Single Fibril Level Research Articles

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

Viscoelastic behavior of discrete human collagen fibrils

Whole tendon and fibril bundles display viscoelastic behavior, but to the best of our knowledge this property has not been directly measured in single human tendon fibrils. In the present work an atomic force microscopy (AFM) approach was used for tensile testing of two human patellar tendon fibrils. Fibrils were obtained from intact human fascicles, without any pre-treatment besides frozen storage. In the dry state a single isolated fibril was anchored to a substrate using epoxy glue, and the end of the fibril was glued on to an AFM cantilever for tensile testing. In phosphate buffered saline, cyclic testing was performed in the pre-yield region at different strain rates, and the elastic response was determined by a stepwise stress relaxation test. The elastic stress-strain response corresponded to a second-order polynomial fit, while the viscous response showed a linear dependence on the strain. The slope of the viscous response showed a strain rate dependence corresponding to a power function of powers 0.242 and 0.168 for the two patellar tendon fibrils, respectively. In conclusion, the present work provides direct evidence of viscoelastic behavior at the single fibril level, which has not been previously measured.

Branching in Amyloid Fibril Growth

Using the peptide hormone glucagon and A β(1–40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35–40°. Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, A β(1–40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and A β(1–40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth

Structure, Formation and Propagation of Amyloid Fibrils

Amyloid fibrils have been a critical subject in recent studies of proteins since they are associated with the pathology of more than 20 serious human diseases. Moreover, a variety of proteins and peptides not related to diseases are able to form amyloid fibrils or amyloid-like structures, implying that amyloid formation is a generic property of polypeptides. Although understanding the structure and formation of amyloid fibrils is crucial, due to the extremely high molecular weight and insolubility of amyloid fibrils, most of the conventional techniques available for soluble proteins are not directly applicable to these fibrils. However, structural studies using solid-state NMR have shown that the basic motif of amyloid fibrils is a beta-strand-loop-beta-strand conformation often in a parallel beta-sheet assembly. From the hydrogen/deuterium exchange of amide protons, amyloid fibrils have been shown to be stabilized by an extensive network of hydrogen bonds substantiating beta-sheets. Our approach using total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye, enabled monitoring fibril growth in real-time at single fibril level. On the basis of these various approaches, increasingly convincing models of amyloid structures, their formation and propagation are emerging.

Direct Observation of Amyloid Fibril Formation of B 2-Microglobulin and Amyloid B Peptide

Amyloid fibrils form through nucleation and growth. To clarify the mechanism involved, direct observations of both processes are important (1, 2). First, we developed a new technique for the direct observation of amyloid fibrils using total internal reflection fluorescence microscopy (TIRFM) combined with thioflavin T (ThT) fluorescence. Fibril growth of b2-microglobulin (b2-m) and amyloid b peptide was visualized in real-time at the single fibril level revealing various dramatic images. With amyloid b peptide, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 m m on the chemically modified quartz surface, suggesting the underlying physicochemical mechanism of senile plaque formation. Second, using atomic force microscopy, ultrasonication-induced formation of b 2-m fibrils was shown, indicating that ultrasonication is useful to accelerate the nucleation process. Third, with the proteolytic fragment of b 2-m, propagation and a transformation of fibril morphology was demonstrated. These direct observations indicate that templatedependent growth and structural diversity are key factors determining the structure and function of amyloid fibrils. The idea can be represented by a cubic puzzle consisting of 27 small cubes, whose folding and misfolding mimic protein folding and amyloid fibril formation, respectively.

Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

Direct Observation of Amyloid Fibril Growth, Propagation, and Adaptation

Amyloid fibrils form through nucleation and growth. To clarify the mechanism involved, direct observations of both processes are important. First, seed-dependent fibril growth of beta2-microglobulin (beta2-m) and amyloid beta peptide was visualized in real time at the single fibril level using total internal reflection fluorescence microscopy combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. Second, using atomic force microscopy, ultrasonication-induced formation of beta2-m fibrils was shown, indicating that ultrasonication is useful to accelerate the nucleation process. Third, with the proteolytic fragment of beta2-m, propagation and a transformation of fibril morphology was demonstrated. These direct observations indicate that template-dependent growth and structural diversity are key factors determining the structure and function of amyloid fibrils.

Direct Observation of Aβ Amyloid Fibril Growth and Inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.

