| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 35, 27110-27116, September 1, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Peptide Induce
Diverse Conformational Changes and Apoptotic Effects in Human Cerebral
Endothelial Cells*
,,,, and¶
From the Department of Pathology, New York University
School of Medicine, New York, New York 10016 and the
§ Istituto Nazionale Neurologico Carlo Besta,
Milano 20133, Italy
Received for publication, April 13, 2000, and in revised form, May 16, 2000
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Cerebral amyloid angiopathy is commonly
associated with normal aging and Alzheimer's disease and it is also
the principal feature of hereditary cerebral hemorrhage with
amyloidosis Dutch type, a familial condition associated to a point
mutation G to C at codon 693 of the amyloid Parenchymal and vascular amyloid-
(A) precursor
protein gene resulting in a Glu to Gln substitution at position
22 of the A (E22Q). The patients carrying the AE22Q variant
usually present with lobar cerebral hemorrhages before 50 years of age.
A different mutation described in several members of three Italian
kindred who presented with recurrent hemorrhagic strokes late in life, between 60 and 70 years of age, also associated with extensive cerebrovascular amyloid deposition has been found at the same position
22, this time resulting in a Glu to Lys substitution (E22K). We have
compared the secondary structure, aggregation, and fibrillization
properties of the two A40 variants and the wild type peptide. Using
flow cytometry analysis after staining with propidium iodide and
annexin V, we also evaluated the cytotoxic effects of the peptides on
human cerebral endothelial cells in culture. Under the conditions
tested, the E22Q peptide exhibited the highest content of -sheet
conformation and the fastest aggregation/fibrillization properties. The
Dutch variant also induced apoptosis of cerebral endothelial cells at a
concentration of 25 µM, whereas the wild type A
and the E22K mutant had no effect. The data suggest that different
amino acids at position 22 confer distinct structural properties to the
peptides that appear to influence the onset and aggressiveness of the
disease rather than the phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(A)1 deposits and
neurofibrillary tangles composed of hyperphosphorylated tau assembled into paired helical filaments are the hallmarks of Alzheimer's disease
(AD), the most frequent form of amyloidosis in humans. Amyloid peptides
A40 and A42, proteolytically derived from their precursor protein
APP, are the main components of the fibrils found in the vessel
walls and in the neuropil, respectively (1). Missense mutations in the
APP gene either outside or inside the A-coding region usually
associate with early onset familial forms of the disease (2-25).
Within the A40/42 segment, ten distinct nucleotide changes have been
reported at eight different positions (Table
I) (2-13). Of the ten mutations, three
are silent, whereas the rest are translated into amino acid
substitutions; four of them, concentrated in the middle of the A
sequence (positions 21-22, corresponding to codons 692-693 of
APP), have been found associated with extensive cerebrovascular
pathology (3-8). They are (i) the Flemish mutation (C to G at codon
692, Ala to Gly at position 21) (3), (ii) the Arctic mutation (A to G
at codon 693, Glu to Gly at residue 22) (5, 6), (iii) the Dutch
mutation (G to C at codon 693, Glu to Gln at position 22) (4), and (iv) the Italian mutation (G to A at codon 693, Glu to Lys at residue 22)
(7, 8).
Mutations in the APP gene
The autosomal dominant form of cerebrovascular amyloidosis in Dutch
patients designated hereditary cerebral hemorrhage with amyloidosis-Dutch Type (HCHWA-Dutch) was the first mutation identified in the APP gene (4). These patients develop cerebral hemorrhage because of the severe CAA, whereas parenchymal amyloid deposits are
rare, and neurofibrillary tangles are consistently absent, features
that clearly distinguish the Dutch phenotype from those related to the
Flemish and Arctic mutations. A different amino acid substitution also
related to familial cerebral hemorrhage has been recently found at the
same position in several members of three unrelated Italian kindred
(HCHWA-Italian). Neuropathologic examination of one patient revealed
extensive A deposits in leptomeningeal and cortical vessels and, to
a lesser extent, amyloid plaques in the neuropil of the cerebral cortex
(7, 8). Vascular deposits were primarily labeled by anti-A40
antibody, whereas parenchymal deposits were predominantly revealed by
anti-A42 antibody, as in AD. However, neurofibrillary changes were
very mild and restricted to the archicortex. Moreover, despite the large amount of A in the vessel walls, thioflavine S fluorescence was rarely detected, suggesting a nonfibrillar A organization.
In the present work, we analyzed the secondary structure and fibril
formation properties of both mutants, compared these parameters with
those of the WT A40, and correlated the results with the ability of
the peptides to induce cell death in human cerebral endothelial cells.
The data suggest that: (i) different amino acids confer distinct
structural properties to the A peptide, which, in turn, is
translated to distinct fibrillogenic propensities and cytotoxic
effects, correlating with the phenotypic expression, and (ii) the
charge of the amino acids appears to be a factor for the cytotoxic
effects and vascular localization of the amyloid.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Patients-- A comparative histological and immunohistochemical study was carried out on a brain tissue samples from one patient with sporadic CAA, one patient with HCHWA Dutch-type, and one patient from an Italian kindred with familial occurrence of recurrent strokes, linked to a G to A transition at the first position of codon 693 of the APP gene resulting in Glu to Lys substitution. The patient with SCAA was a 82-year-old woman who suffered from minor strokes from age 70 leading to intellectual deterioration and parkinsonism. The patient with HCHWA Italian-type experienced the first cerebral bleeding at age 45, followed by minor strokes with transient neurological symptoms and a fatal cerebral hemorrhage at age 62.
