Researchers led by Dennis Selkoe of Brigham and Women’s Hospital, Boston, offer what he considers the “cleanest, best-controlled evidence to date” for the toxicity of small amyloid-β oligomers. Using extracts purified directly from the brains of Alzheimer’s disease patients, the researchers found that Aβ dimers—applied to primary cultures of rat neurons at physiological, sub-nanomolar concentrations—trigger tau hyperphosphorylation and neuritic degeneration. Selkoe reported the findings 10 March 2011 at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases (AD/PD 2011) in Barcelona, Spain, just days before the study’s publication in this week’s early online edition of the Proceedings of the National Academy of Sciences U.S.A.

There is no shortage of data on the destructive potential of Aβ oligomers. In rodent brain slice cultures, the soluble aggregates block hippocampal long-term potentiation (Lambert et al., 1998), throw off calcium flux (Shankar et al., 2007), and induce oxidative stress (De Felice et al., 2007). Furthermore, antibody-mediated clearance of brain Aβ oligomers prevents LTP and reverses memory deficits in AD model mice (Dodart et al., 2002; Klyubin et al., 2005). In addition, more recent studies have strengthened links between Aβ and downstream neuronal effects that are more closely tied to cognitive loss in AD. In an ex vivo system, researchers showed that tau pathology depends on NMDA receptor activation via Aβ (Tackenberg and Brandt, 2009). In another study, treatment of rat hippocampal cultures with Aβ oligomers caused tau to mislocalize into neuronal dendrites, leading to collapse of microtubules and synaptic spines (Zempel et al., 2010).

The big caveat, in Selkoe’s view, is that most of these effects came from synthetic Aβ peptides applied at non-physiologically high concentrations. “We wanted to use a more reliable and appropriate source of Aβ—natural material in the human brain,” Selkoe said, noting that an AD/PD poster presented by Oliver Kleiner, Eisai Limited, London, U.K., reported that Aβ peptides from various sources had strikingly different mass spectrometry profiles compared with the real stuff from human cortex. In a landmark study published several years ago, Selkoe and colleagues purified Aβ oligomers from the brains of AD patients and showed they caused dendritic spine loss—as well as deficits in long-term potentiation, long-term depression, and cognition (ARF related news story on Shankar et al., 2008). In the current paper, first author Ming Jin and colleagues build upon these earlier findings by asking whether Aβ oligomers can induce AD-like tau changes.

Using the same methodology as previously, the researchers purified soluble Aβ oligomers from postmortem cerebral cortices of AD patients and from age-matched controls. They applied Aβ monomer- and dimer-enriched AD preparations to 18-day primary cultures of rat hippocampal neurons, and three days later immunostained the cells with tubulin and tau antibodies. The researchers saw neuritic degeneration and microtubule cytoskeleton abnormalities in cultures treated with AD dimers, but not in samples exposed to AD monomers or corresponding size-exclusion chromatography fractions from control patients. Neurons treated with dimer-rich AD fractions that had been pre-depleted with polyclonal Aβ antisera (AW7) also looked normal, indicating that the morphological changes were Aβ-dependent.

However, “There is a selectivity,” Selkoe said. Whereas striking cytoskeletal changes were seen in neurons that were cultured for 18 days before Aβ exposure, Aβ-treated neurons from seven-day cultures looked just fine. This is consistent with other research indicating that synthetic Aβ is only toxic to neurons allowed to mature sufficiently in vitro. Moreover, by Western blot analysis, AD dimer-treated neurons showed tau hyperphosphorylation at several AD-relevant epitopes (AT8, 12E8, AT270), but not others (PHF-1 and AT180), again suggesting “we’re not dealing with a general toxicity,” Selkoe said.

Taking a cue from recent work of several other labs showing how tau reduction protects neurons from Aβ’s harmful effects in AD mouse models (see ARF related news story on Roberson et al., 2007; ARF related news story on Ittner et al., 2010; ARF related news story on Vossel et al., 2010), Selkoe’s team used small interfering RNAs to knock down tau in the cultured rat neurons and found that this protected the cells from Aβ-induced cytoskeletal disruptions. Conversely, rat neurons that overexpress human tau showed significant neuritic degeneration after just two days of Aβ exposure (whereas neurons with wild-type tau levels took three days to show neuritic changes).

A growing literature links Aβ and tau at the molecular level. However, much of it has “looked at the tau side,” said Lars Ittner, University of Sydney, Australia, who recently reported that tau mediates Aβ toxicity at synapses by targeting the Src kinase Fyn to the N-methyl-D-aspartic acid (NMDA) receptor (Ittner et al., 2010). The present study “looks at the Aβ side of the Aβ-tau interaction and makes a strong case that Aβ dimers are the link to the tau changes,” Ittner said. “It’s very thoroughly done.”

As confirmation of sorts for their studies using natural Aβ dimers from AD brain, the authors observed similar cytoskeletal and neuritic abnormalities in cultured neurons treated with synthetic human Aβ40 dimers. While this shows that the synthetic Aβ was sufficient to cause the cytoskeletal disruptions, more than a 100-fold higher concentration was needed to see effects comparable to those triggered by natural dimers.

