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The deafening buzz of firing neurons can drop to a dull hum if too much Aβ collects in the brain. That’s because excess amyloid slows synaptic transmission by displacing synaptic AMPA and NMDA glutamate receptors. Scientists now have a better idea how this might occur. In a study posted online November 28 in Nature, Lennart Mucke and coworkers at the Gladstone Institute of Neurological Disease in San Francisco, California, report that Aβ oligomers bind, and promote proteasomal degradation of, EphB2, a receptor tyrosine kinase that regulates NMDA receptor function. Furthermore, the researchers showed that knocking down EphB2 expression in the hippocampus of wild-type mice compromised long-term potentiation (LTP), a synaptic strengthening essential for learning and memory. In contrast, boosting expression of the kinase protected Alzheimer’s disease transgenic mice against LTP and memory deficits. Meanwhile, a report published online this week in Nature Neuroscience proposes a mechanism for loss of AMPA receptors in AD mice that pins the blame on non-apoptotic caspase-3 activity (see ARF related news story). The two studies shed light on how Aβ can disrupt signaling at both types of glutamate receptor, and offer potential new targets for AD therapies.

A wealth of data from Mucke’s and other labs has established that Aβ triggers downregulation of NMDA (see Palop et al., 2005; Palop et al., 2007; Snyder et al., 2005) and AMPA (see Hsieh et al., 2006; Almeida et al., 2005) receptors and associated signaling molecules, eventually causing dendritic spines to shrink and die (see also ARF related news story). However, “the mechanisms for how this may happen have been pretty elusive,” Mucke said in an interview with ARF. “There are speculations that a receptor-mediated process could be involved, but it’s unclear whether Aβ binds directly to NMDA receptors.”

First author Moustapha Cissé and colleagues pondered whether Aβ might stymie glutamatergic transmission by acting through EphB2, a master regulator of NMDA function. EphB2 controls expression of genes important for learning and memory by modulating calcium flow through NMDA receptors (Takasu et al., 2002), and, indeed, EphB2-knockout mice show reduced LTP in hippocampal and dentate granule neurons (Henderson et al., 2001). Furthermore, Aβ oligomers cause loss of EphB2 expression in cultured neurons (see ARF related news story on Lacor et al., 2007), and hippocampal levels of Eph receptors dipped prior to memory loss in several AD mouse strains, and in postmortem brain tissue of people with newly diagnosed AD (Simón et al., 2009).

In the current study, the Gladstone researchers addressed two issues—the mechanism by which Aβ causes EphB2 depletion, and the consequences of manipulating EphB2 expression in mice. To look at mechanism, Cissé and colleagues ran pull-down assays and co-immunoprecipitation on transfected cells and primary neurons, reporting that Aβ oligomers bind the fibronectin repeat domain of EphB2. When spiked into primary neuronal cultures from wild-type rats, Aβ oligomers caused EphB2 to drop within three days, and this reduction was blocked by lactacystin (a proteasome inhibitor) but not bafilomycin (an inhibitor of endosomal acidification). They found that three- to four-month-old APP transgenic mice (J20 strain) also had a dearth of hippocampal EphB2 compared to control mice, as reported previously (see Simon et al., 2009). Taken together, the biochemical experiments suggest that “Aβ interacts with EphB2, leading to its proteasomal degradation,” Mucke said. “Our data suggest that this process could trigger the well-established depletion of synaptic NMDA receptors in the presence of Aβ.”

In the second set of studies, the scientists use lentiviruses to alter EphB2 expression in the mouse dentate gyrus, a brain area important for memory formation. They wanted to see if knocking down the receptor would cause trouble for wild-type mice, and if supplying extra EphB2 could preserve cognition in AD transgenic mice.

To address the former, Cissé and colleagues injected lentiviral vectors expressing short-hairpin (sh) EphB2 RNA into the dentate gyrus of four- to five-month-old wild-type mice. The constructs also expressed green fluorescent protein (GFP) to flag transduction efficiency, which was 50 to 74 percent, typical for this procedure. The procedure worked; infected neurons had expressed less EphB2, and the reduction mattered functionally. Compared to cells expressing scrambled control short-hairpin RNA, EphB2-depleted neurons expressed fewer NMDA NR1 subunits, and failed to upregulate the immediate early gene Fos in response to EphB2 activation. Moreover, EphB2 knockdown impaired NMDA (but not AMPA) receptor function in wild-type hippocampal slices, causing LTP deficits similar to those from untreated J20 mice that overexpress human APP with Swedish and Indiana mutations. These mice develop progressive amyloid deposition and memory loss.

