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
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