25 January 2009. When it comes to the synaptic toxicity and cognitive dysfunction that occur in Alzheimer disease, a preponderance of evidence points to soluble oligomers of amyloid-β peptides as the main villains, at least in rodents. But much remains unknown about how oligomers bind to cells, interfere with synapses, or bring down neuronal systems. Could a new mouse model of oligomer toxicity, described in this week’s PNAS online edition, help move the field along? The model, developed by Gianluigi Forloni and colleagues at the Mario Negri Institute for Pharmacological Research, Milan, Italy, combines cerebroventricular injection of synthetic Aβ preparations with the novel object recognition test. Using this combo, the researchers show that oligomers, but not monomers or fibrils, impair memory in normal mice (not transgenic ones). The researchers go on to use the technique to test the recent hypothesis that the cellular prion protein (PrPc) mediates Aβ toxicity. Contrary to recent in vitro findings (see ARF related news story on Lauren et al., 2009), Forloni and colleagues report that, at least in this in vivo situation, the Aβ effects on memory do not depend on the prion protein.
First author Claudia Balducci set up the new model, which entails injection of synthetic Aβ preparations prior to running mice through the novel object recognition test. In that test, mice are placed into a cage with two identical small objects, in this case a plastic cylinder, cup, or metal cube. Once the mice have become familiar with the particular object, the animals are removed. The next day, the mice encounter the familiar object as well as a novel one. Untreated mice, or mice injected with monomers or fibrils, spend more time exploring the novel object. In contrast, mice that were injected with oligomers explore both objects equally. When the mice were allowed to learn the objects first and were then
injected with Aβ before the second exposure, they responded
normally to the novel object, while ignoring the one they had previously learned. This suggests the deficit is in memory acquisition or consolidation, but not recall, consistent with the memory problems seen in early AD, the authors propose. Balducci also showed that the memory defect was reversible, was prevented by an Aβ antibody, and was associated with neurodegeneration.
Other groups have reported memory deficits in animals injected with oligomers derived from cells (see ARF related news story on Cleary et al., 2005), mouse brain (see ARF related news story on Lesne et al., 2006), and even human brain (see ARF related news story on Shankar et al., 2008), but Forloni said this is the first time that such a deficit has been demonstrated with synthetic material. The benefit of synthetic Aβ, rather than naturally derived oligomers, is to make the assay easier and more reproducible. Forloni told ARF that his group started with cell-derived Aβ but moved to synthetic because they wanted to rule out effects of other proteins that might be present. They prepared oligomers in two different ways, using their own protocol or one identical to Lauren et al. (see below), and in both cases saw the same results.
The group next used the model to test the hypothesis that the cellular prion protein is an important mediator of Aβ oligomer toxicity. Last year, Lauren and colleagues from Stephen Strittmatter’s lab at Yale University, New Haven, Connecticut, reported that Aβ oligomers bind to the cellular prion protein with high affinity, and block synaptic plasticity in hippocampal slices from mice in a prion protein-dependent manner (see ARF related news story). Strittmatter himself gave an update at the Keystone Symposium titled “Alzheimer Disease Beyond Aβ”, held 10-15 January 2010 at Copper Mountain, Colorado. His group has crossed PrPc-negative animals with APP/PS transgenic mice (APPSwe/PS1ΔE9). Strittmatter reported that, unlike APP/PS mice, which die prematurely, APP/PS/PrPc-negative mice live a normal lifespan despite a buildup of amyloid plaque. They also behave normally at 12 months, by which time APP/PS mice show difficulty in spatial memory tasks. Levels of synaptophysin, a marker of synaptic density, go down by up to 20 percent as APP/PS animals age but appear normal in the PrPc-negative background, according to Strittmatter. Finally, the APP/PS/PrPc-negative animals do not lose 5-HT neurons, which degenerate in the parent APP/PS transgenic strain.
