(Received for publication, August 6, 1996, and in revised form, December 6, 1996)
From the Institute for Brain Aging and Dementia, Departments of
Psychobiology and Neurology, University of California
Irvine, Irvine, California 92697-4540
The amyloidogenic peptide -amyloid has
previously been shown to bind to neurons in the form of fibrillar
clusters on the cell surface, which induces neurodegeneration and
activates a program of cell death characteristic of apoptosis. To
further investigate the mechanism of A
neurotoxicity, we synthesized the all-D- and all-L-stereoisomers of the
neurotoxic truncated form of A
(A
25-35) and the
full-length peptide (A
1-42) and compared their physical
and biological properties. We report that the purified peptides exhibit
nearly identical structural and assembly characteristics as assessed by
high performance liquid chromatography, electron microscopy, circular
dichroism, and sedimentation analysis. In addition, both enantiomers
induce similar levels of toxicity in cultured hippocampal neurons.
These data suggest that the neurotoxic actions of A
result not from
stereoisomer-specific ligand-receptor interactions but rather from A
cellular interactions in which fibril features of the amyloidogenic
peptide are a critical feature. The promiscuous nature of these
-sheet-containing fibrils suggests that the accumulation of
amyloidogenic peptides in vivo as extracellular deposits
represents a site of bioactive peptides with the ability to provide
inappropriate signals to cells leading to cellular degeneration and
disease.
Alzheimer's disease (AD),1 vascular
dementia, and hereditary cerebral hemorrhage with the Dutch type are
diseases that share an invariant pathological feature, the accumulation
of an amyloidogenic peptide into insoluble fibrillar extracellular
deposits. In all three cases, the major component of the extracellular
debris is the -amyloid peptide (A
) that is derived from the
proteolytic processing of the large membrane-anchored amyloid precursor
protein (APP) encoded by a single gene located on chromosome 21 (1). However, the biological significance of these amyloid deposits has been
extensively debated as to whether they are a causative factor of each
disease or merely a metabolically inert end product lacking in
biological activity. Evidence in support of a causative role for A
in neuropathology comes from genetic analysis of the APP gene where
several autosomal dominant mutations have been linked with AD and
hereditary cerebral hemorrhage with the Dutch type (2, 3). In a recent
in vitro study, the
-APP717 mutation consistently caused a significant increase in the percentage of the
longer and more amyloidogenic A
1-42 over the shorter A
1-40 (4). Incorporation of this same mutation into a
transgenic mouse model yields A
deposition and neuropathology that
closely parallels that observed in AD (5). Additional in
vivo evidence suggesting that the A
peptide itself may be biologically active comes from a transgenic model overexpressing A
1-42, in which A
transgene expression was detected
in a variety of peripheral tissues but histopathological changes were
restricted to the brain. Moreover, the neurodegeneration was largely
limited to the cerebral cortex, hippocampus and amygdala, all
areas affected in AD, and was essentially undetectable in the
cerebellum, which is typically not affected in AD (6). Finally, the
amount of
-amyloid that accumulates in the brain appears to
correlate well with the decline of brain function (7).
Insights into the inherent biological activity associated with A
have come from in vitro studies that show synthetic A
can spontaneously assemble into
-sheet-containing fibrils (8, 9), that
this fibrillar-A
can induce neuritic dystrophy in neuronal cultures
similar to that seen in the AD brain, and that the mechanism of
A
-triggered degeneration is via programmed cell death (PCD) (10).
These observations have led to the general hypothesis that the
biological activity of A
is dependent on its transformation into a
highly stable protease-resistant antiparallel
-sheet conformation
and higher order quaternary assemblies (11) similar to those found in
senile plaques. This is of fundamental importance because it suggests
that the biological activity of A
is dependent on protein
conformation and the transition into this conformation. The
consequences of such a relationship between biological activity and
protein conformation are critical to understanding the role of A
and
other
-pleated sheet protein assemblies such as prion protein in
disease.
