elagnaf2006 elisa

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The FASEB Journal • Research Communication
Detection of oligomeric forms of ␣-synuclein protein
in human plasma as a potential biomarker
for Parkinson’s disease
Omar M. A. El-Agnaf,1 Sultan A. Salem,* Katerina E. Paleologou,* Martin D. Curran,¶
Mark J. Gibson,§ Jennifer A. Court,† Michael G. Schlossmacher,† and David Allsop*
Department of Biochemistry, Faculty of Medicine and Health Science, United Arab Emirates
University, Al Ain, United Arab Emirates; *Department of Biological Sciences, Lancaster University,
Lancaster, UK; ¶Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory,
Belfast City Hospital, Belfast, UK; §Movement Disorders Clinic, Belfast City Hospital, Belfast, UK;
†
Joint MRC Newcastle University Development for Clinical Brain Ageing, MRC Building, Newcastle
General Hospital, Newcastle upon Tyne, UK; and †Center for Neurologic Diseases, Brigham and
Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA
To date there is no accepted clinical
diagnostic test for Parkinson's disease (PD) based on
biochemical analysis of blood or cerebrospinal fluid
(CSF). ␣-Synuclein (␣-syn) protein has been linked to
the pathogenesis of PD with the discovery of mutations
in the gene encoding ␣-syn in familial cases with
early-onset PD. Lewy bodies and Lewy neurites, which
constitute the main pathological features in the brains
of patients with sporadic PD and dementia with Lewy
bodies, are formed by the conversion of soluble monomers of ␣-syn into insoluble aggregates. We recently
reported the presence of ␣-syn in normal human blood
plasma and in postmortem CSF. Here, we investigated
whether ␣-syn can be used as a biomarker for PD. We
have developed a novel ELISA method that detects only
oligomeric “soluble aggregates” of ␣-syn. Using this
ELISA, we report the presence of significantly elevated
(Pⴝ0.002) levels of oligomeric forms of ␣-syn in
plasma samples obtained from 34 PD patients compared with 27 controls; 52% (95% confidence intervals
0.353– 0.687) of the PD patients displayed signals >0.5
OD with our ELISA assay in comparison to only 14.8%
(95% confidence intervals 0.014 – 0.281) for the control
cases. An analysis of the test’s diagnostic value revealed
a specificity of 0.852 (95% confidence intervals 0.662–
0.958), sensitivity of 0.529 (95% confidence intervals
0.351– 0.702) and a positive predictive value of 0.818
(95% confidence intervals 0.597– 0.948). These observations offer new opportunities for developing diagnostic tests for PD and related diseases and for testing
therapeutic agents aimed at preventing or reversing the
aggregation of ␣-syn.—El-Agnaf, O. M. A., Salem, S. A.,
Paleologou, K. W., Curran, M. D., Gibson, M. J., Court,
J. A., Schlossmacher, M. G., Allsop, D. Detection of
oligomeric forms of ␣-synuclein protein in human
plasma as a potential biomarker for Parkinson’s disease. FASEB J. 20, 419 – 425 (2006)
ABSTRACT
Key Words: CSF 䡠 PD 䡠 Lewy bodies 䡠 ␣-syn fibrils 䡠 oligomers
0892-6638/06/0020-0419 © FASEB
␣-Synuclein (␣-syn) is a small protein (⬃14 kDa)
expressed at high levels in nervous tissue (1). Three
point mutations and multiplication events in the gene
encoding ␣-syn have been associated with rare inherited forms of Parkinson’s disease (PD) (2– 6). These
links between ␣-syn and PD led to the discovery that
Lewy bodies (LBs) and Lewy neurites (LNs), the characteristic lesions in brains of patients with sporadic PD
and dementia with Lewy bodies (DLB), contain ␣-syn
fibrils (7). Several neurodegenerative diseases involve
␣-syn deposition and are hence collectively known as
“synucleinopathy disorders” (reviewed in ref 8). There
is substantial evidence to suggest that the conversion of
␣-syn from soluble monomers to aggregated, insoluble
forms in the brain is a key event in the pathogenesis of
PD and related diseases (reviewed in refs 8, 9). A
specific PD-related problem is the long latency between
the first damage to cells in at-risk nuclei of the nervous
system, including the substantia nigra in the human
brainstem and the onset of clinical symptoms. The
symptoms and signs of PD do not develop until 70 –
80% of dopaminergic neurones have already been lost
(10). To date, there is no readily available serological or
urine analysis-based test that can confirm the diagnosis
of PD in already symptomatic patients. The need for
such diagnostic methods is amply demonstrated by
studies indicating that the clinical diagnosis of this
disease during life is correct in ⬃75% of cases when
made by general neurologists, and in ⬃85% when
made by movement disorder specialists (11, 12). The
development of reliable surrogate markers for the
presence and abundance of ␣-syn lesions (LBs, LNs,
and glial cytoplasmic inclusions) in the brain would
1
Correspondence: Department of Biochemistry, Faculty of
Medicine and Health Science, United Arab Emirates University, Al Ain P. O. Box 17666, United Arab Emirates. E-mail:
[email protected]
doi: 10.1096/fj.03-1449com
419
naturally facilitate a more streamlined work-up during
the early care of movement disorder patients, and allow
for the biologically guided evaluation of future drug
trials aimed at neuroprotection in the synucleinopathies.
