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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 ␤ 2 -microglobulin (␤2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of ␤2-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.Amyloid fibrils are associated with the pathogenesis of more than 20 serious diseases, including Alzheimer, Parkinson, and Huntington diseases, and dialysis-related amyloidosis (1, 2). Moreover, various proteins and peptides that are not related to diseases can also form amyloid-like fibrils, implying that the formation of fibrils is a generic property of proteins and peptides (1). In basic structure, amyloid fibrils are long and often twisted, a few nanometers in diameter, and predominantly composed of cross ␤-sheets (1, 3). Amyloid fibrils are formed spontaneously with a lag phase or seed-dependently without a lag time, indicating that they are formed by nucleation and extension. Further understanding of the structure, mechanism of formation, and roles in amyloidosis is one of the most important issues of protein science today.To study amyloid fibrils, we developed a unique technique for their direct observation in which total internal reflection fluorescence microscopy (TIRFM) 2 is combined with amyloidspecific thioflavin T (ThT) fluorescence (4 -8). This technique provides important information about the morphology, growth rate, and extension direction of fibrils in real time at the single fibril level. We have applied the technique to the fibrils of ␤ 2 -microglobulin (␤2-m) responsible for dialysis-related amyloidosis and amyloid ␤ associated with Alzheimer disease (4 -8).␤2-m is a major component of amyloid fibrils deposited in dialysis-related amyloidosis, a common and serious complication in patients receiving hemodialysis for more than 10 years (9 -11). ␤2-m, a typical immunoglobulin domain made of 99 amino acid residues and seven ␤-stands, is present as the nonpolymorphic light chain of the class I major histocompatibility complex (12). Renal failure disrupts the clearance of ␤2-m from the serum and, moreover, ␤2-m does not pass through the dialysis membrane, resulting in an increase in the concentration of ␤2-m in blood by up to 50-fold. Although an increase in the concentration of ␤2-m is the most important risk factor for fibrillation, how...

Amyloidosis refers to a group of diseases with amyloid fibrils deposited in various organs and is classified into more than 30 diseases in humans based on the kind of amyloid protein.In order to elucidate the molecular pathogenesis of human amyloidosis, we studied the molecular mechanism of amyloid fibril formation in vitro. We first developed a novel fluorometric method to determine amyloid fibrils in vitro based on the unique characteristics of thioflavin T. We next proposed a nucleation-dependent polymerization model to explain the general mechanism of amyloid fibril formation in vitro. Based on this model, we characterized the biological molecular interactions that promote or inhibit amyloid fibril formation in vitro and developed models of pathological molecular environment for inducing human β 2 -microglobulin-related amyloidosis in long-term hemodialysis patients. We also proposed a novel and attractive cytotoxic mechanism of β 2 -microglobulin amyloid fibrils, that is, the disruption of endosomal/lysosomal membranes by endocytosed amyloid fibrils. These findings may be useful to elucidate the molecular pathogenesis of other kinds of human amyloidosis.

The amyloid deposition of amyloid ␤ (A␤) 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 ␤ 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 A␤ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed A␤ 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.

Complex amyloid aggregation of amyloid-β (1–40) (Aβ1–40) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of Aβ1–40 aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of Aβ1–40 induced by low solvent polarity accelerated cytotoxic Aβ1–40 amyloid fibrillation, while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of β-structure-rich protofibrils or oligomers via aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying Aβ1–40 aggregation with conceptual energy diagrams and propose that aggregation-prone partial helical structures are key to inducing amyloidogenesis. We demonstrate that context-dependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.

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Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.