Brain Advance Access originally published online on April 6, 2006
Brain 2006 129(5):1177-1187; doi:10.1093/brain/awl063
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CSF amyloid-ß-peptides in Alzheimer's disease, dementia with Lewy bodies and Parkinson's disease dementia
1 Department of Psychiatry, University of Goettingen, Goettingen, 2 Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, 3 Bruker Daltronics, Leipzig, 4 Paracelsus-Elena Klinik, Kassel, 5 Department of Neurology, University of Ulm, Steinhövelstr, Ulm, Germany and 6 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
Correspondence to: Professor Dr Jens Wiltfang, Molecular Neurobiology Lab, Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany E-mail: Jens.Wiltfang{at}psych.imed.uni-erlangen.de
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As the differential diagnosis of dementias based on established clinical criteria is often difficult, biomarkers for applicable diagnostic testing are currently under intensive investigation. Amyloid plaques deposited in the brain of patients suffering from Alzheimer's disease, dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD) mainly consist of carboxy-terminally elongated forms of amyloid-beta (Aß) peptides, such as Aß1–42. Absolute Aß1–42 levels in CSF have shown diagnostic value for the diagnosis of Alzheimer's disease, but the discrimination among Alzheimer's disease, DLB and PDD was poor. A recently established quantitative urea-based Aß-sodium-dodecylsulphate–polyacrylamide-gel-electrophoresis with Western immunoblot (Aß-SDS–PAGE/immunoblot) revealed a highly conserved Aß peptide pattern of the carboxy-terminally truncated Aß peptides 1–37, 1–38, 1–39 in addition to 1–40 and 1–42 in human CSF. We used the Aß-SDS–PAGE/immunoblot to investigate the CSF of 23 patients with Alzheimer's disease, 21 with DLB, 21 with PDD and 23 non-demented disease controls (NDC) for disease-specific alterations of the Aß peptide patterns in its absolute and relative quantities. The diagnostic groups were matched for age and severity of dementia. The present study is the first attempt to evaluate the meaning of Aß peptide patterns in CSF for differential diagnosis of the three neurodegenerative diseases—Alzheimer's disease, DLB and PDD. The Aß peptide patterns displayed disease-specific variations and the ratio of the differentially altered Aß1–42 to the Aß1–37 levels subsequently discriminated all diagnostic groups from each other at a highly significant level, except DLB from PDD. Additionally, a novel peptide with Aß-like immunoreactivity was observed constantly in the CSF of all 88 investigated patients. The pronounced percentage increase of this peptide in DLB allowed a highly significant discrimination from PDD. Using a cut-off point of 0.954%, this marker yielded a diagnostic sensitivity and specificity of 81 and 71%, respectively. From several lines of indication, we consider this peptide to represent an oxidized
-helical form of Aß1–40 (Aß1–40*). The increased abundance of Aß1–40* probably reflects a disease-specific alteration of the Aß1–40 metabolism in DLB. We conclude that Aß peptide patterns reflect disease-specific pathophysiological pathways of different dementia syndromes as distinct neurochemical phenotypes. Although Aß peptide patterns failed to fulfil the requirements for a sole biomarker, their combined evaluation with other biomarkers is promising in neurochemical dementia diagnosis. It is noteworthy that DLB and PDD exhibit distinct clinical temporal courses, despite their similar neuropathological appearance. Their distinct molecular phenotypes support the view of different pathophysiological pathways for each of these neurodegenerative diseases.
Key Words: Alzheimer's dementia; Lewy-body dementia; Parkinson's disease dementia; cerebrospinal fluid; amyloid-ß peptides
Abbreviations: Aß peptides = amyloid-beta peptides; Aß-SDS–PAGE/immunoblot = amyloid-beta-sodium-dodecyl-sulphate–polyacrylamide-gel-electrophoresis with Western immunoblot; APP = beta-amyloid precursor protein; bicine = N,N'-bis-[2-hydroxyethyl]glycine; C% = percentage of N,N'methylenebisacrylamide (bis) of the total of bis plus acrylamide; DLB = dementia with Lewy bodies; IP = immunoprecipitation; IPG = immobilized pH gradients; MALDI–TOF = matrix-assisted laser desorption ionization mass analysis–time-of-flight modus; MMSE = Mini-Mental-Status Examination; NINCDS–ADRDA = National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's Disease and Related Disorders Association; ND = non-demented; PDD: Parkinson's disease dementia; SKT = Syndrom-Kurz-Test; SPE = solid-phase extraction; T% = percentage of acrylamide of the total of bis plus acrylamide
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Received March 30, 2005. Revised February 14, 2006. Accepted February 20, 2006.
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Introduction |
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Amyloid-beta (Aß) peptides form the major component of amyloid plaques deposited in the brain of patients suffering from neurodegenerative diseases such as Alzheimer's disease (Glenner and Wong, 1984
), dementia with Lewy bodies (DLB) (Jendroska et al., 1997) and Parkinson's disease dementia (PDD) (Jendroska et al., 1996).
Aß peptides derive from a transmembrane amyloid precursor-protein (APP), when cleaved by two enzymes, ß- and 
-secretase (Haas and Selkoe, 1993). Distinct -secretase activities are hypothesized to be responsible for generation of either carboxy-terminally truncated (Ct-truncated) or elongated (Ct-elongated) Aß peptides as referenced to Aß1-40 (Citron et al., 1996). Cleavage of the APP by the 
-secretase results in amino-terminally elongated (At-elongated) Aß peptide species (Simons et al., 1996).
