The topic is important, and the results are intriguing.

The issue of Aβ oligomerization is a complex one, and several methodological questions could be raised:

1. The immunoprecipitation with 6E10 and Western blotting with 6E10 may not distinguish between Aβ and soluble APP fragments. It is not clear from the paper whether the detected species are Aβ rather than an APP fragment. Of note, the levels of soluble APP in the CSF are about 100-fold higher than Aβ (see Nitsch et al., 1995, Table 2), so even a minor APP fragment would give a lot of signal in the 6E10-based assay.

2. It will be important to control for the possibility that the immunoprecipitation and Western blotting assay procedures themselves induce artifactual aggregation of Aβ (see Esparza et al., 2013, Fig. 2 L). Monomeric Aβ can aggregate at high local concentrations, such as those that occur after immunoprecipitation.

3. Additional controls of interest regarding the assay include test-retest reproducibility, dilutional linearity, and spike-recovery linearity.

4. A future direction could...  Read more

The topic is important, and the results are intriguing.

The issue of Aβ oligomerization is a complex one, and several methodological questions could be raised:

1. The immunoprecipitation with 6E10 and Western blotting with 6E10 may not distinguish between Aβ and soluble APP fragments. It is not clear from the paper whether the detected species are Aβ rather than an APP fragment. Of note, the levels of soluble APP in the CSF are about 100-fold higher than Aβ (see Nitsch et al., 1995, Table 2), so even a minor APP fragment would give a lot of signal in the 6E10-based assay.

2. It will be important to control for the possibility that the immunoprecipitation and Western blotting assay procedures themselves induce artifactual aggregation of Aβ (see Esparza et al., 2013, Fig. 2 L). Monomeric Aβ can aggregate at high local concentrations, such as those that occur after immunoprecipitation.

3. Additional controls of interest regarding the assay include test-retest reproducibility, dilutional linearity, and spike-recovery linearity.

4. A future direction could involve quantification of the amount of protein corresponding to the signals detected on Western blot relative to known biochemical standards.

References:
Nitsch RM, Rebeck GW, Deng M, Richardson UI, Tennis M, Schenk DB, Vigo-Pelfrey C, Lieberburg I, Wurtman RJ, Hyman BT. Cerebrospinal fluid levels of amyloid β-protein in Alzheimer's disease: inverse correlation with severity of dementia and effect of apolipoprotein E genotype. Ann Neurol. 1995 Apr;37(4):512-8. Abstract

Esparza TJ, Zhao H, Cirrito JR, Cairns NJ, Bateman RJ, Holtzman DM, Brody DL. Amyloid-β oligomerization in Alzheimer dementia versus high-pathology controls. Ann Neurol. 2013 Jan;73(1):104-19. Abstract

View all comments by David Brody

The measurements of CSF oligomeric Aβ were done by immunoprecipitation/Western blot using as little as 240 μL per determination (ran as triplicate, then averaged). Using such a low volume suggests it may be possible to integrate similar measurements in longitudinal studies. It is disappointing that CSF levels of Aβ dimers could not be determined due to the experimental design (the acrylamide content in the gel cannot resolve small species). Concentrations of Aβ1-42 and tau/pt181-tau were determined by ELISA, as it is traditionally done for biomarker studies.

Both oligomeric Aβ species (Aβ*56 and Aβ trimers) detected in the CSF correlated with tau/ptau concentrations in aged, unimpaired subjects, while presumably monomeric Aβ1-42 did not. By extrapolation, it could indicate that the elevation of trimer-based Aβ oligomers seen in aging and AD is linked to abnormal tau changes. This interpretation is consistent with the notion that Aβ*56 and Aβ trimers may initiate the disease process during the latent phase of AD (i.e., preclinical AD).

In impaired individuals (including...  Read more

The measurements of CSF oligomeric Aβ were done by immunoprecipitation/Western blot using as little as 240 μL per determination (ran as triplicate, then averaged). Using such a low volume suggests it may be possible to integrate similar measurements in longitudinal studies. It is disappointing that CSF levels of Aβ dimers could not be determined due to the experimental design (the acrylamide content in the gel cannot resolve small species). Concentrations of Aβ1-42 and tau/pt181-tau were determined by ELISA, as it is traditionally done for biomarker studies.