incubated with 100 ␮L/well of ExtrAvidin-Alkaline phosphatase (Sigma) diluted 3:5000 in blocking buffer and incubated
for 1 h at 37°C. The wells were then washed 4 times with
PBST, before adding the enzyme substrate Yellow “pNPP”
(Sigma) (100 ␮L/well) and leaving the color to develop for
30 min at room temperature. Absorbance values at 405 nm
were determined.
PATIENTS AND METHODS
Cerebrospinal fluid samples
Preparation of ␣-syn
Recombinant ␣-syn was expressed in Escherichia coli and
purified as described previously (1, 13).
Preparation of aged solutions of ␣-syn
Purified samples of ␣-syn in sterilized phosphate-buffered
saline (137 mM phosphate buffer, 150 mM NaCl, pH 7.4)
(PBS), in parafilm-sealed, 1.5 mL Eppendorf tubes were
incubated at 37°C for 4 days in an Eppendorf Thermomixer
with continuous mixing (1000 rpm). Samples were collected
at various time points and stored at – 80°C until tested by the
ELISA.
Antibodies
The antibody used for the ELISA to detect ␣-syn oligomers
was 211 mouse monoclonal antibody (mAb) (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), which recognizes
amino acid residues 121-125 of human ␣-syn This mAb did
not react with recombinant ␤-synuclein or ␥-synuclein on
Western blots. Some plasma samples were depleted of ␣-syn
prior to analysis in the ELISA. This was achieved by prior
treatment with magnetic dynabeads coupled to antibody
LB509 (a mouse monoclonal antibody to ␣-syn residues
115-12, from Zymed Laboratories, San Francisco, CA, USA)
or to antibody FL-140 (a rabbit polyclonal antibody raised
against full-length ␣-syn, from Santa Cruz Biotechnology), as
described (14).
Preparation of the biotinylated antibody
Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, USA) (200 ␮g)
was reacted with the antibody to be biotinylated (1 mL at 200
␮g/mL) in PBS, then placed on ice for 2 h. The mixture was
desalted on Bio-Spin-6 columns (BIO-RAD, UK) to remove
excess uncoupled biotin. The biotinylated antibodies were
stored at 4°C until used.
An ELISA to measure ␣-syn oligomers
An ELISA plate was coated by overnight incubation with 1
␮g/mL of nonbiotinylated mAb 211 (100 ␮L/well), in 200
mM NaHCO3 (Sigma, St. Louis, MO, USA), pH 9.6, containing 0.02% (w/v) sodium azide at 4°C, washed 4 times with
PBST (PBS containing 0.05% Tween 20), and incubated with
200 ␮L/well of blocking buffer (PBS containing 2.5% gelatin
and 0.05% Tween 20) for 2 h at 37°C. The plate was washed
4 times with PBST, and 100 ␮L of the samples to be tested
were added to each well (fresh or aged ␣-syn solutions were
diluted to 500 nM in PBS; cerebrospinal fluid (CSF) samples
were diluted 1:1 with PBS, whereas plasma samples were used
neat). The plate was incubated at 37°C for 2 h. After washing
4 times with PBST, 100 ␮L of biotinylated mAb 211 diluted to
1 ␮g/mL in blocking buffer was added, and incubated at
37°C for 2 h. The wells were washed 4 times with PBST and
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March 2006
Postmortem CSF samples were obtained from the London
Neurodegenerative Diseases Brain Bank (Department of Neuropathology, Institute of Psychiatry, King’s College, UK),
Queen Square Brain Bank for Neurological Disorders (Department of Molecular Pathogenesis, Institute of Neurology,
University College London, UK), and The Newcastle Brain
Tissue Resource (Joint MRC Newcastle University Development for Clinical Brain Ageing, MRC Building, Newcastle
General Hospital, Westgate Road, Newcastle upon Tyne, UK).
The samples were stored at – 80°C before analysis. Repeat
freeze/thaw cycles were avoided.