Neuropathologic Study--
Samples of cerebrum and cerebellum
were fixed in 4% formaldehyde and embedded in paraplast.
Seven-µm-thick serial sections were stained with hematoxylin-eosin,
Congo red, and thioflavine S, or immunostained with anti-A
antibodies. These included anti-SP28 (1:300 dilution), a rabbit
antiserum to the 28 residue synthetic peptide homologous to the
N-terminal region of A, and anti-A40 (1:2000 dilution), a rabbit
antiserum that specifically recognizes the carboxyl end of the short
forms of A. Before immunostaining, the sections were treated with
98% formic acid for 30 min. The immunoreactions were revealed using
the EnVision Plus-horseradish peroxidase system for rabbit
immunoglobulins (Dako) and diaminobenzidine as chromogen.
Peptide Preparation--
Synthetic A40 peptides (wild type
(WT) and mutants AE22K and AE22Q) were synthesized by the W. M. Keck Foundation (Yale University, CT). All peptides were purified by
reverse-phase high performance liquid chromatography (HPLC), and their
purity was evaluated by amino acid sequence analysis and laser
desorption mass spectrometry. Peptide concentration was accurately
calculated by amino acid analysis.
Aggregation Studies-- Lyophilized aliquots of the peptides were dissolved at a concentration of 250 µM in 5 mM Tris, pH 7.4, 150 mM NaCl (TS), and incubated at room temperature for up to 20 days. After incubation, samples were centrifuged at 10,000 rpm for 15 min, and protein concentration of the supernatant was determined spectrophotometrically (Beckman DU640 spectrophotometer) using standards of the same peptide that had been previously calibrated by quantitative amino acid analysis.
Electron Microscopy-- For fibril formation, peptides were incubated in TS for up to 20 days at room temperature. After incubation, a 5-µl aliquot was absorbed onto 300 mesh carbon-coated copper grids (Ted Pella) and negatively stained with 5 µl of a saturated solution of uranyl acetate in water. Specimens were examined with a transmission electron microscopy (Philips CM 12).
Circular Dichroism Spectra-- For CD spectroscopy, peptides dissolved in TS were centrifuged at 10,000 rpm for 15 min to eliminate large aggregates. The supernatant was loaded into a 0.1-mm-path length quartz cell, and the CD spectra was recorded in the far ultraviolet light using a JASCO 720 spectropolarimeter (JASCO Corporation). Forty scans/experimental conditions were obtained at 0.2-nm intervals over the wavelength range 190-260 nm. Final spectra were obtained after the subtraction of background readings of buffer only blanks.
Transmission-Fourier Transform Infrared Spectroscopy
(T-FTIR)--
For T-FTIR spectroscopy, peptides dissolved in TS at a
concentration of 250 µM, freshly prepared or after 20 days of incubation, were lyophilized and resuspended at a concentration
of 10 mg/ml in D2O. Infrared spectra of the suspensions
were collected in a FTS 6000 FT-IR spectrometer (Bio-Rad) equipped with
a deuterated tri-glycine sulfate detector. Traces of
trifluoroacetic acid remaining from the HPLC purification of the
peptides were removed as described by Janek et al. (26).
Aliquots (20 µl) were placed in demountable cells containing
CaF2 windows separated by 50-µm Teflon spacers. The
solvent spectrum was recorded under identical conditions and subtracted
from the peptide spectra. For each sample, 256 interferograms were
coadded and Fourier-transformed to generate a spectrum with a nominal
resolution of 4 cm
1. Residual water vapor was
interactively subtracted as described (27). Band narrowing of the
spectra by Fourier self-deconvolution, which leads to a better
visualization of the overlapping bands, was carried out using a
half-bandwidth of 16 cm1 and a band narrowing factor of
k = 2.
Attenuated Total Reflection (ATR)-FTIR--
For ATR-FTIR
spectroscopy, peptides were incubated in TS for 20 days. After
incubation, samples were centrifuged at 10,000 rpm for 15 min, and the
pellets were loaded on the surface of a horizontal Golden Gate single
reflection diamond ATR cell (Specac, Smyrma, GA). Dry films of protein
obtained after slow evaporation of the buffer were subjected to 1-h
deuteration as described (28), and ATR-FTIR spectra was recorded at
room temperature on a Bio-Rad FTS 6000 infrared spectrophotometer
continuously purged with dry air obtained from a Whatman 74-5041 FT-IR
purge gas generator. Two hundred fifty-six scans were accumulated to
improve the signal/noise ratio and spectra recorded at a nominal
resolution of 0.5 cm1. An interactive difference routine
was used to subtract the buffer spectrum from that of the sample.
