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

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  1. This is an interesting study that addresses two important issues relating to the role of Aβ in memory impairment, namely biophysical characterization of synthetic memory-impairing assemblies and the requirement of PrPc for memory impairment. Using atomic force microscopy (AFM) and size exclusion chromatography (SEC), Balducci and colleagues characterized four distinct Aβ1-42 preparations. Freshly dissolved unaggregated Aβ, seemingly mostly Aβ monomer; Aβ fibrils formed at low pH; and Aβ were incubated at either 4oC or 22oC for 24 hours and contained different heterogeneous mixtures of Aβ monomer and small prefibrillar Aβ assemblies. The latter mixtures are simply referred to as “oligomers.”

    Each preparation was then tested for its effect using the novel object recognition task. The initial paradigm involved intracerebroventricular injections (icv) of vehicle or one of the Aβ preparations. Injections were made at two hours prior to mice being allowed to explore two identical objects and then again two hours before animals were tested for 24 hours’ recall by exposure to two objects: one familiar and one novel. Animals injected with Aβ monomer or Aβ fibrils behaved in a manner highly similar to those injected with vehicle, whereas mice injected with either the 4oC or the 22oC “oligomers” were unable to discriminate between the novel and the familiar object.

    Importantly, this effect required only tiny amounts of Aβ. For instance, injection of only 30 ng x 2 of the “oligomer” preps was sufficient to impair object recognition. But given that monomer was the most abundant component of the 4oC “oligomer” prep, it seems highly likely that the amount of active Aβ species was much lower, probably in the order of 0.15 to 0.75 ng x 2. Compared to prior studies testing the effect of non-fibrillar Aβ assemblies on memory, the concentrations used by Balducci and colleagues are comparable to those used to demonstrate impairment of ALCR performance by icv injection of ADDLs (Reed et al., 2009), but are substantially lower than the concentration of lipid-induced reverse protofibrils used to induce impairment of avoidance learning and contextual fear conditioning (Martins et al., 2008). Thus, a highlight of the current study is the fact that the relatively simple and fast object recognition task is sensitive to very low concentrations of certain Aβ assembly forms.

    Interestingly, when animals were allowed to recover and tested for recall nine days after the second Aβ injection, the impairment of object recognition did not persist. This finding suggests that animals injected two hours prior to familiarization were able to encode the memory of the familiar object, but that “oligomer” injection two hours prior to 24-hour recall prevented retrieval, whereas the same injection was not able to prevent retrieval at 10 days. However, these data appear to be in conflict with the finding that a single injection of “oligomers” at two hours prior to familiarization impaired recall at 24 hours, whereas injection of “oligomers” at two hours prior to recall had no effect. Thus, further work is required to discern how injection of “oligomers” prior to familiarization, which seemly perturbs encoding, could be overcome by simply testing after a longer duration. Nonetheless, Balducci et al. have clearly demonstrated the utility of this paradigm.

    They next turned their attention to determine whether or not the observed “oligomer”-mediated impairment required PrPc. This they addressed using PrP knockout (KO) mice. Prior work by Lauren et al. (2009) using murine hippocampal slices bathed in medium containing ADDLs (Lambert et al., 1998) demonstrated that PrPc was required for the Aβ-mediated block of LTP; thus, one might have anticipated that Balducci and colleagues would find PrP KO animals resistant to the memory-impairing effects of their “oligomer” prep. However, this was not the case. When PrP KO mice were injected with either 4oC or 22oC “oligomers,” these animals could not discriminate between the familiar and the novel object. Although not required for Aβ-mediated impairment of object recognition, Balducci et al. reported that their “oligomer” preps bound to brain-derived PrPc.

