Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Abstract

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (␣APF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in ␣APF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric ␣-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a ␤ barrel structural motif.Many age-related neurodegenerative diseases are characterized by the accumulation of amyloid deposits derived from a variety of misfolded proteins (1). These diseases typically have both sporadic and inherited forms, and in many cases the mutations associated with the familial forms are in the gene encoding the protein that accumulates or in genes directly related to its production, processing, or accumulation (2). The genetic linkage between the mutant allele and disease is evidence of the causal relationship of amyloid accumulation to pathogenesis, and many of the mutations either destabilize the natively folded state, produce more amyloidogenic protein, or they increase its propensity to aggregate (3). Although fibrillar amyloid deposits are among the most obvious pathognomonic features of disease, their role in pathogenesis is not clear. The extent of fibrillar amyloid plaque deposition does not correlate well with Alzheimer's disease pathogenesis, and there are a significant number of non-demented individuals that have equivalent amounts of amyloid plaques as disease patients (4). Pathological changes are observed in transgenic animals before the onset of amyloid plaque accumulation (5, 6), and it has been reported that soluble A␤ oligomers correlate better with dementia than insoluble, fibrillar deposits (7, 8), suggesting that oligomeric forms of A␤ may represent the primary toxic species. Soluble oligomers have been implicated as the primary ...

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

“…Recently, a 12-16-mer oligomeric species of A␤42 called "globulomers" was reported to be formed independently of the fibril formation pathway (18), and a similar observation was reported for a 9 -15-mer species generated in the presence of SDS (19). Moreover, annular protofibrils (APFs) formed from prefibrillar oligomers (PFOs) failed to convert to fibrils even after an extended period of time, suggesting that these pathogenic oligomers are formed outside the nucleation-dependant fibril formation pathway (20). Reports such as these have shown that there are multiple pathways of aggregation that compete with fibril formation, especially during the prenucleation stage, giving rise to several kinds of oligomers.…”

Transformation of amyloid β(1–40) oligomers into fibrils is characterized by a major change in secondary structure

“…Aggregation intermediates could be on-pathway or off-pathway to give rise to fibrils [9,24]. Moreover, it has been suggested that oligomerization and fibrillization could be independent processes which may be selectively inhibited [23,48,49].…”

“…Assembly of Ab monomers leads to soluble aggregates, namely oligomers, presenting b-structure [6]. Many types of Ab oligomers have been described and linked to AD etiology: dimers [7], trimers, tetramers, dodecamers (Ab*56) [8], Ab-derived diffusible ligands (ADDLs), protofibrils and also annular oligomers [9]. Further aggregation gives rise to insoluble fibrils causing extraneuronal deposits in the brain.…”

Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

“…Metastable Aβ heterogeneous oligomers that can be visualized by TEM and AFM and range in size from ~42 kDa to > 1 mDa 12,13,15,16,18,20,21,93 • Protofibrils Metastable, heterogeneous, -sheet rich prefibrillar structures that have been observed by AFM and TEM during amyloid fibril formation. Different protofibril preparations of Aβ reveal heterogeneous mixtures of discrete β-sheet aggregates of different sizes (50-1,500 kDa) and morphologies (spherical (5-20 nm), annular structures and smooth, curvilinear structures (5 nm in diameter and < 200 nm in length)).…”

“…These β-sheet-rich oligomeric intermediates are collectively referred to as prefibrillar aggregates and disappear on fibril formation [11][12][13] . Some of the well-documented prefibrillar morphologies include protofibrils (curvilinear structures less than 200 nm in length and 4-6 nm in diameter) [12][13][14] , Aβ-derived diffusible ligands (ADDLs; spherical aggregates 3-5 nm in diameter) 15 , Aβ-annular assemblies (doughnut-like structures; outer diameter 8-12 nm and inner diameter 2-2.5 nm) [16][17][18] and LMW oligomers 17,19 (Table 1). Whether some of these species represent off-or on-pathway intermediates to amyloid fibril formation remains unknown.…”