Small2 1 Department of Pathology, University of Melbourne, Victoria, Australia;2Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia;3Department of Ch
Trang 1b-Amyloid protein oligomers induced by metal ions and acid pH
are distinct from those generated by slow spontaneous ageing
at neutral pH
Genevieve M J A Klug1, Dusan Losic2,3, Supundi S Subasinghe1,2, Marie-Isabel Aguilar2,
Lisandra L Martin3and David H Small2
1
Department of Pathology, University of Melbourne, Victoria, Australia;2Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia;3Department of Chemistry, Flinders University, Adelaide, Australia
Amyloid protein (Ab1–40) aggregation and conformation
was examined using native and sodium dodecyl sulfate/
polyacrylamide gel electrophoresis,and the results
com-pared with those obtained by atomic force microscopy,
and with Congo red binding,sedimentation and turbidity
assays The amount of Ab aggregation measured was
different,depending upon the method used Incubation
for 15 min at pH 5.0 or in the presence of Fe2+,Cu2+or
Zn2+did not alter the level of Ab oligomers observed on
SDS and native gels However,the slow aggregation of
Ab to form high molecular mass species over 5 days was
inhibited In contrast,when Ab aggregation was
monit-ored using a Congo red binding assay or sedimentation
assay,a rapid increase in Ab aggregation was observed
after incubation for 15 min at pH 5.0,or in the presence
of Fe2+,Cu2+or Zn2+ The low pH-, Zn2+- or Cu2+ -induced Ab aggregation measured in a turbidity assay was reversible In contrast,a considerable proportion of the
Ab aggregation measured by native and SDS/PAGE was stable Atomic force microscopy studies showed that Ab aged at pH 5.0 or in the presence of Zn2+ produced larger looser rod-shaped aggregates than at pH 7.4 Ab that had been aged at pH 7.4 was more cytotoxic than Ab aged at pH 5.0 Taken together,the results suggest that
Ab oligomerizes via two mutually exclusive mechanisms
to form two different types of aggregates,which differ in their cytotoxic properties
1
Keywords: Alzheimer’s disease; amyloid; Ab aggregation; toxicity; fibril
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder,characterized by the accumulation of amyloid in
the brain in the form of amyloid plaques and cerebral
amyloid angiopathy The major component of the amyloid
plaques,the amyloid-b protein (Ab),is a polypeptide of
39–43 amino-acid residues,which is derived from a larger
amyloid-b protein precursor (APP) [1–4] Ab can
poly-merize via a nucleation-dependent process [5,6] generating
insoluble fibrillar aggregates which form amyloid plaques
Analysis of plaque amyloid has revealed that these
aggre-gates adopt a b-sheet arrangement [7,8] Aggregation of Ab
in vivo may also lead to the formation of ill-formed,
nonfibrillar amorphous aggregates known as the diffuse or
fleecy plaques [9]
There is strong evidence that Ab has a causative role in the development of AD The neurotoxicity of Ab has been demonstrated in neuronal cultures [10–12] and aggregation
of Ab,which can be generated by aging (i.e incubation of the peptide for several days),is required for this effect [10,11] Recent studies have shown that low molecular mass oligomeric species are also neurotoxic [13–15] In contrast, diffuse,amorphous aggregates of Ab do not appear to possess the neurotoxic properties of the fibrillar forms [16] The mechanism by which monomeric Ab is converted
to high molecular mass species in vivo is unknown The influence of metal ions on aggregation in vitro has been investigated extensively Zn2+and Cu2+have been shown
to promote aggregation [17–20] and it has been suggested that the toxicity of Ab involves free radical-induced oxidative damage through the involvement of Cu2+ [20,21] Several studies have demonstrated that the aggre-gation of Ab can occur under acid pH conditions [22,23], such as those which occur in intracellular vesicular com-partments Thus,some Ab aggregation could also occur intracellularly,prior to secretion
Not all studies have yielded similar conclusions about Ab aggregation For example,it has been reported that at
pH 5.0,Ab rapidly aggregates to form fibrils [24] However,
a more recent study suggested that aggregated species generated at low pH are nonfibrillar and are unable to be converted into fibrils or to seed fibril formation [25] The aim of the present study was to examine the effect of
pH and metal ions on the aggregation and conformation of
Correspondence to D H Small,Department of Biochemistry and
Molecular Biology,Monash University,Victoria,3800,Australia.
Fax: + 61 3 9905 3726,Tel.: + 61 3 9905 1563,
E-mail: david.small@med.monash.edu.au
Abbreviations: Ab, b-amyloid protein; AD,Alzheimer’s disease; AFM,
atomic force microscopy; APP,amyloid precursor protein; CR,
Congo red; EDTA,ethylenediaminetetraacetic acid; HOPG,highly
oriented pyrolytic graphite;
MTS,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium;
VSMC,vascular smooth muscle cell.