The differential diagnosis of dementias based on established clinical criteria is often difficult during lifetime and the selective reduction of Aß1–42 in the CSF of Alzheimer's disease patients has widely been investigated as a diagnostic biomarker to support the diagnostic accuracy during lifetime. An expert review recently considered it adequate for applicable Alzheimer's disease diagnostic testing in addition to clinical criteria (Andreasen et al., 2003). Decreased Aß1–42 levels have also been reported for DLB (Andreasen et al., 2001; Mollenhauer et al., 2005) and PDD (Mollenhauer et al., 2005) patients. Thus, the specificity of this finding and consequently its differential diagnostic value in distinguishing between different subtypes of dementias was low (Andreasen et al., 2001, 2003; Mollenhauer et al., 2005).
A quantitative urea-based Aß-sodium-dodecylsulphate-polyacrylamide-gel-electrophoresis with Western immunoblot (Aß-SDS–PAGE/immunoblot) recently revealed the regular abundance of the Ct-truncated Aß peptides 1–37, 1–38, 1–39 in addition to 1–40 and 1–42 in CSF. This Aß peptide pattern displayed disease-specific variations in its absolute and relative quantities in the CSF of patients with Alzheimer's disease, Creutzfeldt–Jakob disease (CJD), chronic inflammatory diseases and other neuropsychiatric diseases (Wiltfang et al., 2002, 2003).
To evaluate the meaning of this finding for the differential diagnosis of dementias, we investigated 88 age-matched patients suffering from Alzheimer's disease, DLB, PDD and various neuropsychiatric diseases for disease-specific Aß peptide patterns.
We were able to demonstrate disease-specific variations of the Aß peptide patterns in CSF for each of the diagnostic groups, which allow a highly significant discrimination and may reflect pathophysiological pathways of dementia subtypes.
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Aß peptide patterns were analysed by Aß-SDS–PAGE/immunoblot (Wiltfang et al., 2002
) in the CSF of patients with Alzheimer's disease, DLB, PDD and non-demented disease controls (NDC). A total of 88 patients were divided into four diagnostic groups according to their clinical diagnosis and tested for significant differences in absolute and relative Aß peptide values. The patients were selected between 1999 and 2004 onward and from the dementia outpatient clinic. The mean age did not significantly differ between the diagnostic groups. Mini-Mental-Status-Examination (MMSE) (Folstein et al., 1975) was performed on patients suffering from cognitive impairments at the time of lumbar puncture. The MMSE score did not significantly differ between the diagnostic groups of dementias (Alzheimer's disease, DLB, PDD) and was significantly different for the NDC group (P = 2.0 x 10–6).
The study was conducted under the guidelines of the Declaration of Helsinki (World Medical Organisation, 1996) and approved by the ethics committee of the University of Goettingen. Investigations were carried out with the informed consent of all patients or, for patients with severe dementia, their next of kin.
Patients with Alzheimer's disease
All 23 patients (8 men and 15 women) of this group fulfilled the Diagnostic and Statistical Manuals (DSM) IV criteria for Alzheimer's disease and the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association (NINCDS–ADRDA) criteria for clinical diagnosis of probable Alzheimer's disease (McKhann et al., 1984). Age of this group was 69.5 ± 11.5 years (mean ± standard deviation). MMSE was available for 22 patients. One patient underwent the Syndrom-Kurz-Test (SKT) instead of the MMSE. The SKT score was 20, indicating a moderate form of dementia. The mean MMSE score was 18.4 ± 5.1 (mean ± standard deviation) in this group.
Patients with dementia with Lewy bodies
All 21 patients (15 men and 6 women) of this group fulfilled the DSM IV criteria for dementia and the McKeith criteria for clinical diagnosis of probable DLB (McKeith et al., 1996). Age of this group was 71.5 ± 6.6 years (mean ± standard deviation). MMSE was available for 20 patients. One patient rejected the cognitive testing. He displayed moderate cognitive impairments at the time of lumbar puncture, and neuropathological postmortem analysis confirmed this patient to be a case of DLB. The mean MMSE score was 18.1 ± 5.3 (mean ± standard deviation) in this group.
Parkinson's disease dementia patients
All 21 patients (16 men and 5 women) of this group fulfilled the DSM IV criteria for dementia and the UK Parkinson's Disease Society Brain Bank clinical diagnostic criteria for idiopathic Parkinson's disease (Gibb and Lees, 1988). All patients in this group presented parkinsonism at least one year before onset of dementia according to the criteria of McKeith et al. (1996). Age of this group was 72.4 ± 6.8 years (mean ± standard deviation). The MMSE score was 17.7 ± 7.3 (mean ± standard deviation) in this group.
Non-demented disease controls
This group consisted of 23 non-demented patients (4 men and 19 women), who underwent lumbar puncture for other differential diagnostic reasons; none of these patients displayed clinical features of neurodegenerative disease. Age of this group was 68.5 ± 9.0 years (mean ± standard deviation). A subgroup of 11 patients suffering from depression with cognitive complaints was assessed by MMSE. The score was 27.7 ± 3.1 (mean ± standard deviation). One patient suffering from major depression rejected cognitive testing. The cognitive impairments of all depressive patients improved after antidepressant medication. The group further included patients suffering from temporal lobe epilepsy (n = 1), normal pressure hydrocephalus (n = 2), cerebral transient ischaemic attacks (n = 1), brain metastasis (n = 1), peripheral herpes zoster infection (n = 1), breast cancer (n = 1), peripheral facial nerve palsy (n = 1), systemic vasculitis (n = 1), spinal cord compression (n = 1) and intervertebral disk herniation (n = 1).
