Aβ Oligomers: A Fatal Attraction for Glutamate Receptors?

Despite the risk of getting burned, moths cannot resist a flame. Do some glutamate receptors find amyloid-β (Aβ) just as dangerously attractive? Clustering around synthetic Aβ oligomers embedded in the cell membrane, metabotropic glutamate receptors unleash an influx of calcium that is toxic to neurons, according to a paper in the June 10 Neuron. The receptor clustering could be one of the earliest events in the pathological cascade that leads to Alzheimer disease (AD) according to lead researcher Antoine Triller at the Ecole Normale Superieur, Paris, France. The work was a collaboration between his lab and that of Bill Klein at Northwestern University, Evanston, Illinois.

The researchers discovered the clustering phenomenon using microscopic flames of their own, that is, quantum dot-labeled Aβ oligomers. Quantum dots are small, intense fluorophores that allow the tracking of single particles, a technique Triller uses to observe how molecules diffuse across cell surfaces. He teamed up with Klein to adapt the strategy to studying the movement of Aβ peptides in neuronal membranes. Joint first authors Marianne Renner and Pascale Lacor and colleagues treated cultured hippocampal neurons with synthetic soluble Aβ oligomers (otherwise known as Aβ-derived diffusible ligands or ADDLs; see Chromy et al., 2003) that they first labeled with quantum dot (QD) fluorophores, or biotin—which they detected with QD-coated streptavidin.

The researchers found that, within an hour of application to neuronal cultures, the synthetic Aβ oligomers (mostly 60 kDa species) stick to the cell membrane and cluster near excitatory synapses. It appears the peptides are drawn into the clusters as they laterally diffuse through the cell membrane, not simply taken up from the culture medium, since the researchers rinsed excess Aβ from the cells shortly after applying the oligomer mixture. In fact, single particle tracking (SPT) confirmed that QD-labeled oligomers initially diffuse rapidly along the cell surface, but then quickly slow down, especially around synapses, where the fluorescence due to clustering grows most intense. The tracking suggests that the oligomers are initially free to flutter about the cell membrane, but then get stuck as they form large clusters around excitatory synapses.

What is it about synapses that attracts Aβ oligomers? Other investigators have previously reported that Aβ oligomers can bind to synaptic proteins such as the NMDA receptor subunit NR1 (see ARF related news story on De Felice et al., 2007). Renner and colleagues now report that NR1 and other synaptic proteins including NR2, GluR1, and the metabotropic glutamate receptor mGluR5, immunoprecipitated with Aβ oligomers added to hippocampal neurons. But whereas antibodies to mGluR5 reduced the amount of Aβ oligomers that bound to cultured neurons when applied together, mGluR1 and mGluR2 antibodies did not. Aβ binding fell by half in mGluR5+/- cells and almost totally disappeared in mGluR5-/- cells, suggesting the metabotropic receptor is—quite literally—a major sticking point for Aβ.

If Aβ oligomers cluster in the presence of mGluR5, what happens to the metabotropic receptor in the presence of Aβ? Renner and colleagues noticed that more of mGluR5 becomes detergent insoluble when Aβ oligomers are added, hinting that the receptor might be doing a little clustering of its own. The scientists tested this idea by labeling the receptor with a yellow fluorescent protein to trace its tracks. In the presence of Aβ, mGluR5 fluorescence became brighter at synapses. Diffusion of extrasynaptic mGluR5, determined by QD single particle tracking, dramatically slowed on addition of Aβ oligomers, and the surface area explored by freely diffusing receptors fell by 90 percent. The authors conclude that membrane-bound Aβ oligomers “by direct or indirect interactions, induce the dynamic redistribution of mGluR5 receptors to synapses.” In contrast, Aβ had no effect on distribution of other receptors, such as the GABA or AMPA glutamate receptors. The work provides a rationale for previous studies from Klein’s lab showing that synthetic Aβ oligomers bind to discrete puncta on neurons and activate Arc, which mediates mGluR5 signaling (see Lacor et al., 2004).

Triller told ARF that he believes that Aβ oligomers are acting like an extracellular scaffold that then recruits other proteins. “If you have a protein like Aβ that sticks or binds to a receptor and it stops diffusing, it may encounter other Aβ molecules and they then form microclusters, which get bigger and recruit more and more receptors,” he said.

One consequence of such clustering could be induction of long-term depression, which is induced by recruitment of mGluR5 to synapses and has been linked to Aβ toxicity (see ARF related news story and ARF news story on Li et al., 2009). To explore this idea, Renner and colleagues examined what happens to ionotropic glutamate receptors when Aβ oligomers are added to neurons. They saw loss of NR1 NMDA receptor subunits, which is in keeping with an LTD effect. In addition, more neurons exhibit spontaneous influx of calcium when treated with Aβ oligomers, which could be due to activation of mGluR5. Interestingly, simply cross-linking mGluR5 receptors in the absence of Aβ ablated NR1 subunits and induced calcium influx. The results suggest that Aβ-induced clustering activates mGluR5, leading to downstream effects. Supporting this idea, the researchers found that the mGluR5 antagonist SIB1757 protects neurons against mGluR5 crosslinking and the sequelae of Aβ clustering, including calcium influx and NMDA receptor loss.