For the rescue experiments, Mucke’s team injected two-month-old J20 mice with EphB2 lentiviral constructs, bringing their EphB2 to wild-type levels, and analyzed the animals’ electrophysiology and behavior two months later. Boosting dentate gyrus EphB2 levels protected these mice from LTP deficits, rescued synaptic function through NMDA receptors, and improved cognition as judged by tests of spatial memory, object recognition memory, and passive avoidance memory. Based on these findings, Mucke proposed that rescuing EphB2 levels restores glutamatergic transmission by countering the Aβ-induced loss of synaptic NMDA receptors. “That’s our favorite working hypothesis,” he said, acknowledging that “EphB2 is a complicated molecule that also affects other pathways.”

Along those lines, Pascale Lacor, a research assistant professor working with William Klein at Northwestern University in Evanston, Illinois, noted that it may be hard to determine if other receptors besides EphB2 are also mediating Aβ’s ill effects at synapses (see full comment below). A recent study by the Klein lab showed Aβ oligomers co-immunoprecipitating with EphB2 alongside other synaptic proteins, including NR2, GluR1, and the metabotropic glutamate receptor mGluR5 (see ARF related news story on Renner et al., 2010).

Still, scientists say the new data could point to new avenues of therapy for AD. “Strategies to manipulate EphB2, either by increasing receptor levels or by designing drugs to activate EphB2 signaling pathways, may help in the treatment of synaptic dysfunction associated with AD,” wrote Joaquin Del Rio and Diana Frechilla in an e-mail to ARF (see full comment below). They and coworkers at the University of Navarra, Pamplona, Spain, reported that EphB2 levels were down in postmortem hippocampal tissue from patients with incipient AD (Simón et al., 2009)—a result confirmed in the present study as data not shown.

Cissé and colleagues want to test next whether EphB2 benefits might hold up in a more physiological context, such as using small molecules to block Aβ oligomer-EphB2 binding and/or prevent EphB2’s degradation. In addition, the scientists are considering a reversion experiment in which they would test J20 mice at six months to quantify their cognitive impairment, then inject EphB2 lentiviral vectors and retest the mice to check if they get better. The current report describes a preventative paradigm, as the transgenic mice had no deficits when injected with EphB2 at two months of age.

Lending hope to future efforts, the present data suggest that even partial normalization of EphB2 had benefits. “We only infected about 60 percent of neurons in the dentate gyrus,” Mucke said. “This shows that one doesn’t necessarily have to normalize molecular alterations in every cell. The brain regions don’t need a full set of neurons to function pretty normally, as long as there are enough neurons to form functional networks.”—Esther Landhuis.

Reference:
Cissé M, Halabisky B, Harris J, Devidze N, Dubal DB, Sun B, Orr A, Lotz G, Kim DH, Hamto P, Ho K, Yu GQ, Mucke L. Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature. 2010 Nov 28. Abstract

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  1. I find this paper interesting, as it reports evidence that Aβ oligomers may cause dementia by ultimately inhibiting functions of NMDA receptor, a factor that functions in memory. Importantly, the described work predicts that additional Alzheimer’s disease (AD)-linked abnormalities in NMDA receptor functions may be found. A potential reservation relates to the animal model used and its relevance to AD: Both recent (Mawuenyega et al., 2010) and older (reviewed by Robakis, 2010) evidence indicates that, in contrast to animal models based on overproduction of APP and Αβ, there is no increased production of either APP or Aβ in AD. In addition, animal models lack important hallmarks of the disease. Thus, further work will determine whether NMDA receptor abnormalities contribute to Aβ oligomers-induced dementia.

    View all comments by Nikolaos K. Robakis
  2. Reply to comment by Nikolaos K. Robakis
    I much appreciate Nick’s comments, and fully agree that all experimental models must be put into proper perspective. In regards to our paper, this perspective should include the fact that Mother Nature herself has carried out some informative overproduction experiments in humans. Overexpression of APP in people with duplication of the APP gene or with trisomy 21 causes syndromes that closely resemble both sporadic and autosomal-dominant familial AD (FAD). What all of these conditions have in common with FAD-mutant hAPP transgenic mice is abnormally elevated cerebral levels of Aβ. In my opinion, these mice are good models not only of amyloid deposition, but also of Aβ oligomer-induced synaptic dysfunction, a feature we have taken advantage of in this and other studies. In addition, we explored the effects of pathogenic Aβ assemblies in cultures of primary neurons from wild-type rats that did not overproduce APP. The results we obtained in these cultures (depletion of EphB2 and NMDA receptors and suppression of NMDA receptor-dependent gene expression [see paper], and reversal of these effects by increased expression of EphB2 [unpublished]) were completely consistent with the results we obtained in vivo in hAPP transgenic mice. As pointed out in our paper, we and others have also found decreased levels of EphB2 in brains of humans with sporadic AD. All of these points underline the potential relevance of our findings to the human condition, which—I agree—does require further corroboration.

