The incubation of CLAC with preformed Ab fibrils led to increased turbidity, indicating that larger aggregates were formed.. Abbreviations Ab, amyloid b-peptide; AD, Alzheimer’s disease;
Trang 1assembles amyloid fibrils into protease resistant
aggregates
Linda So¨derberg1,*, Camilla Dahlqvist1,*, Hiroyoshi Kakuyama1, Johan Thyberg2, Akira Ito3,
Bengt Winblad1, Jan Na¨slund1and Lars O Tjernberg1
1 Karolinska Institutet and Sumitomo Pharmaceuticals Alzheimer Center (KASPAC), Neurotec, Novum, Huddinge, Sweden
2 Karolinska Institutet, Department of Cell and Molecular Biology, Stockholm, Sweden
3 Sumitomo Pharmaceuticals Research Center, Osaka, Japan
Alzheimer’s disease (AD) is characterized by amyloid
deposits of the amyloid b-peptide (Ab) in brain [1] Ab
is a 40–42 amino acid peptide that is proteolytically
derived from the b-amyloid precursor protein (APP)
The longer Ab42 variant aggregates more rapidly than
the more abundant Ab40 [2] and is the species deposited
initially in the brain in AD and Down’s syndrome [3]
Several lines of evidence suggest that APP processing
and Ab levels have a central role in the pathogenesis of
AD The APP gene is located on chromosome 21 (which
is present in triplicate in Down’s syndrome), providing
an explanation for the elevated levels of APP and Ab,
as well as for the AD-like pathology, observed in Down’s syndrome Moreover, mutations in genes linked
to familiar early onset AD generally result in altered APP processing and an increased Ab42⁄ Ab40 ratio [4]
It is not known how the amyloid plaques are formed
In vitro studies indicate that unstructured Ab mono-mers spontaneously form soluble oligomono-mers (seeds), which, in turn, form protofibrils and mature fibrils Other molecules could be of importance for the poly-merization process in vivo, including apolipoproteins E
Keywords
Alzheimer’s disease; amyloid; CLAC; fibrils;
thioflavin T
Correspondence
C Dahlqvist, Karolinska Institutet, Neurotec,
Novum KASPAC pl 5, SE-141 57 Huddinge,
Sweden
Fax: +46 8585 836 10
Tel: +46 8585 836 21
E-mail: Camilla.dahlqvist@neurotec.ki.se
*Note
These authors contributed equally to this
work.
(Received 28 January 2005, revised 25
February 2005, accepted 7 March 2005)
doi:10.1111/j.1742-4658.2005.04647.x
Recently, a novel plaque-associated protein, collagenous Alzheimer amy-loid plaque component (CLAC), was identified in brains from patients with Alzheimer’s disease CLAC is derived from a type II transmembrane colla-gen precursor protein, termed CLAC-P (collacolla-gen XXV) The biological function and the contribution of CLAC to the pathogenesis of Alzheimer’s disease and plaque formation are unknown In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid b-peptide (Ab) Here, we examined the effects of CLAC on Ab fibrils using assays based
on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy The incubation of CLAC with preformed Ab fibrils led to increased turbidity, indicating that larger aggregates were formed In sup-port of this contention, more Ab was sedimented in the presence of CLAC,
as determined by gel electrophoresis Moreover, electron microscopy revealed an increased amount of Ab fibril bundles in samples incubated with CLAC Importantly, the frequently used thioflavin T-binding assay failed to reveal these effects of CLAC Digestion with proteinase K or tryp-sin showed that Ab fibrils, incubated together with CLAC, were more resistant to proteolytic degradation Therefore, CLAC assembles Ab fibrils into fibril bundles that have an increased resistance to proteases We sug-gest that CLAC may act in a similar way in vivo
Abbreviations
Ab, amyloid b-peptide; AD, Alzheimer’s disease; APP, b-amyloid precursor protein; CLAC, collagenous Alzheimer amyloid plaque
component ⁄ collagen XXV; EM, electron microscopy; NAC, non-amyloid-b-component; ThT, thioflavin T.
