Aβ42 Protofibrils Interact with and Are Trafficked through Microglial-Derived Microvesicles

Microvesicles (MVs) and exosomes comprise a class of cell-secreted particles termed extracellular vesicles (EVs). These cargo-holding vesicles mediate cell-to-cell communication and have recently been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). The two types of EVs are distinguished by the mechanism of cell release and their size, with the smaller exosomes and the larger MVs ranging from 30 to 100 nm and 100 nm to 1 μm in diameter, respectively. MV numbers are increased in AD and appear to interact with amyloid-β peptide (Aβ), the primary protein component of the neuritic plaques in the AD brain. Because microglial cells play such an important role in AD-linked neuroinflammation, we sought to characterize MVs shed from microglial cells, better understand MV interactions with Aβ, and determine whether internalized Aβ may be incorporated into secreted MVs. Multiple strategies were used to characterize MVs shed from BV-2 microglia after ATP stimulation. Confocal images of isolated MVs bound to fluorescently labeled annexin-V via externalized phosphatidylserine revealed a polydisperse population of small spherical structures. Dynamic light scattering measurements yielded MV diameters ranging from 150 to 600 nm. Electron microscopy of resin-embedded MVs cut into thin slices showed well-defined uranyl acetate-stained ring-like structures in a similar diameter range. The use of a fluorescently labeled membrane insertion probe, NBD C₆-HPC, effectively tracked MVs in binding experiments, and an Aβ ELISA confirmed a strong interaction between MVs and Aβ protofibrils but not Aβ monomers. Despite the lesser monomer interaction, MVs had an inhibitory effect on monomer aggregation. Primary microglia rapidly internalized Aβ protofibrils, and subsequent stimulation of the microglia with ATP resulted in the release of MVs containing the internalized Aβ protofibrils. The role of MVs in neurodegeneration and inflammation is an emerging area, and further knowledge of MV interaction with Aβ may shed light on extracellular spread and influence on neurotoxicity and neuroinflammation.