In order to understand the degenerative processes induced by A, it
is essential to define the characteristics of A
salient to its
function as a neurotoxic stimulus. In a previous study, we used a
series of synthetic A
peptides with progressively truncated C-termini to demonstrate that the length of this hydrophobic region is
a crucial determinant of peptide ability to both aggregate and induce
neurotoxicity in vitro (12). We have also synthesized a
series of truncated A
peptides to examine the effects of N-terminal heterogeneity, which occurs in vivo on the assembly and
biological activity of A
. The N-terminal truncated isoforms produced
enhanced aggregation into neurotoxic
-sheet fibrils, which suggests
that these truncated peptides may initiate the pathological
neurodegeneration in AD by acting as a nucleation site for A
deposition (13). Thus far, we have observed that assembled, bioactive
A
peptides exhibit
-sheet structure and that amino acid
substitutions that disrupt A
assembly also prevent
-sheet
structure and abolish toxicity (14). Further analysis of peptides will
be useful in elucidating the specific requirements for both the
assembly and bioactivity of A
.
Since numerous ligand-target interactions are stereospecific, one means
to both examine the nature of the A-cellular interactions and assess
the validity of several proposed mechanisms of A
-induced cell death
is to determine whether A
bioactivity exhibits stereospecificity. Similar issues of ligand stereospecificity have been investigated in
several recent studies by comparing the binding and or activities of
D- and L-enantiomers of small peptide ligands
(15, 16). In the current study, we have utilized a comparable paradigm, synthesizing the all-D- and all-L-amino acid
stereoisomers of the truncated biologically active
A
25-35 and the full-length A
1-42
peptide, and compared their physical and biological properties to
determine whether the interaction of A
with biologically relevant
cells is stereospecific.
A1-42,
A
25-35 (GSNKGAIIGLM), and scrambled sequence
A
25-35 (12) were synthesized from either
all-D- or all-L- amino acids using solid phase
Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acid chemistry
and purified by reverse phase HPLC, as described previously (9). The
purified peptides were then routinely analyzed by electrospray mass
spectroscopy (9). The all-D-A
25-35 and
-A
1-42 enantiomers gave mass values of 1060.4 and
4513.9, respectively. HPLC analysis of the
all-D-enantiomers produced identical elution times to the
all-L-A
peptides, and mixtures of
all-D-A
1-42 and
all-L-A
25-35 produced a single peak by
HPLC. Peptides were solubilized in sterile double deionized water as
2.5 mM stock solutions and allowed to aggregate at room temperature for at least 1 h before using. Aliquots of the peptide stocks were then diluted with an equal volume of 2× Dulbecco's modified Eagle's medium (DMEM) and then with DMEM supplemented with
N-2 (17) just prior to treating the cultures.
The mean residue ellipticity of
A25-35 peptides (25 µM in 5 mM potassium phosphate, pH 7.3) was determined using a Jasco J-720 spectropolarimeter equipped with a computerized data processor, as described previously (14). Samples were loaded into a
1.0-cm path length quartz cell and measured over a 190-250-nm wavelength range at 0.5 nm increments. Data from eight scans were averaged and subtracted from base-line values but otherwise are unsmoothed. The instrument was calibrated with a 0.06% (w/v) solution of d-camphorsulfonate.
For ultrastructural analysis, 25 µM samples of A peptides (20 mM MOPS
buffer, pH 7.4) were adsorbed onto 200 mesh formvar grids and stained
with 2% uranyl acetate prior to viewing with a Zeiss 10CR transmission
electron microscope at 80 kV transmission (14).
Levels of peptide aggregation were
quantitatively determined using a sedimentation assay previously
described (14). Briefly, A25-35 peptides (25 µM in 20 mM MOPS, pH 7.3) were
ultracentrifuged for 1 h at 100,000 × g.
Supernatant peptide concentrations between centrifuged and
non-centrifuged samples were compared by fluorescamine assay. Decreased
supernatant peptide concentration in centrifuged samples was used as a
measure of the aggregated peptide fraction. Analyses were conducted in
quadruplicate samples.
Cultures of hippocampal neurons from
gestational day 18 rat pups were prepared as described previously (12).
Cultures were plated at 2.5 × 104
cells/cm2 on poly-L-lysine-treated multiwell
plates and maintained in serum-free DMEM supplemented with N-2
components. After two days in vitro, cultures were exposed
to the various A peptides for 24 h, after which cell viability
was determined on the basis of trypan blue exclusion (12, 18). Raw data
were statistically compared by analysis of variance followed by
Scheffé f-test.
