Ion channel formation by N-terminally truncated Aβ (4-42): relevance for the pathogenesis of Alzheimer's disease

Abstract

Aβ deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aβ_1-40 and Aβ_1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aβ variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aβ_4-42 exhibits a high tendency to form β-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aβ_4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aβ_4-42. An Aβ variant with a double truncation at phenylalanine-4 and leucine 34, (Aβ_4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aβ species in AD pathophysiology.

Keywords: Amyloid; Electrophysiology; Oligomers; Truncated Aβ peptides.

Figure 1:. Biophysical and structural analysis of Aβ_4–42 and Aβ_4–34 truncated peptides.

The purity of the peptides was assessed via MALDI-TOF (A) performed in a Bruker Daltonics Autoflex MALDI-TOF mass spectrometer (Bremen, Germany) in linear mode using standard instrument settings at the New York University Mass Spectrometry Core for Neuroscience. External calibration was performed using human adrenocorticotropic hormone peptide 18–39 (average mass = 2465.68 Da) and insulin (average mass = 5733.49 Da). In all cases MS spectra were processed and analyzed by FlexAnalysis (Bruker Daltonics). The different Aβ homologues, pre-treated in HFIP and reconstituted in physiologic salt concentration containing buffer, were incubated at 37°C for up to 2 hours. (B) CD spectra of Aβ_4–42 (red line) and Aβ_4–34 (black line) incubated 2h under physiological salt concentrations. Graph is representative of three independent experiments; (C) Fluorescence evaluation of Thioflavin T binding to the respective synthetic homologues (50 μM) either freshly reconstituted (open bars) or after 2h incubation (patterned bars). Results are expressed in arbitrary units (A.U.) and represent the mean ± SEM of three independent experiments after subtraction of blank levels. (D) Structural assessment of Aβ4–42 2h oligomerization visualized by EM after negative staining. (E) EM analysis of Aβ4–34 oligomerization after 2h incubation. In both (D) and (E), bar represents 100 nm

Figure 2:. AFM images (height and amplitude error) of Aβ_4–42 and Aβ_4–34 interaction with DOPC bilayer.

A) Aβ_4–42 and B) Aβ_4–34 incubated on plane mica surface show distribution of monomers and oligomers. The white horizontal line depicts a section through the scanned area whose corresponding profile is shown below the image (A: scan size = 1.4um, height scale = 1.9nm; B: scan size = 1.4um, height scale = 3.2nm).

Figure 3:. AFM images of amyloid beta ion channels when reconstituted in lipid bilayer.

A) Aβ_4–42 reconstituted DOPC bilayer showing clusters of protein distributed (scan size = 3.5um, height scale = 9.5nm). B) Aβ_4–34 reconstituted DOPC bilayer showing clusters of protein distributed (scan size = 1.2um, height scale = 5.6nm). C.) Aβ_4–42 reconstituted DOPC bilayer at low peptide concentration (1:1000) shows intact bilayer with oligomeric peptides (blue dotted circles) in pore-like morphology. The inset in C) shows the pore-like structure in isometric view (scan sizes – 1μm, inset scan size 50nm, height scale 2.3nm). D) Amplitude error (which is proportional to the feedback signal and very sensitive to height changes on the surface) image of Aβ_4–34 reconstituted DOPC bilayer at low peptide concentration (1:1000) shows intact bilayer with oligomeric peptides (black dotted circles) in pore-like morphology. The inset in D) shows the pore-like structure in isometric view. (scan sizes = 650nm, inset scan size = 60nm; height scale = 4.6mV)

Figure 4.. Lipid bilayer membrane conductivity initiated by truncated Aβ peptides.

A) Conductivity initiated by Aβ_4–42 peptide. B) Conductivity initiated by Aβ_4–34 peptide. C) Aβ_4–42 conductivity inhibition by monoclonal antibody 18H6. In all cases: chamber solution contains 100 mM NaCl, 20 mM Tris-Citrate buffer pH 7.4. Membrane formed from POPE/POPG 2:1 w/w, final concentration of lipids 1.5% in n-heptane. Top lines represent transmembrane currents, bottom lines - holding potential.