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A new ELISA specific for oligomeric assemblies of amyloid β protein (oAβ) was developed to examine in vivo levels of oAβ vs. monomeric Aβ in sporadic and familial Alzheimer disease (AD) plasma and brain tissue.
To establish the oAβ ELISA, the same N-terminal Aβ antibody was used for antigen capture and detection. Plasmas and postmortem brains from AD and control subjects were systematically analyzed by conventional monomeric Aβ and new oAβ ELISAs.
We measured oAβ species in plasma samples from 36 clinically well-characterized AD patients and 10 controls. In addition, postmortem samples were obtained from brain autopsies of 9 verified AD and 7 control subjects.
The specificity of oAβ ELISA was validated with a disulfide crossed-linked, synthetic Aβ1–40Ser26Cys dimer that was specifically detected before but not after the dissociation of the dimers in β-mercaptoethanol. Plasma assays showed that relative oAβ levels were closely associated with relative Aβ42 monomer levels across all subjects. Analysis of sequential plasma samples from a subset of the AD patients, including a patient with AD caused by a presenilin mutation, revealed decreases in both oAβ and Aβ42 monomer levels over a 1–2 year period. In brain tissue from 9 AD and 7 control subjects, both oAβ and monomeric Aβ42 were consistently higher in the AD cases.
An oAβ-specific ELISA reveals a tight link between oAβ and Aβ42 monomer levels in plasma and brain, and both forms can decline over time in plasma, presumably reflecting their increasing insolubility in the brain.
Alzheimer’s disease (AD) is characterized by the progressive accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles. The protein subunit of the amyloid plaques, amyloid β-protein (Aβ), does not occur as a single molecular species; many different Aβ-containing peptides have been detected in human CSF and/or brain1,2. The most common Aβ isoform in vivo is Aβ1–40, i.e., a peptide that begins at Asp1 and terminates at Val40 of the Aβ region of APP. Increased accumulation of Aβ1–42, a peptide that differs from Aβ1–40 by the inclusion of Ile41 and Ala42, is particularly associated with development of AD. The extra two hydrophobic amino acids of Aβ42 greatly enhance its aggregation propensity 3, leading to accelerated formation of small (low-n) Aβ oligomers (oAβ), larger intermediate assemblies like protofibrils, and eventually the typical ~8 nm amyloid fibrils found abundantly in neuritic plaques and amyloid-bearing microvessels. Small, soluble oligomers of Aβ have been linked to neuronal toxicity and synaptic failure (for review, see 4). How the oligomeric assemblies reach a balance with monomeric Aβ and large protofibrils and fibrils in human brain is under investigation.
Studies of circulating Aβ in blood have provided insights into Aβ equilibrium between the brain and the periphery. A few studies have associated increased levels of plasma Aβ42 with AD at different stages. For example, an increase in plasma Aβ42 was associated with conversion from normal cognition to mild cognitive impairment (MCI) and on to AD, albeit with unsatisfactory sensitivity and specificity 5. Another study found that patients with AD at baseline and those who developed AD later had significantly higher plasma Aβ42 levels; some of the AD patients showed elevated levels of Aβ42 and Aβ40 before and during the early stages of AD, but plasma levels declined thereafter 6,7. Another study showed that non-demented subjects with high levels of plasma Aβ42 were more than twice as likely to develop AD than those with low levels, and that AD patients showed higher Aβ42 levels than non-AD controls 8.
Familial AD (FAD) patients having mutations in the presenilins, the catalytic subunits of the γ-secretase complex that generates Aβ, have increased plasma levels of Aβ42 9. Plasma Aβ42 levels and the Aβ42/Aβ40 ratio were higher even in presymptomatic subjects carrying FAD mutations in PS1 or APP, and Aβ42 may decrease with disease progression prior to symptom onset 10. In some typical (late-onset) AD cases, elevated plasma Aβ42 has been linked to a locus on Chromosome 10 11, and some first degree relatives of late-onset AD patients have elevated Aβ42, suggesting that increased plasma Aβ is a heritable trait 12, 13.