Correct subtraction of residual H2O was judged to yield an
approximately flat baseline at 2100 cm1. Fourier
self-deconvolution of the spectra in the amide I region was performed
using Bessel apodization function with a resolution enhancement factor
of k = 2 and peak half-width of 12 cm1,
in the Win-IR Pro system (Bio-Rad). Individual components of the amide
I mode were resolved from the deconvolved spectra by least squares
iterative curve fitting using Lorentzian-Gaussian curves and Grams/32
software (Galactic, Salem, NH). Assignment of the different components
of the amide I after Fourier self-deconvolution to secondary structure
was performed as described (29).
Cell Culture, Apoptosis Induction, and Flow Cytometry
Assay--
SV40 large T antigen-immortalized human brain microvascular
endothelial cells (HCEC) were kindly provided by Dr.
Stanimirovic (National Research Council of Canada). Cells were grown on
0.5% gelatin-coated dishes (Costar, NY) in M199 medium containing 10% fetal bovine serum and supplements (30). Cultured cells were identified
as HCEC based on the typical morphology, factor VIII immunofluorescence, and uptake of acetylated low density
lipoproteins and low density lipoproteins (30). HCEC (passage
34-36) were grown to confluence on 60-mm polystyrene-coated dishes
(Corning) in culture medium. Just before the addition of the peptides,
the medium was replaced with fresh depleted medium (devoid of serum and
supplements). Cell viability was examined by Trypan Blue exclusion (Sigma). For apoptosis induction, synthetic peptides were suspended in
sterile Dulbecco-phosphate-buffered saline (Sigma) at a concentration of 500 µM. Peptides solutions freshly prepared were added
directly to the culture-depleted medium to a final concentration of
0.5, 5, 10, 25, and 50 µM on day 3 after plating. As
controls, cells were incubated in the absence of peptide, with reverse
A40-1 (Sigma) and with 1 µg/ml actinomycin-D (Sigma). Cells were
incubated for 24 h in a humidified CO2 incubator at
37 °C, harvested by trypsinization, and pooled with culture medium
so that cells that had lost their adherent properties during apoptosis
("floaters") were included in the analysis. The collected target
cells were washed in Dulbecco-phosphate-buffered saline, resuspended in
100 µl of annexin-V binding buffer containing annexin-V-fluorescein and propidium iodide (Roche Molecular Biochemicals), incubated for 15 min at room temperature in the dark, and analyzed by flow cytometer (FACScan, Becton Dickinson).
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Histochemical Stainings
A-immunohistochemistry of brain tissue samples from sporadic
CAA, HCHWA-Dutch type, and HCHWA-Italian type cases yielded similar
immunostaining patterns. In all cases, a large number of leptomeningeal
and parenchymal vessels were strongly labeled by the anti-A
antibodies anti-SP28 and anti-A40 (Fig.
1, a, c, and
e). By contrast, a remarkable difference was observed
following either Congo red staining (not shown) or thioflavine S
treatment (Fig. 1, b, d, and f).
Whereas in sporadic CAA and HCHWA Dutch-type virtually all
A-immunoreactive vessels showed amyloid burden as revealed by
specific staining methods, in HCHWA Italian-type only ~10% of
A-immunoreactive vessels were birefringent after Congo red or
fluorescent after thioflavine S, suggesting a rather amorphous
organization of A aggregates.
|
Aggregation and Fibril Formation Experiments
The capability of A40 WT and mutant peptides to aggregate was
followed spectrophotometrically. As indicated in Fig.
2, more than 25% of E22Q peptide became
insoluble during the first 24 h of incubation, whereas WT and E22K
peptides exhibited a lower aggregation rate. Only after day 5, these
peptides began to aggregate forming a visible pellet after
centrifugation. At day 20 (the end point of the experiment), only
around 50% of the E22Q mutant peptide remained in solution in
comparison with WT (65%) and E22K (80%). Because the decrease in the
absorbance indicated aggregation but not necessarily fibril formation,
we used transmission electron microscopy to evaluate the ability
of the peptides to form amyloid-like fibrils in vitro. After
24 h of incubation in TS, only the preparation containing the E22Q
peptide revealed the presence of short filaments with a ribbon-like
structure, whereas the WT and E22K peptides did not show any fibrous
material (not shown). Amyloid-like fibrils started to appear after 10 days of incubation for the WT peptide and after 15 days of incubation
for the E22K mutant. Fibrillar material was clearly evident for all the
peptides after 20 days of incubation although with different morphology
(Fig. 3). Whereas the E22Q fibrils were
long and twisted, the E22K peptide formed straight fibrils with no
observable twisting; these fibrils were shorter and unbranched when
compared with those formed by the E22Q. The WT peptide formed fibrils
that were similar to those formed by the E22Q mutant, although the E22Q
peptide produced fibrils at a higher rate than the other peptides.
Strong green birefringence under polarized light after Congo red
staining was observed in all peptide preparations at day 20 (data not
shown).
|
|
Structural Analysis
Circular Dichroism Spectrometry--
Freshly dissolved WT peptides
exhibited a CD spectra characteristic of a predominantly unordered
structure with a strong negative peak at 198 nm (Fig.