(Received 17 June 2003,revised 19 July 2003,
accepted 2 September 2003)
Trang 2Ab and to relate this to the peptide’s toxic effects Most
techniques for studying Ab aggregation do not easily
discriminate between different oligomeric species Some
studies have shown that Ab can form SDS-resistant
oligomers,which can be measured by SDS-gel
electrophor-esis [11,24,26] However, it is unclear whether the formation
of SDS-resistant species accurately reflects the overall state
of Ab aggregation For this reason,in the present study,we
examined Ab aggregation using both SDS and non-SDS
(native) PAGE We also examined Ab aggregation using
atomic force microscopy (AFM),Congo red (CR) binding,
sedimentation and turbidity assays Our results show that
some oligomeric Ab species are sufficiently stable to allow
their measurement by gel electrophoresis However,we
show that not all forms of oligomeric Ab are observed by
PAGE and that different patterns of Ab aggregation are
observed,depending upon the method by which
aggrega-tion is measured Taken together these results suggest that
Ab aggregates by two separate pathways One pathway,
which is inhibited at pH 5.0 or by metal ions,slowly
generates stable species that can be measured by PAGE
The other pathway generates unstable species that rapidly
disaggregate and therefore cannot be measured by PAGE
Furthermore,the results of toxicity studies suggest that the
slow aggregation at pH 7.4 can produce more toxic forms of
Ab than the rapid aggregation at pH 5.0
Materials and methods
Materials
Electrophoretic molecular mass markers and reagents for
enhanced chemiluminescence (ECL) were purchased from
Amersham Pharmacia Biotech,Sydney,NSW,Australia
Electrophoretic reagents and Trans-Blot nitrocellullose
membranes were obtained from Bio-Rad Laboratories,
North Ryde,NSW,Australia A mouse monoclonal
antibody (mAb) WO2,which recognizes the N-terminal
region (residues 1–5) of Ab,has been described previously
[27] Congo red was obtained from Sigma Chemical Co
(St Louis,MO,USA) Highly oriented pyrolytic graphite
(HOPG) was purchased from Group Scientific Pty Ltd
(Adelaide,Australia)
Synthesis and solubilization of Ab1–40
Ab1–40 was synthesized utilizing manual solid-phase
N-tert-butoxycarbonyl (Boc) amino-acid chemistry as
des-cribed by He and Barrow [23] Briefly,peptides were
synthesized using manual solid-phase Boc amino-acid
chemistry with in situ neutralization Peptide purification
was achieved using an acetonitrile/water (0.01%
trifluoro-acetic acid) gradient on a reverse-phase preparative Zorbax
HPLC column heated to 60C Peak fractions were
lyophilized and the purity (‡ 95%) and identity of the
peptide were analysed by analytical HPLC,electrospray
mass spectroscopy and amino-acid analysis Preliminary
studies using PAGE demonstrated that Ab peptides
dissolved and stored at)80 C in dimethylsulfoxide were
less aggregated than when dissolved and stored in water
Furthermore the results of PAGE experiments were found
to be more reproducible when the stock Ab was made up in
dimethylsulfoxide Therefore,routinely Ab1–40 was dis-solved in 100% dimethylsulfoxide at a concentration of
2 mMand sonicated for 20 min Sonication was used to help dissolve the peptide and was not found to have any effects
on the final outcome of the experiment as those experiments performed in the absence of sonication showed similar results Peptide solutions were then filtered using 0.22 lm centrifuge tube filters (Costar) for 3 min at 10 000 g to remove particulate matter Filtration did not cause any significant loss of Ab as there was no significant change in the concentration of UV-absorbing material following filtration The peptide was stored at)80 C Under these conditions,Ab was stable and no aggregation was observed during storage Furthermore,no significant differences were observed in the ability of different batches of Ab1–40 stored for different periods of time to aggregate
Just prior to use,all peptide solutions were diluted to 1–2.5% (v/v) dimethylsulfoxide with deionized water or
20 mM sodium phosphate buffer,pH 7.4 that had been prefiltered using 0.45 lm filter units (Millipore,Bedford, MA,USA)
Electrophoresis and Western blotting Samples were analysed on 15% native [28] or SDS/ polyacrylamide gels using a Tris/tricine buffer system over 1.5 h [29] The duration of electrophoresis was 1.5 h in the presence of SDS or 2 h for the native gels (in the absence of SDS) After electrophoresis,Ab was detected by Western blotting,which yielded similar results to silver staining,but was much more sensitive for the detection of higher molecular mass complexes Protein was electrophoretically transferred from the gels onto nitrocellulose at a constant current of 300 mA overnight Membranes were then pre-blocked with 0.5% (w/v) casein in NaCl/Pi,pH 7.4 with gentle agitation for 1 h at room temperature The blocking solution was replaced with primary monoclonal mouse antibody,WO2 (1 : 50 dilution in blocking solution) and incubated with gentle agitation for 2 h at room temperature Blots were then probed with a secondary polyclonal rabbit anti-(mouse IgG) Ig conjugated to horseradish peroxidase (1 : 5000 dilution in blocking solution) (Amersham Phar-macia Biotech,Sydney,NSW,Australia) with gentle agita-tion for 1 h and then developed by the ECL detecagita-tion system SDS and non-SDS/PAGE in two dimensions
Stock solutions of 2 mMAb1–40 were thawed and diluted into 20 mMsodium phosphate buffer to a final concentra-tion of 10 lM Samples were incubated at 37C for 15 min and then loaded (2 lg per lane) onto 1 mm thick,15% Tris/ tricine gels prepared with or without 0.1% SDS and separated in the first dimension After electrophoresis,gels were removed and single lanes excised,bathed in freshly prepared stacking gel (for SDS/PAGE slices) or separating gel (for non-SDS/PAGE slices) and then loaded horizon-tally onto a second gel with or without 0.1% SDS The buffer for electrophoresis was the same in the second dimension as in the first Proteins were separated in the second dimension,after which slab gels were electroblotted onto nitrocellulose and then analysed by Western blotting with the mAb WO2
Trang 3Congo red binding assay
Ab1–40 was diluted into NaCl/Pi(pH 7.4) in the presence
or absence of 1 mM MgSO4,CaSO4,CuSO4,FeSO4 or
ZnSO4to give a final peptide concentration of 10 lM CR
