Amyloid Growth Research Articles

PLoS Biology Issue Image | Vol. 2(10) October 2004

Recent discoveries show that prion phenomena in both humans and yeast are determined by amyloid-like conformations of misfolded proteins, and similar conformations are associated with noninfectious diseases such as Alzheimer's. Shown are AFM images of new amyloid growth from the ends of epitope-labeled seeds encapsulated in diagrams of yeast cells. (See Collins, et al.) Image Credit: Image by Angela DePace

Mechanism of prion propagation: amyloid growth occurs by monomer addition.

Abundant nonfibrillar oligomeric intermediates are a common feature of amyloid formation, and these oligomers, rather than the final fibers, have been suggested to be the toxic species in some amyloid diseases. Whether such oligomers are critical intermediates for fiber assembly or form in an alternate, potentially separable pathway, however, remains unclear. Here we study the polymerization of the amyloidogenic yeast prion protein Sup35. Rapid polymerization occurs in the absence of observable intermediates, and both targeted kinetic and direct single-molecule fluorescence measurements indicate that fibers grow by monomer addition. A three-step model (nucleation, monomer addition, and fiber fragmentation) accurately accounts for the distinctive kinetic features of amyloid formation, including weak concentration dependence, acceleration by agitation, and sigmoidal shape of the polymerization time course. Thus, amyloid growth can occur by monomer addition in a reaction distinct from and competitive with formation of potentially toxic oligomeric intermediates.

Seeding Specificity in Amyloid Growth Induced by Heterologous Fibrils

Over residues 15-36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid beta (Abeta) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Abeta-(1-40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Abeta-(1-40) elongation, exhibiting weight-normalized efficiencies of only 1-2% compared with Abeta-(1-40) fibrils. Amyloid fibrils of peptides with sequences completely unrelated to Abeta also exhibit poor to negligible seeding ability for Abeta elongation. Fibrils from a number of point mutants of Abeta-(1-40) exhibit intermediate seeding abilities for wild-type Abeta elongation, with differing efficiencies depending on whether or not the mutation is in the amyloid core region. The results suggest that amyloid fibrils from different proteins exhibit structural differences that control seeding efficiencies. Preliminary results also suggest that identical sequences can grow into different conformations of amyloid fibrils as detected by seeding efficiencies. The results have a number of implications for amyloid structure and biology.

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We demonstrate the real-time monitoring of the growth of amyloid-protein aggregates in a semi-rigid gel environment constructed from a 5% w/v gelatin solution. The kinetics of amyloid fibril growth from reduced and carboxy-methylated κ-casein occurring in the gel medium was contrasted against that obtained in a regular solution assay. Aggregation kinetics were recorded using Thioflavin T fluorescence. Transmission electron microscopy was used to confirm the aggregates' existence and morphology. The current demonstration of controlled amyloid growth in a gel environment represents the first step towards development of an experimental model for investigating the role of spatial and medium factors in the kinetics of aggregation-based proteopathies.

Apolipoprotein E affects amyloid formation but not amyloid growth in vitro: Mechanistic implications for ApoE4 enhanced amyloid burden and risk for Alzheimer's disease

The transition from the partially folded soluble Aβ monomer to insoluble Aβ amyloid fibrils is seminal to the formation and growth of amyloid plaques in Alzheimer's disease (AD). A detailed understanding of the role of AD risk factors in these processes is essential to understanding the physiochemical nature of this conformational rearrangement- The apolipoprotein E ϵ4 allele, a risk factor for AD, affects AD pathology by increasing amyloid burden relative to the much more common ϵ3 allele. In the present study, in vitro models were employed to probe the effect of these proteins on kinetically distinct steps in Aβ fibrillogenesis. Formation of Aβ amyloid was faster in the presence of apo E4 than apoE3, while growth of existing plaques was unaffected by either isoform. Further, experiments with Aβ stereoiso-mers establish that this effect ofapoE3 is mediated through interaction with oligomeric fibrillogenic intermediates rather than through specific contacts with monomeric Aβ. Consistent with the altered pathology and enhanced risk for AD associated with inheritance of the ϵ4 allele, we conclude that APOE ϵ4 is a risk factor for AD not due to a pathological gain of function of apo E4 but to a loss of protective function of apo E3.

In vitro studies of amyloid beta-protein fibril assembly and toxicity provide clues to the aetiology of Flemish variant (Ala692-->Gly) Alzheimer's disease.