Plasma samples
Blood samples were obtained from 34 clinically diagnosed PD
patients (50% female, 50% male, age range 54 – 82 years,
mean age 65.4) attending an outpatients’ clinic in the Neurology Department of Belfast City Hospital. For each case, the
diagnosis of idiopathic PD was made by a consultant neurologist (M.J.G.), based on the UK Parkinson's Disease Society
Brain Bank criteria for idiopathic Parkinson's disease (15).
The diagnosis was made on the basis of a progressive history
and more than two of the cardinal signs of PD being present
(resting tremor, bradykinesia, rigidity, postural instability). In
addition, there was an absence of any features, suggesting an
alternative cause for parkinsonism. All subjects had been
followed up in a Movement Disorders Clinic for at least 2
years. Ethical Committee approval was obtained and written
consent given by all subjects. Age-matched control samples
were obtained from the Haematology Department at Blackpool Victoria Hospital (50% female, 50% male, age range
55– 85 years, mean age 68.0). The control samples were from
individuals suffering from disorders such as cancer, heart
disease, stroke, diabetes, and chronic renal failure. For statistical analysis, the controls were not individually matched with
each PD case. Ethical Committee approval was also obtained
for these control samples. Blood (10 mL) was collected in
plastic tubes containing either sodium citrate or potassiumEDTA from all subjects, and plasma was separated by centrifuging the blood at 3000 rpm at 4°C for 20 min. Plasma was
collected in 0.5 mL plastic tubes and stored frozen at – 80°C.
The samples were thawed at room temperature directly
before analysis.
Size exclusion chromatography
Sephadex G-75 superfine gel (Amersham Pharmacia Biotech,
Uppsala, Sweden) was packed into a 44 cm long and 1 cm
internal diameter column. CSF or plasma samples were
cleared by centrifugation (3000 rpm or 20 min), then 0.5 mL
loaded onto the column and eluted with 50 mM ammonium
acetate (pH 7.4) at a flow rate of 0.25 mL/min. Absorbance of
the eluted material was monitored at 215 nm. Fractions of 1
mL were collected, concentrated to 300 ␮L by centrifugation
under vacuum, and analyzed by the ELISA for the presence of
␣-syn oligomers.
The FASEB Journal
EL-AGNAF ET AL.
Statistical analysis
Fisher’s exact probability tests were performed to assess
whether the frequency differences observed between the
levels of ␣-syn oligomers found in the cohort of PD subjects and the age-matched control group were statistically
significant using the computer package “StatsDirect” (www.
statsdirect.com). It was not possible to individually match the
disease/control samples. Three arbitrary cutoff points were
chosen (0.2, 0.3, and 0.5 OD absorbance at 405 nm) to
discern the threshold of positive correlation between the
clinical working diagnosis of PD and the signal obtained for
the ELISA of plasma results, with cases above this point
deemed to possess high levels of ␣-syn oligomers.
RESULTS
We recently reported the detection of ␣-syn in human
plasma, postmortem CSF, and conditioned medium of
neuronal cell cultures by immunoprecipitation followed by sodium dodecyl sulfate PAGE (SDS-PAGE)
and Western blot methods (14). In this previous work,
we found considerable overlap in the amount of monomeric ␣-syn detected in CSF and plasma samples
from normal individuals and those from patients with
PD and DLB. However, this work involved immunocapture of ␣-syn on magnetic dynabeads, followed by
elution from the beads and separation by SDS-PAGE.
This protocol thereby involves denaturing the proteins
with heating and strong detergent (SDS), which could
result in disassociation of any oligomeric forms of ␣-syn
that we hypothesized to be present in vivo (16). Therefore, we developed a specific and sensitive novel ELISA
method that uses a nondenaturing approach designed
to recognize only the oligomeric species of human
␣-syn. Here, we have refined this method to detect
oligomeric forms of ␣-syn in human CSF and plasma.
The ELISA is based on a conventional sandwich system
with capture of ␣-syn by highly specific anti-␣-syn monoclonal antibody (mAb) 211, followed by detection with
a biotinylated form of the same mAb (Fig. 1A). The
biotinylated mAb is subsequently detected with ExtrAvidin-Alkaline phosphatase, followed by a colorimetric
enzyme substrate. Monomeric ␣-syn cannot give a signal in this assay because the capture mAb occupies the
only antibody binding site available on the protein, but
in the case of oligomeric forms of ␣-syn, multiple mAb
binding sites are available, permitting both capture and
detection (Fig. 1A). Recently, we successfully developed a similar ELISA for the detection of oligomeric
A␤ peptide, associated with Alzheimer’s disease (AD)
(17).