(100 lMstock in NaCl/Pi,pH 7.4) was then added to the
peptide solution to give a final concentration of 10 lMCR
and 9.09 lMAb1–40 This ratio of CR to Ab was required
for maximum saturation of all CR binding sites on Ab1–40
aggregates [30] Solutions of 10 lMCR lacking Ab were also
prepared Solutions were vortexed briefly and then
incuba-ted at room temperature for 15 min Absorbance values at
403 and 541 nm were recorded for samples and CR alone
preparations using a Bio-Rad SmartSpec 3000
spectro-photometer in a cuvette with a 1-cm path cuvette length
Background absorbance values of buffer (with or without
metal ion) alone were subtracted from the values obtained
for each sample The concentration of aggregated Ab in
each preparation was determined as described by Klunk
et al [30] using the formula
Aggregated AbðlgmL1Þ¼ð541nmAbs=4780Þ
ð403nmAbs=6830Þ
ð403nmAbsCR alone=8620Þ The amount of aggregated Ab monomer was then
calculated assuming a molecular mass for Ab1–40 of
4330 All preparations were prepared in triplicate and the
assay was conducted independently three times with similar
results in each experiment
Sedimentation assay of Ab aggregation
Ab aggregation was essentially measured using a
sedimen-tation assay as described by Atwood et al [19] Ab1–40
(100 lM) was diluted to a final concentration of 10 lMin
20 mMsodium phosphate buffer (pH 7.4 or 5.0) containing
1 mMZnSO4,FeSO4,CuSO4,MgSO4,CaSO4or no metal
After incubation for 15 min or 120 h at 37C,the samples
were centrifuged at 12 000 g in a Z160M microcentrifuge
(Hermle Labortechnik,Wehingen,Germany) for 10 min
After centrifugation,the supernatant fractions were removed
and the pellets were resuspended in sample buffer (100 lL)
containing 0.5 M Tris/HCl,pH 6.8,5% (v/v) glycerol,
0.005% (w/v) bromophenol blue,2% (w/v) SDS and 5%
2-mercaptoethanol Samples were boiled for 5
min,centri-fuged and then analysed by 15% SDS/PAGE Ab was
blotted electrophoretically onto nitrocellulose sheets and Ab
immunoreactivity was visualized by ECL The total
immu-noreactivity in each lane was then quantified by densitometry
usingSCION IMAGESoftware (Scion Corporation,Frederick,
MD,USA) Mean values of total lane immunoreactivity
were then determined from the analyses of the triplicate
samples (3 lanes) The percentage increase in
immunoreac-tivity in the pellet fraction compared with control incubation
(no metal,pH 7.4,00.25 h) pellet fraction was then calculated
Atomic force microscopy
Ab1–40 (2 mM in dimethylsulfoxide) was diluted to
10 l with 20 m sodium phosphate buffer,pH 5.0,
pH 7.4 or pH 7.4 with 1 mM of Zn2+ (as ZnSO4) Solutions were incubated at room temperature for 15 min and 120 h without agitation Immediately prior to AFM imaging,the solutions were diluted 50–100 times using same buffer solution Five lL of the prepared solution was applied to the substrate (HOPG),left for one minute, and then rinsed with 100 lL of water twice This sample was dried with stream of nitrogen for one min and used for imaging immediately Aged solutions were prepared in the same manner following incubation for 120 h Some samples were left to age while on the substrate,in air for
120 h
AFM imaging was performed using a MultiMode microscope in conjunction with a Nanoscope IV system (Digital Instruments,Santa Barbara,CA) Tapping mode
in air was used for the experiments reported in this work, but contact mode was also used to obtain higher resolution images of fibrils Silicon cantilevers (Digital Instruments, Santa Barbara,CA,model TESP),which operate at frequencies of the 300–400 kHz were used Height and phase data were simultaneously collected at a scan rate between 1 and 3 Hz Typical images were acquired from several regions on the substrate Data processing (particle size measurement) and cross section analysis of Ab oligo-mers was performed usingNANOSCOPE III software (Veeco Instruments Inc.,Santa Barbara,CA,USA)
Turbidity assay of Ab aggregation
Ab aggregation was measured using a turbidity assay as described by Huang et al [31] To examine the effect of metal ions,solutions of 50 lMFeSO4,Zn SO4and CuSO4
in 40 mM sodium phosphate buffer,pH 7.4 were pre-pared Ab1–40 (2 mM in dimethylsulfoxide) was diluted with H2O to a 50-lM concentration Metal and Ab solutions were combined to give a final ratio of 25 lM Ab1–40–25 lM metal ion in 20 mM sodium phosphate buffer Solutions (200 lL) were immediately added to flat-bottomed microtitre plate wells (Nunclon,Nunc,Den-mark) in triplicate Plates were incubated at room temperature The absorbance at 405 nm was monitored
at 1-min intervals using a Wallac 1420 Multilabel counter and 1420 software 2.0,release 8 (Perkin Elmer Life Sciences,Turku,Finland) Plates were agitated by orbital shaking every 30 s between measurements to resuspend peptide aggregates After 4 min,20 lL aliquots of either
10 mM ethylenediaminetetraacetic acid (EDTA),10 mM metal ion or H2O were added to each well After each addition of metal or chelator,samples were equilibrated for 2 min at room temperature with agitation every 30 s (equilibration period) and then absorbance measurements were recorded
To assess the stability of Ab1–40 oligomers formed at low pH,the turbidity of an Ab1–40 solution was examined at pH 5.5 and after conversion to neutral pH Ab1–40 (2 mM in dimethylsulfoxide),was diluted to
25 lM with H2O Samples (200 lL) were added to microtitre plate wells (in triplicate) and absorbance measured at 405 nm at 1-min intervals The pH was adjusted as appropriate by addition of 10 lL of 100 mM sodium acetate buffer,pH 5.5,H2O or 40 lL of 500 mM sodium phosphate buffer,pH 7.8
Trang 4Cell viability (MTS) assay
Cell lines of Wistar-Kyoto (WKY) rat aortic VSMC were
obtained from G Dusting (Howard Florey Institute of
Experimental Physiology,Melbourne,Australia) Cells were
grown in 96-well titre plates in DMEM with 10% fetal
bovine serum and 1% (v/v) penicillin/streptomycin until
80% confluent before treatment with Ab40 preparations
Ab (100 lM) was aged for 15 min or 120 h in 20 mMNaPO4
buffer at pH 5.0 or 7.4 Aged Ab was then diluted in culture
medium and added to VSMC cultures at 10 lMfor 24 h To
determine cell viability after treatment,a 10-lL aliquot of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added to
100 lL cell culture medium per well Culture plates were
covered in foil and then allowed to incubate for 2 h at 37C
Absorbance values were then determined using a Wallac
