The chaperone domain BRICHOS prevents CNS toxicity of amyloid-β peptide in Drosophila melanogaster

Abstract

Aggregation of the amyloid-β peptide (Aβ) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer's disease (AD). Mutations of the BRICHOS chaperone domain are associated with amyloid disease and recent in vitro data show that BRICHOS efficiently delays Aβ42 oligomerization and fibril formation. We have generated transgenic Drosophila melanogaster flies that express the Aβ42 peptide and the BRICHOS domain in the central nervous system (CNS). Co-expression of Aβ42 and BRICHOS resulted in delayed Aβ42 aggregation and dramatic improvements of both lifespan and locomotor function compared with flies expressing Aβ42 alone. Moreover, BRICHOS increased the ratio of soluble:insoluble Aβ42 and bound to deposits of Aβ42 in the fly brain. Our results show that the BRICHOS domain efficiently reduces the neurotoxic effects of Aβ42, although significant Aβ42 aggregation is taking place. We propose that BRICHOS-based approaches should be explored with an aim towards the future prevention and treatment of AD.

Keywords: Alzheimer’s disease; Amyloid; Chaperone; Protein misfolding.

Fig. 1.

BRICHOS suppresses the toxic effects of Aβ42 on Drosophila lifespan. The fraction of 100 living flies over time is plotted for (A) control flies, and flies expressing BRICHOS, Aβ42×1 and Aβ42×2; (B) control flies, and flies expressing Aβ42×1 and Aβ42×1 + BRICHOS; and (C) control flies (Gal4-elav^c155), and flies expressing Aβ42×2 and Aβ42×2 + BRICHOS. Survival plots were calculated using the Kaplan-Meier method and differences between groups were tested using the log-rank test, ****P<0.0001. See supplementary material Table S2 for statistical analyses of differences between all groups.

Fig. 2.

BRICHOS reduces the toxic effects of Aβ42 on locomotor activity. Aβ42×1, Aβ42×2, Aβ42×1 + BRICHOS, Aβ42×2 + BRICHOS and control (Gal4-elav^c155) flies were analyzed in a climbing assay. Flies were analyzed at (A) day 10, (B) day 15 and (C) day 25. The number of flies passing a line (8 cm above ground) within 10 seconds was counted and expressed as a percentage for each group of five flies. Twenty-five flies of each genotype divided into five tubes were analyzed five times, and statistical analyses were made using the Mann-Whitney test. The boxes extend from the 25th to 75th percentiles, the whiskers are drawn from the 10th percentile to the 90th percentile and the black dots represent outliers. The red line represents the median value. ****P<0.0001, **P<0.01, *P<0.05. See supplementary material Table S2 for statistical analyses of differences between all groups.

Fig. 3.

BRICHOS delays Aβ42 deposition in the brain. Confocal images of fly brains immunostained with an anti-Aβ42 antibody (green) and for presynaptic zones (bruchpilot antibody; red), obtained at age 10, 15 and 25 days: Aβ42×1 (A,B,E,F,I,J), Aβ42×1 + BRICHOS (C,D,G,H,K,L), Aβ42×2 (M,N,Q,R,U,V), Aβ42×2 + BRICHOS (O,P,S,T,W,X). For each specimen, two figures are shown: the one to the right showing Aβ42 staining only and the one to the left showing Aβ42 staining merged with presynaptic-zone staining. Arrows mark examples of dense granular deposits and arrowheads mark examples of fiber-like structures. The results are representative for three independent experiments. Scale bars: 40 μm. The white dotted line marks the border between the two brain hemispheres. The localization in the fly brain of the area shown here is given in supplementary material Fig. S4A.

Fig. 4.

BRICHOS delays aggregation of Aβ42 and increases soluble Aβ42 in the fly brain. Quantification of soluble and insoluble Aβ42 protein, from five fly heads of each of the different groups studied, was determined using MSD immunoassay. The levels of soluble Aβ42 in HEPES buffer pH 7.3 (A) and the levels of insoluble Aβ42 (dissolved using 5 M guanidinium-HCl in HEPES buffer pH 7.3) (B) (referred to as insoluble in the text), as well as the ratio (C) between soluble and insoluble Aβ, were plotted for day 10, 15 and 25 for the different groups. Error bars in A and B correspond to the standard deviation of the sample. The results are representative for three independent experiments.

Fig. 5.

BRICHOS colocalizes with Aβ42 in the fly brain and binds to Aβ42 fibrils formed in vitro. Immunostaining for (A) Aβ42 (6E10 antibody; green), (B) BRICHOS (anti-BRICHOS antiserum; red) and (C) both Aβ42 and BRICHOS in the brain of an Aβ42×1 + BRICHOS fly, using confocal microscopy at 63× magnification, with DAPI (blue) as counterstaining for nuclei. (D) 3 μM of Aβ42 was co-incubated with 0.7 molar equivalents of recombinant BRICHOS at 37°C overnight and an aliquot was analyzed by immuno-gold labeling for BRICHOS and electron microscopy. Scale bars: (C) 20 μm; (D) 200 nm.