It’s no surprise that the artificial Aβ was less toxic, said Brigita Urbanc, a biophysicist at Drexel University, Philadelphia, Pennsylvania. The synthetic dimers were made from mutant Aβ40 peptides, whereas the dimers in AD brain are predominantly Aβ42. At last year’s Society for Neuroscience meeting in San Diego, Urbanc reported on a computational analysis suggesting that Aβ42 may be the more toxic of the two species because its N-terminal end has greater exposure to solvent, presumably making Aβ42 more prone to enzymatic cleavage and other modifications such as N-terminal truncations that are increasingly seen as the most harmful of Aβ oligomers (see ARF related conference story). In line with Urbanc’s structural data (ARF related conference story), Selkoe and colleagues showed that an infusion of monoclonal antibodies to Aβ’s N-terminus (but not to its C-terminal end) protected rat neurons from the neuritic dystrophy induced by natural Aβ dimers.

“The outstanding question” from the current study is the precise chemical composition of the dimer, Torleif Härd noted in an e-mail to ARF after hearing Selkoe’s AD/PD presentation. “It could be two Aβ monomers that are covalently linked. It could be two monomers that stick together without covalent bonds. Or It could be a single Aβ monomer that is linked to something else, which makes it appear to be a ‘dimer.’” Härd and colleagues at Swedish University of Agricultural Sciences, Uppsala, have generated synthetic Aβ monomers that form toxic oligomers with detergent-stable dimer/trimer patterns similar to those seen in AD (ARF related news story on Sandberg et al., 2010).

Structural studies of natural Aβ oligomers are hard, given their low nanomolar concentrations in the brain, Selkoe said. Still, his group is trying to gain some insight on structure by comparing the natural oligomers to well-defined synthetic Aβ assemblies using size exclusion chromatography, mass spectrometry, and conformation-specific Aβ antibodies. “We are committed to using all of these approaches, but the low concentrations and difficulty of fully purifying them from brain tissue limit the structural characterization that one can do,” Selkoe noted.

Another unresolved issue is whether it’s the Aβ dimer itself, or something bigger made from the dimers, that causes toxicity. In a recent study, coauthor Dominic Walsh of University College Dublin, Ireland, showed that the synthetic Aβ40 dimers could block LTP in mouse hippocampus, but only after aggregating into protofibrils (ARF related news story on O’Nuallain et al., 2010). Even with the new data, “we don’t yet know whether AD brain dimers can cause direct effects (on tau and neurites), or whether they aggregate into larger assemblies of dimers before inducing neural changes, like (Walsh’s) crosslinked dimers seem to require,” Selkoe said. “We focused on the dimer—the smallest oligomer—and it is definitely bioactive (see also ARF related news story on McDonald et al., 2010; Pham et al., 2010). But we don’t know that multimers of dimers aren’t also bioactive.”

The next challenge will be figuring out what Aβ oligomers bind to on cells. Other studies have suggested that Aβ binds cellular prion protein (ARF related conference story on Laurén et al., 2009) and various receptors (e.g., α7 nicotinic acetylcholine, NMDA, and insulin receptors). However, those studies used synthetic Aβ, and Selkoe said he does not think these relatively hydrophilic receptors are the principal initial binding sites of Aβ. Given that hydrophobic amino acids are more exposed in the dimer structure of Aβ, relative to the monomer, “the most likely target of Aβ oligomers would be membrane lipids, and then those other receptors get engaged,” Selkoe said.

His team will use a multi-step chromatography approach to pull out and successively enrich for even purer Aβ oligomers, which could then be used for a “really unbiased binding experiment,” he told ARF. “We all need to find the target of these Aβ oligomers. That’s going to be a lot of hard work.”—Esther Landhuis


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News Citations

  1. Paper Alert: Patient Aβ Dimers Impair Plasticity, Memory
  2. APP Mice: Losing Tau Solves Their Memory Problems
  3. Honolulu: The Missing Link? Tau Mediates Aβ Toxicity at Synapse
  4. The Plot Thickens: The Complicated Relationship of Tau and Aβ
  5. San Diego: Pilin’ on the Pyro, Aβ Going Rogue
  6. San Diego: Flexible N-Termini Key to Aβ42 Oligomer Toxicity?
  7. Stable Aβ Oligomers?—A Little Protein Engineering Goes a Long Way
  8. Aβ Neurotoxicity—Is it the Dimer? No, and Yes
  9. Bad Guys—Aβ Oligomers Live Up to Reputation in Human Studies
  10. Keystone: Partners in Crime—Do Aβ and Prion Protein Pummel Plasticity?

Paper Citations

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  4. . Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci. 2002 May;5(5):452-7. PubMed.
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  7. . Abeta oligomers cause localized Ca(2+) elevation, missorting of endogenous Tau into dendrites, Tau phosphorylation, and destruction of microtubules and spines. J Neurosci. 2010 Sep 8;30(36):11938-50. PubMed.
  8. . Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008 Aug;14(8):837-42. PubMed.
  9. . Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science. 2007 May 4;316(5825):750-4. PubMed.
  10. . Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models. Cell. 2010 Aug 6;142(3):387-97. Epub 2010 Jul 22 PubMed.
  11. . Tau reduction prevents Abeta-induced defects in axonal transport. Science. 2010 Oct 8;330(6001):198. PubMed.
  12. . Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15595-600. PubMed.
  13. . Amyloid beta-protein dimers rapidly form stable synaptotoxic protofibrils. J Neurosci. 2010 Oct 27;30(43):14411-9. PubMed.
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  15. . Progressive accumulation of amyloid-beta oligomers in Alzheimer's disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins. FEBS J. 2010 Jul;277(14):3051-67. PubMed.
  16. . Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature. 2009 Feb 26;457(7233):1128-32. PubMed.

External Citations

  1. AD/PD poster

Further Reading

Primary Papers

  1. . Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5819-24. PubMed.