Efficient reversal of Alzheimer's disease fibril formation and elimination of neurotoxicity by a small molecule

Abstract

The Abeta1-42 peptide that is overproduced in Alzheimer's disease (AD) from a large precursor protein has a normal amino acid sequence but, when liberated, misfolds at neutral pH to form "protofibrils" and fibrils that are rich in beta-sheets. We find that these protofibrils or fibrils are toxic to certain neuronal cells that carry Ca-permeant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Disrupting the structure of the Abeta1-42 fibrils and protofibrils might lead to the discovery of molecules that would be very useful in the treatment of AD. A high-throughput screen of a library of >3,000 small molecules with known "biological activity" was set up to find compounds that efficiently decrease the beta-sheet content of aggregating Abeta1-42. Lead compounds were characterized by using thioflavin T (ThT) as a beta-sheet assay. The most effective of six compounds found was 4,5-dianilinophthalimide (DAPH) under the following conditions: DAPH at low micromolar concentrations abolishes or greatly reduces previously existing fully formed Abeta1-42 fibrils, producing instead amorphous materials without fibrils but apparently containing some protofibrils and smaller forms. Coincubation of the Abeta1-42 peptide with DAPH produces either amorphous materials or empty fields. Coincubation of DAPH and Abeta1-42 greatly reduces the beta-sheet content, as measured with ThT fluorescence, and produces a novel fluorescent complex with ThT. When the Abeta1-42 peptide was coincubated with DAPH at very low micromolar concentrations, the neuronal toxicity mentioned above (Ca(2+) influx) was eliminated. Clearly, DAPH is a promising candidate for AD therapy.

Fig. 1.

Schematic representation of neurotoxicity. Misfolding of peptide Aβ42 produces a neurotoxic molecule.

Fig. 2.

Time course of aggregation of Aβ42_30k at 10 μM in Tyrode's solution/2 mM Ca (pH 7.4) incubated at 37°C for the indicated times; negative-staining EM is shown. (A) t = 0 h. Arrow indicates one of the many donuts shown, which is enlarged in E. A larger and rarer aggregate is also shown. (B) t = 6 h. The “beads” are roughly the same size as the beads that make up the donuts in A and E. (C) t = 16 h. (D) t = 48 h. (E) t = 0 h. A portion of A (arrow) is enlarged to show a donut. The image is black/white-inverted for clarity.

Fig. 10.

DAPH eliminates Ca influx of Aβ42_Total in CATH.a cells. (A) As described in the legend to Fig. 8, solutions were sequentially exchanged with 10 μM aggregated Aβ42_Total, then Aβ42_Total plus DAPH (coincubated at 10 μM plus 10 μM), and then Aβ42_Total. Because DAPH is soluble only in DMSO, DMSO was present in all solutions. This experiment was repeated three times. (B) Effect of DAPH (30-0.25 μM) on Ca influx. The protocol used was as in A, except with DAPH concentrations ranging from 30-0.25 μM. (Variance is high; see Materials and Methods.) The corresponding concentration of DMSO was used for the controls.

Fig. 3.

DAPH reverses Aβ42_30k fibrils. (A) Aβ42_30k at 10 μM in Tyrode's solution/2 mM Ca (pH 7.4), incubated at 37°C for 24 h. (B) Incubation continued for another 24 h in the presence of vehicle (1% DMSO). (C) Incubation continued for another 24 h in the presence of 10 μM DAPH in DMSO.

Fig. 4.

Time course of reversal of fibril, formed by incubating 10 μM Aβ42_Total in Tyrode's solution/2 mM Ca (pH 7.4) at 37° for 24 h. The β-sheet content was measured with ThT (see Materials and Methods). To obtain net values, the fluorescence of ThT in Tyrode's buffer was subtracted. ▪, Aggregated Aβ42_Total plus 10 μM DAPH; ○, aggregated Aβ42_Total plus 20 μM DAPH. The 0-h time points shown were Aβ42_Total with only DMSO (the vehicle).

Fig. 5.

DAPH prevents Aβ42_30k fibril formation. Aβ42_30k was incubated without (A) and with (B) DAPH at a 10:10 μM dilution in Tyrode's solution/2 mM Ca (pH 7.4) at 37°C for 24 h. Samples were prepared by using negative-staining EM.

Fig. 6.

ThT spectrum of Aβ42_Total plus DAPH suggests a new β-sheet conformation. To measure β-sheet content, ThT was added to Aβ42_Total plus DAPH and coincubated at a dilution of 10:10 μM for 48 h at 37°C. An emission spectrum at E_x = 435 nm was measured with an f4500 spectrofluorimeter.

Fig. 7.

The IC₅₀ value for the inhibition by DAPH of Aβ42_Total, measured with ThT fluorescence, is 15.2 μM DAPH. Each data point is the mean of four experiments of 10 μM aggregated Aβ42_Total plus DMSO, with varying DAPH concentrations. A logistics curve was fitted and then used to obtain the IC₅₀ value. Parallel experiments using DiBAC₄ (3) fluorescence are also shown; this IC₅₀ value is ≈4.5 μM DAPH.

Fig. 8.

AMPA antagonists NBQX and CNQX eliminate Ca influx. CATH.a cells on a coverslip were loaded with the Ca-sensitive fluorescent dye fura-2 and measured at E_x = 340 and 380 nm and E_m = 510 nm by using a microscope-photometry system obtained from Photon Technology International. Solutions were exchanged sequentially with 10 μM aggregated Aβ42_Total, then Aβ42_Total plus 50 μM CNQX (A) or 50 μM NBQX (B), and then Aβ42_Total again. The NBQX effect is partially reversible.

Fig. 9.

Ca influx by aggregated Aβ42_Total into CATH.a cells is concentration-dependent. (A) We incubated 10 μM aggregated Aβ42 _Total at 37°C for 0, 24, and 48 h. (B) Aβ42_Total at 5,10, and 20 μM (n = 1, 15, and 3, respectively) and Aβ42_30k at 10 and 20 μM (n = 5 and 4, respectively) were incubated for 48 h at 37°C, then applied to neuronal CATH.a cells, and cytosolic Ca was measured by fura-2 fluorescence (n = 1, 15, and 3, respectively).