Pre-analytical treatment of CSF for Aß-SDS–PAGE immunoblot
CSF was drawn from patients by lumbar puncture, sampled in polypropylene vials, and centrifuged (1000g, 10 min, 4°C), and aliquots of 200 µl were stored at –80°C within 24 h for subsequent one- and two-dimensional Aß-SDS–PAGE/immunoblot analysis.
Pre-analytical concentration of CSF by immunoprecipitation (IP) was performed as recorded previously (Wiltfang et al., 2002). The amino-terminal-selective mouse monoclonal antibodies 6E10 (Senetec Drug Delivery Technologies Inc, USA) and 1E8 were used in comparison with the carboxy-terminal-selective 13E9 and 6D5 directed against the carboxy-terminus of Aß1–40 and Aß1–42, respectively. The latter three antibodies were provided by Schering AG, Berlin, Germany.
One-dimensional Aß-SDS–PAGE/immunoblot
For separation of Aß peptides and subsequent detection, 10 µl of unconcentrated CSF was boiled in a sample buffer for SDS–PAGE, and Aß-SDS–PAGE/immunoblot was conducted as published elsewhere (Wiltfang et al., 1997, 2002).
Samples were run as triplicates and each gel carried a four-step dilution series of the synthetic Aß peptides Aß1–37, Aß1–38, Aß1–39, Aß1–40 and Aß1–42. Synthetic peptides Aß1–38, Aß1–40 and Aß1–42 were obtained from Bachem (Bubendorf, Switzerland); Aß1–37 and Aß1–39 were synthesized automatically according to Janek et al. (Janek et al., 2001). Standard preparations of synthetic Aß peptide mixture were created as described previously (Bibl et al., 2004) and bands were quantified from individual blots of each patient relative to this dilution series using a charge coupled device camera (CCD-camera) and the Quantity-ONE software (BIORAD).
The inter- and intra-assay coefficients of variation and sensitivity of detection of the Aß-SDS–PAGE/immunoblot have been published elsewhere (Wiltfang et al., 2002; Bibl et al., 2004).
Two-dimensional Aß-SDS–PAGE/immunoblot-Aß-IPG-2D-PAGE
For isoelectric focussing (IEF) on immobilized pH gradients (IPG) followed by Aß-SDS–PAGE/immunoblot (Aß-IPG-2D-PAGE), 25 µl of immunoprecipitated CSF were equilibrated in IPG sample buffer, and Aß-IPG-2D-PAGE was performed as published previously (Wiltfang et al., 2002).
Two-dimensional non-urea/urea-SDS–PAGE
Bicine/bistris/tris/sulphate SDS–PAGE (without urea) on 12% T (acrylamide)/5% C (N,N'methylenebisacrylamide) gels (Wiltfang et al., 1991) was used for the first dimension to achieve a separation that solely depends on the effective molecular radii of the peptides. In this separation gel, monomeric Aß peptides migrate in one single band close to the moving boundary, whereas oligomeric forms can be separated as a result of their higher molecular radii. Five microlitres of immunoprecipitated CSF were applied per lane. After electrophoresis at a constant current of 12 mA/gel, the whole lane was cut out and placed horizontally on a 0.75 mm thick separation gel of the same composition but containing 8 M urea. The gel was run at 18 mA/gel constant current for separating the different Aß peptide species on the basis of urea-induced peptide-specific shifts in binding of SDS.
Solid-phase extraction of Aß 1–40
HLB Extraction Cartridges (Waters, Nr. 186000115) were activated with 50% v/v methanol and subsequently washed with 8 ml 5% methanol prior to sample loading. Ten microlitres of Aß peptide (0.1 mg/ml) in SDS–PAGE sample buffer were mixed with 490 µl of phosphate-buffered saline (PBS) and loaded into one cartridge. The Aß peptide was eluted with 83% acetonitrile/0.08% trifluoroacetic acid (TFA). Four fractions of 1.5 ml each were collected in polypropylene vials. From each fraction, 50 µl were vacuum-dried and re-dissolved in binding buffer for further IP according to the protocol of the manufacturer (Bruker Immunocapturing Kit #233794) and subsequent matrix-assisted laser desorption ionization mass analysis–time-of-flight modus (MALDI–TOF) analysis. Samples for Aß-SDS–PAGE were dissolved in SDS–PAGE buffer according to Wiltfang et al. (1997, 2002).
Localization and excision of bands corresponding to Aß 1–40 and Aß 1–40* from unstained SDS–PAGE
To localize Aß 1–40 and Aß 1–40* in unstained gels, contact blots were produced immediately after electrophoresis by placing pre-wetted PVDF blotting membranes on each gel for 5 min at room temperature. After a brief rinse in double distilled water (H2Odd), peptides and proteins were visualized on the PVDF membrane by colloidal silver-staining [2% (w/v) trisodium citrate dihydrate, 0.8% ferrum(II) sulphate heptahydrate, 0.2% nitrated silver] according to van Oostveen et al. (1997). The stained membranes served as templates for cutting Aß 1–40 and Aß 1–40* out of the gel.