Plasma Aβ is a potentially promising but understudied candidate marker for diagnosis and pre-clinical prediction. However, plasma Aβ40 or Aβ42 was found not to be an optimal candidate in unbiased proteomic searches for AD fluid biomarkers 14,15. In one study, increased plasma Aβ42 was detected in MCI patients, but a significant association was only observed in women 16. In a cohort of men at age 70, plasma Aβ40 and Aβ42 were not associated with incident AD at follow-up, whereas low plasma Aβ40 in another cohort of men at age 77 was associated with higher incidence of AD 17. Yet another study found that subjects with low plasma Aβ42/Aβ40 ratios had higher risk of MCI or AD and greater cognitive decline 18. A correlation between increased plasma Aβ40 and increased risk of dementia has also been reported 19. A recent studies of MCI patients followed up to 7 years showed no significant difference of plasma Aβ species between MCI patients that later developed AD and stable MCI patients or healthy controls 20.
These often inconsistent reports on the association of plasma Aβ levels with AD may reflect the fact that measurements to date only represent the pools of monomeric Aβ and were measured by different Aβ ELISAs 21. To understand the relationship among different Aβ species in vivo, we developed an ELISA that can detect oAβ and simultaneously measured both Aβ monomers and oAβ in human plasma or in postmortem brain tissue. Levels of human oAβ detected by our new oAβ-specific ELISA were closely associated with the levels of monomeric Aβ42. Levels of both soluble and insoluble Aβ42 and oligomeric Aβ species were significantly higher in AD brains compared to controls. We also observed decreases in plasma Aβ in follow up samples from the same patients 1–2 years later. The development of an oligomer-specific ELISA applicable to plasma extends Aβ measurement to a highly relevant neurotoxic form of this pathogenic peptide.
Blood samples were collected in K2-EDTA-containing collection tubes and centrifuged at 1600 g for 15 min. The plasma supernatant was aliquoted and stored at −80°C until measured. Average ages for AD patients and control subjects at time of blood drawing were 72 (n=36) and 62 years old (n=10), respectively. Brain lysates from postmortem human brains (n=16) were prepared as recently described 22.
Sandwich ELISAs for monomeric Aβ were performed as described 23. The use of C-terminal capturing antibody and N-terminal or mid-region detecting antibodies has been a standard format for measuring monomeric Aβ species in many studies 9,24–26. The capture antibodies 2G3 (to Aβ residues 33–40) and 21F12 (to Aβ residues 33–42) were used for Aβ40 and Aβ42 species, respectively. The detecting antibodies were biotinylated 3D6 (to Aβ residues 1–5) for Aβ1–40/42 or biotinylated 266 (to Aβ residues 13–28) for Aβx-40/42 species. These antibodies were kindly provided by P. Seubert and D. Schenk (Elan, plc).
To detect oAβ species, the same N-terminal antibody, either 82E1 (to Aβ residues 1–16; Immuno-Biological Laboratories, Inc., Minneapolis, MN) or else 3D6, was used for both capture and detection. An identical sandwich ELISA procedure as for the traditional monomer assays was followed to measure relative oAβ levels, which were calculated using standard curves of synthetic Aβ1–40 peptide captured by 2G3 antibody and measured by same detecting antibodies (82E1 or 3D6).
CHO cells stably expressing wt human APP751 plus either wt or mutant PS1 [resulting in lines PS1wt-1, PS1wt-2, PS1wt-3, PS1M146L-1, PS1M146L-2 PS1M146L–M146L missense mutation), and PS1C410Y-1, PS1C410Y-2, PS1C410Y-3 (C410Y missense mutation)] were maintained in 200 ug/ml G418 (InVitrogen) plus 25 ug/ml puromycin (for PS1). We also examined CHO lines singly transfected with wt APP751 or APP751 bearing the V717F (“Indiana”) missense mutation. Cells were incubated in methionine-free, fetal bovine serum-free media for 45 min before labeling with 200 uCi/ml [35S]-methionine for 38 hr. Conditioned media were collected, and immunoprecipitation was performed as described 25.
