David B. Teplow

Amyloid β-Protein Fibrillogenesis STRUCTURE AND BIOLOGICAL ACTIVITY OF PROTOFIBRILLAR INTERMEDIATES

Alzheimer’s disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid β-protein (Aβ). Fibrils, both in vitro andin vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Aβ production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Aβ fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364–22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Aβ (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer’s disease and for our understanding of fibril assembly are discussed.

Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate.

Fibrillogenesis of the amyloid β-protein (Aβ) is a seminal pathogenetic event in Alzheimer’s disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of Aβ fibrillogenesis. We report results of studies designed to examine the initial stages of Aβ oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 m Tris-HCl, pH 7.4, and removal of pre-existent seeds, Aβ chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 ± 0.2 nm, consistent with the predicted size of dimeric Aβ. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6–8 nm in diameter and <200 nm in length, which we have termed “protofibrils.” The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of Aβ-(1–40), Aβ-(1–42), and Aβ-(1–40)-Gln22, peptides associated with both sporadic and inherited forms of Alzheimer’s disease, suggesting that protofibril formation may be a general phenomenon in Aβ fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.

Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinsons disease and Alzheimers disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimers disease.

Structure–neurotoxicity relationships of amyloid β-protein oligomers

Amyloid β-protein (Aβ) oligomers may be the proximate neurotoxins in Alzheimers 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. Aβ monomers were largely unstructured, but oligomers exhibited order-dependent increases in β-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 Aβ oligomers of specific order.

Presenilin‐dependent γ‐secretase processing of β‐amyloid precursor protein at a site corresponding to the S3 cleavage of Notch

The presenilin (PS)‐dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar γ‐secretase cleavage of the β‐amyloid precursor protein (βAPP) results in the secretion of amyloid β‐peptide (Aβ). However, little is known about the corresponding C‐terminal cleavage product (CTFγ). We have now identified CTFγ in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFγ is facilitated by PSs, since a dominant‐negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, γ‐secretase inhibitors, including one that is known to bind to PS, also block CTFγ generation. Sequence analysis revealed that CTFγ is produced by a novel γ‐secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

Amyloid β-protein assembly and Alzheimer disease

The biochemistry of amyloid proteins has been a fascinating and important area of research because of its contribution to our understanding of protein folding dynamics and assembly and of the pathogenetic mechanisms of human disease. One such disease is AD,2 the most common neurodegenerative disorder of aging. In AD, Aβ (Fig. 1A), which is expressed normally and ubiquitously throughout life as a 40–42-residue peptide, forms fibrils that deposit in the brain as “amyloid plaques.” This pathologic deposition process led researchers to investigate fibril formation as a target for therapeutic intervention. In doing so, an increasing number of fibril precursors and non-fibrillar Aβ assemblies have been identified, the majority of which are neurotoxic. These findings have altered prevailing fibril-centered views of the pathobiology of amyloid diseases (1) and intensified efforts to understand the early folding and assembly dynamics of Aβ. In the discussion that follows, we seek to introduce the reader to the complex world of Aβ assembly and biological activity, a goal we hope will provide a conceptual framework upon which further knowledge or experimentation may be built. FIGURE 1. Aβ assembly. A, the sequence of Aβ42 is shown in one-letter amino acid code. The side chain charge at neutral pH is color-coded (red, negative; blue, positive). B, nucleation-dependent polymerization, reflecting the unfavorable self-association ...

Presenilin Proteins Undergo Heterogeneous Endoproteolysis between Thr291and Ala299and Occur as Stable N- and C-Terminal Fragments in Normal and Alzheimer Brain Tissue

Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimers disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.

Enhanced Production and Oligomerization of the 42-residue Amyloid β-Protein by Chinese Hamster Ovary Cells Stably Expressing Mutant Presenilins

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of early onset familial Alzheimers disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or mutant (M239V) PS2 genes were stably transfected into Chinese hamster ovary cells that overexpress the β-amyloid precursor protein (APP). The identity of the 43-45-kDa PS1 holoproteins was confirmed by N-terminal radiosequencing. PS1 was rapidly processed (t1/2 = 40 min) in the endoplasmic reticulum into stable fragments. Wild-type and mutant PS2 holoproteins exhibited similar half lives (1.5 h); however, their endoproteolytic fragments showed both mutation-specific and cell type-specific differences. Mutant PS1 or PS2 consistently induced a 1.4-2.5-fold increase (p < 0.001) in the relative production of the highly amyloidogenic 42-residue form of amyloid β-protein (Aβ42) as determined by quantitative immunoprecipitation and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in Aβ42/Aβtotal ratios, spontaneous formation of low molecular weight oligomers of Aβ42 was observed in media, suggesting enhanced Aβ aggregation from the elevation of Aβ42. We conclude that mutant PS1 and PS2 proteins enhance the proteolysis of β-amyloid precursor protein by the γ-secretase cleaving at Aβ residue 42, thereby promoting amyloidogenesis.

On the nucleation of amyloid β‐protein monomer folding

Neurotoxic assemblies of the amyloid β‐protein (Aβ) have been linked strongly to the pathogenesis of Alzheimers disease (AD). Here, we sought to monitor the earliest step in Aβ assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease‐resistant segments within monomeric Aβ(1–40) and Aβ(1–42). The results revealed a 10‐residue, protease‐resistant segment, Ala21–Ala30, in both peptides. Remarkably, the homologous decapeptide, Aβ(21–30), displayed identical protease resistance, making it amenable to detailed structural study using solution‐state NMR. Structure calculations revealed a turn formed by residues Val24–Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val‐Gly‐Ser‐Asn and Gly‐Ser‐Asn‐Lys sequences to form a β‐turn, long‐range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24–Lys28 region of Aβ nucleates the intramolecular folding of Aβ monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C‐terminal peptide segments within Aβ(1–40) and Aβ(1–42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Aβ(1–42) production. Our results suggest that therapeutic approaches targeting the Val24–Lys28 turn or the Aβ(1–42)‐specific C‐terminal fold may hold promise.

Grape-derived polyphenolics prevent Aβ oligomerization and attenuate cognitive deterioration in a mouse model of Alzheimer's disease

Alzheimers disease (AD) is a neurodegenerative disorder characterized by progressive impairments in memory and cognition. Extracellular accumulation of soluble high-molecular-weight (HMW) Aβ oligomers has been proposed to be largely responsible for AD dementia and memory deficits in the Tg2576 mice, a model of AD. In this study, we found that a naturally derived grape seed polyphenolic extract can significantly inhibit amyloid β-protein aggregation into high-molecular-weight oligomers in vitro. When orally administered to Tg2576 mice, this polyphenolic preparation significantly attenuates AD-type cognitive deterioration coincidentally with reduced HMW soluble oligomeric Aβ in the brain. Our study suggests that grape seed-derived polyphenolics may be useful agents to prevent or treat AD.