4A). Under identical
conditions, the E22K variant exhibited a similar predominantly
unordered secondary structure (Fig. 4A). On the contrary,
the E22Q mutation induced -sheet structure to the peptide; a
characteristic CD spectra with a negative peak at 220 nm and a positive
peak at 195 nm was evident (Fig. 4A). After 24 h of
incubation, the intensity of the CD signal started to decrease as a
consequence of the formation of aggregates and the disappearance of the
peptide in solution. The signal continued to decrease up to the end of
the experiment although no changes in the spectra were seen in any of
the peptides (data not shown).
|
T-FTIR and ATR-FTIR Measurements--
Peptides, freshly dissolved
or after 20 days of incubation in TS, were resuspended at a
concentration of 10 mg/ml in D2O and analyzed by T-FTIR. As
indicated in Fig. 4B, the deconvolved amide I profile
spectra of a freshly prepared suspension of the WT peptide exhibited a
principal component at 1648 cm1 characteristic of random
coil conformations, which contributed ~40% of the final structure.
Other contributions were associated with 
-helix (22%),
-sheet/aggregated strands (21%), and -turn (17%) structures.
Similarly, the E22K peptide suspension freshly prepared exhibited a
comparable spectra, with a main component at 1652 cm1 and
a final structure composed of 50% random coil, 10% -helix, 30%
-sheet/aggregated strands, and 10% -turn conformations (Fig. 4B). Detectable spectral changes were noted in E22Q mutant
peptide, showing two predominant components at 1652 cm1
(26% random coil) and 1630 cm1 (55%
-sheet/aggregated strands). These results correlated with those
obtained by CD spectroscopy and depicted in Fig. 4A. When peptides were incubated for 20 days, an increase in the intensity of
the bands between 1620 and 1640 cm1 was noticeable for
all the peptides spectra (WT, E22K, and E22Q) with a proportional
decrease in the intensity of the other bands, indicating an increment
in the content of -sheet structures during the time of incubation
(Fig. 4C). In comparison, E22Q presented with the highest
amount of -structures; the contribution of -sheets plus -turns
accounted for ~67% of the total conformation of the peptide when
freshly prepared (Fig. 4B), raising to ~95% when incubated for 20 days in TS (Fig. 4C). The peptide pellets
obtained after 20 days of incubation were analyzed by ATR-FTIR
spectroscopy. Aggregated peptides were collected by centrifugation and
spread evenly on the surface of a horizontal single reflection diamond ATR plate. In all cases, the deconvolved amide I profile of the spectra
showed a main absorbance between 1635 and 1625 cm1
corresponding to more than 90% -sheet structure, with a little contributions by the other conformations (Fig. 4D).
Apoptosis Assay
HCEC were incubated for 24 h with WT and mutant A peptides
at a concentration range between 0.5 and 50 µM, and
induction of apoptosis was evaluated by flow cytometry after annexin V
staining. Representative dot plot profiles generated by flow cytometry
analysis of cells are shown in Fig. 5.
Fig. 5A illustrates the negative (no addition of peptide)
and positive (actinomycin D) controls, respectively. Under the
conditions tested, ~80 and 4% of the cells were viable (lower
left quadrants), with 17 and 94% of the cells respectively
exhibiting positive staining for annexin V (both, upper and lower right quadrants). HCEC treated
with WT peptide at a concentration of 0.5 and 50 µM
showed 18 and 17% of the cells staining positive for annexin V,
respectively (Fig. 5B), whereas cells treated with the E22K
peptide at identical concentrations exhibited 15 and 13% of the cells
staining positive for annexin V, respectively (Fig. 5C). In
both cases, the values were similar to the value observed in the
negative control. Different results were obtained when cells were
treated with the E22Q peptide. Incubations at the same concentrations
of 0.5 and 50 µM showed that 26 and 64% of the cells
were positively stained for annexin V. HCEC treated with the reverse
A40-1 peptide showed similar results to those obtained when no
peptide was added to the cells (not shown). The difference in mean
fluorescence intensity between the negative control and E22Q-treated
cells was significant at 25 µM (p = 0.01).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
CAA is a significant risk factor for hemorrhagic strokes in the
elderly, is a frequent component of AD and related disorders, and is
the dominant pathology in several hereditary conditions including
HCHWA-Icelandic type (31), the vascular variant of PrP (32), the
Hungarian and Ohio kindred of meningocerebrovascular amyloidosis (33,
34), the gelsolin-related spinal and cerebral CAA (35), familial
British dementia (36), familial Danish dementia (37) and selected A
mutants, particularly E22Q and E22K (4, 7, 8). The E22Q mutant is
associated with HCHWA-Dutch type, an autosomal dominant form of severe
CAA that presents clinically between the age of 45 and 60 (mean 50.4 years). Survivors to the first stroke (~60% of the patients) have
further strokes that lead to cognitive deficits. Neuropathologically,
extensive Congo red positive amyloid deposits are seen in the walls of
small cerebral arteries and arterioles. In these patients, parenchymal
deposition is largely in the form of preamyloid lesions or diffuse
plaque-like structures that are Congo red negative and lack the dense
amyloid cores commonly present in AD (38, 39). Diffuse plaques are occasionally surrounded by dystrophic neurites, although
neurofibrillary tangles are consistently absent (40) as described in
others species such as aged dogs (41) and non-human primates
(42). Several in vitro studies have demonstrated
that the full-length E22Q peptide, as well as fragments
containing the mutation, exhibit increased aggregation rates
(38, 39), form amyloid-like fibrils at a faster rate than wild
type A, and are toxic to cultured human leptomeningeal smooth muscle
cells (HCSM) (43, 44). We and others have studied the aggregation of
the Dutch peptide and found that the presence of the mutation might not