A β dimers were generated by atmospheric oxidation of a 20 uM solution of synthetic Aβ1–40Ser26Cys in 20 mM ammonium bicarbonate, pH 8.0, for 4 days at room temperature. To facilitate disassembly of aggregates formed during the oxidation reaction, the peptide solution was lyophilized and the lyophilate incubated in 5 M GuHCl, 50mM Tris-HCl, pH 8.0, for 4 hours. Disulfide crossed-linked Aβ dimers were isolated from unreacted monomer and higher aggregates by size exclusion chromatography using a Superdex 75 10/30 HR column eluted with 50 mM ammonium acetate, pH 8.5, at a flow rate of 0.8 ml/min. Fractions (0.5 ml) were collected, an aliquot of each electrophoresed on 16% Tris-tricine polyacrylamide gels, and protein detected by silver staining. SEC fractions found to contain exclusively dimeric Aβ were pooled and used as the dimer stock. The concentration of peptides in stock solutions was determined by comparison to wild type Aβ1–40 of known concentration. Once collected, all samples were stored at −80oC until used. To disrupt the disulfide bonds linking the monomers, dimeric Aβ was treated with 3% β-mercaptoethanol (βME), followed by serial dilutions for quantification by ELISA. The diluted, residual amount of βME did not interfere with the capture and detecting antibodies. Aβ1–40Ser26Cys was synthesized by the Biopolymer Laboratory in the Department of Neurology at UCLA Medical Center and the correct sequence and purity confirmed by amino acid analysis, reverse-phase HPLC and mass spectrometry.
To analyze oAβ species in human samples, we established a sensitive and specific oAβ ELISA. The assay relies on the use of a single monoclonal antibody for both capture and detection. Thus, for oAβ to be detected by this sandwich ELISA, an Aβ assembly must contain at least two exposed copies of the same epitope that is accessible by the identical capturing and detecting antibody 27, 28. This means that this assay will recognize only Aβ assemblies that contain at least two Aβ molecules. Two monoclonal antibodies, 82E1 and 3D6 that each recognize the extreme N-terminus of human Aβ, were tested.
To confirm the specificity of the ELISA, we utilized a synthetic Aβ peptide, Aβ1–40 Ser26Cys, that is capable of reversibly forming covalently cross-linked Aβ dimers (dAβ) under oxidizing conditions 22, 29. Disulfide crossed-linked Aβ dimers were separated from unreacted monomer and higher aggregates by size exclusion chromatography. The concentration of dAβ in stock solutions was determined by comparison to wild type (wt) synthetic Aβ1–40 of known concentration; both dAβ and monomeric wt Aβ peptides were detected by SDS-PAGE and silver staining (Fig. 1A). Based on the densitometric signal obtained from synthetic wt Aβ1–40 peptide, we estimated that the concentration of our synthetic dAβ stock was 81 uM. In the presence of 3% β-mercaptoethanol (βME), the disulfide bonds linking the monomers that form dAβ were disrupted, producing a reduced monomer (Fig. 1B). In the absence of βME, no monomeric Aβ was detected, and the vast majority of Aβ species was dAβ (Fig. 1B).
Serial dilutions of dAβ were made after the dAβ stock was treated with or without βME, and the diluted samples were assayed using either antibody 82E1 (Fig. 1C) or 3D6 (Fig. 1D) in a sandwich format. When the relative levels of the diluted dAβ (800 pM) of the dAβ stock were normalized to 1, either monoclonal antibody detected a clear linear reduction in the levels of dAβ when further diluted to 400 and 200 pM (Fig. 1C). In the presence of βME, dissociation of the disulfide bond markedly reduced the amount of dAβ signal, and the levels of remaining dAβ were less than 10% of the same fraction without βME, with 82E1 showing even greater specificity than 3D6 in this regard (Fig. 1C, D). Therefore, our new ELISA accurately measured the levels of dAβ in a linear fashion and is highly sensitive to the dissociation of dAβ to monomers.
