Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm

Abstract

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Abeta) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Abeta specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Abeta oligomers (approximately >60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Abeta oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Abeta channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.

Figure 1

Ion channel hypothesis for AD pathology. A. Aβ, a 39–43 AA long peptide is cleaved from the single transmembrane spanning precursor, called amyloid precursor protein (APP). B. Aβ undergoes multi-step oligomerization, ranging from the formation of small oligomers to large oligomers (commonly referred to as the ADDLs) to protofibrils to fibrils and plaques. In normal physiological conditions, most of the secreted amyloids remain monomers or small oligomers. C. Several mechanisms for neurotoxic activity of Aβ have been proposed, such as activation of a signaling pathway by extracellular aggregates (fibrils, plagues), induction of oxidative stress due to protein aggregates, or recruitment of factors by intracellular aggregates. Large oligomers (or the ADDLs) may be toxic by non-selectively disrupting cell membrane viability, like a hammer hitting a leaf). Small oligomers (mono- and dimers) fold into the membrane forming ion channels. D. A common denominator for AD toxicity is mainly the gain of intracellular calcium, the severity of toxicity depends upon the level of calcium loading as well as the cell’s defense capability. See [4, 14, 26, 47, 48, 50].

Figure 2

Morphological changes induced in neuronal cells by Aβ(1–42). Cells are loaded with calcein AM. Images A, C, E and G are taken as control before online addition of Aβ(1–42). Images B, D, F and H are taken 45 minutes after addition of Aβ(1–42). No morphological degeneration is observed for cells not treated with Aβ(1–42) (panels A and B). Significant degeneration is observed for cells treated with 50 nM Aβ(1–42) (panel D), indicated by arrows. Loss of cell-cell contact and neurite beading is observed after treatment with 500 nM Aβ(1–42) (panel F), and 5 μM Aβ(1–42) leads to loss of neuronal processes and fragmented neurites [11]. Scale bars 10 μm.

Figure 3

Cell viability assay of neuronal cells treated with Aβ(1–42). Cellular toxicity induced by Aβ(1–42) was blocked by Zn²⁺ and by removal of extracellular calcium, but not by tachykinin or NMDA antagonist. Fluorescence images of cells treated with calcein (live cells, left panels) and ethidium homodimer 1 (dead cells, right panels) are shown after Aβ(1–42) treatment. Panels A and B show control cells not treated with Aβ(1–42), panels C and D show cells treated with 10 μM Aβ(1–42), panels E and F show cells after Aβ(1–42) treatment in the absence of extracellular calcium, panels G and H show cells after Aβ(1–42) treatment in the presence of 50 μM ZnCl₂, panels I and J show cells after Aβ(1–42) treatment in the presence of 20 μM tachykinin (physalaemin), and K and L show cells after Aβ(1–42) treatment in the presence of 20 μM MK-801, an NMDA receptor antagonist [11].

Figure 4

Confocal Calcium Green fluorescence images of endothelial cells before (A), and after addition of 0.22 μM (B), and 2.2 μM (C) of Aβ(1–42). Observed Calcium waves are consistent with extracellular calcium uptake and amyloid [6].

Figure 5

Amyloid beta(1–42) deposited on mica after incubation in PBS for 3 hours (left) and 48 hours (right). Even after long incubation time the majority of peptide is still in globular form, although some fibrils are observed [11].

Figure 6

Electrophoresis of British amyloid (ABri), Danish Amyloid (ADan), Alpha-synuclein, Amylin, Serum Amyloid A, and Amyloid Beta (1–40), showing membrane induced oligomerization. In solution monomers, and sometimes dimers (ABri, Amylin, Aβ(1–40)) are observed. In the membrane oligomers up to octamers are observed. The insets show circular dichroism spectra of the amyloids in solution.

Figure 7

Uptake of ⁴⁵Ca²⁺ in lipid vesicles reconstituted with Aβ(1–40). Uptake is blocked partially by anti- Aβ(1–40) antibody, and completely blocked by Zinc.

Figure 8

AFM images of liposomes prepared by bath sonication, reconstituted with (left) and without (right) Aβ(1–40) [25].

Figure 9

AFM images of Aβ(1–40) and Aβ(1–42) reconstituted in bilayers. Images are amplitude images acquired in tapping mode. Donut shaped features are observed with sizes ranging from 8–15 nm [11, 26]. The inset shows a larger scale membrane patch.

Figure 10

High resolution AFM images of different amyloids reconstituted in lipid bilayers. The image sizes are 20 nm. Different conformations and subunit arrangements can be observed, consistent with the results from parallel electrophoresis studies [26].

Figure 11

Structural evidence for amyloid ion channel paradigm and its relevance to AD pathophysiology. An array of studies using combination of AFM, biochemical analysis, fluorescence microscopy and electrical recording supports the ion channel model. A. Small oligomers of Aβ formed by enzymatic cleavage of APP remain globular up to 24 hours of incubation. B. AFM image of globular/monomeric amyloids on mica surface. C. AFM image of an array of ion channels randomly distributed in a lipid bilayer, the inset shows a larger scale bilayer patch. D. Ion channel structure and activity. AFM images of individual ion channels for Aβ (left) and α-Synuclein (right) with a central pore as well as their single channel currents recorded in parallel. Below the electrical recording graphs are shown theoretical models of Aβ and α-Synuclein ion channels as well as their membrane insertion [–49], with a top view and a cross-sectional view of transmembrane insertion. E. Toxicity by calcium uptake and disruption of ionic homeostasis through Aβ- or pre-existing ion channels ultimately leads to neuronal degeneration.