Figure 1.
Analysis of Aβ42cc morphology using atomic force microscopy (AFM).
(A) AFM image of Aβ42cc protofibrils on dry mica surface. (B) Average z-heights and cross-sections of Aβ42cc (black) and wild type Aβ42 (red) protofibrils (grey lines represent measurements of 20 Aβ42cc protofibrils). (C-F) High magnification AFM images of single protofibrils of Aβ42cc (C) and wild type Aβ42 (D; identified in aggregation reaction mixtures, Fig. S2), and of amyloid fibrils of Aβ40 (E) and Aβ42 (F). Measured z-heights of particles are indicated in panels C-F.
Figure 2.
Size distribution of Aβ42cc protofibrils measured using different methods.
The blue and red lines/symbols represent data from atomic force microscopy. One sample (blue) was washed briefly with deionized water, while a second sample (red) was washed extensively. The lengths of ca. 1500 protofibrils were measured in each case. The gray dashed line reflects an expected distribution corresponding to the analytical ultracentrifugation measurements (Fig. 3) assuming that Aβ42cc protofibrils have a dehydrated diameter of 3.1 nm. The black line represents the distribution of apparent hydrodynamic radius obtained from nanoparticle tracking analysis using a NanoSight microscope.
Figure 3.
Size distribution of Aβ42cc protofibrils in solution monitored by analytical ultracentrifugation.
(A) A subset of the raw sedimentation velocity centrifugation data of 300 µM Aβ42cc protofibrils at 20°C recorded over a period of 20 h. (B) Sedimentation coefficient distribution of Aβ42cc protofibrils analyzed using a continuous c(s) distribution model.
Figure 4.
Aβ42cc protofibrils expose binding sites for the ANS dye.
Fluorescence emission spectra of 50 µM free ANS (red) and of ANS in the presence of Aβ42cc protofibrils (black) or monomeric Aβ42cc (green). Peptide concentrations are in both cases 10 µM monomer units.
Figure 5.
The fibril specific OC serum recognizes Aβ42cc protofibrils and wild type Aβ42 fibrils, but not monomeric Aβ42cc or protofibrils that have been denatured by boiling in SDS.
Figure 6.
SDS-PAGE showing the separation of protein interaction partners of Aβ42cc protofibrils (PF) extracted from human serum (M = molecular mass markers).
The arrow indicates the band corresponding to apolipoprotein E. Essentially no binding is observed in control experiments with glycine-coated beads (-PF). The strong bands around 15 kDa are SDS-stable dimers and trimers of Aβ42cc, as reported previously [16].
Figure 7.
Effect of Aβ42cc protofibrils (red) and wild type Aβ42 oligomers (blue) on spontaneous synaptic activity in mouse primary hippocampal neurons grown on a multielectrode array (MEA) chip.
Changes in firing rates are normalized to the initial electrical activity in the absence of treatment and compared to buffer-treated neurons: ** – p<0.0015, * – p<0.026 (Student's t-test); the difference between Aβ42 oligomers and Aβ42cc protofibrils is not significant.