Both oligomeric Aβ species (Aβ*56 and Aβ trimers) detected in the CSF correlated with tau/ptau concentrations in aged, unimpaired subjects, while presumably monomeric Aβ1-42 did not. By extrapolation, it could indicate that the elevation of trimer-based Aβ oligomers seen in aging and AD is linked to abnormal tau changes. This interpretation is consistent with the notion that Aβ*56 and Aβ trimers may initiate the disease process during the latent phase of AD (i.e., preclinical AD).

In impaired individuals (including MCI and AD groups), CSF Aβ trimers and Aβ1-42 correlated to tau/ptau concentrations (in opposite fashion). These observations could indicate that: 1) soluble Aβ trimers might still drive abnormal tau changes in the AD brain, but their effects are reduced (based on comparing correlation coefficients); 2) Aβ*56 might not be as "active" a toxin in AD as in preclinical AD; and 3) tau changes become less dependent on oligomeric Aβ as disease progresses (none of these scenarios are mutually exclusive). Whether these scenarios would apply to other oligomeric forms of Aβ remains to be examined.

Overall, the data presented are consistent with the hypothesis that Aβ*56 and Aβ trimers (and presumably trimer-based oligomers) play important roles in the prodromal stages of the disease. These findings are also in agreement with alterations in CSF Aβ levels occurring early in asymptomatic individuals.

View all comments by Sylvain Lesne

Development of valid and quantitative assays for oligomeric Aβ species is considered by many to be the Holy Grail in the AD fluid biomarker field. Such assays are technologically challenging for many reasons. Several groups have reported such assays, but establishing an assay’s validity has been problematic, and none has stood the test of time. Dr. Ashe’s group has been interested in oligomeric Aβ species for several years, reporting in 2006 the presence of the Aβ*56 form in Tg2576 mice and its memory-disrupting ability when injected into rats. In this current paper, they report the presence of Aβ trimers as well as the Aβ*56 species in human CSF samples using a combined immunoprecipitation and immunoblotting procedure. Although the assay appears not to be quantitative (instead, semi-quantitative), the reported percent coefficient of variation among triplicates was good (  Read more

Development of valid and quantitative assays for oligomeric Aβ species is considered by many to be the Holy Grail in the AD fluid biomarker field. Such assays are technologically challenging for many reasons. Several groups have reported such assays, but establishing an assay’s validity has been problematic, and none has stood the test of time. Dr. Ashe’s group has been interested in oligomeric Aβ species for several years, reporting in 2006 the presence of the Aβ*56 form in Tg2576 mice and its memory-disrupting ability when injected into rats. In this current paper, they report the presence of Aβ trimers as well as the Aβ*56 species in human CSF samples using a combined immunoprecipitation and immunoblotting procedure. Although the assay appears not to be quantitative (instead, semi-quantitative), the reported percent coefficient of variation among triplicates was good (<15 percent), especially given the fact that it is an immunoblotting method. I would have liked to be able to view an example of a representative blot (to confirm that the assay does not recognize other cleaved APP fragments), and know the details of their quality control (QC) procedures (e.g., how they control for potential blot-to-blot differences)—important things to evaluate in biomarker studies. Hopefully, there will be a detailed, method-based paper forthcoming from this group.

Assuming the assay is indeed valid for detecting these specific oligomeric species, the data are very interesting and support several current hypotheses regarding Aβ metabolism in AD. The observation that these species were elevated in cognitively normal individuals at risk for developing AD dementia (defined as those with a high tau/Aβ42 ratio) suggests that they are involved in the very early (preclinical) stages of the disease. It would have been nice to see the relationship between monomeric Aβ42 (the proposed analyte quantified in the INNOTEST ELISA assays) and the various oligomeric species, as well as the distribution of levels of these species in the various clinical groups. It would also be interesting to know whether levels were elevated in MCI (or control) individuals who later progressed to AD, but were not elevated in MCI (or control) individuals who remained stable (hypothesized not to have underlying AD). The analysis of individuals as a function of the tau/Aβ42 ratio at baseline suggests that this would be the case, and I expect future studies that include longitudinal clinical follow-up in this cohort will be able to shed light on this issue.