Using the oAβ-specific ELISA, we screened plasma samples obtained from 36 well-characterized AD patients and 10 control subjects. The ages of the AD subjects ranged from 50 to 90 at the time of blood sampling, with average age at 72. The age of control subjects ranged from 52 to 68, with average age at 62. Conventional Aβ ELISAs were applied to measure monomeric Aβ species.
We analyzed plasma levels of oAβ as a function of subject age but did not observe an age-dependent alteration of oAβ levels (Fig. 2). There were a number of subjects who carried high plasma levels of oAβ-reactive species, with a wide age distribution between 60 and 90. Overall, we found that most (7/10) control subjects had plasma levels of oAβ below our detection limit, whereas more than half (19/36) AD subjects had detectable oAβ levels (Fig. 2).
From the same aliquot of plasma, we measured four monomeric Aβ species using four distinct ELISAs: Aβ1–40, Aβ1–42, and N-terminally heterogeneous Aβ40 and Aβ42 species (i.e., Aβx-40 and Aβx-42). The levels of Aβ1–40 and Aβx-40 did not reveal a clear separation of AD subjects from control subjects (Fig. 3A and B). On average, the levels of Aβx-40 were higher than those of Aβ1–40 (note scales on the ordinates of Figs. 3A and B), indicating that a variable portion of plasma Aβ40 is N-terminally truncated. When the levels of Aβx-42 (Fig. 3C) and Aβ1–42 (Fig. 3D) were analyzed from the same aliquots of plasma, we found that the levels of Aβ1–42 species were generally lower than those of Aβ1–40 species, and that the levels of Aβx-42 were likewise lower than Aβx-40 (compare Figs 3A, B to C, D).
We calculated the average plasma Aβ levels for each of the four ELISAs in all AD and control subjects. Plasma levels of Aβ 40 and Aβx-42 did not differ significantly between AD and controls (Fig. 4A). However, the average plasma levels of both Aβ1–42 and oAβ were found to be significantly higher in AD subjects than controls (Fig. 4A). Furthermore, the relative levels of Aβ1–42 and oAβ were closely associated. For this, we chose a threshold of 10 pM for Aβ1–42 (i.e., readily detectable above baseline) and identified 7 subjects that carried >10 pM levels of Aβ1–42 (Fig. 4B). Next, we identified 7 subjects that carried higher levels of oAβ than the remaining subjects (Fig. 4C). We found that the subjects that carried >10 pM levels of Aβ1–42 (Fig. 4B) were the same subjects that carried high levels of oAβ (Fig. 4C), and the relative levels of Aβ1–42 and oAβ for each subject were tightly linked. Except for one patient who had the highest oAβ level (Fig. 4C, arrow) but had relative low Aβ1–42 (Fig. 4B, arrow), our oAβ ELISA clearly detected oAβ-reactive species whose levels were tightly associated with those of monomeric Aβ1–42.
To further validate the close association of monomeric Aβ42 with oAβ detected by our oAβ ELISA, we analyzed these Aβ species generated from cell lines expressing FAD-causing mutations in either APP or PS1. These cell lines have elevated Aβ42 monomer levels and secrete SDS-stable low-n Aβ oligomers into the media 25, 30, 31.
