Alzheimer’s disease (AD) is the most common age-related dementia. Unfortunately due to a lack of validated biomarkers definitive
in AD progression and many therapeutic strategies target various aspects of this biology. While A? deposition is the most prominent
methods allowing routine monitoring of levels of such species in living populations. We have used surface enhanced laser desorption
containing fraction of blood provides a new source of biomarkers. There are significant differences in the mass spectra profiles of AD
indicate that fundamental biochemical events relevant to AD can be monitored in blood, and that the species detected may be useful
Alzheimer’s disease (AD) is the most common age-related de-
diagnosis still relies on postmortem histological demonstration
progression of AD, and while A? deposition is the most promi-
with disease severity (McLean et al., 1999). A? peptides are gen-
by the proteases ?- and ?-secretases (Masters et al., 2006).
The clinically useful methods for monitoring A? species in
vivo are either in the CSF via ELISA methods using antibodies
raised against monomeric A? (Sjo ¨gren et al., 2003) or via
tive11C-labeled Pittsburgh compound B (PIB) to measure the
2007). Neither the monomeric nor fibrillar forms of A? are
thought to be responsible for the toxicity associated with A?
(Cappai and Barnham, 2008). Recent studies have implicated
oligomeric forms of A? as the toxic species that induce the neu-
ronal dysfunction associated with AD (Walsh et al., 2002; Walsh
and Selkoe, 2004; Shankar et al., 2008). These species include
sue (Shankar et al., 2008). To date, there is no method for mon-
itoring such species in living subjects, although an ELISA assay
for detecting oligomeric A? has recently been described (Xia et
APP is a ubiquitously expressed type 1 transmembrane pro-
tein found in all tissues including blood. Traditionally, when
blood is collected and fractionated for diagnostic purposes,
plasma and/or serum is analyzed while the pellet containing cel-
discarded. Although a recent longitudinal study found that
higher plasma A?42 levels at the onset of the study were associ-
ated with a threefold increased risk of AD (Schupf et al., 2008),
attempts to use plasma A? levels as a biomarker for AD have, at
best, generated variable results (Zetterberg and Blennow, 2006).
Not only is APP a transmembrane protein but so are the pro-
teases that generate A? and a range of studies have shown that
many of the deleterious biological effects attributable to A? are
cell membranes (Glabe and Kayed, 2006; Hung et al., 2008).
by membrane interactions—and A? oligomers have a higher af-
Correspondence should be addressed to Kevin J. Barnham, Level 4, Bio21 Institute, 30 Flemington Road,
TheJournalofNeuroscience,May5,2010 • 30(18):6315–6322 • 6315
finity for lipid membranes (Hung et al., 2008)—we investigated
whether the usually discarded membrane-rich CE fraction may
contain oligomeric A? species that correlate with markers of AD
Participants. The blood of 118 participants was analyzed for the study.
Fifty-two elderly individuals with well documented normal cognitive
function, 43 patients with mild to moderate AD, and 23 subjects with
MCI were recruited for the study (Table 1). Clinical classification was
and clinical dementia rating (CDR). All AD patients met National Insti-
tute of Neurological and Communicative Dis-
orders and Stroke-Alzheimer’s Disease and
Related Disorders Association criteria for
objective cognitive difficulties, predominantly
affecting memory, in the absence of dementia
or significant functional loss (Petersen et al.,
1999). All patients were recruited from the
Austin Health Memory Disorders and Neu-
robehavioural Clinics or the Healthy Aging
Study (Mental Health Research Institute).
None of the participants were receiving, nor
the Austin Health Human Research Ethics
Blood sample preparation. Whole blood was
Bio-One) and processed within 20 min of procurement. Vacutainers
were spun at 3500 rpm (1.9 g) at 4°C for 30 min. The upper layer of
plasma was then removed and small aliquots of 100 ?l were made and
was then homogenized using a vortex and after this 100 ?l aliquots were
made and stored at ?80°C for future use.
Each aliquot was used just once by combining 10 ?l of this material
by duplicate) 130 ?l of this mixture were added per spot to be analyzed.
Surface enhanced laser desorption ionization-time of flight mass spec-
trometry. PS10 ProteinChip arrays (Bio-Rad) were used for the SELDI-
TOF (surface enhanced laser desorption ionization time of flight) mass
added to the arrays in PBS (0.25 mg/ml). To confirm that the binding
observed was not due to nonspecific binding control spectra using a
overnight at 4°C in a humidity chamber.
