Laser-induced propagation and destruction of amyloid beta fibrils

Abstract

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.

FIGURE 1.

Visualization of the laser-induced propagation of Aβ(1–40) fibrils. A and B, real-time observations revealing explosive propagation from a single fibril. C, propagation of random walk-like fibrils with branching. D, mixed image of the laser-induced destruction and propagation of fibrils. The scale bars represent 10 μm. E–G, time courses of fibril propagation obtained by quantifying TIRFM images shown in A–C. Abscissa represents time after initiation of laser irradiation. The fibrils were monitored by laser irradiation of 3–5 s duration at the observation point. No. 1–3 indicate the numbering of quantified images, all of which are summarized in Fig. 5.

FIGURE 2.

Effects of an interval between laser irradiations on propagation and destruction of Aβ(1–40) fibrils. A, propagation of new fibrils from preformed fibrils. B, time course of fibril propagation obtained by quantifying the regions No. 4–7 at 40 h of A. Detailed images were shown in supplemental Fig. S1. C, destruction of preformed fibrils by intermittent irradiation. The fibrils were monitored by laser irradiation of 3–5 s duration at the observation point. The scale bars represent 10 μm.

FIGURE 3.

Visualization of the laser-induced destruction of Aβ(1–40) fibrils. A, effects of extensive laser irradiation on spontaneous Aβ(1–40) fibril growth. After 14 h, the fibrils were monitored every 1 or 2 h to 25 h, by laser irradiation of 3–5 s duration at the observation point. B, effect of extensive laser irradiation on preformed fibrils. Aβ(1–40) fibrils were prepared on quartz slides in the absence of irradiation. The elongated fibrils were then irradiated intermittently at an intensity of 40 milliwatts for 1 min. The scale bars represent 10 μm. C, dependence of fibril destruction on the interval of laser irradiation. The fibrils were monitored by laser irradiation of 40–55 milliwatts for 1 min at the observation point. Original data are shown in supplemental Figs. S3 and S4.

FIGURE 4.

Analyses of the laser-irradiated Aβ(1–40) fibrils in a glass cell. The irradiation was performed at 442 nm. A, kinetics of the disruption of fibrils monitored using ThT fluorescence (●, ■) and light scattering (○, □) with (●, ○), or without (■, □) laser irradiation. B and C, the sedimentation pattern of fibrils without (B) or with (C) laser irradiation. Sedimentation patterns were recorded at 3,000 rpm (664 × g) (B and C, gray lines) and 53,000 rpm (207,270 × g) (C, black lines) by monitoring the absorbance at 280 nm, and several traces at intervals of 2 min (3,000 rpm) or 30 min (53,000 rpm) are presented. D, identification of chemical modifications by amino acid analysis. E–H, mass spectra. E, the reference spectrum of Aβ(1–40) fibrils without irradiation or ThT, after laser irradiation without ThT (F), without irradiation in the presence of ThT (G), and after irradiation in the presence of ThT (H).

FIGURE 5.

Laser irradiation-dependent propagation and destruction of Aβ(1–40) fibrils leading to a profile with an optimum. All the quantified kinetic data of laser-dependent propagation (No. 1–8) or destruction (No. 9–17) of fibrils are plotted. The plot also includes the data of the dependence on the laser power (No. 18–20). Quantified ThT intensities were normalized to be one for the end points of fibril propagation and for the starting points of fibril destruction. Abscissa in log scale represents a total irradiation energy calculated by multiplying the laser power by the total irradiation period in seconds.