only cause quantitative differences in the kinetics of A
fibrillogenesis and aggregation (45) but could also influence the
assembly of the wild-type A peptides by providing Dutch fibril
nuclei from which the wild-type or mixed fibrils could elongate (38,
39). Also, it has been demonstrated that a short peptide partially homologous to the central hydrophobic region of A (residues 17-21), but containing amino acids that prevent the adoption of a -sheet structure (i.e. proline), binds A and inhibits amyloid
fibril formation in vitro, suggesting an important role of
this region for the fibrillogenesis (46). The E22K mutation associated
with HCHWA-Italian type has been recently described in several members of three Italian kindred who presented with recurrent hemorrhagic strokes. These patients die at 62-75 years of age, following a 10-20-year history of mild cognitive decline, recurrent strokes, and
epilepsy in some cases. There are extensive A deposits in leptomeningeal and cortical vessels and, to a lesser extent, in the
neuropil of the cerebral cortex, in the absence of neurofibrillary tangles. Notably, despite the large amount of A vascular deposits, amyloid-related thioflavine S-fluorescence was rarely detected, suggesting a rather amorphous organization of A (7, 8). Studies
using synthetic peptides recently showed that a change or loss of
charge at position 22 of the A enhances the pathogenic effects of
the peptide toward HCSM cells suggesting that it may contribute to the
pathogenesis of the phenotypically related HCHWA disorders (44).
Our observations clearly indicate that the E22Q peptide has an
increased propensity to self-aggregate and form amyloid-like fibrils
when compared with the WT and E22K variants. Fibrillogenesis with both
WT and E22K progressed at a lower rate, perhaps reflecting the initial
unordered conformation and the stability of the peptides in solution.
The fibrils obtained from each peptide were similar in dimension;
however, the morphology of the fibrils varied considerably. Among the
three peptides, the E22K showed the least propensity to form fibrils;
under our experimental conditions, straight fibrils with no observable
twist and no distinguishable higher level order appeared only after 15 days of incubation. These findings correlate with the observation that
the vascular E22K deposits found in the Italian kindred are
predominantly thioflavine S-fluorescence negative, as described above,
illustrated in Fig. 1, and summarized in Table
II. Assessment of the secondary structure
in suspension by T-FTIR revealed that the process of aggregation of the
peptides during incubation was associated with a spectral change
consistent with an increase in the content of -sheet conformation, a
structure confirmed by ATR-FTIR in the precipitated peptides. However,
the remaining soluble peptides that still were not aggregated and/or fibrillized conserved their original secondary structures. Because E22Q
was the only one exhibiting an important amount of -sheet conformation from the time of dissolution, the findings certainly agree
with many published reports suggesting that the progression from the
soluble to the fibrillar-aggregated form requires the conversion of the
peptide to a predominantly -sheet conformation (47).
|
In vitro WT A40 and A42 are toxic to neurons in cell
culture (48, 49) and an apoptotic effect of A in neurons of AD patients has been observed by in situ DNA end-labeling (50). Wild type A42, A40 E22Q, and A40 E22K at a concentration of 25 µM, but not WT A40 at the same concentration, have
been reported to be toxic, after 6-12 days of incubation, to human
smooth muscle cells (HCSM) (44, 51, 52). We observed that the A40
E22Q peptide produces apoptosis in cultured HCEC at a concentration of
25 µM. The WT A40 and the mutant E22K did not have an
apoptotic effect even at concentrations as high as 50 µM.
Perhaps this lack of effect on HCEC correlates with the inability of
these peptides to aggregate and form fibrils during the 24 h of
incubation with the cells. This suggests that intermediate conformers
and/or the final fibrils, rather than the soluble peptide, are involved
in the cytotoxic mechanisms. It has yet to be determined if longer incubation times of the peptides with the cells will increase the toxic
effect of both WT and E22K mutant peptides.
Among all APP mutants, those localized within the A region are
preferentially vasculotropic, whereas the rest mainly produce deposition of wild type A peptide in the form of parenchymal plaques
(Table I). The structural changes induced by the amino acid
substitutions appear to influence the onset and duration of the disease
rather than the phenotype. Although further studies are needed to
unveil the final mechanism of fibrillization of these mutants, unknown
tissue factors may well be responsible for the preferential vascular
deposition. The fact that the positively charged E22K mutant peptide
produces thioflavine S negative deposits correlates with the
observation that high isoelectric point values have been associated
with nonfibrillar light chain deposition disease cases rather than
light chain amyloidosis (53). Defining the precise steps in the
apoptotic pathway, the factors that could be involved and the possible
regulatory targets, may allow for the development of strategies that
will delay the progression of neurovascular degeneration of AD.
| |
ACKNOWLEDGEMENT |
|---|
We thank Dr. Danica Stanimirovic (National Research Council, Ottawa, Canada) for the SV40 immortalized human cerebral endothelial cells.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants AG08721, NS38777, and AG05891 and by the Italian Ministry of Health, Department of Social Services.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of the New Investigator Development Award from the American Heart Association New York City affiliate. To whom correspondence should be addressed: Dept. of Pathology, TH432, New York University School of Medicine, New York, NY 10016. Tel.: 212-263-5775; Fax: 212-263-6751; E-mail: ghisoj01@popmail.med.nyu.edu.