Trang 2and J, and heparin sulfate proteoglycans [5] These
pro-teins have been suggested to act as ‘pathological
chap-erones’ that bind to Ab and favor its deposition Other
proteins may have the opposite effect and attenuate
amyloidogenesis The amorphous Ab aggregates found
in so-called diffuse plaques might be precursors of the
Ab fibrils found in mature plaques, but the mechanism
for this conversion remains to be determined
Recently, a novel plaque-associated protein,
colla-genous Alzheimer amyloid plaque component⁄ collagen
XXV (CLAC) [6], was observed in brain from subjects
with AD CLAC is derived from a type II
transmem-brane collagen protein, CLAC-P We have recently
shown that the AMY antigen is identical to CLAC [7]
CLAC is a trimer formed from three identical
polypep-tides and includes three triple-helical collagen domains
that are flanked and separated by nonhelical domains
[8] In vitro studies show that CLAC binds to fibrillized
Ab, but not to monomeric Ab [6,8] The biological
function of CLAC, and the contribution of CLAC to
plaque formation and the pathogenesis of AD, are still
unknown Previously, CLAC was shown to colocalize
with the more mature plaques, but not with
Ab42-pos-itive diffuse plaques or with amyloid deposits in
cereb-ral blood vessels [9,10] Recently, it was suggested that
CLAC preferentially binds to plaques composed of
prefibrillar Ab42 and thus may prevent further
matur-ation of amyloid deposits [11] Therefore, there is
cur-rently no clear consensus regarding to which form of
amyloid CLAC binds
Here, we examined the effects of CLAC on Ab fibrils
in vitro using a variety of biochemical techniques We
showed that the addition of CLAC to Ab fibrils assembles
the latter into protease-resistant fibril bundles We suggest
that CLAC could have a similar effect on amyloid fibrils
in vivoand thus increase the amyloid burden
Results and Discussion
Incubation of Ab1–40 fibrils with CLAC results
in further aggregation
The newly discovered plaque-associated protein, CLAC,
binds to Ab fibrils but not to monomeric Ab [6] (H
Kakuyama, L So¨derberg, K Horigome, C Dahlqvist,
B Winblad, J Na¨slund & LO Tjernberg, unpublished
results) In order to investigate the effect of CLAC on
fibrillar Ab, fibrils formed from 45 lm Ab1–40 were
incubated in the presence or absence of 100 nm CLAC
The turbidity of the samples was measured at different
time-points and found to be increased after only
30 min in the presence of CLAC (Fig 1A) As we use
preformed fibrils that are separated from soluble Ab
by centrifugation, the total amount of aggregated Ab cannot increase Therefore, we conclude that the increase in turbidity arises from the formation of lar-ger aggregates To verify this result, we centrifuged the samples and subjected them to SDS⁄ PAGE In the presence of CLAC, the amount of Ab was increased in the pellet and decreased in the supernatant as com-pared to the control (Fig 1B) This effect occurred after only 30 min of incubation and remained evident also after a longer incubation (18 h) We suggest that
A
C
B
Fig 1 Incubation of amyloid b-peptide 1–40 (Ab1–40) fibrils with collagenous Alzheimer amyloid plaque component ⁄ collagen XXV (CLAC) results in further aggregation (A) Turbidity measurements
of Ab1–40 fibrils incubated, for 30 min and 18 h, in the presence (100 n M ) or absence of CLAC (B) Coomassie staining of Ab1–40 fibrils incubated in the presence of CLAC (100 n M ) or NaCl ⁄ Tris The pellet samples (P) were sedimented, washed and dissolved in 70% (v ⁄ v) formic acid (FA) The supernatant samples (Sup) were sedimented from the supernatant of the first centrifugation and dis-solved in 70% (v ⁄ v) FA (C) Electron micrographs of mixtures con-taining preformed Ab1–40 fibrils incubated in the presence of CLAC (100 n M ) or NaCl ⁄ P i for 24 h at room temperature Samples were prepared for electron microscopy as described in the Experimental procedures Scale bars, 200 nm.