The relationship between levels of Aβ trimers/Aβ*56 and tau/ptau in cognitively normal individuals and its attenuation in symptomatic cases is arguably the most interesting finding of this study. Such a relationship is not observed between levels of monomeric Aβ42 and tau, suggesting a potential unique role of oligomeric species in the pathologic cascade at the earliest stages of the disease. While interesting in its own right to those striving to understand the normal evolution of AD pathobiology, this finding also has potentially important implications for the design and evaluation of therapies targeting Aβ and/or amyloid. Hopefully, the assay can be developed into a truly quantitative platform with higher throughput that will permit the evaluation of large numbers of samples from individuals who have been well characterized clinically over time and with multiple biomarker assessments. Then, perhaps, we can add a line to the left of the Aβ42/amyloid line in the hypothesized biomarker trajectory schematic.

View all comments by Anne Fagan

It is our hope that the publication of our JAMA Neurology paper and the accompanying Alzforum story will motivate other laboratories to study Aβ*56. We certainly recognize that the existence of this species as an authentic oligomer that occurs in vivo is controversial. Perhaps, though, the following considerations will encourage skeptics and believers alike to take a closer look at Aβ*56.

The existence of specific Aβ oligomers as real entities, rather than artifacts, has been questioned because of the possibility that they are artificially generated through exposure to detergents, such as SDS. Several lines of evidence argue against this possibility.

1. When proteins in undiluted CSF are first separated by size-exclusion chromatography (SEC) and then analyzed by Western blot, Aβ*56 and Aβ monomers are seen in separate fractions eluted from the SEC column. If Aβ*56 was artifactually generated from monomers during the process of gel electrophoresis, one would expect to see both of these species in the same fractions from the SEC column.

2. Using the same extraction...  Read more

It is our hope that the publication of our JAMA Neurology paper and the accompanying Alzforum story will motivate other laboratories to study Aβ*56. We certainly recognize that the existence of this species as an authentic oligomer that occurs in vivo is controversial. Perhaps, though, the following considerations will encourage skeptics and believers alike to take a closer look at Aβ*56.

The existence of specific Aβ oligomers as real entities, rather than artifacts, has been questioned because of the possibility that they are artificially generated through exposure to detergents, such as SDS. Several lines of evidence argue against this possibility.

1. When proteins in undiluted CSF are first separated by size-exclusion chromatography (SEC) and then analyzed by Western blot, Aβ*56 and Aβ monomers are seen in separate fractions eluted from the SEC column. If Aβ*56 was artifactually generated from monomers during the process of gel electrophoresis, one would expect to see both of these species in the same fractions from the SEC column.

2. Using the same extraction and detection protocols to measure the oligomers, we have observed that different mouse lines overexpressing APP consistently display line-specific sets of oligomeric Aβ species.

3. The Aβ oligomers that are particular to a given mouse line accumulate with age in an orderly fashion (Lesné et al., 2006; Larson and Lesné, 2012).

4. Aβ dimers and Aβ*56 in micro-dissected tissue are differentially distributed (Liu et al., 2011a).

5. Finally, perhaps the most compelling evidence in favor of the existence of Aβ*56 is the strong correlation between levels of this oligomer and markers of compromised neuronal function in both brain and CSF, and in humans as well as APP transgenic mice.

Levels of brain Aβ*56 correlate with cognitive impairment in multiple lines of transgenic mice (Lesné et al., 2006; Cheng et al., 2007); a transient (~three-week) dip in the levels of this oligomer during the period of the most rapid plaque deposition in Tg2576 mice is accompanied by a temporary recovery of cognitive function (Lesné et al., 2008) . In human subjects who were cognitively normal at the time of death, Aβ*56, but not other Aβ oligomers, correlated negatively with the postsynaptic markers drebrin and Fyn kinase, and positively with pathological conformers of tau (Lesné et al., in press). In the CSF of clinically unimpaired subjects, Aβ*56 correlated strongly with levels of total tau and tau phosphorylated at threonine 181—putative markers of neuronal injury (Handoko et al., 2013). It seems to us very unlikely that an entity generated artificially during extraction or blotting would consistently correlate with other indicators of an unhealthy brain.