Preparation of gel pieces for MALDI–TOF analysis
Pre-analytical concentration of CSF by IP with the amino-terminal-selective mouse monoclonal antibody 1E8 non-covalently coupled to M280-Dynal-Beads was performed as described previously (Wiltfang et al., 2002). In brief, excised gel pieces were incubated overnight at 4°C in 40 µl of 5-fold concentrated RIPA detergent buffer (RIPA5x: 2.5% Nonidet P-40, 1.25% sodium desoxycholate, 0.25% SDS, 750 mM NaCl, 250 mM HEPES, one tablet of Protease Inhibitor Cocktail Complete Mini per 2 ml of RIPA5x, pH adjusted to 7.4 with NaOH) and 160 µl H2Odd in addition to 20 µl of 1E8-M280-Dynal-Beads. The magnetic beads were then captured on a magnetic stand and washed twice with PBS/0.1% bovine serum albumin (BSA) and once with 10 mM Tris-HCL (pH 7.4). Finally, the bound proteins were eluted in 10 µl of elution buffer (Bruker Immunocapturing Kit #233794) under constant agitation at 37°C for 1 h and for an additional 2 min in an ultrasonic bath.
MALDI–TOF mass spectrometry
A thin layer of saturated -cyano-hydroxycinnamic acid in acetone containing 3% H2O and 10 mM NH4H2PO4 was prepared on a ground steel target. An aliquot of 0.5 µl sample was directly spotted onto the matrix and dried. Subsequently, the preparation was washed twice by adding 5 µl 10 mM NH4H2PO4/0.1%TFA and removing it after 10 s, dried and analyzed using an autoflex II TOF/TOF mass spectrometer (Bruker Daltonics, Germany) in the positive reflectron mode. Three hundred individual shots were aggregated for each spectrum. Spectra were calibrated using ClinProt Standard (Bruker Daltonics, Germany).
Statistical analysis
Aß peptide levels were scanned and calculated as absolute values (ng/ml) as well as percentages of the total Aß peptide concentration of all Aß peptide species that were detected. The data on peptide levels were obtained from individual blots of each patient. For comparison of the patient groups, mean concentrations and standard deviation (SD) were calculated.
The Mann–Whitney U-test was applied to evaluate the significance of differences between the groups.
The two-sided level of significance was defined as P < 0.05. A P-value < 0.01 was considered as highly significant.
Receiver operating characteristic (ROC) curve analysis was used to determine cut-off points. The cut-off level for dichotomizing values was selected as the situation optimizing sensitivities, specificities and the Youden index.
Computations were performed using the statistical software package SPSS, version 10.0.
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An additional peptide with Aß-like immunoreactivity: its specification and quantification
A highly conserved pattern of three Ct-truncated Aß peptides in addition to Aß1–40, 1–42 could be shown by the Aß-SDS–PAGE/immunoblot in the CSF of all investigated patients. Additionally, a previously undescribed peptide with Aß-like immunoreactivity was found to regularly migrate cathodically of Aß1–37 in all CSF samples analysed (see Supplementary Fig. 1). All peptides migrate as a single band of approximately 4 kDa in a conventional SDS–PAGE, where urea is absent in otherwise unchanged separation gels (Fig. 1). In contrast, synthetic
-synuclein was found to migrate at a molecular mass of approximately 16 kDa in a non-urea SDS–PAGE (data not shown).
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The novel peptide displayed no electrophoretic co-migration with the synthetic Aß peptides Aß1–43, Aß1–36, Aß1–35, Aß1–34, Aß1–33 and Aß1–28 in the Aß-SDS–PAGE/immunoblot (data not shown).
The peptide was detected by the anti-Aß peptide-specific monoclonal antibody 1E8 during immunoblot and was not visualized on account of unspecific reactions of the secondary antibody or biotin-mediated affinity to the streptavidin complex (data not shown). A cross-reactivity of the 1E8 to synthetic -synuclein could be ruled out also in the immunoblot (see Supplementary Fig. 2). During IP, the novel peptide was enriched by the amino-terminal-specific antibodies 1E8 (Fig. 1) and 6E10 against Aß peptides and by the 13E9, directed against the carboxy-terminus of Aß1–40, respectively. The carboxy-terminal-specific antibody against Aß1–42 6D5 did not recognize the peptide (data not shown).
A two-dimensional combination of a conventional SDS–PAGE followed by urea-based Aß-SDS–PAGE/immunoblot (non-urea/urea-SDS–PAGE) revealed that the novel peptide migrated in one band with the other five Aß peptides in the conventional SDS–PAGE and was subsequently separated cathodically of Aß1–37 in the urea-based Aß-SDS–PAGE/immunoblot (Fig. 2A and B).
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The Aß-IPG-2D-PAGE revealed an identical isoelectric point for the Aß peptide quintet and the novel peptide (Fig. 2C).
