Fig 1.
Age-related increase in the amount of Aβ aggregates in the midgut of Aβ-expressing flies.
(A) Illustration of longevity analyses of the Arctic-, T22Aβ1-42- and control w1118 flies, from a previous study [40]. The fluorescence acquisition was carried out when the flies were 4 and 8 days old, see markings. (B-C) Age study of flies expressing either (B) the tandem dimeric construct T22Aβ1-42 or (C) the Arctic mutant of Aβ1-42 in the enterocytes using the Myo31DF driver line. (C) Arrowheads indicate vague outlines of aggregates. The gut tissue is stained with LCOs HS-84 (green) or HS-169 (orange). The age of the flies is indicated in the figure. Scale bars are 20 μm.
Fig 2.
The smurf phenotype indicates gut leakage.
(A) A fly with the smurf phenotype to the left compared to a non-smurf fly to the right. (B) The smurf phenotype was evaluated in Arctic-, T22Aβ1-42- and control w1118 flies, 8 days post eclosion.
Fig 3.
Several smaller Aβ species detected by the antibody and HS-169 in T22Aβ1-42 flies are not detected by HS-84.
Detection of Aβ species in the anterior midgut of Drosophila flies expressing the tandem construct T22Aβ1-42 in the enterocytes using the Myo31DF driver. The confocal microscope single-plane images show gut tissue co-stained with Mabtech anti-human Aβ antibody (magenta) and LCO ligands HS-84 (green) and HS-169 (orange) 8 days post eclosion. The white, dotted line outlines the edges of the intestine. White circles: Aβ species labeled by the antibody and HS-169. White arrowheads: Aβ species labeled by all three staining agents. Scale bar, 20 μm.
Fig 4.
Several Aβ species detected by the antibody and HS-84 in Arctic flies are not detected by HS-169.
Detection of Aβ species in the anterior midgut of Drosophila flies expressing the Arctic mutant of Aβ1-42 in the enterocytes using the Myo31DF driver. The confocal microscope single-plane images show gut tissue costained with Mabtech anti-human Aβ antibody (magenta) and LCO ligands HS-84 (green) and HS-169 (orange) 8 days post eclosion. The white, dotted line outlines the edges of the intestine. White circles: Aβ species labeled by the antibody and HS-84. White arrowheads: Aβ species labeled by all three staining agents. Scale bar, 20 μm.
Fig 5.
Quantifications of the co-staining in T22Aβ1-42- and Arctic flies.
Nonbiased scoring of Aβ1-42 species per mm2 in Arctic- and T22Aβ1-42 flies at day 8, n = 6. Only Aβ1-42 species where HS-84 or HS-169 colocalized with the antibody were counted. Data represented as ± SD. **** represents P < 0.0001, respectively, as determined by an unpaired t test. ns equals non-significant.
Fig 6.
The proteotoxic effect is substantially stronger in Arctic flies compared to T22Aβ1-42 flies.
MSD analyses were performed on the body of Myo31DF-derived flies expressing the Arctic mutant of Aβ1-42 or tandem construct T22Aβ1-42 to measure the levels of Aβ1-42. Data represented as mean (±s.d). (A) The levels of soluble Aβ1-42. (B) The levels of insoluble Aβ1-42. (C) The Aβ1-42 proteotoxic effect was assessed by dividing the reduction in median survival time of the Aβ genotypes relative to the control flies, with the total level of Aβ1-42 per fly (see A-B). Data represented as ± SD. **** represents P < 0.0001 as determined by a one-way ANOVA followed by Tukey’s test. ns equals non-significant.
Table 1.
Statistic data from MSD analysis where the stability of Aβ aggregates produced in Arctic and T22Aβ1-42 was tested against Gua-HCl. SD: standard deviation. Aβ1-42 levels in the 0 M-, 2.5 M-, 4 M-, and 5 M fractions were measured (graph illustrated in Fig 7A).
Fig 7.
MSD analyses show different Aβ species with varying stability.
MSD analyses were performed on the body of Myo31DF-derived flies expressing the Arctic mutant of Aβ1-42 or tandem construct T22Aβ1-42 to measure the levels of Aβ1-42. Data represented as mean (±s.d). (A) Samples were treated with increasing concentrations of Gua-HCl: 0 M; 2.5 M; 4 M; and 5 M, to obtain the level of Aβ1-42 in each fraction (named according to the Gua-HCl concentration). Data represented as ± SD. **** represents P < 0.0001 as determined by a one-way ANOVA followed by Tukey’s test. ns equals non-significant. (B) The amount of Aβ1-42 is calculated in percentage for each fraction relative to the total level of Aβ1-42. (C) The difference in Aβ1-42 levels between Arctic and T22Aβ1-42 flies is calculated for all Gua-HCl fractions (0 M-, 2.5 M-, 4 M-, and 5 M) (data is seen in Fig 7A and Table 1).
Fig 8.
Proposed illustration of the Aβ fibril formation in relation to the MSD analysis using a Gua-HCl gradient.
Aβ can assemble through primary- or secondary nucleation. The type of Aβ species residing in each Gua-HCl fraction is indicated in the figure.