We would expect an Aβ species that initiates the amyloid cascade to stimulate downstream processes (network dysfunction, generation of toxic tau species, neuroinflammation, aberrant cell-cycle re-entry?) that eventually lead to neuron death. We find it difficult to reconcile the slow progression of AD with acute Aβ-induced toxicity in cell culture models. We would argue that such acute toxicity is an experimental artifact, caused by biologically irrelevant (perhaps artificially generated?) species of Aβ, unrealistically high concentrations of Aβ, spatial or temporal patterns of exposure to Aβ that are not reflective of those that occur in situ, or elevated susceptibility to Aβ toxicity. Consistent with what we would expect of an Aβ species that triggers the amyloid cascade, Aβ*56 does not kill cells—APP transgenic mice that generate Aβ*56 do not exhibit widespread neurodegeneration, and exogenous administration of Aβ*56 to healthy host animals results in reversible memory dysfunction. These observations suggest that Aβ*56 interferes with synaptic function or plasticity; elucidating the mechanisms of action of Aβ*56 is a major focus of our laboratory.

We fully acknowledge that detecting Aβ*56 on blots can be difficult. Many other proteins run at ~55 kDa on SDS-PAGE, including the IgG heavy chain. It is critical to immunodeplete samples of endogenous mouse IgG when using mouse anti-Aβ antibodies followed by secondary anti-mouse antibodies for detection. Blocking of blots and protein extraction methods are also key factors. We (Liu et al., 2011b) and others (Sherman and Lesné, 2011) have published detailed methods that should ensure success, if followed.

Dr. Ashe has already alluded to the approaches used to determine whether Aβ*56 is an oligomer—some of these have been published (Lesné et al., 2006) and others will be found in a paper currently in press in Brain (Lesné et al., in press). We continue to refer to Aβ*56 as a “putative dodecamer,” recognizing that its identity is not yet firmly established. Whatever this band represents, it correlates with cognitive deficits in multiple APP transgenic mouse lines, and now with markers of disease/synaptic dysfunction in humans. We strongly believe that these findings make Aβ*56 worthy of further study.

To really test the hypothesis that Aβ*56 is necessary to trigger the amyloid cascade, we must determine whether selectively decreasing the levels of Aβ*56 or interfering with its interactions with its cellular targets reduces the risk of symptomatic AD. Such studies await the identification of the targets of Aβ*56 and/or development of reagents that selectively target specific Aβ oligomers.

References:
Cheng IH, Scearce-Levie K, Legleiter J, Palop JJ, Gerstein H, Bien-Ly N, Puoliväli J, Lesné S, Ashe KH, Muchowski PJ, Mucke L. Accelerating amyloid-beta fibrillization reduces oligomer levels and functional deficits in Alzheimer disease mouse models. J Biol Chem. 2007 Aug 17;282(33):23818-28. Abstract

Handoko M, Grant M, Kuskowski M, Zahs KR, Wallin A, Blennow K, Ashe KH. Correlation of Specific Amyloid-β Oligomers With Tau in Cerebrospinal Fluid From Cognitively Normal Older Adults. JAMA Neurol. 2013 Mar 11;:1-6. Abstract

Larson ME, Lesné SE. Soluble Aβ oligomer production and toxicity. J Neurochem. 2012 Jan;120 Suppl 1:125-39. Abstract

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. Abstract

Lesné S, Kotilinek L, Ashe KH. Plaque-bearing mice with reduced levels of oligomeric amyloid-beta assemblies have intact memory function. Neuroscience. 2008 Feb 6;151(3):745-9. Abstract

Lesné, S.E., Sherman, M., Grant, M., Kuskowski, M., Schneider, J.A., Bennett, D.A., and Ashe, K.H. (in press). Brain amyloid-β oligomers in aging and Alzheimer’s disease. Brain.

Liu P, Forster C, Kotilinek L, Paulson J, Chen G, Bennett D, Cleary J, Zahs K, Lesné S, Ashe K. (2011a). Aβ dimers mediate plaque-associated cytopathology without affecting cognition. Alzheimer's & Dementia: the journal of the Alzheimer's Association 7:e23.

Liu P, Kemper LJ, Wang J, Zahs KR, Ashe KH, Pasinetti GM. Grape seed polyphenolic extract specifically decreases aβ*56 in the brains of Tg2576 mice. J Alzheimers Dis. 2011b;26(4):657-66. Abstract

Sherman MA, Lesné SE. Detecting aβ*56 oligomers in brain tissues. Methods Mol Biol. 2011;670:45-56. Abstract

View all comments by Kathleen Zahs