The initial experiments to investigate whether an
all-D-enantiomer of an amyloidogenic peptide retains
biological activity were performed with
all-D-A25-35. This is the smallest commonly
studied fragment of A
that retains both the ability to form
-sheet-containing fibrils and neurotoxicity (19). An additional
advantage of A
25-35 is that it can be modeled relatively easily, and information on the alignment of the antiparallel strands as well as the importance of various side chain interactions in
formation of the A
fibrils and in defining the surface topography necessary for neurotoxicity can be investigated. In the antiparallel
-sheet conformation, the surface topography is determined by the
amino acid sequence and the alignment of adjacent strands in the
-sheet. In a computer-generated model of A
25-35 in
which the two peptides are maximally overlapped, a cluster of
positively charged lysine residues is observed on one face of the
-sheet while the other face of the
-sheet contains primarily hydrophobic residues. Previous studies with A
25-35
containing single amino acid substitutions have confirmed the
importance of the sequence for retaining the properties of A
(14).
Two separate lots of the all-D-A25-35 were
synthesized to control for lot to lot variability in the biological
activity of the peptides. Comparison of the all-D and
all-L forms of A
25-35 by electrospray mass
spectroscopy gave essentialy identical mass values, and HPLC analysis
showed that the two enantiomers had similar elution times. Mixed
samples of both enantiomers eluted as a single peak from the HPLC (Fig.
1A). All-D-A
25-35 was allowed to assemble in parallel with the
all-L-A
25-35, and then both peptides were
subjected to a series of commonly used assays to monitor the properties
of fibrillar A
. Both enantiomers rapidly produced visible aggregates
in aqueous solution (Fig. 2, A-D),
and analysis of negatively stained specimens by electron microscopy
showed similar fibrillar structures (Fig. 1, B and C). Sedimentation analysis was then used to compare the
extent of peptide assembly for both enantiomers. Three different lots of the all-L-A
25-35 and two lots of the
all-D-A
25-35 were used, and both forms gave
similar results (data not shown). The above results show that both
peptides have similar physical properties and can only be distinguished
by CD analysis where mirror image spectra are generated (data not
shown).
The biological properties of the two enantiomers were then tested by
applying the enantiomers to primary cultures of rat hippocampal and
cortical neurons that have previously been used to assay the neurotoxic
activity of A (12, 18). The all-D-A
25-35 produced visible aggregates in the tissue culture wells and appeared to
bind to the surface of neurons equally as well as the
all-L-A
25-35 (Fig. 2,
A-D). Noticeable neuronal degeneration was
apparent at 12 h, and extensive cell death was observed at 24 h for both enantiomers. In order to compare the levels of neurotoxicity
between the two enantiomers, a dose response curve was generated. As
can be seen in Fig. 2E, the
all-D-A
25-35 produced similar toxicity to
the all-L-A
25-35 over the entire range of
concentrations tested. The specificity of the neurotoxicity was
determined by analyzing peptides with scrambled sequences of both the
all-L and the all-D enantiomers. Neither
scrambled sequence produced detectable neurotoxicity over the entire
range of concentrations utilized for the dose response curve (data not
shown).
During the comparison of computer-generated models of
A25-35 in antiparallel
-sheet conformation, we
discovered, that with perfect alignment of the individual strands of
peptide, that a pseudo-axis of symmetry was generated do to the planar
nature of the
-sheet such that the distribution of the surface
groups produced topochemically similar enantiomers. Other cases of
topochemically similar peptides that bind to stereoselective receptors
and posses similar activities have been reported (20, 21). Based on the high level of bioactivity associated with the
all-D-A
25-35, we next determined whether
the all-D-enantiomer of the full-length A
1-42 peptide would also bind to cells and produce
similar neurotoxicity to the all-L-A
1-42.
Although the
-sheet-containing fibrils of the A
1-42
are predicted to form planar sheets, the longer length of the
A
1-42 peptide would reduce the probability of formation
of topochemically similar enantiomers since the surface topography of
A
1-42 would be far more complex than with
A
25-35.
Highly purified all-D-A1-42 was subjected
to CD, and the spectra were compared with the
all-L-A
1-42 (Fig.
3A). The all-D-enantiomer
produced the expected mirror image spectra, indicating similar
secondary structure for the enantiomers. The peptides were then
examined by electron microscopy, and, while the filamentous structures
were different from those observed with A
25-35, the
A
1-42 enantiomers produced fibrils that were
indistinguishable from each other (Fig. 3, B and
C). The ability to bind certain dyes, such as Congo red and
thioflavine T, is a characteristic property of amyloidogenic peptides
(22-24) and can be used to measure the amount of peptide in
-sheet-containing fibrils (25). Analysis of assembled peptides of
both enantiomers indicates that the
all-D-A
1-42 binds thioflavine with
intensity equal to that of the all-L-A
1-42
(data not shown).