“This is a very high-tech paper and a beautiful use of quantum dots,” Gunnar Gouras, Weill Cornell Medical Center, New York, told ARF. He also thought the link between mGluR5 recruitment in response to Aβ and LTD was interesting (see full comment below), but cautioned that other receptor dysfunction might be involved as well. Triller agreed that the clustering could be more widespread. “I think other proteins are likely involved because mGluR5 binds Homer, and Homer may be bound to other elements. So there is probably a molecular complex,” he told ARF. “In that complex, Aβ oligomers could be bound to prion proteins and other receptors.”

Whether the reported interactions may be an early event in AD pathology remains to be seen. Triller told ARF that one next step would be to repeat the experiments in organotypic slices and eventually in vivo to see if the findings hold up in physiological settings. As Gouras wrote in his comment, much of the focus nowadays is on intracellular Aβ, and the relevance of added extracellular Aβ is not clear. The other issue that researchers are grappling with is what form of Aβ is the most important physiologically (see ARF related news story). Different sizes and species of oligomers have been studied, and the 60 kDa oligomers made in vitro may or may not be the most relevant to AD. Dimers isolated from patient tissue are known to be toxic, for example (see ARF related news story on Shankar et al., 2008). Interestingly, those dimers also seem to work in a metabotropic glutamate receptor-dependent manner (see ARF related news story on Li et al., 2009). Triller suggested that mGluR5 might be worth examining as a potential therapeutic target for AD. Some mGluR5 antagonists are already being tested for other conditions, such as fragile X mental retardation (see ClinicalTrials.gov) and Parkinson disease (see Rylander at al., 2010).—Tom Fagan

Comments

  1. These are cutting-edge experiments building on fascinating prior work from the Klein lab showing that extracellular Aβ oligomers appear to selectively bind to excitatory synapses. The focus of the paper is mGluR5; it is quite interesting that there is differential Aβ-related lateral diffusion in and out of synapses among the synaptic receptors that they assayed. They consistently used excellent controls. The Aβ-related mGluR5 recruitment to synapses, which also occurs with LTD, is certainly interesting. A challenge is that they show many different synaptic receptors binding to Aβ42 oligomers and one is again left wondering about the relevance of any given receptor to AD. When we initiated this line of work looking for Aβ-related changes in synapses (Almeida et al., 2005), we used various antibodies against pre- and post-synaptic markers. Some changed (PSD95, glutamate receptors) while others didn’t. The challenge is to figure out which Aβ-related synapse change comes first, or is most critical. I am not sure how much headway we are really making on this front. I will add that I also remain a bit uncertain about the relevance to AD of adding extracellular Aβ, since in my view more evidence currently points to less Aβ secretion and more intraneuronal accumulation with AD pathogenesis (Gouras et al., 2010).

    References:

    Almeida CG, Tampellini D, Takahashi RH, Greengard P, Lin MT, Snyder EM, Gouras GK. Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses. Neurobiol Dis. 2005 Nov;20(2):187-98. PubMed.

    Gouras GK, Tampellini D, Takahashi RH, Capetillo-Zarate E. Intraneuronal beta-amyloid accumulation and synapse pathology in Alzheimer's disease. Acta Neuropathol. 2010 May;119(5):523-41. PubMed.

    View all comments by Gunnar Gouras

  2. The very interesting report by Renner et al. demonstrates Aβ-induced clustering of mGluR5 receptors and suggests that membrane-bound Aβ oligomers interact with mGluR5. Since chemical crosslinking of the receptor and application of Aβ oligomers produce similar synaptotoxic effects of mGluR5, the data point to (direct or indirect) Aβ-mediated crosslinking of mGluR5 receptors and thus support the idea that Aβ can function as a receptor crosslinker (cf. Aβ-crosslinker-hypothesis).

    Both mGluR5 and the neurotrophin receptor p75 influence NMDAR-dependent long-term depression elicited by low-frequency stimulation; while an mGluR5 antagonist largely prevents both induction and maintenance of LTD (e.g., Popkirov and Manahan-Vaughan, 2010), it is maintenance of LTD that is principally impaired in p75 knockout mice (Rösch et al., 2005). Modulation of mGluR5-induced LTD by p75 would agree with the crucial involvement of caspase-3 in LTD (Li et al., 2010), because stimulated p75 can induce caspase-3 activation. Modulation of mGluR5-induced LTD also fits with a likely interaction between p75 and NMDAR via PSD-95 and an inhibitory influence of p75 on NMDAR currents (Sandoval et al., 2007), which would also protect against excitotoxicity.

    P75 importantly contributes to Aβ-induced hippocampal degeneration (Sotthibundhu et al., 2008; Knowles et al., 2009), is activated by Aβ oligomers via an Aβ-binding site within its neurotrophin-binding region, and has a second binding site for Aβ within its juxtamembrane stalk region that should serve Aβ-mediated crosslinking. Although a functional cooperation of mGluR5 and p75 without direct association of the two receptors is conceivable, the idea of an Aβ-mediated interaction of mGluR5 and p75 is appealing in view of the above arguments; such an interaction could also link mGluR5 with the NMDAR complex.