    View all comments by Lennart Mucke
  3. The study by Mucke et al. supporting the role of EphB2 in Aβ oligomer binding and synaptotoxicity is interesting and has been conducted with elegant approaches using both knockdown and overexpression of EphB2 in in vitro and animal models of AD. The study first confirms the withdrawal of surface EphB2 resulting from cultured neurons exposed to Aβ oligomeric species and demonstrates that loss of EphB2 is due to an increase of the proteosomal degradation of the receptor. Additionally, they demonstrate the interaction between EphB2 and oligomers, first by using EphB2 constructs to demonstrate that, in test tubes, EphB2 binds Aβ oligomers in the extracellular fibronectin type III repeats domain, and also by doing co-immunoprecipitation experiments using cultured neurons.

    Surface EphB2 withdrawal induced by Aβ oligomers was originally reported by us (see Lacor et al., 2007 and ARF related news story). A different interpretation is made as surface EphB2, but not total EphB2, is largely decreased after six hours’ incubation with ADDLs while NR1 and NR2B decrease earlier (three hours), whereas Mucke et al. see loss over several days. Timing for NR1 loss was also in the order of hours for Paul Greengard’s studies (see Snyder et al., 2005). Mucke's study and ours also differ in the type of Aβ oligomer preparations used. Mucke uses alternately Aβ from 7PA2-conditioned media and ADDL preparations. Plus, changes in the ADDL protocol may have generated different Aβ oligomeric species. It cannot be ruled out that the extended exposure time of the Aβ oligomeric preparations in culture would generate oligomeric species that have different characteristics than what was initially added. In this regard, we recently demonstrated that addition of low-molecular-weight Aβ oligomers for a long period of time most probably trigger similar events to those induced by high-molecular-weight Aβ oligomers (MW >50kDa), as the smaller species will accumulate and cluster at synapses and ultimately also get immobilized there where it can trigger synaptotoxicity (Renner et al., 2010).

    Another difference between our study and Mucke’s is cell culture age. We conducted our experiments when synapses are fully mature, whereas experiments here might be looking at a developmental regulation of NMDAR by EphB2. While they conclude that EphB2 is responsible for NMDAR withdrawal, that prediction cannot be drawn from our study, as NMDARs are massively lost prior to EphB2 decrease.

    Co-IP of Aβ oligomers with EphB2 alongside many other receptors was also previously shown by our team (Renner et al., 2010). The co-immunoprecipitation experiments are only an indirect method to determine ligand receptor interaction. The number of potential receptors described to date, such as nAchR, PrP, and mGluR5, and their intricate regulation make it difficult to pinpoint which receptor(s) (and maybe we can expect different receptors for different oligomeric species) are the first to interact with Aβ and trigger synaptotoxicity. In fact, we have also found that competition using antibodies against the extracellular portions of NMDAR1, mGluR5, PrP, or EphB2 are unable to fully abolish the binding of ADDLs, yet prevent different aspects of synaptotoxicity. This strongly suggests that more than one receptor is involved in the Aβ oligomer-receptor complex, and that matters are complicated by different species binding different receptors. In the present study, there seems to be a contradiction between the test tube experiment and the cell experiment, for example, where it is noticeable that in one case monomers are pulled down, while in the other, remarkable amounts of oligomeric species larger than trimers are pulled down.

    The rest of the study very nicely demonstrates that EphB2 overexpression reverses specifically Aβ-triggered synaptic deficits and restores memory function originating in the dentate gyrus of an animal model of AD without a major change in Aβ expression. This report increases the evidence that Aβ oligomers are synaptotoxic ligands, and that major changes occur at synaptic levels prior to amyloid deposition, as initially proposed by Mucke's group.

    View all comments by Pascale Lacor
  4. Synapse loss in the hippocampus and other brain regions is today considered the best neuropathological correlate of cognitive decline in Alzheimer’s disease. Eph receptors, the largest family of tyrosine-kinase receptors, stabilize synaptic structure, regulate glutamatergic neurotransmission, and influence synaptic plasticity and memory (Murai and Pasquale, 2004; Klein, 2009). Little attention has been paid, however, to the role of Eph receptors in Alzheimer’s disease synaptic dysfunction. EphB2 is one of the Eph receptors more intensively studied in the adult brain, where it is located in dendritic shafts in the hippocampus and cerebral cortex (Bouvier et al., 2008). In the current paper by Mucke’s group, an early reduction of EphB2 mRNA and protein levels was found in the hippocampus of transgenic hAPP mice, a result consistent with a previous report (Simon et al., 2009).