Excess antibodies were then removed and blocking buffer (0.5 M eth-
anolamine in PBS) was added (5 ?l) and arrays were incubated for 30
min. After the removal of the blocking buffer, each array was washed for
5 min with 50 ?l of 0.5% Triton X-100/PBS (wash-buffer). The solvent
was then removed, and the arrays were washed for 5 min with 50 ?l of
PBS. All biological samples were analyzed in triplicate. Individual sam-
ples (130 ?l) were added to each spot and the arrays were incubated at
spot was washed twice with 100 ?l of wash-buffer for 10 s, followed by a
wash with 100 ?l of PBS twice for 10 s as well. Finally, the arrays were
washed twice with 100 ?l of HEPES 1 mM for 10 s. The array was then
air-dried. One microliter of sinapinic acid (SPA, 50% saturated in 50%
(v/v) acetonitrile and 0.5% in TFA) was applied to each spot twice. The
were performed on a shaking table.
spectra analyzed using Ciphergen ProteinChip software 3.1. The distri-
butions of the mass to charge ratio (m/z) peak intensities in the spectra
showed skewness to either left or right. By taking the logarithm of the
peak intensities, skewness was substantially reduced, and the distribu-
tions met criteria for normality. To demonstrate that the detected peak
intensities are dependent on sample concentration, linear standard
curves of concentration versus peak intensity were constructed for syn-
thetic A? and the A?1-42Met35(O) dimer (see supplemental material,
available at www.jneurosci.org).
Preparation of A?1-42Met35(O) dimer. Resin-bound A?11-42Met35(O)
et al., 2003). Dityrosine was prepared according to the previously re-
of dityrosine and incorporation into SPPS of the A?1-42Met35(O) dimer
was performed according to the previously reported method (Kok et al.,
Genotyping. ApoE genotype was determined by PCR amplification of
Neuropsychological assessments. All subjects undertook a variety of
neuropsychological tasks, designed to assess a broad range of cognitive
6316 • J.Neurosci.,May5,2010 • 30(18):6315–6322Villemagneetal.•Blood-BorneA?DimerinAlzheimer’sDisease
domains commonly affected by AD and aging. In addition to the MMSE
and CDR, episodic memory was assessed using delayed recall of the Cal-
ifornia Verbal Learning Test–Second edition (CVLT-II), and the Rey
Complex Figure Test (RCFT), while executive function was measured
using letter fluency, category fluency, verbal fluency switching task, and
ical test results of 65 healthy older people with negative PiB and normal
MRI scans as the reference, a composite episodic memory score was
generated by taking the average of the Z scores
for the memory tasks (Pike et al., 2007), and a
composite executive function score was gener-
executive function tasks.
Neuroimaging. All subjects underwent a 3D
spoiled gradient echo (SPGR) T1-weighted
MRI acquisition for screening, quantification
of gray matter atrophy, and subsequent coreg-
istration with the PET images. As described
gray matter (GM), white matter (WM), and
CSF using an implementation of the expecta-
tion maximization segmentation algorithm.
The Montreal Neurological Institute (MNI)
single-subject MRI brain template (Collins et
al., 1998) and corresponding Automated Ana-
tomical Labeling (AAL) template (Tzourio-
Mazoyer et al., 2002) and tissues priors were
spatially normalized to each participant to au-
tomatically obtain a parcellation for each se-
lected atlas into GM, WM, and CSF. The
measured gray matter volumes were normal-
ized for head size using the total intracranial
volume, defined as the sum of GM, WM, and
as the proportion of total intracranial volume.
Production of11C-PiB and PiB-PET scans
were performed at the Centre for PET, Austin
Hospital, as previously described (Rowe et al.,
2007). Briefly, a 30 min acquisition emission
PET scan was acquired starting at 40 min after
gional Standardized Uptake Value (SUV), de-
concentration, corrected for injected dose and
body weight, was normalized to the cerebellar cortex to obtain SUV
as the average SUVR of the area-weighted mean for the following
cortical regions of interest: frontal (consisting of dorsolateral pre-
frontal, ventrolateral prefrontal, and orbitofrontal regions), superior
parietal, lateral temporal, lateral occipital, and anterior cingulate and
normality of distribution using the Shapiro–Wilk test and visual inspec-
tion of variable histograms. Statistical evaluations were performed
(Benjamini and Hochberg, 1995) to select the peaks that were different
between groups. The selected peaks for each group were then compared
using ANOVA, followed by a Dunnet’s test to compare each group with
controls, and a Tukey-Kramer HSD test to establish differences between
test. Pearson product-moment correlation analyses were conducted be-
tween the different variables. Assessment of the robustness of the corre-
lations was performed via tenfold leave-10%-out cross-validation. To
trum peaks, a hierarchical cluster analysis using an average linkage
method was performed (Sokal and Michener, 1958). In all instances
statistical significance was defined as p ? 0.05. Multiple comparisons
Demographic, clinical, neuropsychological, and neuroimaging
characteristics of the 118 participants are reported in Table 1.