A band co-migrating with Aß1–40* was detected after the application of synthetic Aß1–40 to solid-phase extraction (SPE) and subsequent acetonitrile/TFA elution in the same fractions as Aß1–40 (synthetic Aß1–40*) as shown by Aß-SDS–PAGE/immunoblot analysis (see Supplementary Fig. 3). Moreover, synthetic Aß1–40* was present after picking of Aß1–40 from a coomassie stained gel, but was not found when Aß1–40 was picked from unstained gels and analysed subsequently by one-dimensional Aß-SDS–PAGE/immunoblot (data not shown). The synthetic Aß1–40* was purified through separation in Aß-SDS–PAGE/immunoblot and subsequent picking (purified synthetic Aß1–40*). This purified synthetic Aß1–40*, an SPE fraction of synthetic Aß1–40, synthetic standard preparations of Aß peptides (mixture of five Aß peptides) and an immunoprecipitated CSF pool of five representative DLB patients were comparatively applied to the urea-based Aß-SDS–PAGE/immunoblot, where the band corresponding to Aß1–40* co-migrated among all four samples (see Supplementary Fig. 3). In the absence of urea and in otherwise unchanged separation gels, one single band in the molecular weight range of monomeric Aß (
4 kDa) was detected in all samples. The SPE fraction, synthetic standard mixture of five Aß peptides and an immunoprecipitated CSF pool of five representative DLB patients were applied to IEF by IPG. After IEF, the region corresponding to the isoelectric point of N-terminally unchanged Aß peptides (e.g. 5.37) was cut out of each strip and analysed in the Aß-SDS–PAGE/immunoblot on parallel lanes. This approach revealed one- and two-dimensional co-migration of the peptide generated from Aß1–40 during SPE, with Aß1–40* occurring in CSF and the respective band detected in standard preparations (see Supplementary Fig. 3). Moreover, the synthetic Aß1–40* was captured by the carboxy-terminally-specific antibody against Aß1–40 (13E9) during IP and detected by the amino-terminally-specific antibody against Aß1–40 (1E8) in the immunoblot (see Supplementary Fig. 3). Thus, the synthetic Aß1–40* exhibited similar electrophoretic and immunological features to Aß1–40* that occurs in vivo. The direct mass analysis of the SPE fractions using MALDI–TOF analysis revealed two mass peaks of 4329.9 and 4345.2 Da, respectively. The expected mass of Aß1–40 and oxidized Aß1–40 (Aß1–40ox) would be 4329.6 and 4345.6 Da, respectively. MALDI–TOF analysis of each respective band, picked from the Aß-SDS–PAGE and enriched by IP revealed that the Aß1–40* band comprises exclusively a mass peak corresponding to the expected mass of Aß1–40ox (see Supplementary Fig. 4). Otherwise, the Aß1–40 band exhibits predominantly a mass peak corresponding to the expected mass of unoxidized Aß1–40 (see Supplementary Fig. 4). The expected mass for Aß1–40ox could be inconsistently detected herein and, if present, its intensity was minor as compared with the one expected for Aß1–40.
We conclude from the data that the novel peptide is a monomeric Aß peptide with electrophoretic and immunological features of Aß1–40, but it migrates at a different position in the Aß-SDS–PAGE/immunoblot. A similar band originates from Aß1-40 in vitro and exhibits a mass corresponding to Aß1–40ox. We consequently named the novel peptide Aß1–40*
Another unknown peptide migrated cathodically of Aß1–40* and exhibited similar electrophoretic properties to Aß1–40*. We consequently named this peptide Aß1–40**. Aß1–40** was not consistently detectable in all investigated samples and, if present, its concentration was close to the level of detection. Therefore, Aß1–40** was not systematically quantified in CSF.
The absolute values of Aß1–40* given below were measured arbitrarily relative to the Aß1–37 standard peptide. The abundance of Aß1–40* in absolute and relative forms was the lowest of all Aß peptides quantified.
Aß-SDS–PAGE/immunoblot: Aß peptide patterns and their use for neurochemically supported differential diagnosis of dementias
Each dementia group, DLB, Alzheimer's disease and PDD, displayed decreased absolute levels of Aß1–42 relative to the NDC group, whereas Aß1–37 levels increased slightly in Alzheimer's disease as compared with the other dementia groups (Fig. 3). The decrease of Aß1–42 was most pronounced for the Alzheimer's disease group and not statistically significant for the PDD group (P > 0.05).
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By introducing the ratio of Aß1–42 to Aß1–37, the NDC group could be differentiated at a highly significant level from Alzheimer's disease (P = 5.2 x 10–7), DLB (P = 2.0 x 10–4) and PDD (P = 1.2 x 10–3). The Alzheimer's disease group was highly significantly differentiated from the DLB group (P = 3.2 x 10–3) and the PDD group (P = 1.1 x 10–4), respectively (Fig. 4). Using a cut-off value of 0.848, Alzheimer's disease could be discriminated from NDC with a sensitivity and specificity of 87%.
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The DLB group could be differentiated from the PDD group by a percentage increase of Aß1–40* relative to the sum of all Aß peptides (Aß1–40*%) in the DLB group at a highly significant level (P = 6.0 x 10–4) (Fig. 5). Aß1–40* was also elevated in the Alzheimer's disease group, but failed the level of significance.
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The absolute and relative abundances of Aß peptides of each diagnostic group are summarized in Table 1. The cut-off points, sensitivities and specificities of the best discriminating Aß peptide ratio (i.e. Aß1–42/37 or Aß1–40*%, respectively) for each differential diagnostic testing are summarized in Table 2.