Similar to our AD and control cases that carry different levels of plasma Aβ, we have found variable levels of Aβ produced from multiple stable cell lines expressing wt or mutant (M146L or C410Y) PS1 and wt or mutant (V717F) APP. These lines were metabolically labeled with [35S]-Met, and the conditioned media were immunoprecipitated with antibody 21F12 (to Aβ ending at residue 42) or 1282 (a pan-Aβ polyclonal antibody) and analyzed by gel fluorography. Antibody 21F12 immunoprecipitated both monomeric Aβ42 and the p342 species (generated by sequential α- and γ-secretase cleavages of APP) from the conditioned media of all cell lines. In media from APPV717F and PS1M146L-2 cells, additional Aβ42-immunoreactive bands migrating at ~5 kDa, 8–10 kDa and 12–14 kDa (collectively designated SDS-stable low-n oligomers) were also detected (Fig. 5, upper panel). Light exposure of blots separating Aβ42 monomer from oAβ indicated that more Aβ42 was produced from cells expressing APPV717F or mutant PS1 (PSM146L-2) (Fig. 5, middle panel). The levels of oAβ were correlated to the levels of monomeric Aβ42, i.e. the cell lines secreting elevated Aβ42 also had higher levels of oligomers in their media. We further examined the levels of total Aβ precipitated by antiserum 1282 but did not observe an elevation in total Aβ signal corresponding to those of Aβ42 and oAβ. Thus, the amounts of total Aβ generated by cells expressing APPV717F or mutant PS1 (PSM146L-2) were comparable to those from the other cell lines (Fig. 5, bottom panel). Moreover, no oAβ species were detected in these immunoprecipitates of total Aβ, suggesting that the oAβ species made by the cells are principally composed of Aβ42. These results in well-defined cultured cell lines support our finding in human plasma that levels of oAβ are closely associated with those of monomeric Aβ42.
The close association of monomeric Aβ42 and oAβ levels suggests a dynamic conversion between these two Aβ species. To examine further the relationship of Aβ42 and oAβ in vivo, we obtained sequential blood samples from a symptomatic FAD patient carrying a mutant PS1 gene. Blood samples were drawn at ages 58 and 60. Aβ levels were quantified by separate ELISAs for oAβ and the four monomeric Aβ species (Table 1). For oAβ species, we observed a significant decrease, so that oAβ was no longer detectable at age 60. For monomeric Aβ, the plasma concentrations of Aβ1–40 and Aβx-40 were 48 and 84 pM, respectively, at age 58. The concentration of Aβ1–42 was 7 pM, and that of Aβx-42 was 36 pM. The plasma Aβ42/Aβtotal ratio was in the same range as those reported in patients carrying FAD-causing PS1 mutations 9. Plasma samples collected 22 months later had significantly lower Aβ concentrations: the level of Aβ1–40 measured 36 pM (25% reduction), and Aβ1–42 was now undetectable (Table 1). Aβx-40 and Aβx-42 declined to 48 pM (43% reduction) and 15 pM (58% reduction), respectively. Thus, while the levels of both Aβ species dropped over two years, Aβ42-ending species were reduced more dramatically than Aβ40-ending species. The complete loss of the Aβ1–42 and oAβ signals is consistent with our findings above that the levels of these two species are closely associated.
We also obtained sequential blood samples from a subset of our AD patients (n=12, including the FAD patient described above) and compared their Aβ levels over a 1–2 year span. All five Aβ species were again measured by ELISA, and the change in Aβ levels calculated. A similar number of subjects showed increased or decreased levels of Aβ1–40 over the course of 1–2 years (Fig. 6A), failing to show a clear temporal trend in Aβ1–40. Comparison of Aβx-40 levels revealed 3 out of 11 subjects with an increased Aβx-40 and the remaining 8 subjects had a reduced Aβx-40 (Fig. 6B). In the 4 AD subjects with detectable plasma Aβ1–42 levels (Fig. 6C), three subjects showed a dramatic reduction in Aβ1–42 after one year, whereas one subject displayed almost identical levels of Aβ1–42 at both times. The latter subject also maintained similar levels of oAβ after one year, whereas 3 AD subjects with detectable oAβ levels had a reduction in relative plasma oAβ (Fig. 6E). Thus, the drop of Aβ1–42 levels occurred in subjects who also showed a similar drop in oAβ levels. Strikingly, all but one subject showed decreases in Aβx-42 in the second plasma samples (Fig. 6D).