pathways for APP: an amyloidogenic pathway that is elevated in AD and a non-amyloidogenic pathway that is elevated in the
Correlation between A?42 monomer and dimer. The intensities of the peaks
Villemagneetal.•Blood-BorneA?DimerinAlzheimer’sDisease J.Neurosci.,May5,2010 • 30(18):6315–6322 • 6317
MMSE and cognitive composite scores.
The AD group had a higher prevalence
(?70%) of ApoE ?4 allele carriers. There
was no significant difference in age or in
gender distribution between groups.
Blood was collected from all participants
were then extracted with an aqueous solu-
break up any potential protein/protein or
protein/membrane interactions involving
any APP/A? fragments. A variety of dena-
turants and detergents were assessed over a
large concentration range to identify the
conditions that reproducibly gave the best
signal-to-noise in the mass spectra. The ex-
cate by SELDI-TOF MS, with the operator
blinded to the disease status of the subjects,
using antibody capture (WO2 epitope A?
residues 4–8 and 4G8 epitope A? residues
17–21). These are generic anti-A? antibod-
subjects contained a large number of
peaks; with m/z ranging from 3.5 to 16.0
kDa; consistent with a variety of APP/A?
fragments present in the CE. Moreover,
the spectra obtained from AD subjects
were substantially different to that of the
HC, and ?10 peaks showed significant
shows representative examples). The m/z
values of the peaks with intensities that were found to be signifi-
2. SELDI-TOF MS of the plasma fraction did not resolve any
peaks that were significantly different between the control and
disease groups; nor were any peaks due to species normally asso-
ciated with AD (i.e., A?) observed (data not shown).
2A. At one end of the cluster analysis are a number of peaks that
were elevated in AD with m/z ratios that are consistent with spe-
cies prominent in the amyloidogenic pathway commonly associ-
an oxidized form of A?; i.e., the calculated molecular weight of
the peaks due to A?42 are 16% higher in the AD subjects com-
increase in monomeric A? levels did not reach statistical signifi-
in AD compared with control subjects (35% higher p ? 0.001)
(Fig. 2B). There was a strong correlation between the amount of
monomeric A?42 detected and the corresponding dimer (r ?
0.79, p ? 0.0001) (Fig. 3). The dimer peak was also significantly
antibody (Table 2). While definitive identification of the species
responsible for the various peaks will require high resolution
MS/MS data, to more confidently characterize that the peaks at
m/z 4529 and 9058 corresponded to A?42 species, SELDI-TOF
spectra were collected using the antibodies G210 (A?40 specific)
and G211 (A?42 specific) (Ida et al., 1996). As shown in Figure
4A, the peaks that were assigned to A?42 and the correspond-
WO2, G211) but were not detected by the A?40-specific anti-
body G210. To further characterize the A? dimer we synthe-
sized an oxidized A? dimer (the sulfur atom of Met35 is
oxidized to a sulfoxide) where the two A? peptide chains are
covalently cross-linked with a dityrosine moiety at residue
number 10. As can be seen from Figure 4B the SELDI MS of
the Mwt of this synthetic dimer is the same as the m/z of the
dimer elevated in AD blood.
of peaks that were decreased in AD compared with control; the
largest difference being for a peak with a m/z of 9962 Da which
was significantly decreased in AD compared with HC by 56%
result of ?-secretase activity, and too big to be the result of
?-secretase activity, suggesting an alternative, non-amyloido-
genic processing pathway. The observed distribution in the clus-
ter analysis (Fig. 2A) is consistent with the notion that there are
6318 • J.Neurosci.,May5,2010 • 30(18):6315–6322Villemagneetal.•Blood-BorneA?DimerinAlzheimer’sDisease
differential processing pathways for APP between diseased and
Not only are the A? monomer and dimer elevated in AD blood
compared with controls, but there are correlations between the
levels of these species in the blood and other clinical, neuropsy-
chometric, and biological markers of AD. These include MMSE
(r ? 0.35 and 0.36, p ? 0.0002 and 0.0001, for monomeric and
dimeric A?, respectively), memory impairment (r ? 0.27 and
0.37, p ? 0.004 and ?0.0001, for monomer and dimer respec-
tively), executive function (r ? 0.30 and 0.35, p ? 0.0012 and
0.0002, for monomer and dimer respectively), gray matter vol-
as measured by PET imaging using11C-PiB (r ? 0.19 and 0.22,
p ? 0.04 and 0.02, for monomer and dimer respectively) (Fig.
