Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer's disease and related disorders

Abstract

Several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, are characterized pathognomonically by the presence of intra- and/or extracellular lesions containing proteinaceous aggregates, and by extensive neuronal loss in selective brain regions. Related non-neuropathic systemic diseases, e.g., light-chain and senile systemic amyloidoses, and other organ-specific diseases, such as dialysis-related amyloidosis and type-2 diabetes mellitus, also are characterized by deposition of aberrantly folded, insoluble proteins. It is debated whether the hallmark pathologic lesions are causative. Substantial evidence suggests that these aggregates are the end state of aberrant protein folding whereas the actual culprits likely are transient, pre-fibrillar assemblies preceding the aggregates. In the context of neurodegenerative amyloidoses, the proteinaceous aggregates may eventuate as potentially neuroprotective sinks for the neurotoxic, oligomeric protein assemblies. The pre-fibrillar, oligomeric assemblies are believed to initiate the pathogenic mechanisms that lead to synaptic dysfunction, neuronal loss, and disease-specific regional brain atrophy. The amyloid beta-protein (Abeta), which is believed to cause Alzheimer's disease (AD), is considered an archetypal amyloidogenic protein. Intense studies have led to nominal, functional, and structural descriptions of oligomeric Abeta assemblies. However, the dynamic and metastable nature of Abeta oligomers renders their study difficult. Different results generated using different methodologies under different experimental settings further complicate this complex area of research and identification of the exact pathogenic assemblies in vivo seems daunting. Here we review structural, functional, and biological experiments used to produce and study pre-fibrillar Abeta assemblies, and highlight similar studies of proteins involved in related diseases. We discuss challenges that contemporary researchers are facing and future research prospects in this demanding yet highly important field.

Fig. 1. Comparison of photo-cross-linking using Aβ peptides from different sources

Synthetic Aβ from Global Peptide (G) and the UCLA Biopolymers Laboratory (U), and recombinant Aβ from rPeptide (R) were prepared in 10 mM sodium phosphate, pH 7.4 at 2 mg/ml nominal concentration and filtered through a 10-kDa molecular-weight cut-off filter [77]. Each filtered peptide was cross-linked using PICUP [73]. The resulting cross-linked oligomers were subjected to SDS-PAGE and silver-staining. The data suggest that Aβ40 from Global peptide contained contaminants that prevented cross-linking and that Aβ42 from rPeptide aggregated during the filtration step and was hardly detectable.

Fig. 2. Punctate ADDL-binding to neurons

ADDLs isolated from AD brain or prepared in vitro show identical punctate binding to neuronal cell-surface proteins. Cultured hippocampal neurons were incubated with soluble extracts of human brain or synthetic ADDLs. Immunoreactivity against ADDLs was visualized by microscopy using M93 antibody. Soluble AD-brain proteins (a), soluble control-brain proteins (b), synthetic ADDLs (c), and synthetic ADDLs pre-treated (1 h) with oligomer-specific antibody M71 (d) are shown. Small puncta distributed along neurites, are evident for AD extracts and synthetic ADDLs, but not for control extracts or antibody-preadsorbed ADDLs (Scale bar = 10 µm). Adopted with permission from [27].

Fig. 3. Hierarchical aggregation process of HypF-N

(a) Tapping AFM images taken under liquid (height data, scan size 670 nm, Z range 20 nm) of HypF-N globular aggregates observed a few hours after the onset of the aggregation process in the presence of 30% trifluoroethanol (TFE). Scale bar = 100 nm. Inset, STM image (height data, scan size 42 nm, Z range 15 nm) of globular aggregates obtained under the same conditions, but diluted 1:50 prior to deposition onto the substrate; the globules are apparently asymmetric and tend to form a defined mutual orientation. Scale bar = 10 nm. (b) HypF-N crescents and a ring observed after 3 days of incubation in 30% TFE (scan size 5.3 µm, Z range 45 nm). Inset, high resolution image of a ring (scan sizel.9 µm, Z range 120 nm), revealing its globular components, taken after 5 days of incubation in 30% TFE. Scale bars = 400 nm. (c) Tapping-mode AFM image taken in air (height data) after three days of incubation in 30% TFE showing the co-existence of annular structures with thin and wide ribbon-like fibrils. Scan size 4.8 µm, Z range 40 nm. Scale = 500 nm. (d) Tapping-mode AFM images taken in air (height data) of mature fibrils obtained in 30% TFE showing supercoiled fibrils after eight days (scan size 3.6 µm, Z range 80 nm). Adopted with permission from [252].