Since plaque level appeared similar in PrPc-negative and positive animals, the data support the idea that PrPc modulates toxicity downstream of Aβ. The peptide has been linked to glutamate receptor dysfunction (see ARF related news story, ARF news story, and ARF related news story); however, Strittmatter reported that Xenopus oocytes expressing AMPA or NMDA receptors with or without PrPc show no sensitivity to Aβ oligomers. “If PrPc mediates Aβ effects on glutamate receptors, then it may be through some other molecule,” he said. He suggested one potential mediator might be fyn kinase, which colocalizes with the prion protein and has been linked to toxicity in APP transgenic mice by work from Lennart Mucke’s lab at the University of California, San Francisco (see Chin et al., 2005). Strittmatter reported that fyn and PrPc immunoprecipitate when coexpressed in non-neuronal cells and that Aβ stimulates phosphorylation of the kinase. Whether fyn kinase turns out to be a major player in Aβ toxicity remains to be seen. In the meantime, Strittmatter believes that the APP-PrPc interaction could be a therapeutic target, and has screened a library of 20,000 compounds to find two, so far, that disrupt the interaction.
In their paper, Forloni and coworkers confirm that Aβ and the prion protein bind avidly in vitro, but in vivo they saw no difference in behavior after oligomer injection in normal versus prion knockout mice. In vitro, the Aβ oligomers were toxic to primary neurons in culture, whether the cells came from wild-type or prion-knockout mice.
“In the animals, the data are very clear. The behavior of the knockout mice without prion was exactly the same as the control, so they respond the same way and they develop the deficit when we inject oligomers,” Forloni said. He noted that his study and that of Lauren and colleagues use different techniques. Nonetheless, based on his own data, Forloni said, “We feel it is possible that an interaction exists between oligomers and prions, but the idea that the prions could mediate the functional activity of oligomers is almost impossible.”
However, in an e-mail to ARF Strittmatter noted that the PrPc-negative and
control mice do not behave in quite the same way when treated with Aβ.
The latter show reduced objective memory, as in they explore the novel
object less than untreated mice, but the former actually show a preference
for the familiar object. "Since novelty-seeking is reversed, any assessment
of memory function is tenuous but, at face value, memory is intact in the
Aβ oligomer-injected PrPc-null mice," wrote Strittmatter. "These data tend
to support, rather than refute, the hypothesis that PrPc is required for
memory impairment by Aβ. Clearly, Aβ has an unexplained action in this
experiment that alters preference for novelty versus familiarity, possibly
via arousal or anxiety."
Because object recognition behavior is easy to measure, the mouse model could give researchers a new way to study the mechanism of oligomer action and test potential treatments. Forloni argues it is more physiological than many transgenic mouse AD models that express amyloid protein at high levels. Unlike other memory tests, the novel object recognition is a natural behavior and puts the mice under less stress than, for example, the water maze. The lab is already at work dosing mice with potential therapeutics, and Forloni reports they have some data in preparation on tetracycline, which he has studied in prion diseases for its anti-amyloid effects (see, e.g., De Luigi et al., 2008).
In a related paper in the January 10 online edition of Nature Chemical Biology, another group of Italian researchers reports new insights into the basis for oligomer toxicity. Fabrizio Chiti and colleagues at the University of Florence studied the bacterial HypF-N protein, an amyloidogenic peptide that they find forms two kinds of stable oligomers. The assemblies look similar by atomic force microscopy and thioflavin T staining, yet one is toxic to cultured cells and one is not. The difference, the researchers find, is that the toxic oligomer has a looser packing and more solvent exposure of hydrophobic interactions between adjacent proteins. This correlates with a higher ability to penetrate the cell membrane and cause a deadly influx of calcium ions. The results indicate that while many peptides can form amyloid structures, whether or not they will be pathogenic depends on their specific structural features, namely the flexibility and hydrophobic exposure of proteins in oligomers. The findings suggest that, natural or synthetic, the secret of Aβ may lie in its exposed hydrophobic residues, and that may provide a molecular target for neutralization.—Pat McCaffrey and Tom Fagan.
Balducci C, Beeg M, Stravalaci M, Bastone A, Sclip A, Biasini E, Tapella L, Colombo L, Manzoni C, Borsello T, Chiesa R, Gobbi M, Salmona M, Forloni G. Synthetic amyloid-beta oligomers impair long-term memory independently of cellular prion protein. PNAS Online, 2009 January 11. Abstract
Campioni S, Mannini B, Zampagni M, Pensalfini A, Parrini C, Evangelisti E, Relini A, Stefani M, Dobson CM, Cecchi C, Chiti F. A causative link between the structure of aberrant protein oligomers and their toxicity. Nature Chemical Biology. 2010 January 10. Abstract