. Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples. Acta Neuropathol. 2013 Apr;125(4):549-64. PubMed.

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  1. Important to note.

  2. Based on ~15 years of discovery and experimental evidence, there is a wide consensus that oligomers of amyloid-β protein (Aβ), particularly Aβ42, initiate the pathologic cascade that leads to Alzheimer’s disease (AD). The evidence is abundant, yet circumstantial, because visualizing Aβ oligomers in the brain, let alone in the act of causing harm to synapses or neurons, currently is not possible. In Aβ oligomers, monomers are held together by weak forces that may be disrupted even by subtle manipulations. Thus, similar to Heisenberg’s Principle of Uncertainty, any attempt to detect the oligomers, measure their size or internal organization, or quantify them may affect the very quality or quantity measured. This is true for any and all of the techniques used today for examining Aβ oligomers in the brain. They all bias the measured outcome.

    In particular, one of the methods used most commonly to attest for the existence of Aβ oligomers, SDS-PAGE, is infamous for creating artifacts. We reported in 2003 that synthetic Aβ42 species with electrophoretic mobilities corresponding to trimers and tetramers likely were artifacts (Bitan et al., 2003). We showed in a follow-up study using size-exclusion chromatography that the presence of SDS, both below and above the critical micelle concentration, induced artificial oligomerization of Aβ42 (Bitan et al., 2005). Similar findings were reported by Hepler et al., who analyzed unaggregated and fibrillar preparations of synthetic Aβ42 and found the exact same pattern: monomers, in addition to the artifactual “trimer” and “tetramer” bands in both cases (Hepler et al., 2006). These and multiple other studies (reviewed in Rahimi et al., 2008) have demonstrated that SDS induces artificial assembly of Aβ and suggested that SDS-PAGE was not a reliable method for characterizing Aβ oligomers.

    In their new paper, Watt et al. went a step further to examine populations of Aβ species in brains of patients who died with diagnoses of sporadic AD, familial AD, or frontotemporal lobar degeneration, and non-demented, age-matched control brains. To minimize oligomer disruption, Watt et al. used a gentle extraction protocol, excluding the use of detergent, harsh acids, or chaotropes. To control for biases induced by the method of detection, they used three different techniques. In agreement with previous studies cited in the paper, they observed predominant Aβ dimer bands following SDS-PAGE fractionation of their extracts and detection by Western blots using a monoclonal antibody specific for Aβ residues 5-8. In contrast, there was no evidence for the existence of dimers in measurements of the same extracts using surface-enhanced laser-desorption ionization-time-of-flight (SELDI-TOF) mass spectrometry. The discrepancy between the results obtained by the two methods let the authors to “draw into question the utility of oligomeric Aβ as a therapeutic target.”

    I agree with the authors that in view of the tendency of SDS-PAGE to induce artifactual Aβ assembly, the observation of Aβ dimers in their brain extracts does not necessarily mean that Aβ dimers exist in the AD brain. Interestingly, the central role ascribed to Aβ dimers was proposed by Walsh et al. in 2002, yet a more recent re-examination of the role of dimers by Walsh and coworkers suggested that dimers needed to assemble into larger structures, namely protofibrils, in order to cause toxicity (O'Nuallain et al., 2010). The correlation found by Watt et al. between the abundance of the dimer band in brain extracts and the presence of AD suggests that the dimer band is a good proxy for a pathologic process in the AD brain involving Aβ, even though the dimers themselves might be SDS-induced artifacts.

    Having said that, it is also important to consider the limitations of SELDI-TOF mass spectrometry for detection of Aβ oligomers, including dimers. First, as Watt et al. point out, the use of urea and organic solvents in the preparation of the brain extract samples for SELDI-TOF analysis might have disrupted Aβ oligomers and precluded their detection. Second, mass spectrometric detection relies on the ratio between mass and charge (m/z), which may be identical for monomers and oligomers, i.e., a singly charged monomer would have the same m/z value as a doubly charged dimer, necessitating additional manipulations for unambiguous distinction between the two. It should be noted that this problem is more common in electrospray ionization (ESI) mass spectrometry than in techniques relying on laser-desorption ionization and time-of-flight detectors. Third, whereas the presence of SDS micelles causes artificial high local concentration of Aβ, promoting self-assembly, the high-vacuum conditions in the mass spectrometer may induce dissociation of Aβ oligomers and bias the readout in the opposite direction from SDS-PAGE, namely, no observation of dimers. Of note, none of these problems would apply to the covalently crosslinked Aβ dimer Watt et al. used as a control. From their experiments, we learn that the SELDI-TOF system detects covalently crosslinked dimers, but not if this system detects non-covalently crosslinked dimers.

    Studies examining the validity of current methods used to detect Aβ oligomers in human samples are highly important. Artifactual data may lead to a tremendous waste of resources and time in the battle against AD. It is important to conduct such studies using methods that are as non-invasive and non-disruptive as possible to minimize the potential for dissociation of the metastable oligomers. In view of the inherent technical difficulties in such studies, Watt et al. are to be congratulated for their cautious and thoughtful interpretation of the data they obtained. At the same time, questioning Aβ oligomers as therapeutic targets based on these data seems to go too far. In my view, a more conservative conclusion would be to question the usefulness of methods relying on SDS-PAGE, or using SDS in the extraction buffer, for studies of Aβ oligomers from any source.

    See also:

    Heisenberg W. (1927) Ueber den anschaulichen Inhalt der quantentheoretischen Kinematik and Mechanik, Zeitschrift für Physik 43, 172-98.

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    . Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer's disease and related disorders. Curr Alzheimer Res. 2008 Jun;5(3):319-41. PubMed.

    . Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples. Acta Neuropathol. 2013 Apr;125(4):549-64. PubMed.

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