    Why PrPc binds certain forms of Aβ and appears to be required for Aβ-mediated impairment of LTP (an observation we have replicated in my lab), but not the impairment of object recognition, is unclear. Experimental differences including differences in the actual Aβ species used, their effective concentration, and the genetic background of the PrP KO animals could contribute to these divergent results. Whatever the reason, using PrP KO mice, it will be important to simultaneously test the effect of well-defined Aβ assemblies on both LTP and hippocampal-dependent behavioral paradigms known to parallel LTP (e.g., avoidance learning, Whitlock et al., 2006). Regardless of the outcome of future studies, it is already clear that the link between Aβ, PrP, and Alzheimer disease is not as simple as it first appeared.

    References:

    . Cognitive effects of cell-derived and synthetically derived Aβ oligomers. Neurobiol Aging. 2011 Oct;32(10):1784-94. PubMed.

    . Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice. EMBO J. 2008 Jan 9;27(1):224-33. PubMed.

    . Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature. 2009 Feb 26;457(7233):1128-32. PubMed.

    . Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6448-53. PubMed.

    . Learning induces long-term potentiation in the hippocampus. Science. 2006 Aug 25;313(5790):1093-7. PubMed.

    View all comments by Dominic Walsh
  2. This study further explores interaction between Aβ oligomers and cellular prion protein (PrPC) as proposed by Laurén et al., 2009. In the current study, the authors incubated Aβ1-42 to generate oligomers, which were assayed for the ability to bind PrPC and to affect recognition memory. While this synthetic Aβ preparation did bind endogenous PrPC in a dose-dependent fashion, the effect of Aβ oligomers on recognition memory was not significantly different between wild-type and PrPC knockout mice. This convincing study by Balducci et al. has a number of controls, such as comparing the effects of fibrils and rescuing the effects on memory with an anti-Aβ antibody.

    The critical question that remains, though, is how to reconcile these findings with those by Laurén et al. One key point is that the physiologic assay used in these studies is different—long-term potentiation by theta burst stimulation in Laurén et al. and recognition memory and toxicity in Balducci et al. The former is an in vitro electrophysiologic phenomenon studied in a well-characterized and simplified acute hippocampal slice preparation. It is unclear how LTP in hippocampus precisely correlates with recognition memory, which is tested in the context of completely intact neural circuitry. Given the complexities in the vast literature describing Aβ interactions with neuronal receptors, it remains highly plausible that the various pathophysiologic effects of this hydrophobic peptide are driven by a number of receptor pathways. Consequently, this study affirms that dissecting mechanisms of AD pathology is made even more difficult by our incomplete understanding of the requirements for normal neural physiology.

    References:

    . Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature. 2009 Feb 26;457(7233):1128-32. PubMed.

    View all comments by Ganesh M Shankar

References

News Citations

  1. Keystone: Partners in Crime—Do Aβ and Prion Protein Pummel Plasticity?
  2. “Natural” Aβ Oligomers Cause Transitory Cognitive Disruptions
  3. Aβ Star is Born? Memory Loss in APP Mice Blamed on Oligomer
  4. Paper Alert: Patient Aβ Dimers Impair Plasticity, Memory
  5. Amyloid-β Zaps Synapses by Downregulating Glutamate Receptors
  6. AMPA Receptors: Going, Going, Gone in Aβ-exposed Synapses, PSD95 Knockouts
  7. Neuronal Glutamate Fuels Aβ-induced LTD

Paper Citations

  1. . Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature. 2009 Feb 26;457(7233):1128-32. PubMed.
  2. . Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005 Jan;8(1):79-84. PubMed.
  3. . A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. PubMed.
  4. . Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008 Aug;14(8):837-42. PubMed.
  5. . Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer's disease. J Neurosci. 2005 Oct 19;25(42):9694-703. PubMed.
  6. . The efficacy of tetracyclines in peripheral and intracerebral prion infection. PLoS One. 2008;3(3):e1888. PubMed.

Further Reading

Primary Papers

  1. . A causative link between the structure of aberrant protein oligomers and their toxicity. Nat Chem Biol. 2010 Feb;6(2):140-7. PubMed.
  2. . Synthetic amyloid-beta oligomers impair long-term memory independently of cellular prion protein. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2295-300. PubMed.