Structure–neurotoxicity relationships of amyloid β-protein oligomers

Abstract

Amyloid ␤-protein (A␤) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). ''Oligomer'' is an ill-defined term because many kinds have been reported and they often exist in rapid equilibrium with monomers and higher-order assemblies. We report here results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied by using a combination of CD spectroscopy, Thioflavin T fluorescence, EM, atomic force microscopy (AFM), and neurotoxicity assays. A␤ monomers were largely unstructured, but oligomers exhibited order-dependent increases in ␤-sheet content. EM and AFM data suggest that dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. The structure-activity correlations reported here significantly extend our understanding of the conformational dynamics, structure, and relative toxicity of pure A␤ oligomers of specific order.Alzheimer's disease ͉ toxicity A lzheimer's disease (AD) is the most common form of late-life dementia. Current estimates of AD incidence are Ͼ24 million worldwide, a number that is expected to double every 20 years, reaching 81 million in 2040 (1). AD is a slowly progressive disorder with insidious onset and progressive impairment of episodic memory and executive function coupled with aphasia, apraxia, and agnosia (1).The amyloid ␤-protein (A␤) appears to play an essential role in the pathogenesis of AD. A␤ is produced throughout life through posttranslational processing of the A␤ precursor (APP). Familial forms of AD increase A␤ production or the propensity of A␤ to aggregate (2). The ''amyloid cascade hypothesis'' proposes that assemblies of A␤ initiate a process leading to neuronal dysfunction and cell death (2). The most potent neurotoxic assemblies appear to be oligomeric, rather than fibrillar, in nature (3, 4). For example, oligomers extracted from AD brain potently impair synapse structure and function (5). The smallest of these oligomers appears to be dimeric (5). However, a systematic correlation of oligomer structure and neurotoxic potency has not been reported. This correlation is critical for the targeting and design of disease-modifying therapeutic agents.Efforts to establish rigorous structure-toxicity correlations have been hindered by the complex, dynamic equilibria displayed by A␤ (for recent reviews, see refs. 6 and 7). To enable determination of the oligomer frequency distribution, we have used in situ chemical cross-linking to prevent oligomer dissociation or growth (8-10). Oligomers thus stabilized can be visualized and quantified by SDS/PAGE. However, in theory, the method also could be used to produce pure populations of oligomers of defined order † , enabling the biophysical and biological studies necessary to establish structure-toxic...

Crucial role of nonspecific interactions in amyloid nucleation

“…From a biological and biomedical perspective, it is important to understand the conditions under which oligomeric clusters form, because such species exhibit high cytotoxicity (1,(29)(30)(31). Indeed, there is strong evidence that the disordered oligomers rather than fully grown fibrils are the main pathogenic species in protein aggregation diseases (31)(32)(33).…”

Lipoproteins and Diseases of the Brain

“…It has been suggested that the physical properties of proteins (e.g., aggregate pattern and size) is a crucial determinant in mediating pathogenic toxicity [57,58]. This toxicity occurs independent of their sequences or lengths [59] in a manner that is similar to the aggregation of Aβ in Alzheimer's disease [60] or α-synuclein in Parkinson's disease [61]. Previous studies showed that Pam3-Cys, the synthetic N-terminus of ospA, self-assembled and showed aggregating potential in vitro assays [58,62], which can be related to brain tissue damage including the disruption of synaptic function.…”

Natural Compounds May Open New Routes to Treatment of Amyloid Diseases

“…Small oligomers of Aβ secreted from cell lines that overproduce Aβ 42 inhibit long-term potentiation in hippocampal synaptic transmission [32] and induces neuronal toxicity in mice [35]. Oligomeric forms of αS and Aβ inhibit mitochondrial activity [31,36]. Toxicity of Aβ 42 aggregates ebbs as monomers are depleted, but can be restored by the addition of fresh monomeric protein [31].…”

“…Methionine and tyrosine are easily modified by ROS. Oxidation of methionine 35 increases Aβ aggregation propensity [45] and tri-and tetrameric Aβ oligomers that are cross-linked by radical-induced dityrosine bridges are toxic to cells [36]. It is not clear whether oxidative stress and the generation of ROS is the primary pathologic event or whether it is a consequence of protein misfolding [46].…”

“…Since then, it has been shown that EGCG binds directly to a large number of proteins that are involved in protein misfolding diseases and that EGCG inhibits their fibrillization. Examples include αS [81,82], Aβ [36,82], transthyretin [83], lysozyme [84], the prion protein PrP [85], and the yeast prion Sup35 [86].…”