Tadato Ban

Metal Ion-dependent Effects of Clioquinol on the Fibril Growth of an Amyloid β Peptide

Although metal ions such as Cu2+, Zn2+, and Fe3+ are implicated to play a key role in Alzheimer disease, their role is rather complex, and comprehensive understanding is not yet obtained. We show that Cu2+ and Zn2+ but not Fe3+ renders the amyloid β peptide, Aβ1–40, nonfibrillogenic in nature. However, preformed fibrils of Aβ1–40 were stable when treated with these metal ions. Consequently, fibril growth of Aβ1–40 could be switched on/off by switching the molecule between its apo- and holo-forms. Clioquinol, a potential drug for Alzheimer disease, induced resumption of the Cu2+-suppressed but not the Zn2+-suppressed fibril growth of Aβ1–40. The observed synergistic effect of clioquinol and Zn2+ suggests that Zn2+-clioquinol complex effectively retards fibril growth. Thus, clioquinol has dual effects; although it disaggregates the metal ion-induced aggregates of Aβ1–40 through metal chelation, it further retards the fibril growth along with Zn2+. These results indicate the mechanism of metal ions in suppressing Aβ amyloid formation, as well as providing information toward the use of metal ion chelators, particularly clioquinol, as potential drugs for Alzheimer disease.

αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β2-microglobulin

AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric alpha-crystallins in preventing Abeta-(1-40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on alpha-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Abeta-(1-40). alphaB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Abeta-(1-42), as well as the fibril growth of Abeta-(1-40) when seeded with the Abeta-(1-42) fibril seed. Sedimentation velocity measurements show that alphaB-crystallin does not form a stable complex with Abeta-(1-40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. alphaB-crystallin binds to the amyloid fibrils of Abeta-(1-40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that alphaB-crystallin prevents the fibril growth of beta2-microglobulin under acidic conditions. It also retards the depolymerization of beta2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimers disease.

Branching in Amyloid Fibril Growth

Using the peptide hormone glucagon and Abeta(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 degrees . Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Abeta(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 Abeta(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth.

Lipocalin-type prostaglandin D synthase/β-trace is a major amyloid β-chaperone in human cerebrospinal fluid

The conformational change in amyloid β (Aβ) peptide from its monomeric form to aggregates is crucial in the pathogenesis of Alzheimers disease (AD). In the healthy brain, some unidentified chaperones appear to prevent the aggregation of Aβ. Here we reported that lipocalin-type prostaglandin D synthase (L-PGDS)/β-trace, the most abundant cerebrospinal fluid (CSF) protein produced in the brain, was localized in amyloid plaques in both AD patients and AD-model Tg2576 mice. Surface plasmon resonance analysis revealed that L-PGDS/β-trace tightly bound to Aβ monomers and fibrils with high affinity (KD = 18–50 nM) and that L-PGDS/β-trace recognized residues 25–28 in Aβ, which is the key region for its conformational change to a β-sheet structure. The results of a thioflavin T fluorescence assay to monitor Aβ aggregation disclosed that L-PGDS/β-trace inhibited the spontaneous aggregation of Aβ (1–40) and Aβ (1–42) within its physiological range (1–5 μM) in CSF. L-PGDS/β-trace also prevented the seed-dependent aggregation of 50 μM Aβ with Ki of 0.75 μM. Moreover, the inhibitory activity toward Aβ (1–40) aggregation in human CSF was decreased by 60% when L-PGDS/β-trace was removed from the CSF by immunoaffinity chromatography. The deposition of Aβ after intraventricular infusion of Aβ (1–42) was 3.5-fold higher in L-PGDS-deficient mice and reduced to 23% in L-PGDS-overexpressing mice as compared with their wild-type levels. These data indicate that L-PGDS/β-trace is a major endogenous Aβ-chaperone in the brain and suggest that the disturbance of this function may be involved in the onset and progression of AD. Our findings may provide a diagnostic and therapeutic approach for AD.

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

In Alzheimer disease, amyloid β, 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 μm 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.

Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation

To understand the mechanism by which amyloid fibrils form, we have been making real-time observations of the growth of individual fibrils, using total internal fluorescence microscopy combined with an amyloid-specific fluorescence dye, thioflavin T (ThT). At neutral pH, irradiation at 442 nm with a laser beam to excite ThT inhibited the fibril growth of β2-microglobulin (β2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of β2-m showed that the inhibition of fibril growth and moreover the destruction of preformed fibrils were coupled with the excitation of ThT. Several pieces of evidence suggest that the excited ThT transfers energy to ground state molecular oxygen, producing active oxygen, which causes various types of chemical modifications. The results imply a novel strategy for preventing the deposition of amyloid fibrils and for destroying preformed amyloid deposits.

Direct observation of amyloid growth monitored by total internal reflection fluorescence microscopy

Most morphological investigations of amyloid fibrils have been performed with various microscopic methods. Among them, direct observation of fibril growth is possible using atomic force microscopy and fluorescence microscopy. Direct observation provides information about the rate and direction of growth at the single fibril level, which cannot be obtained from averaged ensemble measurements. In this chapter, we describe a new technique for the direct observation of amyloid fibril growth using total internal reflection fluorescence microscopy (TIRFM) combined with amyloid-specific thioflavin T (ThT) fluorescence. TIRFM has been developed to monitor single molecules by effectively reducing the background fluorescence in an evanescent field. One of the advantages of TIRFM is that one can selectively monitor fibrils lying along a glass slide, so that one can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent fibril growth of amyloid beta (1-40). The fibril growth was a highly cooperative process, with the fibril ends extending at a constant rate. Because ThT binding is common to all amyloid fibrils, the present method will have general applicability to the real-time analysis of amyloid fibrils.

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.