Alzheimer's disease (AD) is an archetype of a class of diseases characterized by abnormal protein deposition. In each case, deposition manifests itself in the form of amyloid deposits composed of fibrils of otherwise normal, soluble proteins or peptides. An ever-increasing body of genetic, physiologic, and biochemical data supports the hypothesis that fibrillogenesis of the amyloid beta-protein is a seminal event in Alzheimer's disease. Inhibiting A beta fibrillogenesis is thus an important strategy for AD therapy. However, before this strategy can be implemented, a mechanistic understanding of the fibrillogenesis process must be achieved and appropriate steps selected as therapeutic targets. Following a brief introduction to AD, I review here the current state of knowledge of A beta fibrillogenesis. Special emphasis is placed on the morphologic, structural, and kinetic aspects of this complex process.

According to Thomas Kuhn, the success of 'normal science,' the science we all practice on a daily basis, depends on the adherence to, and practice of, a paradigm accepted by the scientific community. When great scientific upheavals occur, they involve the rejection of the current paradigm in favor of a new paradigm that better integrates the facts available and better predicts the behavior of a particular scientific system. In the field of Alzheimer's disease, a recent example of such a paradigm shift has been the apparent rejection of the 'amyloid cascade hypothesis,' promulgated by Hardy and Higgins in 1992 to explain the etiology of Alzheimer's disease, in favor of what has been referred to as the 'oligomer cascade hypothesis'. This paradigm shift has been breathtaking in its rapidity, its pervasiveness in the Alzheimer's disease field, and its adoption in an increasing number of other fields, including those of Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and the prionoses. However, these facts do not mean, a priori, that the experiments extant, and any re-interpretation of them, should be accepted by rote as support for the new paradigm. In the discussion that follows, I consider the foundational studies leading to the oligomer cascade hypothesis and evaluate the current state of the paradigm. I argue here that, more often than not, insufficient rigor has been applied in studies upon which this new paradigm has been based. Confusion, rather than clarity, has resulted. If the field is to make progress forward using as its paradigmatic basis amyloid β-protein oligomerization, then an epistemological re-evaluation of the amyloid β-protein oligomer system is required.

Amyloid proteins cause a number of progressive, degenerative diseases. Among these is Alzheimer's disease (AD), the etiology of which is linked to the formation of neurotoxic assemblies by the amyloid beta-protein (Abeta). The clinical importance of AD has stimulated intense interest in the mechanisms of Abeta folding and self-assembly. Studying these phenomena in vitro requires the preparation of Abeta peptide stocks that are well defined and display reproducible biophysical and biological behaviors. Unfortunately, the propensity of Abeta to self-assemble has made this goal difficult. I discuss here a biphasic strategy for preparing Abeta for structural and functional studies. The strategy involves sodium hydroxide pretreatment of synthetic Abeta, followed by size fractionation procedures. This approach produces Abeta solutions that have been used successfully in a variety of in vitro and in vivo experimental systems.

More than 20 diseases have been identified which are caused by the deposition of amyloid. Natural and chemically synthesized amyloidogenic proteins are used widely to study the structure, assembly, and physiologic effects of both oligomeric and fibrillar forms of these proteins. In many cases, conflicting results arise in these studies, in part owing to difficulties in reproducibly preparing amyloidogenic proteins in a well-defined assembly state. To avoid these problems, several methods have been devised that provide reliable means of preparing amyloid-forming proteins for experimental use. Here, we discuss methods that have been used successfully to prepare one such protein, the amyloid beta-protein (Abeta), involved in Alzheimer's disease. Methods for reproducible preparation of Abeta in a well-defined assembly state include isolation of low molecular weight (LMW) Abeta by size exclusion chromatography, filtration through LMW cut-off filters, and solubilization/lyophilization in the presence of reagents which facilitate disassembly of Abeta. These reagents include strong bases and acids, and fluorinated alcohols. These methods, which were originally developed for Abeta, are generally applicable to amyloidogenic peptides and proteins. In this chapter, we describe the preparation of LMW Abeta using size exclusion chromatography and filtration. The advantages and disadvantages of each method are discussed.

