Amyloid-β protofibrils: size, morphology and synaptotoxicity of an engineered mimic

Abstract

Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ42cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. Aβ42cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put Aβ42cc protofibrils into the same class of species as fibrillar oligomers of wild type Aβ. Aβ42cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aβ42cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aβ42cc and aggregates of wild type Aβ42. We suggest that Aβ42cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aβ.

Figure 1. Analysis of Aβ₄₂ cc morphology using atomic force microscopy (AFM).

(A) AFM image of Aβ₄₂ cc protofibrils on dry mica surface. (B) Average z-heights and cross-sections of Aβ₄₂ cc (black) and wild type Aβ₄₂ (red) protofibrils (grey lines represent measurements of 20 Aβ₄₂ cc protofibrils). (C-F) High magnification AFM images of single protofibrils of Aβ₄₂ cc (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 Aβ₄₂ cc 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 Aβ₄₂ cc 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 Aβ₄₂ cc protofibrils in solution monitored by analytical ultracentrifugation.

(A) A subset of the raw sedimentation velocity centrifugation data of 300 µM Aβ₄₂ cc protofibrils at 20°C recorded over a period of 20 h. (B) Sedimentation coefficient distribution of Aβ₄₂ cc protofibrils analyzed using a continuous c(s) distribution model.

Figure 4. Aβ₄₂ cc protofibrils expose binding sites for the ANS dye.

Fluorescence emission spectra of 50 µM free ANS (red) and of ANS in the presence of Aβ₄₂ cc protofibrils (black) or monomeric Aβ₄₂ cc (green). Peptide concentrations are in both cases 10 µM monomer units.

Figure 5. OC serum dot blot.

The fibril specific OC serum recognizes Aβ₄₂ cc protofibrils and wild type Aβ₄₂ fibrils, but not monomeric Aβ₄₂ cc or protofibrils that have been denatured by boiling in SDS.

Figure 6. SDS-PAGE showing the separation of protein interaction partners of Aβ₄₂ cc 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 Aβ₄₂ cc, as reported previously .

Figure 7. Effect of Aβ₄₂ cc 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 Aβ₄₂ cc protofibrils is not significant.