. Single-channel Ca(2+) imaging implicates Aβ1-42 amyloid pores in Alzheimer's disease pathology. J Cell Biol. 2011 Oct 31;195(3):515-24. PubMed.

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  1. This is an elegant paper that uses the “optical patch clamp” method to resolve individual calcium transients resulting from incorporation of Aβ42 oligomers into the membrane. Previous electrophysiological recordings of Aβ42-induced channels in lipid bilayers already supported the hypothesis that Aβ42 oligomers form calcium-permeable channels in the membranes. Imaging experiments in this paper by Demuro at al. demonstrate that Aβ42 oligomers can form calcium-permeable pores in native membranes. Moreover, the use of imaging methods enabled the authors to perform parallel single-channel studies of Aβ42-formed channels. The results provide important quantitative data about the kinetic and conductance properties of Aβ42-formed pores. This information is useful for many researchers who study mechanisms of Aβ42 toxicity.

  2. Over the past 18 years, since the proposal of the channel hypothesis of Alzheimer's disease by Arispe et al. (1993), considerable controversy has surrounded this theory. Despite repeated demonstrations of the channel-forming ability of various amyloid peptides in planar lipid bilayers, liposomes, and cells, there has been much discussion over whether the ion permeability induced by Aβ and other amyloid peptides is due to a channel or non-channel mechanism. The present paper uses total internal reflection fluorescence (TIRF) microscopy to image calcium influx in Xenopus laevis oocytes. The results are clear, and are a dramatic confirmation of channel formation by Aβ1-42. The authors find that Aβ1-42 oligomers evoke single-channel calcium fluorescent transients, which resemble those of classical ion channels, but cannot be due to endogenous channels since the oocyte membrane lacks them. Because these transients are of clear and distinct but variable magnitudes resembling those caused by native calcium channels, they rule out a membrane-thinning mechanism such as that proposed previously (Sokolov et al., 2006). Because the interaction of Aβ with endogenous ion channels is also ruled out in oocyte membranes, this clearly leads to the conclusion that Aβ1-42 oligomers must be forming the calcium permeable channel pathway. Since the oligomers used here are essentially identical to those used by Sokolov et al., the “non-channel” hypothesis is no longer tenable.

    Intriguingly, the single-channel fluorescence transients are variable in amplitude and open probability, similar to the reports of channels recorded by electrophysiological techniques. Furthermore, the channels are blocked by sub-millimolar levels of zinc ion, a property exhibited not only by Aβ channels, but by virtually all amyloid peptide channels (Kagan et al., 2004). The Aβ used in this study incorporates a wide range of oligomers with masses from 35 to 300 kiloDaltons which would consist of five to 40 monomers per oligomer and were recognized by sequence and fibrillar-specific Aβ antibodies. Thus, these oligomers appear to essentially overlap with the sizes of oligomers previously implicated in Aβ neuronal toxicity. The growth with time of these calcium transients in the experiment is also consistent with aggregation of oligomers and incorporation of new monomers into pre-existing oligomers previously observed in planar lipid bilayer studies. Although this work provides strong evidence favoring direct channel formation by Aβ oligomers over other mechanisms, it does not directly address whether this is the mechanism by which Aβ works in vivo, nor does it address which cellular membrane—plasma or intracellular—is attacked by Aβ pores in vivo. Nevertheless, the ability of this technique to observe many pores simultaneously makes those in-vivo experiments more feasible, both for Aβ1-42 and for other amyloid peptides.

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    . Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):567-71. PubMed.

    . Giant multilevel cation channels formed by Alzheimer disease amyloid beta-protein [A beta P-(1-40)] in bilayer membranes. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10573-7. PubMed.

    . Soluble amyloid oligomers increase bilayer conductance by altering dielectric structure. J Gen Physiol. 2006 Dec;128(6):637-47. PubMed.

    . Amyloid peptide channels. J Membr Biol. 2004 Nov;202(1):1-10. PubMed.