    References:

    Knowles JK, Rajadas J, Nguyen TV, Yang T, Lemieux MC, Vander Griend L, Ishikawa C, Massa SM, Wyss-Coray T, Longo FM. The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo. J Neurosci. 2009 Aug 26;29(34):10627-37. PubMed.

    Li Z, Jo J, Jia JM, Lo SC, Whitcomb DJ, Jiao S, Cho K, Sheng M. Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization. Cell. 2010 May 28;141(5):859-71. PubMed.

    Popkirov SG, Manahan-Vaughan D. Involvement of the metabotropic glutamate receptor mGluR5 in NMDA receptor-dependent, learning-facilitated long-term depression in CA1 synapses. Cereb Cortex. 2011 Mar;21(3):501-9. PubMed.

    Rösch H, Schweigreiter R, Bonhoeffer T, Barde YA, Korte M. The neurotrophin receptor p75NTR modulates long-term depression and regulates the expression of AMPA receptor subunits in the hippocampus. Proc Natl Acad Sci U S A. 2005 May 17;102(20):7362-7. PubMed.

    Sandoval M, Sandoval R, Thomas U, Spilker C, Smalla KH, Falcon R, Marengo JJ, Calderón R, Saavedra V, Heumann R, Bronfman F, Garner CC, Gundelfinger ED, Wyneken U. Antagonistic effects of TrkB and p75(NTR) on NMDA receptor currents in post-synaptic densities transplanted into Xenopus oocytes. J Neurochem. 2007 Jun;101(6):1672-84. PubMed.

    Sotthibundhu A, Sykes AM, Fox B, Underwood CK, Thangnipon W, Coulson EJ. Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor. J Neurosci. 2008 Apr 9;28(15):3941-6. PubMed.

    View all comments by Rudolf Bloechl

  3. In this paper, it is observed that membrane-attached oligomers of amyloid-β cause clustering of metabotropic glutamate receptors (mGluR5), which leads to an increase in intracellular calcium and causes synapse deterioration. Since mGluR5 is widely distributed in brain regions crucial for memory, such as the hippocampus, this study provides an explanation for selective amyloid-β toxicity and synaptic failure in specific brain regions.

    References:

    Renner M, Lacor PN, Velasco PT, Xu J, Contractor A, Klein WL, Triller A. Deleterious effects of amyloid beta oligomers acting as an extracellular scaffold for mGluR5. Neuron. 2010 Jun 10;66(5):739-54. PubMed.

    View all comments by Mordhwaj Parihar

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References

News Citations

  1. Aβ Oligomers and NMDA Receptors—One Target, Two Toxicities
  2. AMPA Receptors: Going, Going, Gone in Aβ-exposed Synapses, PSD95 Knockouts
  3. Neuronal Glutamate Fuels Aβ-induced LTD
  4. San Diego: Oligomers Live Up to Bad Reputation, Part 3
  5. Paper Alert: Patient Aβ Dimers Impair Plasticity, Memory

Paper Citations

  1. Chromy BA, Nowak RJ, Lambert MP, Viola KL, Chang L, Velasco PT, Jones BW, Fernandez SJ, Lacor PN, Horowitz P, Finch CE, Krafft GA, Klein WL. Self-assembly of Abeta(1-42) into globular neurotoxins. Biochemistry. 2003 Nov 11;42(44):12749-60. PubMed.
  2. De Felice FG, Velasco PT, Lambert MP, Viola K, Fernandez SJ, Ferreira ST, Klein WL. Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol Chem. 2007 Apr 13;282(15):11590-601. PubMed.
  3. Lacor PN, Buniel MC, Chang L, Fernandez SJ, Gong Y, Viola KL, Lambert MP, Velasco PT, Bigio EH, Finch CE, Krafft GA, Klein WL. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J Neurosci. 2004 Nov 10;24(45):10191-200. PubMed.
  4. Li S, Hong S, Shepardson NE, Walsh DM, Shankar GM, Selkoe D. Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake. Neuron. 2009 Jun 25;62(6):788-801. PubMed.
  5. Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008 Aug;14(8):837-42. PubMed.
  6. Rylander D, Iderberg H, Li Q, Dekundy A, Zhang J, Li H, Baishen R, Danysz W, Bezard E, Cenci MA. A mGluR5 antagonist under clinical development improves L-DOPA-induced dyskinesia in parkinsonian rats and monkeys. Neurobiol Dis. 2010 Sep;39(3):352-61. PubMed.

External Citations

  1. ClinicalTrials.gov

Further Reading

Primary Papers

  1. Renner M, Lacor PN, Velasco PT, Xu J, Contractor A, Klein WL, Triller A. Deleterious effects of amyloid beta oligomers acting as an extracellular scaffold for mGluR5. Neuron. 2010 Jun 10;66(5):739-54. PubMed.

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