Spherical aggregates of beta-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3beta

Abstract

beta-Amyloid (Abeta) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of Abeta aggregates, we examined in vitro aggregation conditions by using large quantities of homogenous, chemically synthesized Abeta1-40 peptide. We found that slow rotation of Abeta1-40 solution reproducibly gave self-aggregated Abeta1-40 containing a stable and highly toxic moiety. Examination of the aggregates purified by glycerol-gradient centrifugation by atomic force microscopy and transmission electron microscopy revealed that the toxic moiety is a perfect sphere, which we call amylospheroid (ASPD). Other Abeta1-40 aggregates, including fibrils, were nontoxic. Correlation studies between toxicity and sphere size indicate that 10- to 15-nm ASPD was highly toxic, whereas ASPD <10 nm was nontoxic. A positive correlation between the toxicity and ASPD >10 nm also appeared to exist when Abeta1-42 formed ASPD by slow rotation. However, Abeta1-42-ASPD formed more rapidly, killed neurons at lower concentrations, and showed approximately 100-fold-higher toxicity than Abeta1-40-ASPD. The toxic ASPD was associated with SDS-resistant oligomeric bands in immunoblotting, which were absent in nontoxic ASPD. Because the formation of ASPD was not disturbed by pentapeptides that break beta-sheet interactions, Abeta may form ASPD through a pathway that is at least partly distinct from that of fibril formation. Inhibition experiments with lithium suggest the involvement of tau protein kinase I/glycogen synthase kinase-3beta in the early stages of ASPD-induced neurodegeneration. Here we describe the identification and characterization of ASPD and discuss its possible role in the neurodegeneration in Alzheimer's disease.

Fig. 1.

ASPD as a neurotoxin in Aβ_1–40 aggregates. (A)Aβ_1–40 solutions (350 μM) were aggregated, and their toxicity was estimated by MTT assay (n = 6). Aβ_1–40 solution was rotated in Eppendorf tubes (•), glass tubes (▵), carbon-coated tubes (○), gold-coated tubes (⊞), and silicon-coated tubes (▴). Aβ_1–40 solution without rotation also is shown (). Hereafter, we designated toxic Aβ_1–40 aggregates formed by slowly rotating Aβ_1–40 solution (350 μMin50% PBS) as SR₃₅₀–Aβ_1–40. (B) The structure and toxicity of Aβ_1–40 aggregates formed by slow rotation. At the indicated time, aliquots were removed from SR₃₅₀–Aβ_1–40 for transmission electron microscopy and MTT assay (n = 9) (5 μM). (C) Aβ_1–40 solutions [350 μM (SR₃₅₀–Aβ_1–40), 50 μM (SR₅₀–Aβ_1–40), and 1 μM (SR₁–Aβ_1–40)] each were rotated slowly for 7 days, and the toxicity was assessed (n = 7). Thioflavine T assay (20 μM; Sigma; excitation at 445 nm and emission at 485 nm; ref. 48) determined fibril content in freshly dissolved Aβ_1–40 and in each SR–Aβ_1–40 (n = 6). (D) SR₃₅₀–Aβ_1–40 was prepared with or without KLVFF or LPFFD. The toxicity was determined by MTT assay (n = 9) or propidium iodide staining (Right). Image of SR₃₅₀–Aβ_1–40 with LPFFD is shown (bar, 100 nm). Also shown are SR₃₅₀–Aβ_1–40 (○) with KLVFF (•) and with LPFFD (). Data represent the mean ± SE. *, Significant difference from the control. The results suggest that toxicity of SR₃₅₀–Aβ_1–40 is associated with the spherical structures (B, 4 h), which we call ASPD.

Fig. 2.

ASPD morphology and toxicity. (A) Transmission electron microscopic images of isolated ASPD (bars, 100 nm in Lower and 20 nm in Inset). The width (x) and length (y) of each ASPD (n = 101) were measured (Upper). (B) Images of horizontal sections (a) and cross sections (b) of ASPD in the resin as described in Materials and Methods. (C) Fluid-phase atomic force microscopy of ASPD as described in Materials and Methods (n = 100). (D) Toxicity of ASPD. (Top and Middle) After a 40-h ASPD treatment (210 nM), cultures were stained with anti-MAP2 (green) and fillipin (blue). ASPD killed ≈40% neurons, with shrunken or fragmented nuclei stained pink with propidium iodide and Hoechst 33258 (Inset). Cell death in the control culture was <3%. (Bottom) ASPD toxicity was estimated by MTT activity (n = 6). Data in D represent the mean ± SE. *, Significant difference compared with control.

Fig. 3.

Purification of toxic ASPD by the glycerol gradient. (A) ASPD and fibrils in SR₃₅₀–Aβ_1–40 were fractionated as shown. Transmission electron microscopic images of representative fractions are shown. Aβ recovery and percentage of the total toxicity in glycerol gradient are indicated. (B) Immunoblottings with 4G8 of each fraction and of freshly dissolved Aβ_1–40 are shown (Upper). Fraction 2 induced apoptotic cell death as shown by red arrows in propidium iodide and Hoechst 33258 staining, whereas fraction 11 did not (Lower Inset). Mini-Bradford assay quantitated Aβ content, using freshly dissolved Aβ_1–40 as a standard, the concentration of which was determined accurately in advance with a Waters AccQTag system. Comparison of results from mini-Bradford assay with the AccQTag system data by using unfractionated SR₃₅₀–Aβ_1–40 proved a good correlation at concentrations >1 μM. The specific cell death activity was calculated from the Aβ content and the apoptotic activity (n = 16); one unit is defined as the activity inducing 1% apoptotic cell death. The specific cell death activity values of unfractionated SR₃₅₀–Aβ_1–40 and fibrils were 0.37 ± 0.09 and 0.07 ± 0.003, respectively. Data represent the mean ± SE. *, Significant difference compared with control. (C) The particle distribution of each fraction. (D Left) Transmission electron microscopic images of ASPD formed in Aβ_1–42 solution (0.1 μMin50% PBS) after an 8-h slow rotation (bar, 20 nm). (Right)Aβ_1–42-ASPD was separated by a glycerol gradient as in A. The particle distribution of fraction 2, in which the highest toxicity was recovered in repeated experiments (n = 7), is shown.

Fig. 4.

Lithium suppression of ASPD toxicity. (A Upper) After 210-nM ASPD treatment, TPKI/GSK-3β activity was assayed at the indicated time (n = 6). (Lower) Lithium (2 mM) was added to ASPD-treated culture at the indicated time, and the toxicity was assayed (n = 6). (B) Dose-dependent inhibition of ASPD toxicity by lithium. Effect of lithium on 210-nM ASPD toxicity was examined (n = 6). (C) Lithium protection against ASPD-induced apoptosis. After the treatment, as indicated, propidium iodide and Hoechst 33258 staining was performed (n = 9). Data represent the mean ± SE. Significant difference compared with control (*) or with ASPD-treated culture (**).