1420 Workstation at a wavelength of 560 nm
Results
Analysis of Ab by PAGE
Previous studies [26] have shown that Ab oligomers are
stable enough to be analysed by SDS/PAGE Therefore,
initially,we examined the state of aggregation of freshly
prepared Ab by SDS/PAGE A stock solution of Ab1–40
(2 mMin dimethylsulfoxide) was diluted into 20 mMsodium
phosphate buffer,pH 7.4 to achieve a final concentration of
100 lM Ab was incubated for 15 min at 37C and then
analysed by electrophoresis (2 lg per lane) for 1.5 h on a
15% Tris/tricine polyacrylamide gel in the presence of 0.1%
SDS After electrophoresis,proteins were blotted onto
nitrocellulose,which was stained for Ab immunoreactivity
Ab-immunoreactive bands with apparent molecular masses
of 4-,8-,and 12- kDa were observed (Fig 1A) Trace
amounts of higher molecular mass species (> 50 kDa) were
also observed
To determine the stability of the oligomeric Ab species
observed upon SDS/PAGE,a two dimensional gel
electro-phoresis approach was used Ab was incubated for 15 min as
before,separated by Tris/tricine SDS/PAGE and then
sub-jected to an identical electrophoresis step in the second
dimension over 1.5 h The time taken between the two
electrophoresis steps was 30 min After electrophoresis in the
second dimension,the gel was electroeluted onto
nitrocellu-lose and stained for Ab immunoreactivity (Fig 1B)
Analysis showed that a proportion of the Ab
immuno-reactivity migrated at the same relative molecular mass in
the second dimension as that seen in the first dimension,
indicated by the presence of a diagonal band of Ab
immunoreactivity running across the gel (Fig 1B) Some
immunoreactivity was spread in a horizontal staining
pattern,indicating that little oligomeric Ab had dissociated
to lower molecular mass forms From this experiment,it was
evident that a proportion of SDS-resistant Ab oligomeric
species are relatively stable over the time course required to
perform the two electrophoretic steps (a total of 3.5 h)
Ab was also analysed by native PAGE in the absence of
SDS (Fig 1C) In contrast to SDS/PAGE,the majority of
the Ab immunoreactivity was seen in the higher molecular
mass region of the gel To assess the stability of Ab
oligomers seen by native PAGE,PAGE was again performed using the same two-dimensional approach as that for SDS/PAGE,except that SDS was omitted from the electrophoresis buffer in both dimensions (Fig 1D) The total time for electrophoresis on non-SDS/PAGE in each dimension was 2 h and the time taken between each electrophoretic step was 30 min Similar to the SDS/PAGE, the relative mobility of the Ab bands on native PAGE performed in two dimensions remained constant over the period of electrophoresis (4.5 h) A small amount of dissociation was also observed in the second dimension, indicated by horizontal bands directly below the higher molecular mass species However,most of the Ab migrated
at the same relative mobility,producing a diagonal staining pattern This finding showed that a proportion of the Ab oligomers seen on non-SDS/PAGE systems were stable over at least a 4.5-h period
Time course of Ab oligomerization Previous studies have shown that when Ab is incubated for several days, the peptide aggregates [10,16,24,32] To examine the time course of oligomerization of Ab using PAGE,Ab1–40 (10 lM) was dissolved in 20 mM sodium phosphate buffer,pH 7.4 and incubated at 37C for up to
120 h After 0.25,24,48 and 120 h of incubation at 37C, aliquots (15 lL) were collected and analysed by SDS- and non-SDS/PAGE After electrophoresis,the gels were elec-troblotted onto nitrocellulose and stained using the mAb WO2
The time-dependent aggregation of Ab was observed on both native and SDS gel systems (Fig 2) On SDS/PAGE,
Fig 1 Western blot analysis of Ab1–40 analysed by SDS and native PAGE Ab1–40 (100 l M ) was incubated for 15 min in 20 m M sodium phosphate buffer at 37 C and then aliquots (2 lg) were loaded per lane onto the SDS (A) or native (C) gels After electrophoresis in one dimension,the gels were blotted onto nitrocellulose and stained for Ab immunoreactivity with the Ab-specific mAb,WO2 In a second experiment,after electrophoresis in the first dimension,single lanes were excised and loaded horizontally onto a second gel of the same type,either a SDS (B) or a native gel (D) After separation in the second dimension,protein was transferred to nitrocellulose and then probed with the mAb WO2 m,molecular mass in kDa R f (relative mobility with respect to the bromophenol dye front),distance from the origin/distance migrated by the dye front.
Trang 5Ab-immunoreactive bands with apparent molecular masses
of 4-,8-,12- and 95-kDa were observed in samples
incubated for 15 min (Fig 2) After 24 h incubation,an
increase in the 12- and 95-kDa species and the appearance
of a 16-kDa species was observed These changes were
accompanied by a decrease in the 4-kDa species Between 24
and 120 h a reduction in the 4-,8- and 12-kDa bands and an
increase in high molecular mass species was observed,
suggesting that the majority of low molecular mass species
had been converted to higher molecular mass aggregates
Loss of the low molecular mass species was not due to
proteolysis by contaminating proteases,because inclusion of
a cocktail of broad spectrum protease inhibitors did not
block the disappearance of low molecular mass Ab species
(data not shown)
In contrast to the results for SDS/PAGE,both high and
low molecular mass oligomeric species were observed on
native PAGE,even after 15 min of incubation (Fig 2) After
24 h,little of the lower molecular mass species was observed
An immunoreactive band at the top of the gel was also
diminished over the time course This apparent loss of high
molecular mass immunoreactivity was due to Ab
aggrega-ting to such an extent that it was unable to enter the gel
Taken together,the results from both gel systems
demonstrated that initially,most of the Ab1–40 was present
in an aggregated form,although these aggregates were not
stable in the presence of SDS However,with increasing
time of incubation over several days,the proportion of SDS-stable aggregates increased
Effect of pH and metal ions – PAGE analysis The effect of pH on Ab1–40 aggregation was examined by PAGE Despite several reports [22,23] demonstrating that
