A Diagnostic Test for Large Aβ Oligomers?

Researchers in Japan have developed an assay for amyloid-β (Aβ) oligomers that they suggest might be a useful diagnostic test for Alzheimer disease. The assay seems specific for very high-molecular-weight oligomers. The researchers, led by Takahiko Tokuda, Kyoto Prefectural University of Medicine, claim that their test is not only more sensitive than current methods of detecting monomeric Aβ42, but that their data support a role for large Aβ oligomers in AD pathology. First author Hiroaki Fukumoto and colleagues report that levels of these oligomers are higher in cerebrospinal fluid (CSF) from AD patients compared to CSF from control subjects. “If these findings can be replicated, the present paper represents a breakthrough in the field of AD biomarkers,” suggest Lars Lannfelt and Frida Ekholm Pettersson, Uppsala University, Sweden (see full comment below). The findings appeared in the March 25 FASEB Journal online.

This is not the first test for Aβ oligomers. A similar assay developed by Weiming Xia and colleagues at Harvard Medical School also detects oligomeric species and is similar in design—a single-antibody sandwich ELISA that uses one antibody for both capture and detection. That assay detects primarily low-molecular-weight oligomers (see ARF related news story). It is not clear why the two tests have different oligomeric specificities, though the antibodies are different in each case. Xia and colleagues chose monoclonal antibodies 82EI and 3D6, raised to Aβ amino acids 1-16 and Aβ 1-5, respectively, for their tests, whereas Fukumoto and colleagues used BAN50, also raised against Aβ1-16.

Other novel approaches have been taken to assay oligomers. Researchers led by Bill Klein at Northwestern University used antibodies laced with DNA to amplify, à la the polymerase chain reaction, signals from minuscule amounts of oligomers in biological fluids (see ARF related news story), and an antibody-based biosensor method to detect oligomers (see ARF related news story). Though both methods seem to detect Aβ oligomers in AD patients but not normal controls, they have not yet been approved as diagnostic tests. Aileen Funke and Dieter Willbold at the Forschungszentrum Jülich, near Düsseldorf, Germany, have capitalized on the confocal microscope to detect fluorescing antibodies bound to even a single Aβ oligomer (see ARF related news story).

The Japanese researchers tested their ELISA on Aβ oligomers separated by size-exclusion chromatography. They found that the test failed to detect Aβ monomers, dimers, or small oligomers (up to hexamers), even though these species comprised 99 percent of the total amount of Aβ. The test did detect Aβ oligomers that were 40-200 kDa, with most of them being 45-90 kDa (10-20mers). The assay could detect these species at concentrations down to 1 picomolar.

Turning to biological samples, the researchers used the assay to measure oligomers in human cerebrospinal fluid. They detected more oligomers in samples from people diagnosed with AD (18 patients) or mild cognitive impairment (nine patients), compared to a group of 25 controls, which comprised normal people and people with other neurological conditions, such as peripheral neuropathy. The elevation of high-molecular-weight (HMW) oligomers is in contrast to the well-documented drop in CSF monomeric Aβ42 normally seen in AD. In addition, levels of HMW Aβ oligomers inversely correlated with MMSE scores.

Compared to Aβ42 monomeric analysis, the sensitivity/specificity profile of this ELISA is better, according to the authors. More importantly, a test for HMW oligomers might give a better readout of disease progression and response to treatment. As Lannfelt and Ekholm Pettersson point out, CSF Aβ42 does not correlate with disease severity. That HMW oligomers inversely correlate with MMSE scores “holds promise for foreseeing any bioefficacy of drugs in clinical trials,” suggested Weiming Xia (see full comment below), who is now at Satori Pharmaceuticals Inc., Cambridge, Massachusetts. It might also help researchers figure out whether the known drop of CSF Aβ42 in AD occurs because the peptide is getting trapped in the brain parenchyma, or because it is aggregating in the CSF.

Whether this ELISA truly has the makings of a diagnostic needs further testing. “The biggest challenge in developing this kind of assay is validating that it is actually recognizing just oligomers and not monomers, too,” said Anne Fagan, Washington University, St. Louis. It is also not clear if the test is specific for Aβ oligomers. Some antibodies, such as the X11 antibody raised by Charlie Glabe’s lab at the University of California, Irvine, recognize common conformations adopted by different oligomeric proteins (see ARF related news story). Other researchers pointed out that there is considerable overlap between levels of HMW oligomers found in control and AD/MCI CSF samples, which might limit the clinical usefulness of such a test.—Tom Fagan

Comments

  1. For the first time an ELISA has been developed that is sensitive enough for detecting high-molecular-weight oligomers in human CSF, which might be an important biomarker for Alzheimer disease (AD).