Trang 3the increased amount of sedimented material is caused
by an increase in the size of the aggregates in the
pres-ence of CLAC No CLAC could be detected in the
supernatant after incubation, and CLAC was recovered
in the carefully washed pellet, as analyzed by western
blot and mass spectrometry (data not shown) These
data correlate with the turbidity measurements and
sup-port a function for CLAC in fibril assembly The
con-trol protein, aldolase, which does not bind to Ab fibrils,
had no such effect (data not shown) To investigate
whether the effect of CLAC was specific for Ab fibrils,
we performed the same set of experiments on fibrils
formed from another amyloid forming protein,
non-amyloid-b-component (NAC) [12] CLAC was found to
have a similar effect on NAC fibrils, indicating that
CLAC could affect other fibrils in a similar way (data
not shown) Preformed fibrils incubated for 24 h in the
presence or absence of CLAC were subjected to electron
microscopy (EM) analysis A significant increase in the
number of large aggregates was observed in the presence
of CLAC (Fig 1C) Thus, EM data also support the
notion that CLAC can assemble Ab fibrils into larger
aggregates A similar function has not been described
for other proteins that are known to bind Ab fibrils,
such as apolipoprotein E or laminin
CLAC protects fibrillar Ab1–40, but not soluble
Ab1–40, from proteolysis
To investigate the biological relevance of CLAC’s
assembly of preformed fibrils, we used proteinase K and
trypsin to determine whether the interaction between
CLAC and Ab fibrils results in protection of the peptide
from proteolysis Both fibrillar Ab1–40 and soluble
Ab1–40 were tested for protease resistance in the
pres-ence or abspres-ence of CLAC CLAC and fibrillar Ab were
incubated at room temperature for 2 h before the
addi-tion of proteases The extent of Ab proteolysis was
eval-uated by SDS⁄ PAGE and western blot analysis When
the fibrils were incubated without CLAC, only a small
amount of Ab remained after 2 h of digestion with
pro-teinase K, and the samples were completely degraded
after 18 h of digestion (Fig 2A) In contrast, when the
fibrils were preincubated with CLAC, Ab was still
pre-sent after 18 h of digestion (Fig 2A) Digestion with
trypsin was less efficient but, in this case, CLAC had a
protective effect (Fig 2B) In contrast, CLAC did not
have a protective effect on soluble Ab1–40 (Fig 2C)
Our findings offer an explanation for the increased
pro-tease resistance of CLAC positive plaques [13]
There-fore, we speculate that CLAC assembles Ab fibrils
into protease-resistant aggregates in vivo and thereby
obstructs the clearance of amyloid
Fibril assembly results in decreased thioflavin T (ThT) fluorescence
One of the most frequently used methods for studying
Ab polymerization is based on the altered fluorescence
of ThT upon binding to amyloid aggregates Therefore,
we investigated whether the ThT assay could be used to study the CLAC-induced assembly of Ab fibrils When
Ab fibrils are incubated in the presence of CLAC there
is a striking reduction in ThT fluorescence (Fig 3A), which is not accompanied by a reduction in the amount
of sedimented Ab, as analyzed by SDS⁄ PAGE and staining with Coomassie blue (Fig 3B) Similar results were obtained after incubation with the positive control, laminin, while incubation with the negative control, aldolase, had no effect on ThT fluorescence (data not shown) When freshly solubilized Ab1–40 was incubated for 5 days, a similar reduction in ThT fluorescence was observed in the presence of CLAC (Fig 4A) No reduc-tion in the amount of fibrils was observed by EM, but the fibrils appeared to aggregate more in the presence of CLAC (Fig 4B) As CLAC binds to Ab fibrils, but not
to freshly dissolved Ab, CLAC does not affect the lag-phase (Fig 4A), and the reduction in ThT fluorescence
at the end-point is probably a result of the effect of
A
B
C
Fig 2 Collagenous Alzheimer amyloid plaque component ⁄ collagen XXV (CLAC) protects fibrillar amyloid b-peptide 1–40 (Ab1–40) from proteolysis (A) Fibrillar Ab1–40 was preincubated with CLAC (at a final concentration of 200 n M ) for 2 h before the addition of protein-ase K (PK) at an enzyme ⁄ Ab ratio of 1 : 10 (w ⁄ w) Samples were taken at the time-points indicated and analyzed on a 10–20% (w ⁄ w) tricine gel, stained with Coomassie blue, and immunoblotted using 4G8 and 6E10 antibodies (B) Fibrillar Ab1–40 treated with trypsin (C) Freshly solubilized Ab1–40 (50 l M ) was preincubated with CLAC (at a final concentration of 200 n M ), digested with PK and analyzed as described in A.