First, we studied the time-dependent oligomerization
of recombinant ␣-syn using this novel ELISA and found
that freshly dissolved ␣-syn (715 ng/well in a 96-well
plate assay) gave a signal of 0.01 optical density (OD)
units, whereas ␣-syn that had been allowed to aggregate
at 37°C for 4 days in PBS at pH 7.4 gave a signal of 2.3
OD (Fig. 1B). Detection of a large signal in the ELISA
correlated with the presence of soluble aggregates or
“oligomeric” forms of ␣-syn (Fig. 1B, C). Solutions of
OLIGOMERIC ␣-SYNUCLEIN AS BIOMARKER
Figure 1. Oligomerization of ␣-syn measured by the ELISA.
A) Principle of ELISA for oligomeric ␣-synuclein. B) Characterization of the ELISA. Solutions of ␣-syn in PBS at 50
␮M, 10 ␮M, and 5 ␮M were incubated at 37°C, diluted to
0.5 ␮M, and transferred to a microtiter plate already coated
with immobilized mAb 211. Additional epitopes formed by
oligomerization of ␣-syn during the preincubation step
were measured by the subsequent binding of biotinylated
mAb 211. Data shown are representative of 5 independent
experiments, from 3 different preparations of ␣-syn. Measurements were taken in triplicate, and the results show the
mean ⫾ standard deviation for each point. C) Effect of the
concentration of ␣-syn oligomers on the ELISA response. A
solution of ␣-syn in PBS at 50 ␮M was incubated for 4 days
at 37°C, diluted to 1000 – 0.1 nM in PBS, and transferred to
a microtiter plate coated with immobilized mAb 211. Assays
were performed in triplicate; mean ⫾ standard deviations
are shown. The mid-range of the assay for ␣-syn incubated
for 4 days was 500 nM of the starting concentration of the
fresh sample. At 500 nM, the within-assay variance was
5.3%. A day-to-day variance of 4.9% at 500 nM was found in
5 assays calibrated with standard solutions of 50 ␮M ␣-syn
incubated in PBS for 4 days, diluted to 0.5 ␮M in PBS,
divided into aliquots, and stored at – 80°C.
421
␣-syn showed a gradual increase in the formation of
oligomers as the concentrations and time of preincubation were increased (Fig. 1B). The ELISA gave a poor
signal when ␣-syn was predominantly in the form of
mature fibrils or “insoluble aggregates,” as revealed by
a thioflavin-T binding assay and by electron microscopy
(data not shown). We estimated the lower limit of
detection of recombinant protein by the ELISA was
⬃14.3 ng/well, based on the starting concentration of
the fresh ␣-syn sample (Fig. 1C).
We investigated whether our ELISA could detect any
␣-syn oligomers in postmortem CSF samples at the time
of autopsy from patients with PD and related disorders
(based on clinical diagnosis and postmortem histopathology). Two of the three PD samples tested, and two
of the eight DLB samples tested, gave a high signal
(0.2–2.0 OD units), whereas very low signals (⬍0.1 OD)
were obtained from 40 controls, including 26 nonneurological disease controls, 11 cases of AD, and 3
cases of motor neurone disease (data not shown).
Given these results, which suggested the presence of
soluble oligomers of ␣-syn in the extracellular space, we
used the ELISA to test for the presence of soluble ␣-syn
oligomers in a larger number of samples of peripheral
plasma from PD cases and controls. We screened the
plasma samples and observed that 18 of 34 (52%) PD
samples gave a relatively high signal in the ELISA (⬎0.5
OD), whereas only 4 of 27 (14.8%) control samples
gave a similar signal (Fig. 2). Overall the results generated a statistically significant difference between the PD
and control samples (P⫽0.002), suggesting that the
concentration of oligomeric ␣-syn is increased dramatically in PD plasma. This difference is further exaggerated (P⫽0.0003) if one extends the analysis to cases
displaying levels above 0.3 OD, with 22 PD cases (64%)
and 5 control samples (18%). A similar level of statistical significance is also achieved with a cutoff of 0.2 OD
(P⫽0.0003) with 26 PD cases (76%) and 8 control cases
(29%). Similar results were obtained whether plasma
was collected in citrate or in EDTA tubes. When the
reliability of the test is assessed by determining the
established measurements of specificity (proportion of
individuals without the disease who are correctly iden-
tified by the test) and sensitivity (proportion of individuals with the disease who are correctly identified by the
test) for these data, it is clear the test has significant
diagnostic promise. Its specificity of 0.852 is relatively
high, while the sensitivity of 0.529 is moderate for the
selected cutoff of ⬎0.5 OD. This is again reflected in
the positive and negative predictive values for the test,
0.818 (95% confidence intervals 0.597– 0.948) and
0.589 (95% confidence intervals 0.421– 0.744), respectively. However, a reduction of the cutoff of 0.5 OD
