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

Abstract

In Alzheimer disease, amyloid ␤, 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 m on the chemically modified quartz surface. Realtime 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.In Alzheimer disease, amyloid ␤ (A␤) 3 peptide forms amyloid fibrils that deposit in the extracellular space of the brain as senile amyloid plaques, pathological hallmarks of Alzheimer disease, and also in the walls of cerebral blood vessels (1-5). The formation of A␤ amyloid fibrils is considered to be a nucleationdependent process in which A␤ peptides slowly associate to form a nucleus, which then grows via an extension reaction involving the sequential incorporation of A␤ peptides, producing rigid and straight morphology consisting of several layers of cross-␤ sheets (6, 7). This process is influenced by several factors, i.e. peptide concentration, pH, ionic strength, and interactions with other components (8, 9). The interactions with lipid membranes in particular have received attention because the membrane surface might be responsible for both neurotoxicity and senile plaque formation (10 -12).For several proteins including A␤, amyloid fibrils prepared on the solid substrates such as mica or quartz produce radial assemblies (13)(14)(15). Considering that in several neurodegenerative diseases, radial and spherical aggregates of amyloid fibrils are found in tissue deposits (5, 16), surface interaction may play dominant roles in the formation of amyloid fibrils and their deposition in vivo (7,17). Moreover, amyloid deposits are found in a specific tissue region, suggesting that a specific surface chemistry is involved in the fibril formation and deposition processes in general. However, the behavior of amyloid fibrils on solid surfaces is still far from clear. To obtain further insight into the mechanism of senile plaque formation and the effects of surface, direct observations are needed.We previously developed a unique approach to monitoring fibril growth in real time at the single fibril level (18,19), in which TIRFM was combined with the use of thioflavin T, an amyloid-specific fluorescence dye (20). With this approach, we focused on the effects of the physicochemical prope...

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits

“…For example, if fibrils become nucleated on the surface of a preexisting fibril (38), it is possible that the seed partially orients the daughter filament. Furthermore, total internal reflection fluorescence microscopy revealed the outgrowth of amyloidlike fibrils in vitro and the formation of fibril bundles due to fibril branching reactions (39) or the formation of star-like spherulites due to a radial extension of fibrils from a seed (40,41).…”

Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40)

“…We previously reported the amyloid fibrillation of A␤- in real time at a single fibrillar level at pH 7.0 with TIRFM combined with the use of ThT (62). We used the surfaces of various chemically modified substrates.…”

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1–42)

“…In previous reports, the Aβ aggregation was induced on the negatively charged surface owing to the repulsion to the net charge of Aβ and the electrostatic interaction to the positive residues in Aβ [23,26,27]. The results corresponded to the Aβ amyloidosis on the cell surfaces.…”

“…In addition, since Aβ has a negative net charge, the interactions of Aβ with 6-sulfo-GlcNAc and sialic acid were affected by the electrostatic repulsion [23]. Actually, the amyloidosis of Aβ on the cell surfaces is affected not only by saccharide-Aβ interaction, but also by many other interactions such as Aβ-Aβ, Aβ-protein (other proteins) and electrostatic interaction [23,24]. Therefore, the amyloidosis of Aβ on the cell surfaces was not fully understood from the results of our simple experiments.…”