Ab aggregation is promoted at pH 5.0,analysis of Ab aggregation at pH 5.0 revealed that the slow aggregation over 5 days of Ab to higher molecular mass species that can
be seen by PAGE was less than at pH 7.4 (Fig 2) Initially, after 15 min of incubation,there was little difference between the pH 7.4 and pH 5.0 incubation However,at later time points,there was a greater proportion of lower molecular mass forms in the pH 5.0 incubation than in the
pH 7.4 incubation A small increase in a 12-kDa Ab oligomer on SDS/PAGE was seen at pH 5.0 However,no increase was seen in the level of other oligomeric species at
pH 5.0,and the loss of the 4 kDa Ab monomer was less
at pH 5.0 than at 7.4 A similar result was obtained by non-SDS/PAGE,as the rapid conversion of low to high molecular mass species seen at pH 7.4 was not observed at
pH 5.0
Previous studies have shown that metal ions,notably
Zn2+ and Cu2+ can stimulate Ab aggregation [17–19] However,similar to the results obtained at acid pH,metal ions were found to inhibit Ab aggregation analysed by PAGE (Fig 3) Once again,little difference was seen in the extent of oligomerization in the presence of metal ions after
15 min of incubation However,on SDS/PAGE,the slow aggregation of low molecular mass species to larger,SDS-stable oligomeric species was found to be strongly inhibited
by Zn2+,Cu2+,and slightly less so by Ca2+and Fe2+
Mg2+appeared to have little effect on the aggregation of
Ab to SDS-stable species Similarly,the slower aggregation
of Ab over several days observed on non-SDS/PAGE was strongly inhibited by Zn2+and Cu2+,weakly inhibited by
Ca2+and Fe2+and largely unaffected by Mg2+
Effect of pH and metal ions – CR binding and sedimentation analysis
Because of the apparent discrepancy between our results using PAGE,which showed that metal ions and acid pH inhibited aggregation,and previous studies which reported increased aggregation [17–20,22–25], we compared the results obtained by PAGE with those obtained using CR binding and sedimentation assays of Ab aggregation Ab1–
40 was incubated in the presence or absence of 1 mM CuSO4,FeSO4,CaSO4,ZnSO4,or MgSO4at pH 7.4 for
15 min and then the amount of fibrillar Ab was measured using a CR binding assay (Fig 4) In agreement with the previous studies,and in contrast to our PAGE results (Figs 2 and 3) a significantly greater concentration of CR-binding material was observed in the presence of all metals than in incubations lacking metal ions Preparations with Zn2+,Cu2+and Fe2+showed the highest concentra-tions of Ab aggregates (P < 0.05) compared with the control preparation It was not possible to measure the effect of pH using the CR binding assay,because lowering the pH to 5.0 altered the absorbance spectrum of CR (data not shown)
Fig 2 Western blot analysis of the time course of Ab1–40 aggregation
at pH 7.4 and pH 5.0 Ab1–40 (10 l M ) was incubated in 20 m M
sodium phosphate buffer,pH 5.0 or 7.4 at 37 C for 120 h Aliquots
(15 lL) were removed at 0.25,24,48 and 120 h,added to an equivalent
volume of 2 · tricine sample buffer and analysed by 15% Tris/tricine
SDS- and native PAGE Proteins were electrophoretically transferred
to nitrocellulose and Ab-immunoreactivity was detected using the
mAb WO2 All incubations were performed in triplicate m,molecular
mass in kDa R f (relative mobility with respect to the bromophenol dye
front),distance from the origin/distance migrated by the dye front.
Trang 6A sedimentation assay was also employed to examine the
effect of low pH and metal ions on Ab aggregation Ab1–40
(10 lM) was freshly prepared in 20 mMsodium phosphate
buffer (pH 7.4 or 5.0) containing 1 mM ZnSO4,FeSO4, CuSO4,MgSO4 and CaSO4 or no metal Samples were incubated for 15 min or 120 h and then centrifuged at
12 000· g (10 min) to separate aggregated from soluble material After centrifugation,the supernatant fractions containing soluble Ab were removed and the pellets containing aggregated Ab species were resuspended in sample buffer and analysed by SDS/PAGE and Western blotting The results obtained with the sedimentation assay method were similar to those from the CR assay (Fig 5) In the presence of Ca2+,Fe2+,Zn2+,Cu2+and at pH 5.0, significantly more Ab was precipitated than at pH 7.4 and
in the absence of metal ions at the initial time point No significant increase in the amount of precipitated Ab was observed in the presence of Mg2+ After 120 h of incuba-tion,a significant increase in sedimentable material was observed at pH 7.4 in the absence of metal ions In the presence of all metal ions and at pH 5.0,a significant increase in sedimentable material was also observed Analysis of Ab oligomerization by AFM
AFM has proven to be a valuable technique for the study of
Ab aggregation [33–38] We therefore used the morpholo-gical information obtained by AFM to probe the aggrega-tion process over time at pH 7.4 and pH 5.0 Topographic AFM images of Aß1–40 in phosphate buffer at pH 7.4 were taken (Fig 6) Fresh Ab samples (incubated 00.25 h) showed spherical,globular structures of 15–20 nm in diameter evenly dispersed across the substrate (panel A) The height of these globules was less than 5 nm,suggesting that the Ab collapses on the surface and interestingly,many
Fig 3 Western blot analysis demonstrating the effect of divalent cations on Ab1–40 aggregation Ab1–40 (10 ll) was incubated in 20 m M sodium phosphate buffer,pH 7.4 in the presence or absence of 1 m M MgSO 4 ,CaSO 4 ,FeSO 4 ,ZnSO 4 ,or CuSO 4 for 0.25,24,48 or 120 h at 37 C Aliquots (15 lL) were removed at each time point and analysed by SDS and native PAGE Ab immunoreactivity was detected by Western blotting using the mAb WO2 All experiments were performed in triplicate m,molecular mass in kDa R f (relative mobility with respect to the bromophenol dye front),distance from the origin/distance migrated by the dye front.
Fig 4 Congo red (CR) spectrophotometric analysis of Ab1–40
aggre-gation in the presence or absence of 1 m M MgSO 4 , CaSO 4 , FeSO 4 ,
ZnSO 4 , or CuSO 4 at pH 7.4 CR (20 lL) was added to the peptide
solution to give a final concentration of 10 l M CR and 9.09 l M Ab1–
40 Solutions were allowed to incubate for 15 min at room
tempera-ture Incubations of CR alone were also prepared Absorbance values
were then read at 403 and 541 nm The concentration of aggregated
Ab was calculated from the equation,Ab (lgÆmL)1) ¼ ( 541nm
Abs/
4780) ) ( 403nm Abs/6830) ) ( 403nm Abs CR alone /8620) from Klunk et al.