units (which reflects an arbitrary decision by us to
segregate the data) to 0.3 OD units improves the
sensitivity to 0.647 (95% confidence intervals 0.465–
0.802) and only slightly compromises the specificity i.e.,
0.814 (95% confidence intervals 0.619 – 0.937). A further drop in the cutoff to 0.2 OD units reduces the
specificity further (0.7037, 95%CI 0.498 – 0.862) and
again increases the sensitivity (0.765, 95%CI 0.588 –
0.892). While a sensitivity and specificity that are both
as close to 1 as possible is ideal, in practice (as displayed
here), sensitivity is gained at the expense of specificity
and vice versa. To avoid making a false positive diagnosis a high specificity would be favored. This, together
with the fact that the likelihood ratios for the 0.5 and
0.3 OD units cutoff points are similar, 3.57 (95%
confidence intervals 1.50 –9.33) and 3.49 (95% confidence intervals 1.65– 8.14), respectively, and higher
than the 0.2 OD cutoff of 2.58 (95% confidence
intervals 1.49 – 4.93), makes either a good choice as a
cutoff value for the test in its current form. When
plasma samples were first immunoprecipitated with
anti-␣-syn antibodies LB509, FL-140, or syn-1 coupled
to magnetic beads (or to protein G Sepharose beads in
the case of syn-1) (14), then tested by the ELISA, only
traces of signals could be detected above background
when compared with non-␣-syn-directed, “negative control IgG”-mediated capturing, suggesting that immunodepletion removed the antigen from the initial sample (data not shown). These data further demonstrated
the specificity of the ELISA and show, for example, that
the signal generated was not due to nonspecific binding of the biotinylated antibody to other proteins in our
specimens.
Figure 2. Detection of ␣-syn oligomers in human plasma. Plasma
samples from Parkinson’s disease
(PD) and control patients (C) (100
␮L) were analyzed by the ELISA
for oligomeric ␣-syn. Data shown
are representative of at least 4 independent experiments. The assays were performed in triplicate;
mean ⫾ standard deviations are
shown.
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The FASEB Journal
EL-AGNAF ET AL.
To confirm that our ELISA detects only soluble
oligomers and not monomeric forms of ␣-syn in our
CSF and plasma specimens, we used size exclusion
chromatography (SEC) to determine the molecular
weight (MW) of the immunoreactive protein (Fig. 3).
Recently, Sephadex G75 columns have been used successfully to detect soluble oligomers of ␣-syn in extracts
from PD and DLB brains (18). The Sephadex G75
column used in our study gave a linear elution profile
for a set of protein standards with MWs of 8 to 67 kDa,
similar to those in the previous report (18). Postmortem CSF and plasma samples from those PD patients
that gave a robust signal in the ELISA revealed immunoreactive material with an elution peak in SEC fractions corresponding to MWs of 55–70 kDa. Much of the
immunoreactive material was eluted at the void volume,
which indicated a MW of ⬎70 kDa (Fig. 3A, C). These
SEC fractionation results are consistent with the detection of ␣-syn oligomers, since the protein material
detected by the ELISA has a MW much greater than
that of the monomeric form of ␣-syn. As expected, no
significant immunoreactivity was detected in SEC fractions from control samples with low signals in the
ELISA (Fig. 3B, D). Experiments are in progress in our
laboratory to further characterize and analyze the structure and nature of the oligomeric species of ␣-syn and
to discern any modifications of ␣-syn detected by the
ELISA (i.e., nitrated, phosphorylated, dimers, or trimers, etc.). This information will be useful in order to
improve both the sensitivity and selectivity for ␣-syn
protein species in our future ELISA variants.
DISCUSSION
Our recent studies have shown that neuronal cells in
culture constitutively secrete ␣-syn into the culture
medium and that ␣-syn is normally present in CSF and
peripheral plasma (14). There is some evidence that
certain toxic metabolites, including the A␤ peptide
implicated in the pathogenesis of AD, can be cleared
from the brain by drainage from the interstitial fluid
into the CSF, then into the blood plasma (19). Further
support for this idea has come from studies with a
transgenic mouse model of AD, where peripheral administration of a monoclonal antibody to A␤ was found
to induce a rapid increase in plasma A␤, and the
magnitude of this increase was highly correlated with
amyloid burden in the hippocampus and cortex (20,
21). These results demonstrated that A␤ can efflux
from the brain into the plasma pool, and therefore, a
similar mechanism could operate for ␣-syn. At present,
it is unclear whether this “effluxed” A␤ is an oligomeric
form of the peptide.
