Growth of β-Amyloid(1−40) Protofibrils by Monomer Elongation and Lateral Association. Characterization of Distinct Products by Light Scattering and Atomic Force Microscopy

Abstract

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.

Amyloid-β Protofibrils Differ from Amyloid-β Aggregates Induced in Dilute Hexafluoroisopropanol in Stability and Morphology

“…Aliquots of the water stocks were flash frozen in ethanol/dry ice, stored at Ϫ80°C, and thawed at room temperature when needed. Any preformed aggregates were removed from stock solutions by SEC on Superdex 75, and concentrations of A␤ (shown under "Results" to be monomeric) were determined by absorbance with a calculated extinction coefficient of 1450 cm Ϫ1 M Ϫ1 at 276 nm as previously described (35). In some experiments, A␤ peptides were radiolabeled by reductive methylation of primary and secondary amino groups (46) as described previously (35).…”

“…Any preformed aggregates were removed from stock solutions by SEC on Superdex 75, and concentrations of A␤ (shown under "Results" to be monomeric) were determined by absorbance with a calculated extinction coefficient of 1450 cm Ϫ1 M Ϫ1 at 276 nm as previously described (35). In some experiments, A␤ peptides were radiolabeled by reductive methylation of primary and secondary amino groups (46) as described previously (35). [ 3 H]HCHO was used directly (98 mCi/mmol; PerkinElmer Life Sciences) or after dilution (10 Ci/mmol; ARC) with unlabeled HCHO.…”

“…In this process, monomeric A␤ associates noncovalently to form nuclei or "seeds," which then grow into soluble protofibrils and insoluble fibrils. Nucleus formation is considered rate-limiting in this process, but this rate is highly variable even in homogeneous aqueous solutions and is sensitive to many conditions including pH, ionic strength, temperature, and agitation (33)(34)(35). If different nuclear structures form, the molecular structure of fibrils formed by polymerization will vary with the structure of the nucleus.…”

“…One measure that has received little attention is aggregate stability. In this report, we document the stability of A␤-(1-40) protofibrils prepared in dilute buffer at neutral pH (35) by measuring their disaggregation rates following dilution. Isolated protofibrils became progressively more stable over time and reached a point where no disaggregation could be detected.…”

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

“…Based on this analysis, the molecular mass of LFAOs was estimated to be between 60 and 200 kDa. In addition, protofibrils (PFs) of A␤42, which are high molecular mass species (Ͼ200,000 Da; Ͼ500-mers), were also generated following a previously established protocol (25,26) and subjected to fractionation under similar conditions. The PF sample exhibited two elution peaks, the PFs at the void volume (V 0 ; Fig.…”