Synaptotoxicity of Alzheimer beta amyloid can be explained by its membrane perforating property

Abstract

The mechanisms that induce Alzheimer's disease (AD) are largely unknown thereby deterring the development of disease-modifying therapies. One working hypothesis of AD is that Abeta excess disrupts membranes causing pore formation leading to alterations in ionic homeostasis. However, it is largely unknown if this also occurs in native brain neuronal membranes. Here we show that similar to other pore forming toxins, Abeta induces perforation of neuronal membranes causing an increase in membrane conductance, intracellular calcium and ethidium bromide influx. These data reveal that the target of Abeta is not another membrane protein, but that Abeta itself is the cellular target thereby explaining the failure of current therapies to interfere with the course of AD. We propose that this novel effect of Abeta could be useful for the discovery of anti AD drugs capable of blocking these "Abeta perforates". In addition, we demonstrate that peptides that block Abeta neurotoxicity also slow or prevent the membrane-perforating action of Abeta.

Figure 1. Aβ peptide induced formation of membrane perforation in hippocampal neurons.

A, currents induced by a 5 mV depolarizing pulse recorded with control solution at two times in cell-attached mode. B, effect of application of 500 nM (2.2 µg/ml) Aβ via the patch pipette on capacitative membrane current. C, effect of gramicidin (100 µg/ml) on membrane current. D, confocal image shows a neuron stained for 15 minutes with 500 nM fluorescent Aβ. Western blot shows the time dependent association of low molecular weight oligomers with hippocampal cell membranes. E, effect of increasing Aβ concentrations on the time to establish the perforated configuration. F, effects of Aβ gramicidin, and amphotericin on the transferred membrane charge induced by 5 mV depolarization. Each point (mean ± SEM) was measured in a least 6 different hippocampal neurons. * denotes a P<0.05 (n = 6–7 neurons).

Figure 2. Aβ peptide produced cationic perforates comparable to gramicidin.

A, currents were recorded using a cell-attached configuration at the beginning (30 s) and after 15 min of Aβ application via the patch pipette. AMPA and GABA (50 µM) applied to the extracellular membrane induced membrane currents after formation of membrane perforation. B, gradual appearance of synaptically mediated membrane currents after establishing the cell-attached conformation. C, GABA-induced anionic current-voltage relationships obtained during perforated mode with either gramicidin, amphotericin-B or Aβ. D, AMPA induced cationic current using gramicidin, amphotericin -B or Aβ. Each point (mean ± SEM) was measured in a least 5 different hippocampal neurons.

Figure 3. Aβ induced a non channel-like increase in microscopic membrane conductance.

A, current traces show high sensitivity patch recordings obtained in the presence of 500 nM Aβ. The red line represents zero current. B, graph shows an all-point histogram obtained from the recordings in A. C, confocal micrograph shows the peripheral association of fluorescent Aβ to HEK cells. D current trace showing typical single channel behavior from a cell expressing alpha 1 human glycine receptors. Unlike Aβ the current expansion shows clear transitions between closed and open states. E, All point histogram fitted to a single conductance of 92 pS. F–H, traces show either partial or full membrane perforation in the presence of Aβ.

Figure 4. Aβ perforation causes entry of a small organic molecule in parallel with the increase in membrane conductance.

A, the time dependent increase in cellular fluorescence associated with entry of ethidium bromide in presence of Aβ in the pipette. B, the effect of Aβ was blocked by the Na7 peptide. C, Ethidium bromide was unable to enter into the cell in the absence of Aβ. D–E, effect of Aβ on membrane current transferred and fluorescence in the absence and presence of Na7. * denotes a P<0.05 (n = 7–8 neurons).

Figure 5. The effects of Aβ on membrane conductance and synaptotoxicity can be inhibited by small peptides.

A, effect of Aβ(•) on membrane resistance in the absence and presence of Na7 (○ 1µM, ▪ 3 µM, □ 7 µM, ▴ 100 µM). B, effect of Aβ application on intracellular calcium increase and its inhibition by Na7, but not by other ion channel blockers. C, effects of Na7 and low calcium on Aβ-induced destaining of FM1–43. The insets show destaining of FM1–43 (arrows) at 20 min. D, time dependent reduction of synapsin and SV2 induced by Aβ and its inhibition by Na4a and Na7 (50 µM). Each point (mean ± SEM) was measured in a least 5 different hippocampal neurons.

Figure 6. The scheme is a simplified model for association, micro and macro perforation induced by Aβ in cellular membranes.

A, aggregation and binding (association) of Aβ to the neuronal membrane B, smaller perforations are associated to a selective ion influx (gramicin-like ion influx). C, larger perforations allow the entry of large molecules, which include EtBr (∼1.3 nm). All these Aβ effects are blocked by application of anti- Aβ antibody.