[30] The amount of aggregated Ab monomer was then calculated
assuming a molecular mass of 4330.9 **Significantly different
(P < 0.001) from control incubations with no added metal ion.
*Significantly different (P < 0.05) from control incubations with no
added metal ion (two-tailed Student’s t-test).
Trang 7doughnut-shaped annuli were observed Images of samples aged for 5 days (120 h) showed considerable aggregation in solution The aggregates observed after aging were larger than in the fresh solutions (panel B) The addition of Zn2+ ions to the solution during ageing at pH 7.4 resulted in some alignment of the Ab with branched fibril-like structures (panel C) However,although the branched fibril-like arrangements were visible,these structures were funda-mentally small aggregates of Ab,which were somewhat aligned in an organized manner
Solutions of Ab,aged 120 h at pH 5.0,showed medium-sized spherical structures with some similarities
to those at pH 7.4,although the spheres were much better defined and reproducible (panel D) However at
pH 5.0 the tendency to form linear fibers was clearly apparent and these fibers resembled those observed with
Zn2+ ions (pH 7.4) Distinct mature fibrils comprised of spherical aggregates apparently irreversibly attached to each other were also seen (panel E)
Reversibility of Ab aggregation – turbidity assay
As low pH,Zn2+ or Cu2+ rapidly stimulated Ab aggregation in the CR binding and sedimentation assays but inhibited aggregation on PAGE,we examined the possibility that the aggregated Ab measured by the sedimentation assay and CR binding assay may be unstable and consequently not detectable by PAGE The reversibility
of Ab aggregation was examined using a turbidity assay Ab1–40 (25 lM) was incubated in the presence of 25 lM metal ions in 200 lL of 20 mMsodium phosphate buffer in microtitre plate wells Absorbance was monitored at
Fig 6 AFM images of aggregates and fibrils of Ab1–40 on HOPG substrate after incubation at 15 min and 120 h in phosphate buffer at pH 7.4, or at
pH 5.0 for 120 h or at pH 7.4 with Zn2+ions (A) pH 7.4 incubated for 15 min,inset figure of observed Ab structure with characteristic doughnut shape (B) pH 7.4 incubated for 120 h,inset figure of typical Ab aggregates (C) pH 7.4 with Zn 2+ ions incubated for 120 h,showing formation of long linear aggregates,inset figure of typical small branched Ab fibrils (arrows) (D) and (E) pH 5.0 incubated for 120 h,assemblies of Ab aggregates are seen Panel E shows shows a mature fibril All large topographic images are 2 lm · 2 lm in size and with a height range from 5 nm
to 10 nm Inset image in (A) is 100 nm · 100 nm in size while inset images in (B),(C) and (D) are 200 nm · 200 nm in size Image in (E) is
100 nm · 500 nm with size.
Fig 5 Analysis of the effect of pH 5.0 and divalent cations on Ab1–40
aggregation using a sedimentation assay The percentage increase of
total immunoreactivity of Ab aggregation on SDS/PAGE is shown.
Ab1–40 (10 l M ) was incubated in 20 m M sodium phosphate buffer,
pH 7.4 containing 1 m M MgSO 4 ,CaSO 4 ,FeSO 4 ,ZnSO 4 ,or CuSO 4 or
in 20 mm sodium phosphate buffer,pH 5.0 for 15 min (0.25 h) or
120 h at 37 C Samples were then centrifuged at 12 000 g for 10 min
after which time supernatants were removed The peptide pellet was
resuspended in sample buffer and analysed by 15% Tris/tricine SDS/
PAGE Total immunoreactivity in each lane was determined using
SCION IMAGE software Percentage increase from the control
prepar-ation (20 m M sodium phosphate buffer,pH 7.4,15 min) was
calcula-ted for each incubation type Bars show the mean of three
determinants ± SEM **Significantly different (P < 0.001) from
control incubations with no added metal ion *Significantly different
(P < 0.05) from control incubations with no added metal ion
(two-tailed Student’s t-test).
Trang 8405 nm Initially there was slightly more aggregated Ab in
the presence of metal ions than in their absence (Fig 7A)
EDTA (20 lL,10 mM) was then added to each well but
caused no change in the turbidity of the preparations
Subsequent addition of 20 lL of 10 mM Zn2+,or Cu2+
caused a sharp increase in the amount of Ab aggregation In
comparison,addition of H2O had little effect on the
aggregation of Ab After a second addition of 10 mM
EDTA (20 lL),the turbidity of the solutions containing
Zn2+ and Cu2+ decreased rapidly indicating that the
induced aggregates were unstable and dissociated after
chelation of the metal ions A further addition of 10 mM
Zn2+and Cu2+(20 lL) increased Ab aggregation,which
was rapidly reversible with further addition of EDTA
To examine the stability of Ab oligomers induced at low
pH,Ab1–40 (25 lM) was dissolved in distilled water After
an initial absorbance measurement was recorded,a 10-lL
aliquot of 100 mMsodium acetate buffer,pH 5.5 was added
to reduce the pH The effect of this addition was a marked
and steady increase in turbidity over a 20-min period,
suggesting a rapid promotion of aggregation at low pH
(Fig 7B) The subsequent addition of water (10 lL) had
little effect on Ab aggregation The turbidity of the solution
remained stable over a 25-min period After this,an aliquot
of 500 mMsodium phosphate buffer,pH 7.8 (40 lL) was
added to each well to raise the pH The absorbance sharply
declined after the pH change and remained lower over a
15-min period This result showed that low pH promoted
aggregation but that the aggregation was readily reversible
at higher pH
Cytotoxicity of oligomeric Ab
We also examined which forms of oligomeric Ab are toxic
to vascular smooth muscle cells (VSMC) Preliminary
experiments demonstrated that metal ions (Cu2+,Zn2+)
were very toxic to VSMC (data not shown) Therefore we
did not determine the effect of metal-ion pretreatment on
Ab cytotoxicity However,we were able to examine the
effect of pH on the generation of cytotoxic Ab species
VSMC were treated with Ab incubated (aged) for 15 min or
5 days at pH 5.0 or 7.4 After treatment,the pH of the Ab
solution was adjusted to 7.4 as appropriate and the effect of
the peptide solution on VSMC viability was measured
Using the MTS assay,cell viability was reduced 20% and
40% when treated with Ab aged at pH 7.4 for 15 min and
5 days,respectively (Fig 8) There was a significant increase
in cytotoxicity after aging for 5 days In contrast,Ab was
significantly less toxic when aged at pH 5.0 at both time
points These results indicate that Ab1–40 oligomers