Published, JBC Papers in Press, May 19, 2000, DOI 10.1074/jbc.M003154200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
A, amyloid ;
AD, Alzheimer's disease;
APP, A precursor protein;
HCHWA, hereditary cerebral hemorrhage with amyloidosis;
CAA, cerebral amyloid
angiopathy;
WT, wild type;
HPLC, high pressure liquid chromatography;
T-FTIR, transmission-Fourier transform infrared spectroscopy;
ATR, attenuated total reflection.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Wisniewski, T., Ghiso, J., and Frangione, B. (1997) Neurobiol. Dis. 4, 313-328 |
| 2. | Hendriks, L., and Van Broeckhoven, C. (1996) Eur. J. Biochem. 237, 6-15 |
| 3. | Hendriks, L., van Duijn, C. M., Cras, P., Curts, M., Van Hul, W., van Harskamp, F., Warre, A., McInnis, M., Antonarakis, S. T., Martin, J. J., Hofman, A., and Van Broeckhoven, C. (1992) Nat. Genet. 1, 218-221 |
| 4. | Levy, E., Carman, M. D., Fernandez-Madrid, I. J., Power, M. D., Lieberburg, I., van Duinen, S. G., Bots, G. T., Luyendijk, W., and Frangione, B. (1990) Science 1, 1124-1126 |
| 5. | Kamino, K., Orr, H. T., Payami, H., Wijsman, E. M., Alonso, M. E., Pulst, S. M., Anderson, L., O'dahl, S., Nemens, E., White, J. A., et al.. (1992) Am. J. Hum. Genet. 51, 998-1014 |
| 6. | Nilsberth, C., Forsell, C., Axelman, K., Gustafsson, C., Luthman, J., Naslund, L., and Lannfelt, L. (1999) Soc. Neurosci. 25, 120.4 (abstr.) |
| 7. | Bugiani, O., Padovani, A., Magoni, M., Andora, G., Sgarzi, M., Savoiardo, M., Bizzi, A., Giaccone, G., Rossi, G., and Tagliavini, F. (1998) Neurobiol. Aging 19, S238 (abstr.) |
| 8. | Tagliavini, F., Rossi, G., Padovani, A., Magoni, M., Andora, G., Sgarzi, M., Bizzi, A., Savoiardo, M., Carella, F., Morbin, M., Giaccone, G., and Bugiani, O. (1999) Alzheimer's Rep. 2, 23 (abstr.) |
| 9. | Forsell, C., and Lannfelt, L. (1995) Neurosci. Lett. 184, 90-93 |
| 10. | Adroer, R., Lopez-Acedo, C., Oliva, R., Hardy, J., and Fidani, L. (1993) Neurosci. Lett. 150, 33-34 |
| 11. | Jones, C. T., Morris, S., Yates, C. M., Morffoot, A., Sharpe, C., Brock, D. J. H., and St. Clair, D. (1992) Nat. Genet. 1, 306-309 |
| 12. | Carter, D. A., Desmarais, E., Bellis, M., Campion, D., Clerget-Darpoux, F., Brice, A., Agid, Y., Jaillard-Serradt, A., and Mallet, J. (1992) Nat. Genet. 2, 255-256 |
| 13. | Peacock, M. L., Warren, J. T., Roses, A. D., and Fink, J. K. (1993) Neurology 43, 1254-1256 |
| 14. | Balbin, M., Abrahamson, M., Gustafson, L., Nilsson, K., Brun, A., and Grubb, A. (1992) Hum. Genet. 89, 580-582 |
| 15. | Murrell, J., Farlow, M., Ghetti, B., and Benson, M. D. (1991) Science 254, 97-99 |
| 16. | Zubenko, G. S., Farr, J., Stiffler, J. S., Hughes, H. B., and Kaplan, B. B. (1992) J. Neuropathol. Exp. Neurol. 51, 459-463 |
| 17. | Naruse, S., Igarashi, S., Kobayashi, H., Aoki, K., Inuzuca, T., Kaneko, K., Shimizu, T., Ihara, K., Kojima, T., Miyatake, T., and Tsuji, S. (1991) Lancet 337, 978-979 |
| 18. | Goate, A., Chartier-Harlin, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M., and Hardy, J. (1991) Nature 349, 704-706 |
| 19. | Chartier-Harlin, M. C., Crawford, F., Houlden, H., Warre, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, P., Hardy, J., and Mullan, M. (1991) Nature 353, 844-846 |
| 20. | Fidani, L., Rooke, K., Chartier-Harlin, M. C., Hughes, D., Tanzi, R., Mullan, M., Roques, P., Rossor, M., Hardy, J., and Goate, A. (1992) Hum. Mol. Genet. 13, 165-168 |
| 21. | Ancolio, K., Dumanchin, C., Barelli, H., Warter, J. M., Brice, A., Campion, D., Frebourg, T., and Checler, F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4119-4124 |
| 22. | Eckman, C. B., Mehta, N. D., Crook, R., Perez-Tur, J., Prihar, G., Pfeiffer, E., Graff-Radford, N., Hinder, P., Yager, D., Zenk, B., Refolo, L. M., Prada, C. M., Younkin, S. G., Hutton, M., and Hardy, J. (1997) Hum. Mol. Genet. 6, 2087-2089 |