In a Flemish kindred, an Ala(692)-->Gly amino acid substitution in the amyloid beta-protein precursor (AbetaPP) causes a form of early-onset Alzheimer's disease (AD) which displays prominent amyloid angiopathy and unusually large senile plaque cores. The mechanistic basis of this Flemish form of AD is unknown. Previous in vitro studies of amyloid beta-protein (Abeta) production in HEK-293 cells transfected with cDNA encoding Flemish AbetaPP have shown that full-length [Abeta(1-40)] and truncated [Abeta(5-40) and Abeta(11-40)] forms of Abeta are produced. In an effort to determine how these peptides might contribute to the pathogenesis of the Flemish disease, comparative biophysical and neurotoxicity studies were performed on wild-type and Flemish Abeta(1-40), Abeta(5-40) and Abeta(11-40). The results revealed that the Flemish amino acid substitution increased the solubility of each form of peptide, decreased the rate of formation of thioflavin-T-positive assemblies, and increased the SDS-stability of peptide oligomers. Although the kinetics of peptide assembly were altered by the Ala(21)-->Gly substitution, all three Flemish variants formed fibrils, as did the wild-type peptides. Importantly, toxicity studies using cultured primary rat cortical cells showed that the Flemish assemblies were as potent a neurotoxin as were the wild-type assemblies. Our results are consistent with a pathogenetic process in which conformational changes in Abeta induced by the Ala(21)-->Gly substitution would facilitate peptide adherence to the vascular endothelium, creating nidi for amyloid growth. Increased peptide solubility and assembly stability would favour formation of larger deposits and inhibit their elimination. In addition, increased concentrations of neurotoxic assemblies would accelerate neuronal injury and death.

Activation Barriers to Structural Transition Determine Deposition Rates of Alzheimer's Disease Aβ Amyloid

Brainamyloid composed of the ≈40-amino-acid human β-amyloid peptide Aβ is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Aβ during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of Aβ congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Aβ congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type Aβ. The hyperactive peptide, which is linked to a heritable Aβ amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type Aβ. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.

Stereochemical specificity of Alzheimer's disease beta-peptide assembly.

The formation and growth of insoluble amyloid deposits composed primarily of the human beta-amyloid peptide (A beta) in brain is an essentially invariant feature of Alzheimer's disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of A beta in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble A beta peptide onto preexisting A beta aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-A beta aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-A beta aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-A beta isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble A beta onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association.

Stereochemical specificity of Alzheimer's disease β‐peptide assembly

The formation and growth of insoluble amyloid deposits composed primarily of the human β-amyloid peptide (Aβ) in brain is an essentially invariant feature of Alzheimer's disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of Aβ in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble Aβ peptide onto preexisting Aβ aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-Aβ aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-Aβ aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-Aβ isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble Aβ onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association. © 1999 John Wiley & Sons, Inc. Biopoly 49: 505–514, 1999

23] Deposition of soluble amyloid-β onto amyloid templates: With application for the identification of amyloid fibril extension inhibitors

Several human diseases are now recognized to be caused by the formation of insoluble amyloid from naturally occurring peptides and proteins. Amyloid-forming monomers are not intrinsically pathological; rather it is their conformation and assembly state that are most important to their cytotoxicity. Because the chemical information for forming these amyloids apparently lies in the primary structure of the monomers, there has been significant interest in using in vitro systems to study amyloid formation and growth. A β deposition, the process of amyloid growth by the association of individual soluble A β molecules with a preexisting amyloid template, can be modeled readily in vitro under physiologically relevant conditions and importantly at physiological (nM) A β concentrations. Such assays may elucidate key molecular interactions in amyloidosis and may be exploitable in the design of antiamyloid therapeutics. High-throughput A β deposition assays may provide a means for the identification and optimization of such compounds. The methods described in this chapter have been developed for the examination of A β deposition, but are adaptable for the study of other amyloids.

Growth patterns and histochemical characterization of bovine mammary corpora amylacea.

Corpora amylacea in bovine mammary tissue were quantified across a range of size differentials for histochemical properties, lactation age, and lactation stage, in an attempt to characterize amyloid nucleation and growth. At all size classifications, corpora stained positively for amyloid, calcium deposits, and glycoprotein, while staining negatively for mucopolysaccharides. Prevalence of corpora amylacea among the size differentials was unrelated to age of lactating animals, although no corpora were observed in quarters of primiparous heifers at parturition. Corpora amylacea were most abundant during the later stages of lactation for all size differentials, and least abundant during late involution and early lactation. The majority of corpora were observed in alveolar lumens at all stages of lactation. Our results suggest that corpora amylacea development is not restricted to a particular stage of lactation, although their nucleation appears to occur within the alveolar lumens. Gradual increases in both size and numbers of corpora from parturition to late lactation suggest that development of these structures accelerates as lactation progresses. Morphological relationships between corpora amylacea and mammary parenchymal tissue during the later stages of lactation suggest that these structures may have a role in the involutionary process. These findings provide the foundation for additional immunocytochemical techniques to determine the origin of amyloid fibril components.