generated at pH 7.4 are more toxic to VSMCs than those
generated at pH 5.0
Discussion
This study demonstrates that some Ab oligomers are
sufficiently stable to enable measurement by SDS- or
non-SDS/PAGE systems Our experiments suggest that,like
SDS/PAGE,non-SDS PAGE can also be used for the
analysis of Ab aggregation Differences between the
amounts of aggregated Ab in the presence or absence of
SDS are probably a reflection,at least in part,of differences
Fig 7 Analysis of the effect of divalent metal ions and low pH on Ab1–
40 aggregation by turbidity assay (A) Ab1–40 (50 l M ),prepared in
H 2 O was diluted in 50 l M ZnSO 4 ,or CuSO 4 and no metal in 40 m M
sodium phosphate buffer,giving a starting ratio of Ab1–40:metal ion
of 25 l M :25 l M Solutions (200 lL) were added to microtitre plate wells and absorbance at 405 nm was measured at four 1-min intervals After the initial reading,a 20-lL aliquot of 10 m M EDTA was added per well and allowed to incubate at room temperature for 2 min before absorbance measurement Following measurement,a 20-lL aliquot of
10 m M metal ion (M 2+ ) or water (control) was added and the absorbance was recorded This sequence was repeated as indicated to determine the effect of repeated metal/chelator doses The data rep-resent the mean difference ± SEM (n ¼ 3) (B) Solutions (200 lL) of Ab1–40 (25 l M ) prepared in H 2 O,were added to microtitre plates After an initial absorbance was measured,a 10-lL aliquot of 100 m M
acetate buffer,pH 5.5 was added to each well to reduce the sample pH Following an equilibration period,three absorbance measurements were made A 10-lL aliquot of H 2 O was then added to each well and absorbance was recorded as for the acetate addition To raise the pH
to neutral,500 m M sodium phosphate buffer,pH 7.8 (40 lL) was then added to each well and two final absorbance measurements were recorded During both assays,plates were agitated every 30 s to resuspend aggregated Ab and each absorbance measurement of four 1-min intervals (except initial) was preceded by a 2-min equilibration period The experiment in panel B was repeated three times with similar results in each experiment.
Trang 9in the sensitivity of specific Ab oligomers to disassembly by
SDS Interestingly,the results of the PAGE experiments
suggested that some of the Ab was aggregated,even after
15 min of incubation in aqueous solution However,some
of this Ab aggregation may have occurred during the
electrophoresis procedure as well
Using PAGE,Zn2+,Cu2+,Fe2+ or low pH were
observed to have little effect on Ab aggregation initially,
although the slow production of oligomeric Ab species was
inhibited In contrast,Zn2+,Cu2+,Fe2+or low pH rapidly
promoted Ab aggregation observed in CR
binding,sedi-mentation and turbidity assays This discrepancy between
the different assay methods is explained by the fact that Ab
can oligomerize via at least two distinct and mutually
exclusive mechanisms to form two different types of
aggregates (Fig 9) The first mechanism is rapid,generates
unstable aggregates,is stimulated at pH 5.0 or by Cu2+and
Zn2+ The second mechanism is slow (occurs over several
days),generates stable aggregates and is inhibited by low
pH,Cu2+or Zn2+
Therefore,our results suggest that some caution is needed
in the interpretation of Ab aggregation data Different
proportions of Ab aggregation may be measured using
different techniques In addition to the differences between
the PAGE assays and the other assays,we also found
discrepancies between the amount of Ab aggregation
measured in a CR binding assay and that obtained with a
sedimentation assay In a CR binding assay,Fe2+,Zn2+
and Cu2+were approximately equipotent in stimulating Ab
aggregation,whereas in a sedimentation assay,Cu2+was
more potent than Zn2+or Fe2+in stimulating aggregation
One possible interpretation of this finding is that the two
assays do not measure exactly the same thing It is likely that
not all of the Ab aggregates measured in the sedimentation assay bind CR Furthermore,it would be expected that the sedimentation assay would favour the measurement of high molecular mass (more readily sedimentable) aggregates, whereas CR might bind less readily to higher molecular mass forms of Ab due to steric hindrance
The results showed that although a proportion of the aggregated Ab measured by PAGE was stable over the time course of the PAGE experiment,most of the Ab aggregation (measured in a turbidity assay) induced by metal ions or by low pH could be easily reversed Once again,this indicated that the two assay methods are measuring different forms of aggregated Ab Of course it was not always possible to exactly match the conditions of incubation in each experi-ment For example,the metal: peptide ratio in the turbidity experiments differed from that used in the other experiments
of the study because it was not possible to easily chelate the metal ion with EDTA at the concentration (1 mM) used in the other studies Therefore a much lower concentration was used Similarly the buffer conditions could not be exactly reproduced in the turbidity experiment looking at the reversibility of pH because of the need to alter the pH during the course of the experiment It was not possible to maintain the same buffer and salt conditions and change the pH Nevertheless,the results of this experiment explain the discrepancies between the other experiments
The conclusion that Ab1–40 can aggregate via distinct mechanisms was supported by the AFM results,which show that aggregates formed slowly at pH 7.4 are distinct in appearance from those formed in the presence of Zn2+or at
pH 5 At pH 7.4,fresh solutions (00.25 h) clearly demon-strated the presence of small aggregates Based on their dimensions on both substrates,it can be estimated that these
Fig 9 A hypothetical model of Ab aggregation and toxicity Ab aggregates via two pathways The first pathway occurs slowly and at neutral pH leading to the generation of stable toxic aggregates [Ab**] n that can be observed on PAGE The second pathway is reversible and leads to a rapid oligomerization of Ab forming unstable nontoxic aggregates [nAb*]
3 that are not observed when analysed by PAGE This pathway is stimulated in the presence of Cu2+and Zn2+and at
pH 5.0 The stimulation of this pathway under these conditions leads
to an inhibition of the generation of stable aggregates as the peptide starting product is redirected toward the generation of an unstable oligomeric species.