Nevertheless, the opposite may be true as well, where
a principally peripheral source of ␣-syn derived from
either corpuscular elements of whole blood (22) or
peripheral organs, (e.g., liver) is responsible for detection of ␣-syn in plasma. The possibility of a peripheral
source is particularly relevant in light of the very low
OLIGOMERIC ␣-SYNUCLEIN AS BIOMARKER
Figure 3. Size exclusion chromatographic analysis of ␣-syn
oligomers in CSF and plasma. 0.5 mL of plasma from a PD
patient (A) and a control (B), and CSF from a PD patient (C),
and a control (D) were fractionated on a Sephadex G75 SEC
column. Fractions (F ) of 1 mL were collected, concentrated
to 300 ␮L, and analyzed by the ELISA for the presence of
␣-syn oligomers. Peak fractions for the molecular weight
(MW) standards are indicated. These were bovine serum
albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A
(25 kDa), myoglobin (18 kDa), and ubiquitin (8 kDa). The
void volume corresponds to MW ⬎ 70 kDa. Freshly dissolved
and preaggregated (aged) recombinant ␣-syn (ra-syn) were
also tested in the ELISA.
levels of total ␣-syn reactivity that are detectable in
human CSF from living persons when compared with
brain and plasma ␣-syn steady states (M. G. Schlossmacher et al., unpublished observation; El-Agnaf et al.,
423
unpublished results). Likewise, future cell biological
research will have to address the mechanism by which
␣-syn protein (and oligomers thereof) can be found in
the extracellular space under physiological conditions
(14), since no alternative splice variant has been published so far that would direct the nascent ␣-syn protein
into and through the secretory pathway, and no such
transcript of the SNCA gene could be found in an
extensive investigation of primate brain specimens
(M. G. Schlossmacher, unpublished data). There is
some evidence for the existence of oligomeric forms of
A␤ and of the prion protein (PrP) that is associated
with transmissible spongiform encephalopathies
(TSEs) in CSF from patients with AD and the TSEs,
respectively, but not in controls (23, 24). These various
studies, together with mounting evidence for soluble
oligomers being the pathogenetic species that drive
neurodegeneration and neuronal cell death (25–31),
led us to hypothesize that detection of soluble oligomers of ␣-syn in biological fluids could have potential use as a biomarker for PD and related diseases (16).
Therefore, we developed a simple and novel ELISA
method that specifically recognizes only oligomeric
species of ␣-syn. We used this method to probe for
oligomeric forms of ␣-syn in human CSF and in plasma.
Based on our preliminary results, the ELISA was able to
detect ␣-syn oligomers in postmortem CSF from some
PD and DLB patients, but only a very low signal was
obtained from all control samples tested. These data
suggested a higher amount of ␣-syn oligomer production in PD patients either in vivo or during postmortem
autolysis. Regrettably, at the writing of this report, no
leftover CSF specimens were available from living PD
patients to address this question.
These promising results led us to carry out an
extensive study of the more accessible peripheral blood
plasma. We found there was a highly statistically significant difference between PD samples and controls, with
most of the PD samples giving high signals, whereas
only a few control samples gave a high signal. Our
control samples consisted of a random selection of
blood samples from a single hematology laboratory.
The variation in signal from both PD and control
subjects in this first cohort could be related to a
number of different factors, including (but not limited
to) differential blood cell count numbers, total plasma
protein concentration, total ␣-syn content, ␣-syn turnover by degradation in the extracellular space, comorbidities, medication, and, last but not least, postphlebotomy processing time.
If ␣-syn oligomerization occurs before the death of
nigral neurons in PD, then our ELISA could potentially
provide a diagnostic tool for the detection of oligomers
in the early stages of the disease. This could lead to
earlier detection and neuroprotective treatment intervention for high-risk subjects in the future. More extensive clinical studies are required to confirm and
extend our results and to validate the ELISA as a
potential diagnostic test for disease state. In fact, we
recently began enrolment of subjects in a prospective,
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case control study of 300 persons to be conducted over
2 years to monitor their oligomeric and total ␣-syn load
in peripheral plasma. It will be interesting to determine
whether there is any correlation between detection of
␣-syn oligomers and the severity and/or the stage of the
disease and/or the rate of its progression. Studies
performed on blood samples from familial PD cases will
be useful to further validate the ELISA as an early
diagnostic method (32). We also recognize that any
medication, including dopaminergic replacement therapy, taken by previously diagnosed PD patients could
influence the ELISA results (33). Studies of newly
diagnosed patients who are not yet taking any medication (de novo) will indicate whether drugs have any
confounding effect. The ELISA can also be used for
high-throughput screening for modulators of ␣-syn
oligomerization as potential novel drugs for PD and
related disorders during preclinical validation studies
prior to their evaluation in rodent or nonhuman primate models of PD (34). Moreover, the principle of our
ELISA could be applied for the development of similar
sensitive diagnostic tests for the presence of other
forms of oligomeric protein aggregates, such as those
found in AD (A␤) and the TSEs (PrP).
