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Autophagy and Extracellular Vesicle Secretion in the Prion-like Spreading of Amyloid beta

  • Author / Creator
    Viveiros, Anissa M
  • Alzheimer’s disease (AD) is characterized by the accumulation of amyloid beta (Aβ) peptide. It has been proposed that AD pathology is transmissible by a “prion-like” mechanism through extracellular vesicles (EVs) that contain Aβ. In this context, EVs describe both microvesicles and exosomes, which are derived from the outward budding of the plasma membrane and from multivesicular bodies (MVBs) respectively. MVBs can either be targeted to the lysosome as intermediates of autophagic degradation, or to the plasma membrane for exosome release. Autophagy is an essential cellular process and impairments in this pathway occur in AD. Our laboratory has discovered that Aβ inhibits autophagy by reducing protein prenylation: a post- translational modification necessary for lysosomal targeting in autophagy. Despite evolving research in EVs as a vehicle for Aβ spreading, the regulation of EV-mediated Aβ transfer is unknown. We propose that Aβ-induced autophagy blockade increases the cellular release of EVs, thus contributing to the “prion-like” spreading of Aβ aggregates.Prior to assessing the effects of Aβ, we optimized the methods for collection, separation and analysis of EVs derived from cultured cells. We analyzed EVs by image flow cytometry, nanoparticle tracking analysis and transmission electron microscopy. We characterized EV subtypes by image flow cytometry with EV markers CD9 and AnnexinV.We compared differential ultracentrifugation, which is the most common method employed in the field, to size exclusion chromatography for EV separation. Our results corroborated reports that high-speed centrifugation causes aggregation of EVs, thus we determined size exclusion chromatography to be the optimal separation method.We found that Aβ blocked autophagy and increased EV secretion. Further, Aβ treatment induced the secretion of different EV subtypes than untreated cells. We next developed a cell-to-cell transfer paradigm of EVs derived from labeled N2aAPPswe donor cells fed to N2a recipient cells to determine the transmission of fluorescently labeled EVs and Aβ. Image flow cytometry revealed a proportion of recipient cells contained EVs, and this was enhanced by blocked autophagy in the donor cells. However, under our experimental conditions, we could not detect a corresponding increase in Aβ transfer to recipient cells. Nevertheless, based on the indication that different modes of autophagy blockade caused an increase of selective EV subtypes, it is possible that the lack of increased cell-to-cell transfer of Aβ was due to the paradigm that we employed.Overall, we validated the use of Image Flow Cytometry to determine EV secretion and EV subtypes. The novelty of our work is to identify dysfunctional autophagy as critical for the intercellular spreading of Aβ and give insight, based on previous work, as impaired protein prenylation as a possible mechanism. Further, we aimed to identify and address problems in the field, as many conclusions are derived based on ultracentrifugation procedures that may hamper physiological relevance. We proposed image flow cytometry as the optimal technique to derive conclusions on EV secretion.

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  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Master of Science
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