Visualizing the growth of Alzheimer's Aβ amyloid-like fibrils

Polymerization of normally soluble, β-sheet-forming proteins or peptides yielding fibrillar amyloid deposits is a hallmark of many neurodegenerative diseases[1xAmyloid diseases: abnormal protein aggregation in neurodegeneration. Koo, E.H. et al. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9989–9990Crossref | PubMed | Scopus (455)See all References][1]. Alzheimer's disease (AD) is in part neuropathologically defined by senile plaques constituting extracellular deposits of β-amyloid (Aβ) fibrils. Aβ, a 39–43 amino acid peptide is derived from endoproteolytic cleavage of the transmembrane amyloid precursor protein (APP). Mutations in APP and in presenilin, a protein associated with intramembranous cleavage of APP, are linked to early onset forms of inherited AD. Mutations all give rise to increased levels of Aβ peptides, thus suggesting a central role of Aβ in the disease process[2xAlzheimer's disease: genes, proteins, and therapy. Selkoe, D.J. Physiol. Rev. 2001; 81: 741–766PubMedSee all References][2]. Consequently, reduction of Aβ production by blocking the APP cleavage enzymes, stimulating the clearance of amyloid deposits by immunization with fibrillar Aβ, or inhibiting Aβ fibril formation itself[3xApproaches to discovery and characterization of inhibitors of amyloid β-peptide polymerization. Findeis, M.A. Biochim. Biophys. Acta Mol. Basis Dis. 2000; 1502: 76–84Crossref | PubMed | Scopus (156)See all References][3], are all current strategies for the development of therapeutic interventions.With the aim of developing fibril formation inhibitors, the Aβ fibril assembly process and the structures of its products have been characterized in some detail using in vitro assays[4xStudies on the in vitro assembly of Aβ1–40: implications for the search for Aβ fibril formation inhibitors. Goldsbury, C. et al. J. Struct. Biol. 2000; 130: 217–231Crossref | PubMed | Scopus (224)See all References][4]. We have developed an atomic force microscopy (AFM) assay whereby effects can be measured at the single-fibril level. The distinct advantage of AFM over other techniques is its ability to provide direct time-lapse images of changes in the aggregation state of macromolecules in a physiological buffer environment[5xWatching amyloid fibrils grow by time-lapse atomic force microscopy. Goldsbury, C. et al. J. Mol. Biol. 1999; 285: 33–39Crossref | PubMed | Scopus (247)See all References][5]. The assembly of synthetic Aβ1–40 fibrils is carried out in the fluid cell of an AFM, and the growth of individual fibrils attached to a piece of mica is recorded over periods of several hours. To study the effect of inhibitors of fibril elongation, the process is induced by first seeding the surface with pre-assembled fibrils before injecting fresh Aβ1–40. The added monomeric Aβ1–40 is then consumed by the process of elongating the pre-adsorbed fibril seeds (Fig. 1Fig. 1). As well as from direct elongation of pre-adsorbed fibrils, new fibrils often appear to originate from small aggregates on the surface. Aβ fibrils appear to be polymorphic[4xStudies on the in vitro assembly of Aβ1–40: implications for the search for Aβ fibril formation inhibitors. Goldsbury, C. et al. J. Struct. Biol. 2000; 130: 217–231Crossref | PubMed | Scopus (224)See all References][4]. Generally, fibrils appear to follow the same morphology as the adsorbed fibril seed, so that the growing fibril evolves to have the same axial periodicity as the pre-adsorbed seed (Fig. 1Fig. 1, arrows). We are currently in the process of testing potential fibril elongation inhibitors using this assay system. On a related note, the AFM technique could also be applied to determine the fibril-nucleating ability and structural incorporation of other amyloid components, for example heparan sulfate proteoglycans, phospholipids, lipoproteins or metal ions. Such investigations might ultimately aid our understanding of disease-related amyloid-fibril deposition processes.Fig. 1Seeded growth of Aβ1–40 amyloid-like fibrils on a mica surface imaged by atomic force microscopy (AFM). An aliquot of mature Aβ fibrils (‘seeds’) was adsorbed to the mica surface and the surface was then washed several times with buffer (top left panel). Sequential images of the same surface area are shown at the times indicated after injection of a freshly prepared non-fibrillar Aβsolution.View Large Image | Download PowerPoint Slide