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Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia
Published on: November 9, 2018
Amyloid-β42 protofibrils are internalized by microglia more extensively than monomers
Lisa K Gouwens1, Nyasha J Makoni1, Victoria A Rogers1
1Department of Chemistry and Biochemistry, University of Missouri-St. Louis, One University Boulevard, St. Louis, MO 63121, United States.
Abstract:
One pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-β peptide (Aβ) in the affected brain. While there are numerous deleterious effects of Aβ accumulation, there is general agreement that a sustained inflammatory response to aggregated Aβ contributes to progressive neurodegeneration in AD and microglial cells play a significant role in this process. Our laboratory and others have shown that small soluble aggregates of Aβ activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aβ, and this process is likely very sensitive to Aβ structure. In this study we analyzed the proclivity of microglia for internalization of Aβ42 monomers and protofibrils using fluorescently-labeled Aβ. Both Aβ42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aβ42 protofibrils retained their size and morphological properties after labeling but monomers had a much higher stoichiometry of labeling compared to protofibrils. Primary murine microglia internalized AF488-Aβ42 protofibrils rapidly and in significant amounts compared to AF488-Aβ42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aβ42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aβ42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aβ structure in the internalization process and emphasize their affinity for soluble Aβ protofibrils.
Insights
Microglia readily internalize amyloid-beta (Aβ) protofibrils, but not monomers, in Alzheimer's disease research. This structural sensitivity highlights microglial affinity for soluble Aβ aggregates, influencing inflammatory responses.
Area of Science:
- Neuroscience
- Immunology
- Biochemistry
Background:
- Alzheimer's disease (AD) is characterized by amyloid-beta (Aβ) peptide accumulation.
- A sustained inflammatory response to aggregated Aβ, involving microglial cells, contributes to neurodegeneration in AD.
- Microglia-mediated inflammatory responses are activated by small soluble Aβ aggregates, with internalization being a key process sensitive to Aβ structure.
Purpose of the Study:
- To analyze the differential proclivity of microglia for internalizing Aβ42 monomers versus protofibrils.
- To investigate the structural sensitivity of microglia to Aβ during the internalization process.
- To understand the role of Aβ structure in microglial activation and inflammatory cytokine secretion.
Main Methods:
- Fluorescently labeling Aβ42 monomers and protofibrils with Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP).
- Isolation of labeled Aβ42 species by chromatography.
- Incubation of labeled Aβ42 species with primary murine microglia, followed by quantification of internalization via fluorescence microscopy and measurement of tumor necrosis factor α secretion.
Main Results:
- Primary murine microglia rapidly and significantly internalized AF488-Aβ42 protofibrils compared to AF488-Aβ42 monomers.
- Microglial internalization of protofibrils was dependent on time and concentration and correlated with tumor necrosis factor α secretion.
- Competition studies showed no reduction in AF488-protofibril uptake by unlabeled Aβ42 protofibrils, and internalized protofibrils showed limited degradation.
Conclusions:
- Microglia exhibit significant structural sensitivity towards amyloid-beta (Aβ) during the internalization process.
- Microglia demonstrate a strong affinity for soluble Aβ protofibrils over monomers.
- These findings emphasize the importance of Aβ structure in microglial recognition and uptake, potentially influencing AD pathogenesis.
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