5A). Cross-validation of these results showed the robustness of
ing on average 7% and 15% worse than significant non-cross-
validated correlation coefficients (r2) values for monomer and
dimer ratios, respectively, being better for neuropsychometric
Furthermore, all the subjects including the MCI group were
cortical SUVR threshold of 1.45, obtained by unbiased statistical
els for the determination of a Neocortical PiB ‘cutoff’ level ap-
plied to the HC group. Using this threshold, 98% of AD, 57% of
MCI and 35% of HC were classified as PiB-positive. Both the
monomer ( p ? 0.013) and dimer ( p ? 0.0002) are significantly
elevated within the PiB-positive group when compared with the
negative group (Fig. 5B).
There are also significant correlations between m/z 9962 Da
0.006);0.25( p?0.008);?0.21( p?0.028)forMMSE,memory
impairment, and executive function) and brain A? burden r ?
?0.28 ( p ? 0.003) (Fig. 5A). As with the monomer and dimer
peaks, no correlation was found when the clinical groups were
examined separately. The 9962 m/z peak was significantly ( p ?
neuropsychometric and biological markers, such as MMSE, memory performance, executive function, gray matter volume, and brain A? burden as measured by PiB-PET, underlying their
Villemagneetal.•Blood-BorneA?DimerinAlzheimer’sDiseaseJ.Neurosci.,May5,2010 • 30(18):6315–6322 • 6319
higher in controls relative to diseased subjects, the correlations
are of opposite sign to those of monomeric and dimeric A?.
As the 9962 m/z peak and the A? dimer peak reflect a balance
between two different APP processing pathways, i.e., an amyloi-
dogenic pathway and a non-amyloidogenic pathway, we further
9962 Da peak to discriminate between AD and controls. The
ratios was better than when using the peaks intensities indepen-
for monomer and dimer ratios, respectively), memory impair-
ment (r ? 0.36 and 0.42, p ? 0.0001, for monomer and dimer
ratios, respectively), executive function (r ? 0.34 and 0.38, p ?
0.0003 and ?0.0001, for monomer and dimer ratios, respec-
tively), gray matter volume (r ? ?0.18 and ?0.31, p ? 0.16 and
burden as measured by11C-PiB PET (r ? 0.33 and 0.35, p ?
0.0003 and 0.0002, for monomer and dimer ratios, respectively)
(Fig. 6B). This improvement was also reflected in a larger effect
size for the ratio compared with the one obtained with the A?
dimer alone (1.03 and 0.76, respectively).
playing a pivotal role in the development of AD and A? deposi-
tion in the form of amyloid plaque is one of the defining patho-
logical hallmarks of AD. Yet one of the conundrums of AD
disease progression, a finding that has been supported by data
using11C-PiB PET imaging to show that A? burden does not
correlate with cognitive impairment in AD (Rowe et al., 2007),
(Mintun et al., 2006; Rowe et al., 2007; Aizenstein et al., 2008).
oligomers better correlate with disease progression (Lue et al.,
1999; McLean et al., 1999) and that synaptotoxic A? dimers are
elevated in AD brains (Shankar et al., 2008). As a result, there is
targeting soluble oligomers of A?.
Before this study there has been no direct detection of A?
oligomers in blood which probably reflects limitations in the
technology used and in the tissue being examined. None of the
A? biomarker protocols currently used clinically, e.g., PiB PET
imaging or A? ELISAs were designed to specifically detect such
species, although there has been a recent report of an ELISA
method against A? oligomers (Xia et al., 2009).
We have previously shown that SELDI-MS technology is able
to detect an array of A? oligomers and that these oligomers have
a high affinity for lipid membranes (Hung et al., 2008). There-
fore, while most attempts at identifying blood-based biomarkers
6320 • J.Neurosci.,May5,2010 • 30(18):6315–6322 Villemagneetal.•Blood-BorneA?DimerinAlzheimer’sDisease
for AD have concentrated on plasma with disappointing results
which have a high affinity for lipid binding, would be found
Using SELDI-TOF MS technology it is possible to detect a
dimeric form of A? in human blood and show that the levels of
the dimer are significantly elevated in AD (Fig. 2B) and correlate
with clinical markers of the disease (Fig. 5).
