Abstract
Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer’s disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer’s disease.
Highlights
Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer’s disease
To test quantitatively the level of lipid membrane permeability induced by soluble Aβ42 aggregates formed at different stages of the aggregation reaction (Fig. 1a), we used a recently developed high-throughput assay based on the measurement of changes in the localized fluorescence intensity of individual nano-sized lipid vesicles[19]
We immobilized thousands of individual vesicles onto glass cover slides, which act as individual nano-reactors, each containing a reporter of the presence of calcium ions (Ca2+) that enter into the vesicle, the quantity of which can be measured using total internal reflection fluorescence (TIRF) microscopy[19,20,21,22] (Fig. 1b)
Summary
Different Aβ42 aggregates have distinct toxicity mechanisms. To test quantitatively the level of lipid membrane permeability induced by soluble Aβ42 aggregates formed at different stages of the aggregation reaction (Fig. 1a), we used a recently developed high-throughput assay based on the measurement of changes in the localized fluorescence intensity of individual nano-sized lipid vesicles[19]. We observed an opposite trend, for the inhibition of aggregate-induced inflammation in microglia cells, where antibodies designed to bind to the N-terminal region of the sequence were more effective These differences are likely to be due to the favourable accessibility of solvent exposed N-terminal segments in the larger aggregates of Aβ42 that are formed at later stages of the aggregation process. As the aggregation process proceeds, and the Aβ42 aggregates on average increase in size and undergo structural changes, as we have previously observed with α-synuclein[39,48], their ability to disrupt the integrity of lipid bilayers of the type involved in cellular membranes appears to be reduced relative to triggering an inflammatory response These results suggest that different aggregated forms of Aβ42 may differ in their contributions to different mechanisms of toxicity that develop during the progression of AD and other misfolding disorders associate with Aβ self-assembly. We anticipate that effective strategies aimed at targeting pathogenic Aβ42 species formed during the progression of disease may involve the use of cocktails of therapeutic agents targeting the diversity of species that are populated during the aggregation process
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