    In a series of elegant experiments, the authors next found that EphB2 depletion in non-transgenic mice by anti-EphB2 short hairpin RNA induced marked long-term potentiation (LTP) deficits similar to those seen in untreated hAPP mice, probably by impairing NMDA receptor function. Conversely, increasing EphB2 levels in the dentate gyrus of hAPP mice reversed LTP and NMDA-receptor function deficits. The increase in EphB2 expression also ameliorated learning and memory deficits in transgenic hAPP mice. As to the mechanisms involved in EphB2 depletion in hAPP mice, the authors report that Aβ oligomers bind to EphB2 and enhance its proteasomal degradation in primary neuronal cultures. It would, no doubt, be of much interest to assess in future studies the importance of this mechanism in transgenic hAPP mice.

    The study by Mucke’s group is of great value, as it points to a novel target for therapeutic approaches in Alzheimer’s disease. Interestingly, EphB2 receptor levels were also reduced in postmortem hippocampal tissue from patients at an incipient stage of Alzheimer’s disease (Simon et al., 2009). Thus, strategies to manipulate EphB2 receptors, either by increasing receptor levels or by designing drugs able to activate EphB2 signaling pathways, may help in the treatment of synaptic dysfunction associated to Alzheimer’s disease.

    View all comments by Diana Frechilla
  5. Reply to comment by Pascale Lacor
    We much appreciate Pascale Lacor’s interest and comments on our study. As she points out, Aβ binds to multiple proteins and has diverse effects in cell culture models, depending on a plethora of variables. We find it difficult to assess the importance of such findings until the impact of each interaction on brain function has been examined in animal models that more closely simulate the complexity of the intracerebral environment and the chronicity of the human condition. We therefore rely more heavily on the electrophysiological and behavioral analysis of animal models than on the histological or biochemical analysis of cell cultures. Because EphB2 knockdown caused deficits in NMDA receptor function and synaptic plasticity in wild-type mice, while normalizing EphB2 levels prevented such deficits in hAPP transgenic mice, we consider it likely that, at least in the presence of chronically elevated Aβ levels and in vivo, EphB2 depletion is upstream of and mediates NMDA receptor impairments.

    We agree with Dr. Lacor that methodological details could also account for some of the differences observed between pure cell-free conditions and cell cultures, and among cell cultures that differed with respect to source and days in vitro of primary neurons, preparation and concentration of Aβ, and length of Aβ exposure. It is quite possible that the mechanisms underlying acute ADDL-induced loss of NMDA receptors differ from those causing chronic Aβ-dependent NMDA receptor impairments. Additional studies are needed to assess this possibility and the other interesting points Dr. Lacor raised.

    View all comments by Moustapha Cisse

References

News Citations

  1. New Paths to Protect Synapses From Aβ?
  2. Neuronal Glutamate Fuels Aβ-induced LTD
  3. Aβ—Three Places, Three Ways of Wreaking Havoc
  4. Aβ Oligomers: A Fatal Attraction for Glutamate Receptors?

Paper Citations

  1. . Vulnerability of dentate granule cells to disruption of arc expression in human amyloid precursor protein transgenic mice. J Neurosci. 2005 Oct 19;25(42):9686-93. PubMed.
  2. . Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron. 2007 Sep 6;55(5):697-711. PubMed.
  3. . Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci. 2005 Aug;8(8):1051-8. PubMed.
  4. . AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron. 2006 Dec 7;52(5):831-43. PubMed.
  5. . Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science. 2002 Jan 18;295(5554):491-5. PubMed.
  6. . The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function. Neuron. 2001 Dec 20;32(6):1041-56. PubMed.
  7. . Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's disease. J Neurosci. 2007 Jan 24;27(4):796-807. PubMed.
  8. . Early changes in hippocampal Eph receptors precede the onset of memory decline in mouse models of Alzheimer's disease. J Alzheimers Dis. 2009;17(4):773-86. PubMed.
  9. . Deleterious effects of amyloid beta oligomers acting as an extracellular scaffold for mGluR5. Neuron. 2010 Jun 10;66(5):739-54. PubMed.
  10. . Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature. 2011 Jan 6;469(7328):47-52. PubMed.

Other Citations

  1. J20 mice

Further Reading

Papers

  1. . Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature. 2011 Jan 6;469(7328):47-52. PubMed.

News

  1. Amyloid-β Zaps Synapses by Downregulating Glutamate Receptors
  2. Neuronal Glutamate Fuels Aβ-induced LTD
  3. AMPA Receptors: Going, Going, Gone in Aβ-exposed Synapses, PSD95 Knockouts
  4. Do "Silent" Seizures Cause Network Dysfunction in AD?
  5. Aβ—Three Places, Three Ways of Wreaking Havoc
  6. Aβ Oligomers: A Fatal Attraction for Glutamate Receptors?

Primary Papers

  1. . Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature. 2011 Jan 6;469(7328):47-52. PubMed.