AN ASSAY FOR SEEDED PROTEIN AGGREGATION DETECTS ABETA SEEDS IN SERUM

Abstract

Many common neurodegenerative disorders are characterised by progressive protein aggregation. In Alzheimer's disease the principal pathologies of interest are amyloid plaques composed of the amyloid-beta peptide (Aβ) and tangles comprised of tau. The spread of pathology within the brain is thought to be mediated by a prion-like seeding process that accelerates the aggregation of susceptible polypeptides in the neighbourhood of existing deposits. Soluble seeds of Aβ may travel widely: between neurones or even to the CSF and peripheral circulation. We measure seeded Aβ aggregation in brain and blood extracts from animal models of Alzheimer's disease. Firstly we examine brains from Drosophila melanogaster expressing the Aβ peptide versus non-expressing controls. Secondly we assess brain and serum from aged CRND8 mice and compare them to their non-transgenic littermate controls. Brains are homogenised in PBS followed by dilution to give a protein concentration of 3 mg/ml. Serum is diluted to 3 mg/ml in PBS.Our assay uses microfluidic apparatus to compartmentalise these crude biological extracts into aqueous droplets (50 μm diameter) containing fluorescent-labelled Aβ42 peptide along with molten agarose (37°C). The droplets are cooled to room temperature, whereupon the agarose forms a gel, and are then incubated for up to 3 hr. Following this incubation the oil phase is removed and the uncoated agarose microbeads are washed to remove unincorporated fluorescent peptide. Seeded aggregates are marked by their having incorporated fluorescent-labelled Abeta during the incubation step. Aggregates that are larger than MW 1000 kDa are retained within the agarose matrix and are detected and quantified by fluorescence activated cytometry (FACS). Over 10,000 beads are assessed for each assay.In the brain extracts we see significantly higher fluorescence signals in microbeads derived from AD-affected organisms as compared to controls. Remarkably this higher fluorescence signal is also seen in the serum samples from affected mice. Agarose beads alone have a low intrinsic fluorescence (grey). In the presence of fluorescent labelled Aβ peptide (blue) the agarose beads retain a background level of fluorescence. Healthy murine serum resulted in increased fluorescence labelling of beads (orange) but the strongest signal was seen for serum derived from CRND8 mice (green).