    Soluble forms of Aβ correlate better with disease severity than insoluble fibrils (McLean et al., 1999; Näslund et al., 2000). Among the prefibrillar intermediate Aβ species, several oligomeric forms of various molecular sizes have been identified. Some of these have been shown to elicit adverse biological effects both in vitro and in vivo (Walsh et al., 2002), suggesting that they play a central role in the pathogenesis. While an oligomeric 12-mer, Aβ*56, has been claimed to be especially toxic (Lesné et al 2006), other researchers have put the dimer in focus (Shankar et al., 2008). We have previously identified a pathogenic mutation located within the Aβ domain, the Arctic mutation causing early onset Alzheimer disease. The mutation enhances the formation of protofibrils, suggesting that this Aβ species is pathogenic (Nilsberth et al., 2001).

    As part of routine diagnostic procedures, cerebrospinal fluid (CSF) is today often analyzed for levels of Aβ42, tau and phospho-tau, where decreased Aβ42 and increased tau and/or phospho-tau in CSF are indicative of AD. These measures are good predictors for future conversion to AD among subjects with mild cognitive impairment (MCI) (Hansson et al., 2006). However, these biomarkers are neither suitable to follow disease progression nor to monitor drug intervention due to the lack of correlation with disease severity. Thus, we need novel biomarkers that better reflect the continuous disease process and correlate with disease severity.

    Preliminary reports indicate that the concentration of Aβ oligomers in human CSF is very low and close to the detection limit of assays, even using new types of methods including PCR-based amplification steps (Georganopoulou et al., 2005). The presence of aggregated species of Aβ in CSF was demonstrated by us with an indirect method. We calculated an oligomer ratio and could show that this ratio was higher in AD and MCI than in healthy controls (Englund et al., 2009).

    Indirect evidence suggests that the Aβ protofibril could be such a biomarker. In a recent study we demonstrated that Aβ protofibril levels in tgAPP-ArcSwe mice were inversely correlated with spatial learning. In contrast, total Aβ levels of the same mouse brains did not correlate to decreased cognition (Lord et al., 2009). Thus, these data suggest that Aβ protofibrils could be a marker for disease progression.

    Scientists at Takeda Pharmaceutical Company and academic collaborators have now been able to develop a sensitive ELISA for measuring high-molecular-weight Aβ oligomers, i.e., protofibrils, in human CSF. Using the BAN50 antibody as capture and Fab' fragment of the same antibody as detection, their ELISA specifically detects oligomers in the range of 40-200 kDa. In order to get signals from human CSF samples, sensitive chemiluminescent substrate was used instead of the more commonly used colorimetric TMB substrate. However, the standard curve was difficult to interpret for us, and it is surprising that the content of large oligomers in the oligomer mixture is as low as When we prepare large size oligomers, i.e., protofibrils, we usually get 80-90 percent protofibrils and 10-20 percent monomers and low-molecular-weight species. In order to be able to reliably quantify the oligomers present in CSF, the concentration of the high molecular SEC fraction of the synthetic oligomer preparation should be determined and then used as a reference in the ELISA.

    Still, significantly higher levels of protofibrils were found in CSF from AD and MCI as compared to healthy controls, and even more intriguing, there was an inverse correlation between MMSE and the signal in the ELISA; i.e., levels of protofibrils were higher in more demented cases. If these findings can be replicated, the present paper represents a breakthrough in the field of AD biomarkers.

    References:

    Englund H, Degerman Gunnarsson M, Brundin RM, Hedlund M, Kilander L, Lannfelt L, Pettersson FE. Oligomerization partially explains the lowering of Abeta42 in Alzheimer's disease cerebrospinal fluid. Neurodegener Dis. 2009;6(4):139-47. PubMed.

    Georganopoulou DG, Chang L, Nam JM, Thaxton CS, Mufson EJ, Klein WL, Mirkin CA. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease. Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2273-6. PubMed.

    Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L. Association between CSF biomarkers and incipient Alzheimer's disease in patients with mild cognitive impairment: a follow-up study. Lancet Neurol. 2006 Mar;5(3):228-34. PubMed.

    Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. PubMed.

    Lord A, Englund H, Söderberg L, Tucker S, Clausen F, Hillered L, Gordon M, Morgan D, Lannfelt L, Pettersson FE, Nilsson LN. Amyloid-beta protofibril levels correlate with spatial learning in Arctic Alzheimer's disease transgenic mice. FEBS J. 2009 Feb;276(4):995-1006. PubMed.