We are grateful for support from Michael J. Fox Foundation (to O.M.E-A. and M.G.S.) and The Alzheimer’s Research
Trust (to D.A. and S.A.S.). We thank The London Neurodegenerative Diseases Brain Bank (Department of Neuropathology, Institute of Psychiatry, King’s College, UK), The Queen’s
Square Brain Bank for Neurological Disorders (Department
of Molecular Pathogenesis, Institute of Neurology, University
College London, UK) for the human postmortem CSF samples.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
Jakes, R., Spillantini, M. G., and Goedert, M. (1994) Identification of two distinct synucleins from human brain. FEBS Lett. 345,
27–32
Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E.,
Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer,
R., et al. (1997) Mutation in the ␣-synuclein gene identified in
families with Parkinson’s disease. Science 276, 2045–2047
Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M.,
Kosel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O.
(1998) Ala30Pro mutation in the gene encoding ␣-synuclein in
Parkinson’s disease. Nature Gen. 18, 106 –108
Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C., Lezcano, E., Ros,
R., Ampuero, I., Vidal, L., Hoenicka, J., Rodriguez, O., Atares,
B., et al. (2004) The new mutation, E46K, of ␣-synuclein causes
Parkinson and Lewy body dementia. Ann. Neurol. 55, 164 –173
Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S.,
Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum,
R., et al. (2003) ␣-Synuclein locus triplication causes Parkinson's
disease. Science 302, 841
Chartier-Harlin, M. C., Kachergus, J., Roumier, C., Mouroux, V.,
Douay, X., Lincoln, S., Levecque, C., Larvor, L., Andrieux, J.,
Hulihan, M., et al. (2004) ␣-Synuclein locus duplication as a
cause of familial Parkinson's disease. Lancet 364, 1167–1169
Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., and
Goedert, M. (1998) Alpha-Synuclein in filamentous inclusions
of Lewy bodies from Parkinson's disease and dementia with
Lewy bodies. Proc. Natl. Acad. Sci. USA 95, 6469 – 6473
Cookson, M. R. (2005) The biochemistry of Parkinson's disease.
Annu. Rev. Biochem. 74, 29 –52
The FASEB Journal
EL-AGNAF ET AL.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Martin, F. L., Williamson, S. J., Paleologou, K. E., Allsop, D., and
El-Agnaf, O. M. A. (2004) Alpha-synuclein and the pathogenesis
of Parkinson's disease. Protein Pept. Lett. 11, 229 –237
Schapira, A. H. V. (1999) Science, medicine, and the future
Parkinson's disease. Br. Med. J. 318, 311–314
Lang, A. E., and Lozano, A. M. (1998) Parkinson’s disease—first
of two parts. N. Engl. J. Med. 339, 1130 –1143
Lang, A. E., and Lozano, A. M. (1998) Parkinson’s disease—
second of two parts. N. Engl. J. Med. 339, 1144 –1153
El-Agnaf, O. M. A., Jakes, R., Curran, M. D., and Wallace, A.
(1998) Effects of the mutations Ala30 to Pro and Ala53 to Thr on
the physical and morphological properties of ␣-synuclein protein implicated in Parkinson’s disease. FEBS Lett. 440, 67–70
El-Agnaf, O. M. A., Salem, S. A., Paleologou, K. E., Cooper, L. J.,
Fullwood, N. J., Gibson, M. J., Curran, M. D., Court, J. A., Mann,
D. M. A., Ikeda, S. I., et al. (2003) ␣-Synuclein implicated in
Parkinson’s disease is present in extracellular biological fluids,
including human plasma. FASEB J. 17, 1945–1947
Gibb, W. R., and Lees, A. J. (1988) The relevance of the Lewy
body to the pathogenesis of Parkinson's disease. J. Neurol.
Neurosurg. Psychiatry 51, 745–752
El-Agnaf, O. M. A., Walsh, D. M., and Allsop, D. (2003) Soluble
oligomers for the diagnosis of neurodegenerative diseases.
Lancet Neurol. 2, 461– 462
El-Agnaf, O. M. A., Mahil, D. S., Patel, B. P., and Austen, B. A.