In the search for potential biomarkers for AD it has been
found that autoantibodies against A? oligomers are decreased in
In the study by Moir et al. (2005), the decreased autoantibodies
caused by a reaction with copper and resulting in covalently
cross-linked amyloid protein species, the so-called CAPS. The
reaction of A? and copper has been shown to lead to the forma-
tion of dityrosine cross-linked oligomers of A? (Atwood et al.,
autoantibodies is greater accumulation of these A? oligomers in
the blood. Interestingly, we were able to show that the A? dimer
A? dimer were the A? peptide chains that contain a sulfoxide at
residue M35 are covalently crosslinked by a dityrosine moiety.
A peak detected at a m/z of 9962 Da is lower in diseased sub-
jects (Fig. 2B) and is inversely correlated with clinical markers of
AD (Fig. 5). This molecular weight does not correspond to any
from ?-secretase activity, and too big to be the result of
?-secretase activity—nor does the mass correspond to any A?-
alternative, non-amyloidogenic processing pathway. While de-
finitive identification of this fragment will require isolation and
amino acid sequencing, it has previously been reported that the
activity of cathepsin D is decreased in the blood of AD subjects
(Straface et al., 2005). Cathepsin D is an aspartyl protease that
cleaves APP at a number of different sites (Higaki et al., 1996),
including at Ser627, Phe765, Glu766, and Met768. Cleavage at
these sites would give rise to a number of 15 kDa fragments;
subsequent ?-cleavage of these fragments by ?-secretase at
Met722 would give rise to a 10 kDa fragment. There is also a
cathepsin D cleavage site at Val669, subsequent cleavage by
?-secretase at the ? site would give rise to a 5 kDa fragment. As
can be seen from Table 2 and the cluster analysis (Fig. 2A), a
number of related fragments with similar masses are detected as
being elevated in the blood of the control subjects including the
peak at 9962 Da.
The spectrum of APP fragments observed by SELDI-TOF MS
in the cluster analysis is consistent with there being two distinct
processing pathways for APP, an amyloidogenic pathway which
predominates in AD and a non-amyloidogenic pathway which
predominates in healthy subjects (Fig. 2A). Both these pathways
occur physiologically and it could be argued that the progression
to AD is the result of a shift in the processing of APP from the
non-amyloidogenic to the amyloidogenic pathway. The genetics
of early onset AD support this model (Fassbender et al., 2001).
The recent publication that ?-secretase activity in platelets is in-
creased in AD compared with controls (Zainaghi et al., 2007;
Johnston et al., 2008) is consistent with the amyloidogenic path-
way being “favored” in AD.
toms of AD by many years (Price and Morris, 1999), the lack of
valid biomarkers has hampered the development and evaluation
of effective therapies for AD (Clark et al., 2008). A number of
potential AD therapeutic strategies targeting A? and its oli-
gomers (so called disease-modifying drugs) are currently being
is reported to inhibit toxic A? oligomers binding to membranes
(Nitz et al., 2008), and PBT2—a second generation MPAC that
The assessment of outcomes of the clinical trials is often difficult
to define as they rely on highly variable neuropsychometric tests.
To overcome the variability that is inherent in these tests, large
sample sizes and long timeframes are required to observe subtle
changes in subjects’ performance, dramatically increasing the
cost of these trials. The ability to detect preclinical or early stage
disease through reliable laboratory and neuroimaging biomark-
ers for AD would enable more efficient clinical trials to be de-
signed and monitored. Ideally a biomarker should reflect a
disease-specific process and be detected in an easily collected
The most easily accessed tissue is blood and the fractionation
procedures we used were deliberately kept simple to reflect the
standard protocol used in clinical laboratories worldwide to per-
the usually discarded membrane-rich CE fraction. The data pre-
sented here establishes that disease relevant APP/A?-based bi-
omarkers are likely to be found in the membrane fraction of
ease progression they hold the promise of providing a simple yet
effective way of monitoring the success or otherwise of the vari-
ous disease modifying therapies targeting A?/APP processing.
Because the molecular changes occur well before the pheno-
kers for particular traits of the pathological process will permit
early intervention with disease-modifying medications. Further
characterization of the different species in AD and controls is
how these markers change over time and how they relate to cog-
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