed to the doublet of negative peaks near 208
and 222 nm observed in
-helical structure; both structures exhibit
a single positive peak near 200 nm(42, 43) . An
absence of ordered structure, random coil, is indicated by a single
negative peak near 198 nm. Like
1-42, the truncated
n-42 peptides exhibit characteristics of
predominant
-sheet structure (Fig. 3). Although the CD
spectra of all the
n-42 peptides are similar,
8-42 and
17-42 exhibit slightly greater negative
peaks than the other
n-42 peptides; such enhanced
peak values within predominant
-sheet solutions suggest higher
-sheet content resulting from a reduced proportion of random
coil(43) .
Figure 3:
n-42 peptides exhibit
CD spectra characteristic of predominant
-sheet conformation. The
CD spectrum of each
n-42 peptide shows a positive
peak near 200 nm and a single negative peak near 218 nm, defining
characteristics of
-sheet secondary
structure.
These CD observations verify that, like
full-length 1-42, the truncated
n-42
peptide assemblies exist in the predominantly
-sheet conformations
characteristic of amyloid fibrils. In addition, the CD spectra are
consistent with the increased amyloidogenicity of A
peptides with
N-terminal deletions suggested by the sedimentation data. In
particular, the
8-42 and
17-42 peptides exhibited
both the greatest sedimentation levels and the highest apparent
-sheet content. The enhanced relative amyloidogenicity of these
peptides likely reflects the influences of altered primary structure in
promoting and/or stabilizing the
-sheet conformation underlying
A
fibril formation; this conclusion is consistent with recent data
demonstrating significant contributions by N-terminal residues to
A
amyloidogenicity(44) . Further studies are required to
determine the relevant contributing factors (e.g. increased
hydrophobicity, improved interstrand registration).
In order to
compare further the amyloidogenic nature of the truncated
n-42 peptides relative to full-length
1-42, we examined their aggregate structures under electron
microscopy. Previous studies have shown that several synthetic A
peptides form 5-10-nm diameter fibrils in
vitro(25, 38, 39, 40, 41) ,
which are morphologically similar to A
fibrils from AD
plaques(38, 45) . We observed that all truncated
n-42 peptides exhibited negatively stained fibrils
with morphological features similar to
1-42 and consistent
with previous observations of A
fibrils (Fig. 4).
Interestingly, although
17-42 fibrils usually exhibited
morphology comparable with fibrils of the other
n-42 peptides,
17-42 fibrils in some
samples appeared to be relatively shorter in length and narrower in
diameter (data not shown). Similar discrete populations of relatively
wide and narrow A
fibrils have been observed in plaque core
preparations (45) . In addition,
17-42 fibrils were
organized into exceptionally large, dense meshworks (Fig. 4),
consistent with observations of
17-40 fibrils recently
reported by Näslund et al.(46) .
However, these authors also reported (46) that
17-40
aggregates lack the positive thioflavine staining characteristic of all
amyloids. In contrast, using our thioflavine assay(32) , we
have observed that aggregates formed by all the
n-40 and
n-42 peptides exhibit
positive thioflavine staining. (
)
Figure 4:
n-42 peptides exhibit
fibrillar morphology by transmission electron microscopy. A,
negatively stained fibrils formed by all
n-42
peptides exhibit similar morphology under electron microscopy that is
typical of amyloid; representative fibrils from
8-42 are
shown here. B, fibrils formed by
17-42 typically
are organized into a relatively dense meshwork. Scale bar, 25
nm.
In this paper, we have
provided experimental evidence demonstrating a significant influence of
N-terminal length on A peptide assembly. Specifically, A
peptides with N-terminal deletions exhibit enhanced levels of
aggregation in comparison with full-length A
peptides, as
quantitatively assessed by sedimentation assay. Despite the variable
absence of N-terminal residues, the truncated peptides retain the
neurotoxicity and
-sheet, fibrillar structure associated with
aggregated full-length A
. The structure/activity relationship
suggested by these data may impact current concepts regarding the
pathogenic potential of A
peptide fragments. The prevailing
doctrine within the literature has been that
-secretase cleavage,
which cleaves A
PP between A
residues
16 and
17(6) , is the preferred proteolytic pathway in terms of
minimizing disease potential since it generates the theoretically
benign
17-40/42 as opposed to the more pathologic
1-40/42. In contrast to this position, our data predict that
17-40/42 is actually more likely than
1-40/42 to
assemble into deposits in vivo and that the shortened peptide
retains significant neurotoxic activity. Consistent with the idea of
enhanced amyloidogenicity, recent data by Gowing et al.(29) suggest that
17-42 is a primary component
of early stage, diffuse plaques. We suggest that
17-42 and
other prevalent
n-42 peptides may initiate and/or
accelerate plaque formation, perhaps by acting as nucleating centers
that seed the subsequent deposition of relatively less amyloidogenic
but apparently more abundant full-length A
; similar seeding events
have been described previously for A
peptides in
vitro(26) .
In summary, the present findings predict
that N-terminal heterogeneity of A peptides, demonstrated to occur
both in vitro(7, 9, 13, 15) and in AD
brain(16, 18, 21, 22, 23) ,
may accelerate A
deposition into plaques. Thus, proteolytic events
contributing to the cleavage of A
PP within the N-terminal domain
of A
(e.g. activities of
- and
-secretases) may
be of considerable significance in the pathogenesis of AD and related
disorders.