| 23. | Brooks, W. S., Martins, R. N., De Voecht, J., Nicholson, G. A., Shofield, P. R., Kwok, J. B., Fisher, C., Yeung, L. U., and Van Broeckhoven, C. (1995) Neurosci. Lett. 199, 183-186 |
| 24. | Kwok, J. B. J., Li, Q. X., Hallupp, M., Whyte, S., Ames, D., Beyreuther, K., Masters, C. L., and Schofield, P. R. (2000) Ann. Neurol. 47, 249-253 |
| 25. | Peacock, M. L., Murman, D. L., Sima, A. A. F., Warren, J. T., Roses, A. D., and Fink, J. K. (1994) Ann. Neurol. 35, 432-438 |
| 26. | Janek, K., Behlke, J., Zipper, J., Fabian, H., Georgalis, Y., Beyermann, M., Bienert, M., and Krause, E. (1999) Biochemistry 38, 8246-8252 |
| 27. | Fabian, H., Schultz, C., Naumann, D., Landt, O., Hahn, U., and Saenger, W. (1993) J. Mol. Biol. 232, 967-981 |
| 28. | Gasset, M., Baldwin, M. A., Fletterick, R. J., and Prusiner, S. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1-5 |
| 29. | Goornaghtigh, E., Cabiaux, V., and Ruysschaert, J. M. (1994) in Subcellular Biochemistry (Hilderson, H. , and Ralston, G. B., eds), Vol. 23 , pp. 405-450, Plenum Press, New York |
| 30. | Muruganandam, A., Moorhouse Herx, L., Monette, R., Durkin, J. P., and Stanimirovic, D. B. (1997) FASEB J. 11, 1187-1197 |
| 31. | Ghiso, J., Jensson, O., and Frangione, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2974-2978 |
| 32. | Ghetti, B., Piccardo, P., Spillantini, M. G., Ichimiya, Y., Porro, M., Kitamoto, T., Tateishi, J., Seiler, C., Frangione, B., Bugiani, O., Giaccone, G., Prelli, F., Goedert, M., Dlouhy, S. R., and Tagliavini, F. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 744-748 |
| 33. | Vidal, R., Garzuly, F., Budka, H., Lalowski, M., Linke, R., Britting, F., Frangione, B., and Wisniewski, T. (1996) Am. J. Pathol. 148, 361-366 |
| 34. | Petersen, R., Goren, H., Cohen, M., Richardson, S., Tresser, N., Lynn, A., Gali, M., Estes, M., and Gambetti, P. (1997) Ann. Neurol. 41, 307-313 |
| 35. | Kiuru, S., Salonen, O., and Haltia, M. (1999) Ann. Neurol. 45, 305-311 |
| 36. | Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G., and Ghiso, J. (1999) Nature 399, 776-781 |
| 37. |
Vidal, R.,
Revesz, T.,
Rostagno, A.,
Kim, E.,
Holton, J.,
Bek, T.,
Bojsen-M ller, M.,
Braengaard, H.,
Plant, G.,
Ghiso, J.,
and Frangione, B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4920-4925
|
| 38. | Wattendorff, A. R., Bots, G. T., Went, L. N., and Endtz, L. J. (1982) J. Neurol. Sci. 55, 121-135 |
| 39. | Van Duinen, S. G., Castano, E. M., Prelli, F., Bots, G. T. A. B., Luyendijk, W., and Frangione, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5991-5994 |
| 40. | Timmers, W. F., Tagliavini, F., Haan, J., and Frangione, B. (1990) Neurosci. Lett. 16, 223-226 |
| 41. | Cummings, B. J., Head, E., Ruehl, W., Milgram, N. W., and Cotman, C. W. (1996) Neurobiol. Aging 17, 259-268 |
| 42. | Struble, R. G., Price, D. L., Cork, L. C., and Price, D. L. (1985) Brain Res. 361, 267-275 |
| 43. | Davis, J., Cribbs, D. H., Cotman, C. W., and Van Nostrand, W. E. (1999) Amyloid 6, 157-164 |
| 44. | Melchor, J. P., McVoy, L., and Van Nostrand, W. E. (2000) J. Neurochem. 74, 2209-2212 |
| 45. | Wisniewski, T., Ghiso, J., and Frangione, B. (1991) Biochem. Biophys. Res. Commun. 179, 1247-1254 |
| 46. | Soto, C., Kindy, M. S., Baumann, M., and Frangione, B. (1996) Biochem. Biophys. Res. Commun. 226, 672-680 |
| 47. | Wisniewski, T., Aucouturier, P., Soto, C., and Frangione, B. (1998) Amyloid 5, 212-224 |
| 48. | Loo, D. T., Copani, A., Pike, C. J., Whittemore, E. R., Walencewicz, A. J., and Cotman, C. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7951-7955 |
| 49. | Cotman, C. W., and Anderson, A. J. (1995) Mol. Neurobiol. 10, 19-45 |
| 50. | Su, J. H., Anderson, A. J., Cummings, B. J., and Cotman, C. W. (1994) Neuroreport 5, 2529-2533 |