Trang 4CLAC on the fibrils formed during polymerization If
the reduced ThT fluorescence is caused by the increased
fibril assembly in the presence of CLAC, no reduction in
fluorescence should be observed if the fibrils are
immo-bilized prior to the addition of CLAC When Ab fibrils
were immobilized onto microtiter wells, CLAC had no
effect on ThT fluorescence (Fig 4C) Thus, the assembly
of fibrils leads to a reduction in ThT fluorescence,
poss-ibly because of a decrease in the number of ThT-binding
sites and⁄ or quenching owing to a high concentration of
ThT in the bundles This is in keeping with a previous
report [14] Based on ThT fluorescence analysis, a
num-ber of compounds have been reported to depolymerize
fibrillized Ab within a few hours [15,16] In the light of
our present study, we would like to point out that the
results from ThT-binding studies must be evaluated
carefully and supplemented with data obtained by using
alternative techniques In summary, we have shown that
CLAC assembles Ab fibrils into protease-resistant
aggregates We speculate that this process may be of
relevance for AD pathogenesis, offering one explanation
for the accumulation of amyloid plaques in vivo Future
studies in mice with altered CLAC expression will investigate this hypothesis
Experimental procedures
Preparation of fibrillar Ab
Ab1–40 and Ab1–42 were purchased from Bachem (Buben-dorf, Switzerland) Ab fibrils were prepared by mixing lyophilized peptide with ultrapure water to obtain a peptide
A
B
Fig 3 Reduction in thioflavin T (ThT) fluorescence in the presence
of collagenous Alzheimer amyloid plaque component ⁄ collagen XXV
(CLAC) does not correlate with the amount of sedimented amyloid
b-peptide (Ab) (A) Preformed Ab1–40 fibrils incubated in the
pres-ence (100 n M ) or absence of CLAC were mixed with 200 lL of
10 l M ThT for 15 min Each data point represents the mean ± SEM
of a triplicate result of a representative experiment (B) The
sam-ples (one of the triplicates) were centrifuged, washed, dissolved in
70% (v ⁄ v) formic acid and vacuum dried, then loaded onto a
10–20% (w ⁄ w) tricine gel and stained with Coomassie blue after
electrophoresis The samples from time-points 0, 0.5 and 1 h, for
fibrils incubated either with CLAC or with buffer, were analyzed on
the same gel to allow comparison of the fibril amount The samples
from 2, 3 and 4 h were analyzed on another gel.
A
B
C
Fig 4 Effect of collagenous Alzheimer amyloid plaque compo-nent ⁄ collagen XXV (CLAC) on the polymerization of amyloid b-pep-tide 1–40 (Ab1–40) (A) A concentration of 25 l M freshly dissolved Ab1–40 in NaCl ⁄ Tris, pH 7.4, was incubated for 5 days at room temperature, with shaking (600 r.p.m.), in the presence or absence
of CLAC (final concentration 100 n M ) A molar ratio of Ab1–
40 ⁄ CLAC of 250 : 1 was used Fibril formation was monitored by ThT fluorescence Each data point represents the mean ± SEM of three separate experiments (B) Negative stain electron micro-graphs of the 5 day incubation mixtures in (A) Ab1–40 fibrils in the absence of CLAC (left panel) or in its presence (right panel) Scale bar, 100 nm (C) One nanomol of Ab1–42 fibrils was allowed to dry onto microtiter wells and incubated with CLAC (a final concentra-tion of 250 n M ), followed by incubation with a thioflavin T (ThT) solution of 10 or 100 l M Fluorescence was measured with an exci-tation of 440 nm and an emission of 490 nm.
Trang 5concentration of 200 lm This solution was subsequently
stirred gently for 5 min before the addition of 2· NaCl ⁄ Pi,
pH 7.4, to a final concentration of 100 lm and then
vigor-ously stirred for an additional 2–4 days Before use in
assays, fibrils were sedimented by centrifugation at 10 000 g
for 10 min and the supernatant was replaced by an equal
volume of NaCl⁄ Pi
Ab turbidity and detection
Preformed Ab1–40 fibrils were mixed with 100 nm CLAC
The CLAC used in this study was purified from HEK293
cells, as recently described [8] Ab1–40 fibrils and CLAC or
NaCl⁄ Tris, pH 7.4, were incubated at room temperature
with shaking (600 r.p.m.) At the indicated time-points,
sam-ples (50 lL) were subjected to turbidity measurements
Turbidity was measured at 355 nm (FLUOstar Galaxy;
BMG Labtechnologies GmbH, Offenburg, Germany) and
control values of the absorbance of buffer alone were