Intrinsically disordered protein (IDP) conformers occupy large regions of conformational space and display relatively flat energy surfaces. Amyloid-forming IDPs, unlike natively folded proteins, have folding trajectories that frequently involve movements up shallow energy gradients prior to the "downhill" folding leading to fibril formation. We suggest that structural perturbations caused by chiral inversions of amino acid side-chains may be especially valuable in elucidating these pathways of IDP folding. Chiral inversions are subtle in that they do not change side-chain size, flexibility, hydropathy, charge, or polarizability. They allow focus to be placed solely on the question of how changes in amino acid side-chain orientation, and the resultant alterations in peptide backbone structure, affect a peptide's conformational landscape (Ramachandran space). If specific inversions affect folding and assembly, then the sites involved likely are important in mediating these processes. We suggest here a "focused chiral mutant library" approach for the unbiased study of amyloid-forming IDPs.

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid beta-protein.

Alloform-specific differences in structural dynamics between amyloid beta-protein (Abeta) 40 and Abeta42 appear to underlie the pathogenesis of Alzheimer's disease. To elucidate these differences, we performed microsecond timescale replica-exchange molecular dynamics simulations to sample the conformational space of the Abeta monomer and constructed its free-energy surface. We find that neither peptide monomer is unstructured, but rather that each may be described as a unique statistical coil in which five relatively independent folding units exist, comprising residues 1-5, 10-13, 17-22, 28-37, and 39-42, which are connected by four turn structures. The free-energy surfaces of both peptides are characterized by two large basins, comprising conformers with either substantial alpha-helix or beta-sheet content. Conformational transitions within and between these basins are rapid. The two additional hydrophobic residues at the Abeta42 C-terminus, Ile41 and Ala42, significantly increase contacts within the C-terminus, and between the C-terminus and the central hydrophobic cluster (Leu17-Ala21). As a result, the beta-structure of Abeta42 is more stable than that of Abeta40, and the conformational equilibrium in Abeta42 shifts towards beta-structure. These results suggest that drugs stabilizing alpha-helical Abeta conformers (or destabilizing the beta-sheet state) would block formation of neurotoxic oligomers. The atomic-resolution conformer structures determined in our simulations may serve as useful targets for this purpose. The conformers also provide starting points for simulations of Abeta oligomerization-a process postulated to be the key pathogenetic event in Alzheimer's disease.

Amyloidoses comprise a class of diseases characterized pathologically by the presence of deposits of fibrillar, aberrantly folded proteins, known as amyloids. Historically, these deposits were considered the key factors causing disease. However, recent evidence suggests that soluble protein oligomers, which are precursors for amyloid fibrils, are the primary toxic effectors responsible for the disease process. Understanding the mechanism by which these oligomers exert their toxicity requires knowledge of the structure, kinetics, and thermodynamics of their formation and conversion into larger assemblies. Such studies have been difficult due to the metastable nature of the oligomers. For the amyloid beta-protein (Abeta), a consensus about the size and relative abundance of small oligomers has not been achieved. We describe here the application of the method Photoinduced Cross-Linking of Unmodified Proteins (PICUP) to the study of Abeta oligomerization. This approach distinguishes oligomerization patterns of amyloidogenic and nonamyloidogenic proteins, allows quantification of each component in oligomer mixtures, and provides a means of correlating primary structure modifications with assembly characteristics. PICUP thus is a powerful tool for the investigation of small, metastable protein oligomers. The method provides essential insights into the factors that control the assembly of pathogenic protein oligomers, facilitating efforts toward the development of therapeutic agents.

The neuropathology of Alzheimer's disease (AD) includes amyloid plaque formation by the amyloid β-protein (Aβ) and intracellular paired helical filament formation by tau protein. These neuropathogenetic features correlate with disease progression and have been revealed in brains of AD patients using positron emission tomography (PET). One of the most useful positron emission tomography imaging agents has been Pittsburgh Compound-B (PiB). However, since its introduction in 2002, substantial evidence has accumulated suggesting that Aβ oligomerization and protofibril formation, rather than fibril formation per se, may be the more important pathogenetic event in AD. Detecting protofibrils and oligomeric forms of Aβ thus may be of value. We report here the results of experiments to determine whether PiB binds to oligomers or protofibrils formed by Aβ40 and Aβ42. We observed strong binding to Aβ42 fibrils, significant binding to protofibrils, and weaker binding to Aβ42 oligomers. PiB also binds Aβ40 fibrils, but its binding to Aβ40 protofibrils and oligomers is substantially lower than for that observed for Aβ42.