To investigate the association of monomeric Aβ42 and oAβ directly in human brains, we obtained postmortem brain tissues from 9 AD cases and 7 age-matched control cases distinct from the subjects examined in the plasma Aβ studies above. We performed sequential extractions of brain tissues to obtain “soluble” (Tris-buffered saline (TBS)) and “insoluble” (Guanidine (Gu) HCl) extracts and measured their Aβ contents by ELISA.
The soluble pool of brain Aβ extracted by TBS had relatively low levels of Aβ1–40 (Fig. 7A); an average of 1.1 pmol/g of Aβ1–40 was detected in the AD cases vs. 0.7 pmol/g in the controls (not statistically significant). The average level of TBS-soluble Aβ1–42 was significantly higher in AD than control cases (Fig. 7B). Likewise, the relative levels of TBS-soluble oAβ were significantly higher in AD cases, as most control cases had almost undetectable levels of soluble oAβ (Fig. 7C).
After the extraction of soluble Aβ by TBS, the resultant pellets were further extracted in GuHCl to obtain the insoluble pools of Aβ. Overall, insoluble Aβ1–42 levels (Fig. 7E) were several hundred-fold higher than soluble Aβ1–42 levels (Fig. 7B), and they were almost ten fold higher than insoluble Aβ1–40 levels (Fig. 7D). This is consistent with numerous previous reports that amyloid deposits in AD brain are mainly composed of Aβ42. The levels of both insoluble Aβ1–40 and Aβ1–42 were significantly higher in brains from AD cases than those from control cases. Furthermore, a substantial and significant increase in the relative levels of insoluble oAβ was observed in AD brains (Fig. 7F). Importantly, brain levels of insoluble Aβ1–42 were closely associated with the levels of oAβ in both AD patients and control subjects (correlation coefficient, AD=0.88; ND=0.98). For example, one AD case (AD4) showed relatively low levels of Aβ1–42, and accordingly, its oAβ level was low. Another control case (ND3) showed a relatively high level of Aβ1–42, corresponding to a high level of oAβ (Fig. 7F). On average, significantly higher levels of all three insoluble Aβ species (Aβ1–40, Aβ1–42 and oAβ) were found in AD brain tissues, compared to control cases (Fig. 7 D–F).
Given the burgeoning evidence that small oligomeric assemblies of Aβ may be principally responsible for neuronal dysfunction in Alzheimer’s disease, it is important to be able to specifically detect and quantify these species in a range of biological samples. Conventional approaches to measuring the levels of oAβ have focused on semi-quantitative analysis by Western blotting or silver staining of SDS-PAGE gels. Recent advances in nanotechnology have enabled the measurement of sub-picomolar concentrations of oliogmers (also designated amyloid-β derived diffusible ligands (ADDLs)) in human cerebrospinal fluid (CSF) 32. For ELISA-based approaches, several groups have generated antibodies believed to be specific for aggregated Aβ. Among them, monoclonal antibody NAB61 specifically recognizes a complex conformational Aβ1–11 epitope on peroxynitrite- or UV light-treated synthetic Aβ, with much less reactivity for monomeric Aβ. Using NAB61 as a capture antibody and another Aβ antibody (BA27) as a reporter, this sandwich ELISA can differentiate UV crosslinked from monomeric synthetic Aβ with a relative sensitivity of 5:1 33. Another antibody (158) recognizes 50–200 kDa protofibrillar aggregates of synthetic Aβ but not low molecular weight oligomers and monomers of synthetic Aβ 34.
In the current study, we oxidized synthetic Aβ1–40Ser26Cys peptide to generate disulfide crossed-linked dimers in order to test our new oAβ ELISA. The Aβ1–40Ser26Cys dimer preparation was treated with GuHCl and therefore was free of any higher molecular weight aggregates (Fig. 1B). Subsequent separation of the stable dimers from un-crosslinked monomers was achieved by SEC. Detection of the synthetic dimers only in the absence of β-mercaptoethanol clearly demonstrated the specificity of our oAβ ELISA, with far less ability to detect monomers.
