Figure 1.
Removal of protein aggregates by ASR1 decreases Aβ levels in AD brain homogenate.
Aβ levels of an ASR1-treated AD brain homogenate were biochemically evaluated using anti-human Aβ ELISA kits. The quantity of Aβ40 (A) and Aβ42 (B) was measured in different fractions (PBS, SDS, and FA) obtained after serial extraction. Measurements were performed in duplicates and the bars represent the average of the two determinations. Serial extraction was performed in order to assess the content of different aggregated Aβ species (i.e. PBS: monomeric and soluble oligomeric Aβ; SDS: water insoluble oligomers and protofibrils; FA: large fibrilar structures). (C) Coomasie staining of SDS-PAGE fractionated PBS, SDS and FA fractions performed to assess total removal of proteins by the ASR1 compound. PBS: Phosphate Buffer Saline; SDS: Sodium Dodecyl Sulphate; FA: Formic Acid; MW: Molecular Weight Marker. Numbers in the right side of (C) represent molecular weight (in KDa).
Figure 2.
Aggregate-depletion by the ASR1 compound significantly reduces the in vivo seeding effect of an AD brain.
Brains from experimental and control animals were analysed by immunostaining using the 4G8 antibody. (A) Representative pictures of the amyloid deposits found in the hippocampus of non-injected animals, as well as mice injected with AD brain homogenate untreated or treated with ASR1-beads or control-beads. (B) Magnification of the area depicted in (A). The scale bar corresponds to 250 µm in (A) and 100 µm in (B). (C) Image analysis quantification of the 4G8-stained plaques (burden) in the hippocampus. Aβ burden was defined as the area of the brain labeled with the Aβ antibody per total area analyzed and is expressed as percentage. * P<0.05; ** P<0.01. *** P<0.001.
Figure 3.
AD-mice injected with aggregate-depleted samples show lower levels of insoluble Aβ42 in their brains.
Frozen hippocampi were homogenized and the quantity of aggregated Aβ42 was measured by ELISA after formic acid extraction as explained in the Materials and Methods section. The quantity of insoluble Aβ42 was measured using ELISA kits designed to specifically detect this peptide. The values shown correspond to the means ± SEM. * P<0.05.
Figure 4.
Aggregate removal by ASR1 reduces load of ThS-positive amyloid fibrils.
ThS stained brain slices from animals inoculated with different samples were analysed with an epifluorescent microscope. (A) Representative pictures of fibrillar Aβ from each group. (B) Magnification of the areas marked in (A). The scale bar corresponds to 250 µm in (A) and 100 µm in (B). (C) The burden of ThS positive deposits was compared among all different control and experimental groups by image analysis. * P<0.05; *** P<0.001.