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Amyloid-beta aggregation and effects of soluble tetramers on microglia-mediated inflammatory response

University of British Columbia

The presence of Amyloid-β (Aβ) aggregates in the brain is a hallmark of Alzheimer’s disease. However, the heterogeneity of these assemblies and their highly dynamic nature have rendered them challenging to target. It is known that the smaller aggregates are toxic to neurons and show a potential to stimulate a glia-mediated pro-inflammatory response in the brain. We first sought to further characterize the pathway of Aβ aggregation by targeting different stages of assembly with an oligomer-specific antibody (anti-cSNK) and the sugars trehalose and glucosamine. Incubation of Aβ₄₂ with the anti-cSNK antibody and glucosamine lengthened the nucleation phase and diminished the maximum β-sheet signal after 72 hrs, as tracked with a Thioflavin T fluorescence aggregation assay. Incubation with trehalose only affected maximum β-sheet signal. The analysis of samples collected at different timepoints with TEM and immunoblotting revealed a delay in Aβ₄₂ aggregation into insoluble species in presence of the two sugars and the anti-cSNK antibody. In conclusion, we lend further support to the hypothesis that AβOs are part of the fibril formation pathway. To better understand the role that different Aβ soluble aggregates play in microglia activation, we used stabilized Aβ oligomers consisting primarily of tetramers. Upon exposure to AβOs at different concentrations (ranging from picomolar to nanomolar), NO and TNF-α levels in addition to cell morphology were used as indicators of cell activation after 24 hrs. Cell viability was tracked at various time points with different cytotoxicity assays. While the Aβ tetramers did not cause significant NO and TNF-α release, they did induce significant decrease in the signal detected by the MTT assay, usually used as an indirect measure of cell viability. These experiments were conducted in both primary and immortalized murine microglial cells in vitro, yielding consistent results across both systems. However, other measures of cell viability did not reflect the same results, suggesting that the AβOs used in this experiment have an alternative effect on the cells. Although our data do not support our initial hypothesis, they generate a new line of inquiry with regards to the effects of Aβ tetramers on microglia.

Text

2019-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/66644

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/