The biological response to the full-length
all-D-A1-42 by neurons was assayed as
described above for A
25-35. The
all-D-enantiomer clearly binds to the neurons since
clusters of the fibrillar A
1-42 can be seen over much
of the cell surface within 6 h of adding the peptide to the
cultures (Fig. 4, C and D).
Perhaps more importantly, the fibrillar clusters induce extensive
neurodegeneration over a 24-h time course. It should be noted that not
all neurons respond equally to fibrillar forms of A
, as previous
studies have shown that neurons that are immunopositive for GABA are
resistant to the neurotoxic activity of A
(26). A comparison of the
relative neurotoxicity of the two A
1-42 enantiomers is
shown in the dose response curves in Fig. 4E. Both
enantiomers produce noticeable neurotoxic activity at 5 µM, and at 25 µM, approximately half of the
neurons are dead within 24 h.
This study was designed to probe the stereospecificity of the
interaction between A and the plasma membrane of cultured neurons that in vitro leads to programmed cell death (10, 27, 28). According to classic receptor pharmacology, a
D-stereoisomer of an amino acid or peptide would not be
predicted to exhibit bioactivity comparable with the native
L-peptide. For example, glutamate receptors readily
discriminate L- versus
D-antagonistic agents (29). We have analyzed both the
physical and biological properties of the all-D-enantiomers
of A
25-35 and A
1-42 and have compared them with their corresponding all-L-enantiomers. With the
exception of the CD spectropolarimetry study, which produced mirror
image spectrums, both all-D-enantiomers exhibited
essentially identical physical and biological properties to their
all-L-enantiomers.
A number of studies have been done utilizing D-enantiomers
of various ligands to investigate the stereospecific requirements for
binding to their respective receptor proteins. In the cases of three
peptide hormones, bradykinin (30), oxytocin (31), and angiotensin (32),
that must interact with chiral receptors on the plasma membrane, the
all-D-forms of the ligands were inactive. However,
different results were obtained in the case of a synthetic -endorphin analog that contained 18 D-amino acid
residues in the C-terminal portion of the peptide but 5 L-residues within the actual binding site. The
all-D-containing region was designed to form a left-handed
amphiphilic helical segment that was topochemically similar to the
native right-handed amphiphilic helix. The D/L chimeric peptide retained equal ability to bind and to activate the
opiate receptor (21). In some cases, the all-D-peptide
enantiomers can still resemble the parent compound, both in the overall
spatial arrangements and with respect to the electronic nature of the functional groups. In the case of the antibiotic enniatin B,
the topochemically similar enantio-enniatin B possessed
similar antimicrobial activity (20). In two recent studies for example,
the all-D-peptide analogs were found to bind with similar
affinities to their respective receptors. In the first one, two
all-D-amphiphilic helical peptides were shown to interact
with calmodulin in a sterically malleable fashion (16, 33), and the
second example reported that the laminin segment containing the IKVAV
amino acid sequence, which is responsible for cell attachment and
tumor-promoting activities, was retained in the
all-D-peptide. Peptide analogs with either alternating
D-L-substitutions or randomized IKVAV sequence
were inactive, indicating that the sequence and conformational status of the domain contribute to the biological activity but that no stereospecific requirement exists (15).
Although bilayer lipids and membranes are also chiral and contain
numerous asymmetric centers, the partitioning of chiral channel-forming
antibiotic peptides into membranes does not require a specific
chirality. The all-D-analogs of cecropin, magainin II
amide, and melittin were equally effective when tested on achiral synthetic planar bilayers and as antibiotics against bacteria containing chiral membranes (34). The reports that
A1-40 forms giant multivalent cation channels when
incorporated into synthetic bilayers (35, 36) and our findings that the
neurotoxic activity associated with both A
25-35 and
A
1-42 are not chirally dependent are consistent with
the results obtained with the channel-forming antibiotic peptides.
Unfortunately, after many years of extensive study on A
, there is no
definitive evidence that A
forms channels in neurons. Another more
likely possibility is that A
is active as a membrane perturbant,
which may alter the microenvironment between the bilayer and
membrane-bound enzymes or receptors.