    McLean CA, Cherny RA, Fraser FW, Fuller SJ, Smith MJ, Beyreuther K, Bush AI, Masters CL. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol. 1999 Dec;46(6):860-6. PubMed.

    Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Näslund J, Lannfelt L. The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation. Nat Neurosci. 2001 Sep;4(9):887-93. PubMed.

    Näslund J, Haroutunian V, Mohs R, Davis KL, Davies P, Greengard P, Buxbaum JD. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA. 2000 Mar 22-29;283(12):1571-7. PubMed.

    Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008 Aug;14(8):837-42. PubMed.

    Walsh DM, Klyubin I, Fadeeva JV, Rowan MJ, Selkoe DJ. Amyloid-beta oligomers: their production, toxicity and therapeutic inhibition. Biochem Soc Trans. 2002 Aug;30(4):552-7. PubMed.

    View all comments by Lars Lannfelt

  2. An ELISA-based biochemical measurement of oligomeric Aβ (oAβ) species from human fluids such as CSF has been enthusiastically anticipated in the field, in light of extensive in vitro and in vivo studies of neurotoxic oAβ and its direct and indirect effects on synaptic function. Fukumoto et al. have used an N-terminal specific human Aβ antibody, BAN50, as both a capture and a reporter antibody to measure high-molecular-weight oAβ, a similar approach to a previous study by us that used 82E1 and 3D6 (against N-terminus of human Aβ) to measure low-molecular-weight oAβs by ELISA.

    The BAN50-BAN50 ELISA-based measurement of oAβ levels in CSF provides a new readout to separate the control from AD subjects/MCI converters (MCI-C). This is a nice addition to the widely reported approach that quantifies CSF Aβ42, tau, and their ratios. Since a reduction of monomeric Aβ42 in CSF has been associated with AD subjects, it will be critical to understand whether it is due to a deposition of Aβ42 in brains or a formation of high-molecular-weight oAβ (with a low or undetectable molar concentration in CSF measured by monomeric Aβ42 ELISA), or both. Snider and Holtzman have reported a rapid dementia progression in subjects with lower baseline CSF monomeric Aβ42 levels, and the current study reveals that the levels of oAβ in CSF have a significant negative correlation with the MMSE scores of the AD/MCI-C patients. As the report states, this could be a surrogate marker to reflect disease severity.

    A battery of CSF-based biomarkers would be an invaluable tool for selecting and monitoring subjects enrolled in clinical trials that target amyloid. While measuring CSF Aβ42 and tau/phosphorylated tau helps identify antecedent AD/MCI subjects, the sensitivity and specificity of these assays are neither sufficient for monitoring disease progression nor for predicting drug outcome afterwards.

    On the other hand, the levels of CSF oAβ, quantified by BAN50-BAN50 ELISA seem to correlate negatively with the MMSE scores, which holds promise for foreseeing any bioefficacy of drugs in clinical trials. This goal could be achieved once the assay is validated in future studies with a larger number of subjects; e.g., CSF samples collected for the ADNI study could be readily measured by BAN50-BAN50 ELISA and oAβ levels compared to monomeric Aβ42 levels from the same subjects. Longitudinal studies on MCI subjects enrolled in the ADNI study will provide valuable information on potential correlation between the levels of CSF oAβ and disease progression. These MCI subjects from the ADNI study should be more representative of the general population, compared to those MCI converters included in the current report; in this report, two out of seven MCI converters carry the ApoE2 allele, which represents unusually high occurrence.

    Following the temporal sequence of Aβ conversion from monomeric to oligomeric forms, ELISA-based measurement of monomeric Aβ42 (and tau) in CSF will provide a snapshot of pathogenic Aβ42 in CNS even before the disease onset, and quantification of CSF Aβ oligomers may illustrate the ongoing disease progression. Both methods are not sufficient but necessary to capture amyloidogenesis at the molecular level in brains of AD/MCI patients, especially to follow/monitor patients being treated with amyloid directed/targeting therapies.

    References:

    Xia W, Yang T, Shankar G, Smith IM, Shen Y, Walsh DM, Selkoe DJ. A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease. Arch Neurol. 2009 Feb;66(2):190-9. PubMed.

    Snider BJ, Fagan AM, Roe C, Shah AR, Grant EA, Xiong C, Morris JC, Holtzman DM. Cerebrospinal fluid biomarkers and rate of cognitive decline in very mild dementia of the Alzheimer type. Arch Neurol. 2009 May;66(5):638-45. PubMed.