sub-tracted from all measurements To detect the amount of Ab
fibrils after ThT fluorescence measurements, the samples
were transferred into Eppendorf tubes and subjected to
cen-trifugation for 5 min at 10 000 g The resulting pellet was
washed twice with NaCl⁄ Tris, while the supernatant was
transferred to a new tube and centrifuged for 5 min at
10 000 g The supernatant was discarded and both pellets
were dissolved in 70% (v⁄ v) formic acid overnight Samples
were vacuum dried to remove the formic acid and then
resuspended in 100 mm Tris, pH 10.5, containing 9 m urea,
prior to analysis by Coomassie blue staining on a 10–20%
(w⁄ w) tricine gel
Electron microscopy
Samples incubated for 24 h were vortexed, and 10 lL
aliqu-ots of the sample were then applied to formvar-coated
grids After 5 min, excess fluid was withdrawn and the grids
were allowed to dry Buffer salts were removed by dipping
the grids (10 times) into redistilled water The specimens
were negatively stained with 2% (w⁄ v) uranyl acetate in
water, examined in a Philips CM120 electron microscope at
80 kV, and photographed using a MegaView III CCD
cam-era (Soft Imaging System, Mu¨nster, Germany)
Proteolysis of the Ab1–40/CLAC complex
Ab1–40 fibrils were prepared as described above Fibrils
were subjected to proteolysis using proteinase K (Sigma
Aldrich, St Louis, MO, USA; Signet Laboratories,
Ded-ham, MA, USA) and trypsin (Boehringer Mannheim,
Milan, Italy) at an enzyme⁄ protein ratio of 1 : 10 (w ⁄ w) for
all experiments CLAC (at a final concentration of 200 nm)
was added to Ab1–40 fibrils (50 lm) or to freshly dissolved
Ab1–40 (50 lm) in NaCl⁄ Pi and incubated for 2 h before
the addition of proteinase K or trypsin Aliquots were taken after 0, 2 and 18 h of incubation at 37C The reac-tion was stopped by the addireac-tion of formic acid to a final concentration of 70% Samples were treated, as described above, prior to SDS⁄ PAGE analysis on 10–20% (w ⁄ w) tricine gels followed by staining with Coomassie blue and immunoblotting using 6E10 and 4G8 antibodies (Signet Laboratories)
ThT assay
The ThT-binding assay [17] was used to measure the amount of Ab1–40 fibrils in the presence of CLAC Twenty microlitres of Ab, incubated in the presence or absence of CLAC, laminin or aldolase, was aspirated and further incu-bated for 15 min with 200 lL of 10 lm ThT solution (10 mm phosphate buffer, 150 mm NaCl, pH 6.0) Fluores-cence spectra of ThT were acquired using a fluoresFluores-cence spectrometer (FLUOstar Galaxy), with excitation at
440 nm and emission at 490 nm Control spectra of the ThT solution alone were recorded and subtracted from all measurements The amount of Ab in the pellet fraction was determined as described above For the polymerization
assay, a stock solution of 1 mgÆmL )1 Ab1–40 in dimethyl-sulfoxide was diluted in NaCl⁄ Tris to a final concentration
of 25 lm Ab polymerization was carried out in the pres-ence of CLAC (100 nm) or Ab1–40 alone at room tempera-ture with continuous shaking at 600 r.p.m At the indicated time-points, a 50 lL aliquot from each incubation mixture was analyzed for Ab fibril formation by mixing with
200 lL of ThT solution, and the fluorescence was measured
as described above
Solid-phase ThT-binding assay
A competition assay based on solid-phase binding was used, as previously described, with some minor modifica-tions [6,8] Briefly, a dimethylsulfoxide stock solution of Ab1–42 was diluted in NaCl⁄ Pi to a concentration of
20 lm One nanomol of Ab1–42 was allowed to bind to microtiter wells (MaxiSorp; Nunc, Naperville, IL, USA) and dry overnight, at 37C Wells were blocked for 1 h in NaCl⁄ Pi containing 1% (w⁄ v) BSA (blocking buffer) fol-lowed by washing with NaCl⁄ Pi-T [NaCl⁄ Pi containing 0.05% (v⁄ v) Tween-20 (Sigma)] in a microplate washer (ASYS Hitech; Atlantis, GmbH, Eugendolf, Austria) Ab was incubated with 250 nm CLAC in blocking buffer, or in blocking buffer only, for 1 h at room tempera-ture The wells were washed in NaCl⁄ Pi-T and incubated with NaCl⁄ Picontaining 10 or 100 lm ThT for 30 min fol-lowed by washing in NaCl⁄ Pi-T One-hundred microlitres
of NaCl⁄ Pi was added to the wells, and binding of ThT was measured in a microplate reader (FLUOstar Galaxy) with excitation at 440 nm and emission at 490 nm
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