We currently favor a mechanism dependent on the interaction of A
with membrane receptor proteins on the surface of neurons, and other
cell types such as astrocytes, because assembled fibrillar forms of the
amyloidogenic peptides are required for activity (12, 14, 37). It is
possible, for example, that A
acts as a ligand to cross-link
receptors at the cell surface and activates cell death pathways via
activation-induced cell death similar to Fas (38). Consistent with a
mechanism involving membrane receptors, we have shown that the lectin
ConA, which forms clusters of membrane glycoproteins on the cell
surface, also causes neurodegeneration and apoptotic death in cultured
neurons similar to that observed with A
while succinyl ConA, which
binds but does not cross-link, is inactive (39). Recently, Burdick,
et al. (40) have shown that a substantial portion of the
A
that binds to cells can be removed by treatment with trypsin and
several receptors that appear to bind A
peptides have been identifed
(41-46). In the case of the receptor for advanced glycation end
products (RAGE), some evidence has been presented that it may be
directly involved in A
-induced neurotoxicity (45). Experiments with
all-D-A
and these putative A
receptors are in
progress and should provide information on the specificity of these
receptors for A
. Thus, we suggest that extracellular macromolecular
assemblies such as A
can serve as stimuli or agonists that trigger a
particular sequence of cellular reactions in neurons that initiate an
apoptotic program of cell death. These PCD agonists are
characterized in part by
-sheet fibrillar structure but, in
addition, have the common ability to access critical signal
transduction and downstream mechanisms that drive PCD.
Other amyloidogenic proteins, which do not share sequence homology with
A (e.g.. prion and amylin), do form structurally similar
extracellular deposits and have been found to have similar neurotoxic
activity in vitro (47, 48). One possible mechanism that
could explain the common biological activity seen with different amyloidogenic peptides is
-sheet augmentation, whereby a peptide forms a "peptide-surface association" (49) either by inserting itself into a
-sheet-containing domain (50), which has been proposed
for other diseases involving protein conformational changes (51-54),
or by adding to the edge of an anti-parallel
-strand, as has been
implicated in regulating protein associations governing signal
transduction pathways and assembly interactions in certain viral
capsids (49). The
-sheet augmentation mechanism provides much
greater flexibility than classical domain-domain association because
the peptide is not constrained by a rigidly folded domain. The
specificity in this model is dependent on the ability of the peptide to
augment an appropriate
-strand on a protein. Consistent with this
model, A
has shown a pronounced ability to bind to other proteins,
such as
-1-antichymotrypsin, and transthyretin which are rich in
-sheet. Finally, the cell surface contains numerous proteins with Ig
superfamily homology with extensive
-sheet content, which include
receptors (55) and cell adhesion molecules (i.e. NCAM and
N-cadherin) (56), and in several reports, the cell surface
appears to be able to actually nucleate A
assembly (40, 57, 58),
which is also consistent with the model (49).
The fact that the all-D analogs of A retain bioactivity
may present new avenues for therapeutic intervention by allowing the
D-enantiomers of inhibitory peptides to be utilized. An
approach similar to this has recently been used to identify an
all-D-amino acid opioid peptide with analgesic activity
capable of crossing the blood brain barrier using a synthetic
combinatorial library made up of D-amino acid hexapeptides
(59). In addition, the identification of D-peptide ligands
through mirror image phage display using genetically encoded libraries
(60) offers the promise of rapidly screening for D-peptide
ligands that can block assembly and or neurotoxicity of the A
peptide. All-D-ligands are generally resistant to
proteolysis and D-amino acid proteins are reported to have
low immunogenicity, thus making them useful for pharmacological
applications (61).
The results obtained in this study suggest that the neurotoxic activity
of A is independent of the classical stereoisomer-specific ligand-receptor interaction. Rather, A
-induced neurotoxicity is
dependent on the primary sequence of the peptide that regulates both
the ability of the peptides to assemble into active conformations and
bind to cellular surfaces. While mechanisms dependent on the perturbation of cellular membranes or the formation of calcium ion
channels cannot be excluded, the requirement for higher order protein
assemblies by amyloidogenic peptides for biological activity does not
readily support these mechanisms. Further investigation of the
mechanism of A
-induced toxicity will likely benefit attempts to
understand the neurodegeneration that occurs in AD and perhaps other
amyloid-related disorders.
We thank Virany Kreng for excellent technical assistance and Dr. Charles Glabe for synthesis and characterization of the amyloid peptides.