(2000) Oligomerization and toxicity of ␤-amyloid-42 implicated in
Alzheimer's disease. Biochem. Biophys. Res. Commun. 237, 1003–1007
Sharon, R., Bar-Joseph, I., Frosch, M. P., Walsh, D. M., Hamilton, J. A., and Selkoe, D. J. (2003) The formation of highly
soluble oligomers of ␣-synuclein is regulated by fatty acids and
enhanced in Parkinson's disease. Neuron 37, 583–595
Silverberg, G. D., Mayo, M., Saul, T., Rubenstein, E., and
McGuire, D. (2003) Alzheimer’s disease, normal pressure hydrocephalus, and senescent changes in CSF circulatory physiology: a hypothesis. Lancet Neurol. 2, 506 –511
DeMattos, R. B., Bales, K. R., Parsadanian, M., O'Dell, M. A.,
Foss, E. M., Paul, S. M., and Holtzman, D. M. (2002) Plaqueassociated disruption of CSF and plasma amyloid-beta (A␤)
equilibrium in a mouse model of Alzheimer's disease. J. Neurochem. 81, 229 –236
DeMattos, R. B., Bales, K. R., Cummins, D. J., Paul, S. M., and
Holtzman, D. M. (2002) Brain to plasma amyloid-beta efflux: a
measure of brain amyloid burden in a mouse model of
Alzheimer's disease. Science 295, 2264 –2267
Li, Q. X., Campbell, B. C., McLean, C. A., Thyagarajan, D., Gai,
W. P., Kapsa, R. M., Beyreuther, K., Masters, C. L., and Culvenor,
J. G. (2002) Platelet ␣- and ␥-synucleins in Parkinson's disease
and normal control subjects. J. Alzheimers Dis. 4, 309 –315
Pitschke, M., Prior, R., Haupt, M., and Riesner, D. (1998)
Detection of single amyloid beta-protein aggregates in the
cerebrospinal fluid of Alzheimer's patients by fluorescence
correlation spectroscopy. Nat. Med. 4, 832– 834
Bieschke, J., Giese, A., Schulz-Schaeffer, W., Zerr, I., Poser, S.,
Eigen, M., and Kretzschmar, H. (2000) Ultrasensitive detection
OLIGOMERIC ␣-SYNUCLEIN AS BIOMARKER
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
of pathological prion protein aggregates by dual-color scanning
for intensely fluorescent targets. Proc. Natl. Acad. Sci. USA 97,
5468 –5473
Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson,
R. E., and Lansbury, P. T., Jr. (2000) Acceleration of oligomerization, not fibrillization, is a shared property of both alphasynuclein mutations linked to early-onset Parkinson's disease:
implications for pathogenesis and therapy. Proc. Natl. Acad. Sci.
USA 97, 571–576
Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L.,
Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani,
M. (2002) Inherent toxicity of aggregates implies a common
mechanism for protein misfolding diseases. Nature (London) 416,
507–511
Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R.,
Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Naturally
secreted oligomers of amyloid beta protein potently inhibit
hippocampal long-term potentiation in vivo. Nature (London)
416, 535–539
El-Agnaf, O. M. A., Nagala, S., Patel, B. P., and Austen, B. M.
(2001) Nonfibrillar oligomeric species of the amyloid ABri
peptide, implicated in familial British dementia, are more
potent at inducing apoptotic cell death than protofibrils or
mature fibrils. J. Mol. Biol. 310, 157–168
Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M., Takeda, A., Sagara, Y., Sisk, A., and Mucke, L. (2000)
Dopaminergic loss and inclusion body formation in ␣-synuclein
mice: implications for neurodegenerative disorders. Science 287,
1265–1269
Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton,
S. C., Cotman, C. W., and Glabe, C. G. (2003) Common
structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 5618, 486 – 489
Lashuel, H. A., Hartley, D., Petre, B. M., Walz, T., and Lansbury,
P. T., Jr. (2002) Neurodegenerative disease: amyloid pores from
pathogenic mutations. Nature (London) 418, 291
Miller, D. W., Hague, S. M., Clarimon, J., Baptista, M., GwinnHardy, K., Cookson, M. R. and Singleton, A. B. (2004) Alphasynuclein in blood and brain from familial Parkinson disease
with SNCA locus triplication. Neurology 62, 1835–1838
Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury,
P. T., Jr. (2001) Kinetic stabilization of the alpha-synuclein
protofibril by a dopamine-alpha-synuclein adduct. Science 294,
1346 –1349
El-Agnaf, O. M. A., Paleologou, K. E., Greer, B., Abogrein, A. M.,
King, J. E., Salem, S. A., Fullwood, N. J., Benson, F. E., Hewitt,
R., Ford, K. J., et al. (2004) A strategy for designing inhibitors of
␣-synuclein aggregation and toxicity as a novel treatment for
Parkinson's disease and related disorders. FASEB J. 18, 1315–
1317
Received for publication September 1, 2005.
Accepted for publication November 10, 2005.
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