8 June 2012. This, that, and the other form of Aβ have shown themselves harmful to neurons in various settings, but debate continues to rage over which are most menacing in Alzheimer's disease. A paper in this week's Journal of Neuroscience describes a way to determine relative toxicities of Aβ species in vivo. Developed by Luc Buée of INSERM and the University of Lille, France, and colleagues, the approach involves repeated hippocampal injections of synthetic Abta oligomers into freely moving mice—causing tau hyperphosphorylation, neuron loss, and memory deficits. While the authors say their method provides an in vivo readout for evaluating Aβ preparations and potential therapeutics, other scientists for now remain unconvinced of the pathophysiological relevance of the model.
Small, soluble Aβ oligomers—ranging in size from dimers to dodecamers—have been fingered as key drivers of neurotoxicity (Lambert et al., 1998; Lesné et al., 2006; Shankar et al., 2008; Ono et al., 2009). "We wanted to find a way to test all these Aβ oligomers in a controlled animal model," Buée told Alzforum (see also Benilova et al., 2012 review). Analyzing transgenic mice is tricky because "you don’t know which oligomers you will have," he said. Injecting Aβ into the mouse brain is another approach, but past research suggests that anesthesia alone sends phospho-tau levels soaring (ARF news story on Planel et al., 2007).
With these issues in mind, first author Jonathan Brouillette and colleagues set about injecting Aβ into awake, mobile mice. They prepared synthetic Aβ oligomers using a protocol from co-author Bart De Strooper’s lab at the University of Leuven, Belgium (Kuperstein et al., 2010), and injected them through surgically implanted tubes into the hippocampal dentate gyrus of 12-month-old B6 females once a day for six days. The injected material was primarily low-molecular-weight Aβ42 of less than 30 kDa—including monomers, dimers, trimers, and tetramers—as revealed by Aβ immunoblotting (6E10 and 3D6), and by transmission electron microscopy, atomic force microscopy, plus various spectroscopy methods.
Analyzed 24 hours later, the mouse brain showed Aβ oligomers crowding the injection site. It also showed extensive neuron loss, reduced levels of the NMDA receptor subunit NR2B, and elevated levels of cleaved caspase-3. Controls injected with vehicle solution and scrambled Aβ appeared normal. In addition, tau phosphorylation levels rose in the brains of Aβ-injected mice, though only at the S202/T205 site and not at other sites typically affected in AD. If the Aβ oligomers were pre-incubated with the sequestering agent transthyretin (TTR) prior to injection, the mice were spared neuron loss and memory deficits. Previous work suggested TTR protects against AD pathology in transgenic mice (see ARF news story on Buxbaum et al., 2008).
"With this model, we can test which forms of Aβ are most toxic," Buée said, noting it should also be useful for testing therapeutic approaches.
Other scientists wondered how well the data reported to date apply to AD. "The relevance of synthetic Aβ to the disease state is questionable," John Cirrito of Washington University School of Medicine, St. Louis, Missouri, wrote in an email to Alzforum (see full comment below). "It would be interesting to see what naturally derived Aβ oligomers would do in this chronic infusion system."
Moreover, the study used non-physiologic concentrations of Aβ; specifically, an amount that would reach micromolar concentrations if the injected oligomers distributed uniformly throughout one side of the forebrain, according to Karen Ashe and colleagues at the University of Minnesota, Minneapolis (see full comment below). "In comparison, Aβ dimers isolated from AD brains are cytotoxic at sub-nanomolar concentrations (Jin et al., 2011)." The authors used high Aβ concentrations to induce toxic oligomer conformations, and said that doing so accelerates processes that would otherwise take decades—too slow to permit laboratory studies. "Such high Aβ concentrations might actually be quite relevant for what happens in vivo," countered co-author Iryna Benilova of the University of Leuven. "It is suggested that Aβ can concentrate in intracellular compartments, thus creating the conditions for local formation of potentially toxic aggregates." (Hu et al., 2009)
The study did not address the possibility that the oligomers could have formed larger or smaller aggregates after injection. "I would be curious to know whether the Aβ oligomers that wind up in the hippocampus, and can be seen immunohistochemically, go on to become fibrillar (by electron microscopy)," said Lary Walker of Emory University in Atlanta, Georgia. The study did not test if other oligomers besides Aβ could have caused the neuron loss and memory symptoms.
Despite reservations expressed by some researchers, "we believe the authors should be lauded for their careful characterization of the Aβ species present in their preparations before injection and, impressively, the Aβ species actually present in the brain after injection," wrote Hsiao and colleagues.—Esther Landhuis.
Brouillette J, Caillierez R, Zommer N, Alves-Pires C, Benilova I, Blum D, De Strooper B, Buée L. Neurotoxicity and Memory Deficits Induced by Soluble Low-Molecular-Weight Amyloid-beta1-42 Oligomers Are Revealed In Vivo by Using a Novel Animal Model. J Neurosci. 6 June 2012;32(23):7852-7861. Abstract