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Discussion |
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TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
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Differentially expressed Aß peptide patterns in Alzheimer's disease, DLB and PDD—pathophysiological implications
A previously undescribed peptide with Aß-like immunoreactivity (Aß1–40*) aside a highly conserved pattern of the Aß peptides 1–37, 1–38, 1–39, 1–40 and 1–42 was constantly observed in all 88 CSF samples investigated. In summary, Aß1–40* exhibits electrophoretic and immunological features of Aß1–40, but migrates at a different position in the Aß-SDS–PAGE/immunoblot. A similar band originates from synthetic Aß1–40 during SPE under hydrophobic conditions (synthetic Aß1–40*) and exhibits a mass corresponding to the expected mass of Aß1–40ox.
The percentage abundance of Aß1–40* relative to the sum of all investigated Aß peptides (Aß1–40*%) was prominently increased in DLB and to a lesser degree also in Alzheimer's disease as compared with PDD and NDC, respectively.
Significantly decreased CSF Aß1–42 levels relative to NDC were most prominent in Alzheimer's disease and could also be shown for DLB, but not for PDD. In contrast, Aß1–37, Aß1–38 and Aß1–40 were slightly elevated in Alzheimer's disease as compared with the other dementia groups. The introduction of ratios of Aß1–42 to Aß1–37, 1–38, 1–39 and 1–40, respectively, improved the diagnostic test accuracy for each differential diagnostic question relative to the Aß1–42 levels alone. First, this may be due to disease-specific interactions of each ongoing neurodegenerative dementia process with APP metabolism, which cannot be adequately represented by the sole measurement of absolute Aß1–42 levels (Wiltfang et al., 2001). Second, the percentage abundance of each Aß peptide species displayed a lower inter-individual variance of values than its absolute levels (Wiltfang et al., 2003). This corresponds to the observation that the abundances of single Aß peptide species are closely correlated to each other and regulated in narrow limits, whereas the total amount of Aß peptides varies inter-individually (Wiltfang et al., 2002, 2003). The ratio of the differentially altered Aß1–42 to the Aß1–37 levels enabled the best test accuracies and a highly significant differentiation of all diagnostic groups from each other, with the exception of DLB versus PDD. DLB and PDD could then be discriminated at a highly significant level by the specifically increased Aß1–40*% in DLB.
We can only speculate on the pathophysiological implications of these disease-specific Aß peptide patterns
Decreased levels of CSF Aß1–42 have been reported for patients with Alzheimer's disease (Motter et al., 1995; Andreasen et al., 2001, 2003; Wiltfang et al., 2002, 2003; Mollenhauer et al., 2005) and DLB (Andreasen et al., 2001; Mollenhauer et al., 2005). The reduction of Aß1–42 levels in Alzheimer's disease has been explained by an increased clearance of the peptide from CSF into senile amyloid plaques for a long time (Motter et al., 1995). Other studies indicate the existence of alternative mechanisms, including the formation of SDS-stable oligomers (Podlisny et al., 1995) and chaperone complexes of Aß peptides with specific carrier proteins (Wiltfang et al., 2002, 2003; Bibl et al., 2004). However, misfolding and subsequent deposition of proteins is considered to be a major pathological event in both neurodegenerative diseases and amyloid pathology has also been reported for DLB (Merdes et al., 2003).
Alpha-synuclein, the major component of Lewy bodies, has been reported to facilitate the aggregation of Aß peptides, and the interactions between the two peptides essentially involve their respective hydrophobic domains (Yoshimoto et al., 1995). Moreover, the deposition of amyloid plaques in DLB has been most recently shown to be related to the amount of cortical Lewy bodies (Pletnikova et al., 2005). Interactions of hydrophobic templates or domains with Aß can induce a conformational shift of the peptide into an -helix in vitro (Giacomelli and Norde, 2005). Similar interactions of Aß, for example, with -synuclein (Yoshimoto et al., 1995) in the case of DLB, may lead to -helical Aß peptide species in vivo. The transient formation of an -helix has been reported to play a major role in the assembly of toxic oligomers (Klimov and Thirumalai, 2003) and ß-sheet formation, suggesting it to be an on-pathway to aggregation of Aß (Kirkitadze et al., 2001).
Additionally, the -helical structure alters the electronic environment around the sulphur of methionine residue 35 (met-35), making it prone to oxidation (Butterfield, 2003) by a broad variety of oxidizing agents that are abundant in biological systems. The pathogenic role of oxidative stress (e.g. membrane damage and disruption of cellular calcium homeostasis) is well documented for Alzheimer's disease (Butterfield et al., 2003) and DLB (Giasson et al., 2000). On the one hand, oxidation of met-35 to methionine sulphoxide (two-electron oxidation) decreases the cellular toxicity and pro-oxidative potential of Aß (Varadarajan et al., 2001; Butterfield, 2003) and also prevents its fibril aggregation (Watson et al., 1998; Hou et al., 2002; Palmblad et al., 2002). On the contrary, metal ions, such as copper, may react with met-35 to form a sulphuramyl free radical on the sulphur atom (one-electron oxidation), which causes enhanced oxidative stress via DNA/RNA and protein oxidation, lipid peroxidation and formation of reactive oxygen species, respectively (Butterfield, 2003). Moreover, the metal-dependent aggregation of Aß is not affected by the formation of methionine sulphoxide (Barnham et al., 2003) and met-35-oxidized Aß comprises a major component of total brain Aß in senile amyloid plaques (Atwood et al., 2002; Dong et al., 2003). Whilst met-35-oxidized Aß is more hydrophilic, its enhanced release from the neuronal membrane into the synaptic cleft may mediate frequent contact with metal ions, such as zinc and copper, released during neural transmission (Barnham et al., 2003). This may contribute to metal-dependent aggregation and trigger Aß precipitation as a kind of seed at a quite sensitive site of the neuron.