| 51. | Davis, J., and Van Nostrand, W. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2996-3000 |
| 52. | Van Nostrand, W. E., Melchor, J., and Ruffini, L. (1998) J. Neurochem. 70, 216-223 |
| 53. | Gallo, G., Goni, F., Boctor, F., Vidal, R., Kumar, A., Stevens, F. J., Frangione, B., and Ghiso, J. (1996) Am. J. Pathol. 148, 1397-1406 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Baumketner, M. G. Krone, and J.-E. Shea Role of the familial Dutch mutation E22Q in the folding and aggregation of the 15-28 fragment of the Alzheimer amyloid-{beta} protein PNAS, April 22, 2008; 105(16): 6027 - 6032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Tew, S. P. Bottomley, D. P. Smith, G. D. Ciccotosto, J. Babon, M. G. Hinds, C. L. Masters, R. Cappai, and K. J. Barnham Stabilization of Neurotoxic Soluble {beta}-Sheet-Rich Conformations of the Alzheimer's Disease Amyloid-{beta} Peptide Biophys. J., April 1, 2008; 94(7): 2752 - 2766. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. L. Fawzi, K. L. Kohlstedt, Y. Okabe, and T. Head-Gordon Protofibril Assemblies of the Arctic, Dutch, and Flemish Mutants of the Alzheimer's A{beta}1-40 Peptide Biophys. J., March 15, 2008; 94(6): 2007 - 2016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Meinhardt, G. G. Tartaglia, A. Pawar, T. Christopeit, P. Hortschansky, V. Schroeckh, C. M. Dobson, M. Vendruscolo, and M. Fandrich Similarities in the thermodynamics and kinetics of aggregation of disease-related Abeta(1-40) peptides Protein Sci., June 1, 2007; 16(6): 1214 - 1222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Hoos, M. Ahmed, S. O. Smith, and W. E. Van Nostrand Inhibition of Familial Cerebral Amyloid Angiopathy Mutant Amyloid beta-Protein Fibril Assembly by Myelin Basic Protein J. Biol. Chem., March 30, 2007; 282(13): 9952 - 9961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hori, T. Hashimoto, Y. Wakutani, K. Urakami, K. Nakashima, M. M. Condron, S. Tsubuki, T. C. Saido, D. B. Teplow, and T. Iwatsubo The Tottori (D7N) and English (H6R) Familial Alzheimer Disease Mutations Accelerate Abeta Fibril Formation without Increasing Protofibril Formation J. Biol. Chem., February 16, 2007; 282(7): 4916 - 4923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Cabrejo, L. Guyant-Marechal, A. Laquerriere, M. Vercelletto, F. De La Fourniere, C. Thomas-Anterion, C. Verny, F. Letournel, F. Pasquier, A. Vital, et al. Phenotype associated with APP duplication in five families Brain, November 1, 2006; 129(11): 2966 - 2976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Hirsch-Reinshagen, L. F. Maia, B. L. Burgess, J.-F. Blain, K. E. Naus, S. A. McIsaac, P. F. Parkinson, J. Y. Chan, G. H. Tansley, M. R. Hayden, et al. The Absence of ABCA1 Decreases Soluble ApoE Levels but Does Not Diminish Amyloid Deposition in Two Murine Models of Alzheimer Disease J. Biol. Chem., December 30, 2005; 280(52): 43243 - 43256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Cruz, B. Urbanc, J. M. Borreguero, N. D. Lazo, D. B. Teplow, and H. E. Stanley Solvent and mutation effects on the nucleation of amyloid {beta}-protein folding PNAS, December 20, 2005; 102(51): 18258 - 18263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Morelli, R. E. Llovera, I. Mathov, L.-F. Lue, B. Frangione, J. Ghiso, and E. M. Castano Insulin-degrading Enzyme in Brain Microvessels: PROTEOLYSIS OF AMYLOID {beta} VASCULOTROPIC VARIANTS AND REDUCED ACTIVITY IN CEREBRAL AMYLOID ANGIOPATHY J. Biol. Chem., December 31, 2004; 279(53): 56004 - 56013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Remes, S. Finnila, H. Mononen, H. Tuominen, R. Takalo, R. Herva, and K. Majamaa Hereditary dementia with intracerebral hemorrhages and cerebral amyloid angiopathy Neurology, July 27, 2004; 63(2): 234 - 240. [Abstract] [Full Text] [PDF]
|
|