    View all comments by Weiming Xia

  3. Several lines of evidence suggest that Aβ plays a central role in the pathogenesis of Alzheimer disease (AD). Not only Aβ fibrils, but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. Therefore, Aβ oligomers and aggregates might be an interesting disease marker for AD and a method for sensitive and specific detection of such oligomers in body fluids is highly desired.

    Fukumoto et al. developed a novel ELISA specific for high molecular weight (HMW) Aβ oligomers (40-200 kDa) and detected significantly higher amounts of HMW Aβ oligomers in CSF samples from AD and MCI patients as compared to age matched controls. Additionally, they showed a negative correlation with Mini-Mental state examination scores in the AD/MCI group.

    These results further strengthen the theory that Aβ oligomers might be a valuable marker for AD diagnosis and for therapy monitoring as well. Recently, several authors published data gained by different methods showing increased Aβ oligomer/aggregate levels in CSF samples of AD patients vs. controls (1-6). Other authors even developed methods for the detection of Aβ oligomers in blood, but actually no trend can be predicted concerning its diagnostic power (7,8).

    In future, a lot of samples will have to be measured with different assay systems to obtain reliable results. Parallel work is needed to elucidate the nature of the Aβ oligomers relevant to the disease as well as to improve the technical robustness of the applied quantification methods. In addition, the question needs to be addressed, whether methods based on antibodies specific for a certain kind of Aβ oligomer or methods that are able to quantify all kinds of Aβ oligomers (1,4-6) are suitable to deliver the most valuable biomarker readout. Possibly, a combination of these methods or a completely new so far unknown approach will make it at the end into clinical use. In any case, all these approaches benefit from the findings reported by Fukumoto et al. confirming that any effort towards oligomer based diagnostics in neurodegenerative diseases is worth it.

    References:

    Birkmann E, Henke F, Weinmann N, Dumpitak C, Groschup M, Funke A, Willbold D, Riesner D. Counting of single prion particles bound to a capture-antibody surface (surface-FIDA). Vet Microbiol. 2007 Aug 31;123(4):294-304. PubMed.

    Georganopoulou DG, Chang L, Nam JM, Thaxton CS, Mufson EJ, Klein WL, Mirkin CA. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease. Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2273-6. PubMed.

    Haes AJ, Chang L, Klein WL, Van Duyne RP. Detection of a biomarker for Alzheimer's disease from synthetic and clinical samples using a nanoscale optical biosensor. J Am Chem Soc. 2005 Feb 23;127(7):2264-71. PubMed.

    Pitschke M, Prior R, Haupt M, Riesner D. Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy. Nat Med. 1998 Jul;4(7):832-4. PubMed.

    Funke SA, Birkmann E, Henke F, Görtz P, Lange-Asschenfeldt C, Riesner D, Willbold D. Single particle detection of Abeta aggregates associated with Alzheimer's disease. Biochem Biophys Res Commun. 2007 Dec 28;364(4):902-7. PubMed.

    Funke SA, Birkmann E, Willbold D. Detection of Amyloid-beta aggregates in body fluids: a suitable method for early diagnosis of Alzheimer's disease?. Curr Alzheimer Res. 2009 Jun;6(3):285-9. PubMed.

    Santos AN, Simm A, Holthoff V, Boehm G. A method for the detection of amyloid-beta1-40, amyloid-beta1-42 and amyloid-beta oligomers in blood using magnetic beads in combination with Flow cytometry and its application in the diagnostics of Alzheimer's disease. J Alzheimers Dis. 2008 Jun;14(2):127-31. PubMed.

    Xia W, Yang T, Shankar G, Smith IM, Shen Y, Walsh DM, Selkoe DJ. A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease. Arch Neurol. 2009 Feb;66(2):190-9. PubMed.

    View all comments by Susanne Aileen Funke

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References

News Citations

  1. Research Brief: New Methods for Aβ Detection, Production
  2. "Bio-Barcode" Amplifies Aβ Oligomer Signal, Proffers Candidate Diagnostic Test
  3. Nanosensor Sizes up Amyloid-β Oligomers in CSF
  4. Vienna: New Tack to See Amyloid Oligomers in Body Fluids
  5. Amyloid Oligomer Antibody—One Size Fits All?

Further Reading

Primary Papers

  1. Fukumoto H, Tokuda T, Kasai T, Ishigami N, Hidaka H, Kondo M, Allsop D, Nakagawa M. High-molecular-weight beta-amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients. FASEB J. 2010 Aug;24(8):2716-26. PubMed.

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