The initial overexpression of -helical Aß may thus promote both the fibril formation and the metal-dependent aggregation of the peptide. Taken together, the increased abundance of Aß1–40* points to a disease-specific mechanism of amyloid deposition in DLB triggered by conformational transition of Aß upon hydrophobic interactions, probably mediated by -synuclein, and enhanced posttranslational peptide oxidation.
Although neuropathological similarities between DLB and PDD have been reported (Iseki, 2004), one major difference remains that Lewy bodies in PDD are predominantly localized in the brainstem (Jendroska et al., 1996). The less frequent occurrence of cortical Lewy bodies may contribute to the lower extent of cortical amyloid deposition in PDD as compared with DLB (Mastaglia et al., 2003; Pletnikova et al., 2005), which might be reflected by a distinct neurochemical phenotype among both neurodegenerative diseases in CSF. These findings correspond to the observation that the majority of DLB patients show similar clinical features to PDD at an earlier stage of disease with a pronounced dementia syndrome.
Despite some overlap, especially with regard to Alzheimer's disease and DLB, the reported disease-specific neurochemical phenotypes in CSF indicate the existence of distinct pathophysiological mechanisms in the Aß peptide metabolism for Alzheimer's disease, DLB and PDD. We propose the investigation of Aß peptide patterns in CSF and brain homogenates of neuropathologically defined patient groups to further elucidate this aspect.
The specification of a novel peptide with Aß-like immunoreactivity in CSF
The amino acid sequence of the novel peptide and its secondary structure is currently under evaluation, but remains unclear. Nonetheless, four different lines of indication point to an oxidized and -helical form of monomeric Aß1–40 as a probable candidate for Aß1–40*:
First, the two-dimensional combination of a non-urea SDS–PAGE followed by a urea-based Aß-SDS–PAGE/immunoblot (non-urea/urea-SDS–PAGE) demonstrated Aß1–40* to migrate in one single band with other monomeric Aß peptide species at a molecular mass of approximately 4 kDa in the absence of urea and its subsequent electrophoretic separation cathodically of Aß1–37 in the Aß-SDS–PAGE/immunoblot. The same held true for the synthetic Aß1–40* generated from synthetic Aß1–40 via SPE. Like other monomeric Aß peptide species, the peptide can obviously be separated only as a result of urea-induced peptide-specific shifts in binding of SDS during the Aß-SDS–PAGE (Kawooya et al., 2003). Oligomerized Aß peptides can be separated from the monomeric band as a result of their significantly higher mass and correspondingly larger effective molecular radii during a non-urea SDS–PAGE. In accordance with the current literature, synthetic full-length -synuclein was found to migrate at a molecular mass of approximately 16 kDa during a non-urea SDS–PAGE. In brain homogenates from DLB patients, two additional peptides have been reported to migrate in the 12 and 6 kDa range, respectively, in 10% Tris/Tricine gels (Culvenor et al., 1999). These peptides could only be stained with an antibody directed against the NAC region (i.e. non-Aß-component of Alzheimer's disease amyloid) of -synuclein (Culvenor et al., 1999), suggesting that the NAC peptide exhibits a molecular mass of approximately 6 kDa at minimum. These data indicate a monomeric form of Aß peptide to be visualized cathodically of Aß1–37 and make SDS-stable oligomers of Aß peptides or -synuclein and NAC, respectively, unlikely to provoke this band.
Second, the peptide exhibits properties of an amino-terminally unmodified Aß peptide. The Aß-IPG-2D-PAGE revealed an identical isoelectric point for Aß1–40* and the other five Aß peptides. During an IEF, the amino-terminally truncated Aß peptides (Aß2-X, Aß3-X) show a shift in their isoelectric point of one pH unit (5.37 to 6.37) (Wiltfang et al., 2002), whereas amino-terminally elongated Aß peptides (Aß-12-X), which are generated after a cleavage of APP by -secretase, would shift to a more acidic isoelectric point owing to an additional negatively charged amino acid. In contrast, Ct-truncation down to Aß1–28 or Ct-elongation up to Aß1–49 will not influence the isoelectric point. Additionally, a co-migration of Aß1–40* with various carboxy-terminally modified synthetic Aß peptides that have been reported to occur in vivo was excluded in the urea-based Aß-SDS–PAGE/immunoblot.
Third, the amino- and carboxy-terminus of Aß1–40* immunologically react like Aß1–40. Aß1–40* immunoprecipitates with amino-terminally-specific antibodies against Aß peptides (1E8 and 6E10) and with a carboxy-terminally-specific antibody against Aß1–40 (13E9), but not with one against Aß1–42 (6D5). Otherwise, we have ruled out cross-reactions of the detection antibody 1E8 with synthetic -synuclein during immunoblot procedures. As the NAC peptide is considered to be a cleavage product of -synuclein, a cross-reaction with NAC is also unlikely. Neither is there any evidence from the literature that the mAb 1E8 cross-reacts with either -synuclein or the NAC peptide (Culvenor et al., 1999).