Using this new oAβ ELISA and previously established monomeric Aβ ELISAs, we obtained several lines of evidence for a close association between the levels of monomeric Aβ42 and oAβ species in human plasma and brain tissue. First, the close association of Aβ1–42 and oAβ was observed in a subset of AD patients who had readily detectable levels of plasma Aβ1–42 (Fig. 4). The average levels of these two Aβ species were significantly higher in the AD than the control subjects. Indeed, only one of 10 control subjects even showed such readily detectable levels of Aβ1–42. Second, our studies of AD and control brain tissue showed the same pattern, i.e., levels of insoluble Aβ1–42 associated closely with the relative levels of insoluble oAβ across individual cases (Fig. 7). Third, among all AD and control cases that we examined, higher levels of oAβ were not linked to lower levels of monomeric Aβ42, suggesting that the conversion of monomeric Aβ into oAβ per se is not the major contributor to the observed reduction of monomeric Aβ42. Overall, our oAβ-specific ELISA allowed us to establish a close quantitative relationship between the levels of Aβ1–42 and oAβ in human plasma and brain tissues.
This association was also observed in cultured cells expressing human APP. We found that the appearance of soluble oAβ in the medium occurred exclusively in those clonal cell lines with significantly increased Aβ42 monomer production (Fig. 5). FAD mutations in PS1 or APP lead to enhanced Aβ42 generation, and the occurrence of oAβ is attributable to enhanced γ-secretase cleavage of APP at the 42nd residue of Aβ,which is facilitated by AD-causing mutations in either the substrate (APP) or the protease (PS1). Thus, levels of Aβ42 are closely associated with the formation of oAβ.
Our longitudinal comparisons of individual patients with AD provide new insight into the dynamic changes in plasma Aβ levels without the problem of inter-subject variation. Using monomeric Aβ and oAβ ELISAs, we have provided two snap shots of Aβ levels within a relatively short period. Whereas we saw no clear directional change of Aβ1–40 levels, the remaining four Aβ species all showed a reduction in levels over the course of one to two years. About three quarters of cases showed a reduction in Aβx-40. Among the cases with detectable plasma levels of Aβ1–42 and oAβ, all but one case showed a decrease in Aβ levels. In the case of Aβx-42, 9 patients showed a reduction over the 1–2 year period, while one case showed an increase. Importantly, the individuals with decreasing Aβ1–42 monomer levels were the same subjects who showed decreases in oAβ.
While our oAβ ELISA provides an accurate method to measure such species in human blood, our results do not yet validate any one Aβ species as a biomarker for AD. Currently, the CSF tau/Aβ42 ratio has been the best predictor of the development of AD-type cognitive decline in still nondemented subjects 35, 36. Changes in the levels of tau and Aβ42 in CSF may reflect dysfunction in the cerebrum that can be measured by the EEG rhythm 37, and low Aβ42 levels in CSF correlate well with positive PIB uptake by PET scanning 35. Precise measurement of Aβ in plasma and CSF and subsequent correlation with levels in postmortem brain tissue should yield a clearer picture of Aβ metabolism in vivo. Mathematic modeling of the equilibrium between monomeric and oligomeric Aβ would also help elucidate the catabolic turnover of Aβ in the peripheral and central nervous systems 38. Considering a wide range of factors that could contribute to variations in plasma Aβ levels, it is currently difficult to obtain a clear separation of AD from control subjects simply by measuring the levels of plasma Aβ species 39. However, our findings suggest that measuring plasma Aβ and oAβ over 1–2 years or more can reveal a significant reduction in plasma Aβ, especially Aβ42, and this finding raises the possibility of a direct relationship of plasma Aβ to brain amyloid formation.
We thank Drs. Peter Seubert and Dale Schenk for Aβ antibodies, 3D6, 21F12, 266 and 2G3. We also thank Dr. Ganesh Shankar for helpful discussion. This work was supported by the NIH AG015379 (WX and DJS).