Fig 8 Percentage decrease in cell viability of VSMC in the presence of
fresh and 120 h aged Ab40 at pH 7.4 and 5.0 Ab40 (100 l M ) was aged
at 37 C for 0 or 120 h in 20 m M NaPO 4 buffer at pH 7.4 or 5.0 Ab
preparations were diluted in VSMC culture medium to 10 l M which
was then applied to confluent VSMC and incubated for 24 h at 37 C.
Cell viability was then determined using the MTS assay The decrease
in cell viability was calculated as a percentage of the pH 7.4 or 5.0
buffer control Graph shows the mean of three independent
experi-ments ± SEM *Significant difference (P < 0.05) between the pH 7.4
and the corresponding pH 5.0 time point as calculated by the Student’s
two-tailed t-test.
Trang 10aggregates contained up to 4–8 units Aging of the Ab1–40
solution at pH 7.4 caused an increase in aggregation of Ab
in solution
Changes in the oligomeric structure of Ab were evident
upon addition of Zn2+ ions at pH 7.4 or low pH
(pH 5.0) The presence of Zn2+ ions increased
aggrega-tion at pH 7.4 and these aggregates were organized into
proto-fibrils (Fig 6C,D) The proto-fibrils were more
regular in appearance in the low pH solution The data
are consistent with a reversible mechanism functioning at
pH 7.4 in the presence of Zn2+ ions or at low pH,in
which a conformational change in Ab occurs which leads
to the formation of fibrils
The mechanism by which metal ions or low pH stimulate
aggregation is not yet clear Low pH would alter the positive
charge density at the N-terminus of Ab,in the region of the
histidines (residues 6,13 and 14) Furthermore,several
studies have shown that histidine 13 and 14 are involved in
metal-ion binding [19,39,40] Therefore, one possibility is
that the binding of metal ions or protonation of histidines
may induce rapid Ab aggregation by altering the positive
charge density at the N-terminus of the Ab polypeptide
chain This increase in charge density may,in turn,increase
the proportion of b-structure At pH 7.4,Ab1–40 would be
predicted to possess a charge of between)2 and )3 Most
of this negative charge density would be located in the
N-terminal region While it must remain only as
specula-tion,this negative charge might decrease intermolecular
interactions needed for promotion of a b-sheet
configur-ation If this is the case,then protonation or binding of a
metal ion could reduce this charge-charge repulsion and
thereby allow for a b-sheet structure supporting
aggrega-tion Indeed,the circular dichroism studies of He and
Barrow [23] support this view
Several studies [17–20,40–42] suggest that metal ions bind
and promote Ab aggregation and subsequently induce
toxicity via the generation of reactive oxygen species Bush
and coworkers [17] have suggested that Zn2+-promoted
aggregation of Ab may be a key step in the generation of
toxic Ab species However,the role of metal ions in toxicity
is unclear [43] and Mok et al [44] have demonstrated that
the generation of Ab toxicity to VSCMs cannot be blocked
by the antioxidant catalase Acid pH conditions may also
contribute to Ab aggregation,as Ab is first secreted into the
lumen of the endoplasmic reticulum,from which it is
trafficked into the Golgi apparatus,where it is exposed to
the acid pH environment [45]
Our study clearly demonstrates that Ab aggregation
induced by metal ions (or by low pH) occurs via a different
pathway from that which involves the slow aggregation of
stable Ab species Furthermore,cell culture studies suggest
that Ab toxicity can be increased through a process of
aging in which higher molecular mass aggregated forms are
produced [10,11,32] We consistently found that Ab aged at
pH 7.4 over 5 days was more toxic to VSMCs in culture
than Ab that had been incubated for 5 days at pH 5.0
Interestingly,significant toxicity was observed in fresh Ab
solutions (incubated for 15 min at pH 7.4) The lack of
large aggregates in the solution,as observed by AFM,
would suggest that low molecular mass oligomeric forms
of Ab are also toxic,which would be consistent with
pre-vious studies showing that low molecular mass (diffusible)
oligomeric Ab is toxic [13–15,37,46,47] Indeed, studies by Lambert et al [13],Stine et al [37] and Bitan et al [48] suggest that low molecular mass Ab species may be the most toxic form of Ab
In summary,the major conclusion of this study is that Ab can aggregate to form different types of oligomeric complexes and that these complexes may have different toxicities Not all of the Ab in the brain may be toxic,and the mechanism by which Ab aggregates in vivo is likely to be very important in understanding its toxicity So far,very little is known about this mechanism or how toxic species are generated in vivo Ab forms at least two types of plaques
in the brain [49] Amorphous plaques appear to have no associated neurotoxicity,yet fine fibrillar material has been detected in these deposits [50,51] In contrast, neurodegen-eration is more commonly associated with compact amyloid deposits However,even here,not all amyloid plaques may
be toxic,as neuritic pathology is not an invariant feature of all amyloid plaques [49]
The results presented here raise an important issue relating to the development of new therapies for AD While attempts are being made to develop inhibitors of Ab aggregation which may be suitable therapeutic agents,it may not be necessary to inhibit the aggregation of Ab to decrease toxicity As the results of this paper suggest, different forms of Ab may have different toxicities and it may only be necessary to alter the way in which Ab aggregates to reduce toxicity in vivo However,such a possibility must remain speculative until the mechanisms by which Ab aggregates in vivo are more full understood
Acknowledgements
This work was supported by grants from the National Health and Medical Research Council of Australia and from the Monash University Research Fund.
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