Fourth, a band with electrophoretic and immunological features similar to Aß1–40* that occurs in vivo was found after SPE of synthetic Aß1–40 (synthetic Aß1–40*) under hydrophobic conditions. MALDI–TOF analysis of the respective fractions revealed two mass peaks corresponding to the expected molecular mass of Aß1-40 and oxidized Aß1–40 (Aß1–40ox), respectively. MALDI–TOF analysis of each respective band, picked from the Aß-SDS–PAGE and enriched by IP, revealed that the synthetic Aß1–40* band comprises exclusively a mass peak corresponding to the expected mass of Aß1–40ox. The synthetic Aß1–40 band exhibits predominantly a mass peak corresponding to the expected mass of unoxidized Aß1–40, although minor amounts of Aß1–40ox could inconsistently also be detected herein. Accordingly, the oxidation of Aß1–40 probably contributes to its altered migration behaviour in the Aß-SDS–PAGE. Additionally, the above findings strongly suggest that hydrophobic interactions and probably the subsequent formation of a stable -helix of Aß1–40 (Giacomelli et al., 2003, 2005) are thoroughly involved in the generation of Aß1–40*. Two -helices covering the positions 16–24 and 28–36, respectively, have been detected within Aß1–40 (Coles M et al., 1998), the first of which is reportedly discordant and, without sufficient stabilization, is particularly prone to forming ß-stranded structures (Kallberg et al., 2001; Päiviö et al., 2003). The second -helix around position 28–36 becomes destabilized and is abolished completely in the case of met-35 oxidation, whereas the first -helix remains unaffected (Watson et al., 1998). The oxidation and secondary structural transition of Aß1–40 may change the peptide-specific binding of SDS and thus explain its altered migration behaviour during urea-based electrophoresis (Kawooya et al., 2003).
We currently apply circular dichroism spectroscopy to validate our hypothesis.
Aß peptide patterns and their use for neurochemically supported differential diagnosis of dementias
The data clearly demonstrate that CSF Aß peptide patterns vary in a disease-specific manner between Alzheimer's disease, DLB, PDD and neuropsychiatric diseases. However, there is insufficient evidence to suggest Aß peptide patterns as a sole biomarker for differential diagnosis among the three investigated dementias. According to the criteria recommendations of an international consensus group (Wiltfang et al., 2005), Aß peptide patterns come closest, but fail to fulfil the requirements (e.g. both sensitivity and specificity beyond 85%). Nevertheless, using a cut-off value of 0.848 for the ratio of Aß1–42/Aß1–37, a reasonable accuracy in discriminating Alzheimer's disease from NDC (i.e. 87% sensitivity and specificity each) could be obtained. The previously reported sensitivities for Alzheimer's disease detection and specificities for DLB exclusion, respectively, did not exceed 75% in a combined assay of tau and Aß 1–42 enzyme-linked immunosorbent assay (ELISA) (Andreasen et al., 2001). Thus, the actual differential diagnostic value of Aß peptide patterns can be considered to be as relevant as the established ELISAs for tau and Aß1–42. Moreover, since multiparametric approaches are gaining increasing importance in the early and differential diagnosis of dementias (Lewczuk et al., 2004; Wiltfang et al., 2005), the evaluation of Aß peptide patterns may aid neurochemical dementia diagnosis in combination with other biomarkers (e.g. tau and phospho-tau) (Wiltfang et al., 2005).
Although the Aß-SDS–PAGE/immunoblot is a highly sensitive method (Wiltfang et al., 2002), the very low CSF concentration of Aß1–40* comes close to the level of detection in some cases. This can be considered as a major drawback of the test, which most probably contributes to an increased variance of values and consequently to a loss of accuracy. In respect of this concern, the pre-concentration of Aß peptides from CSF using the highly valid and reproducible N-terminally-specific IP (Wiltfang et al., 2002) prior to the Aß-SDS–PAGE/immunoblot promises improved test accuracy. Additionally, it must be taken into consideration that the investigated PDD patients all presented with two or three core features demanded for the diagnosis of probable DLB (McKeith et al., 1996). The differentiation between probable DLB and PDD that lacks further core features of DLB (e.g. fluctuations or hallucinations) might have revealed higher test accuracy.
Hence, the test will have to be re-evaluated using immunoprecipitated CSF samples of neuropathologically defined Alzheimer's disease, DLB and PDD patients to determine whether Aß1–40* will indeed be applicable as a novel neurochemical dementia marker.
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TopSummaryIntroductionPatients and methodsResultsDiscussionSupplementary materialReferences |
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Supplementary data are available at Brain online.
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* These authors contributed equally to this work.
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Acknowledgements |
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M.B., P.L., H.E., J.K., M.O. and J.W. are supported by the BMBF (German Federal Ministry of Education and Science) funded grant, Competence Net Dementia (01GI0102); J.W. and P.L. are supported by University of Erlangen-Nuremberg ELAN-Program Funds; M.B. is supported by the Research program, Faculty of Medicine, Georg-August-Universität Göttingen; and M.O. and J.W. are supported by the CMPB/DFG research center. P.L. and J.W. are supported by the BMBF-funded grant NGFN2 (Project No. PPO-S10T10).
The authors would like to thank Sabine Paul, Birgit Otte, Heike Zech and Nikolaus Kunz for excellent technical assistance.
Funding to pay the Open Access publication charges for this article was provided by German Competence Net Dementias (CND, http://www.kompetenznetz-demenzen.de/) and Research program. Faculty of Medicine, Georg-August-Universität, Göttingen.
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