Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein

Abstract

Misfolding and aggregation of alpha-synuclein (αsyn) resulting in cytotoxicity is a hallmark of Parkinson's disease (PD) and related synucleinopathies. The recent body of evidence indicates that αsyn can be released from neuronal cells by nonconventional exocytosis involving extracellular vesicles (EVs) such as exosomes. The transfer of αsyn between cells has been proposed to be an important mechanism of disease propagation in PD. To date, exosome trafficking mechanisms, including release and cell-cell transmission, have not been fully described. To gain insight into the mechanisms involved, exosomes were purified from conditioned media of stable cells secreting αsyn oligomers. A novel bimolecular protein complementation assay was used to detect exosomes containing αsyn oligomers. Recipient cells were treated with exosomes containing αsyn oligomers or “free” non-exosome-associated αsyn oligomers and internalization was monitored. We demonstrate that cell-derived exosome-associated αsyn oligomers can be efficiently internalized by recipient cells. Interestingly exosome-free αsyn oligomers isolated from conditioned medium were not internalized but remained bound to the extracellular surface. To investigate the endocytic pathway(s) required for the exosome uptake different pharmacological inhibitors of caveolin-dependent, clathrin-dependent, and macropinocytosis pathways were utilized. Surprisingly, none of these pathways appear to play a significant role in the internalization of exosome-associated αsyn oligomers. Finally, the role of heparin sulfate proteoglycans (HSPGs) in exosome-associated αsyn internalization was investigated using genetic approach. Despite previous studies showing HSPGs can modulate internalization of fibrillar αsyn, genetic manipulations did not attenuate internalization of exosome-associated αsyn oligomers in our hands, suggesting that exosome-associated αsyn is internalized via an alternative endocytic pathway(s) that has yet to be elucidated.

A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol

“…Our results after introducing recombinant αSyn to the culture medium suggested that the Reportercells could reliably report the uptake of extracellular αSyn. Similar to other reports 75,77,78 , we did not detect direct cytoplasmic internalization of naked monomeric hαSyn by the Reporter-cells (derived from HEK293 cells). We rather observed that the cytosolic uptake required assistance with lipid-based elements as also had been shown in other studies 75,78 .…”

How is alpha‐synuclein cleared from the cell?

“…Once internalized, exosomal a-Syn escapes the endosomal compartment and is targeted to the lysosome for degradation. Although receptor-mediated endocytosis has been proposed as a main route of exosome internalization, a more recent study using pharmacological inhibitors failed to confirm this (Delenclos et al 2017). According to this study, the entry of exosome-associated a-Syn in recipient H4 neuroglioma cells could be attributed neither to caveolin-mediated endocytosis nor macropinocytosis.…”

In vitro models of synucleinopathies: informing on molecular mechanisms and protective strategies

“…Recent evidence suggests that membrane binding is a crucial step for a-Syn internalization, which is mediated by Rab proteins (Masaracchia et al 2018). A recent study characterizing the internalization mechanisms of a-Syn containing exosomes found that the process was an active as opposed to a passive process, yet resistant to conventional blockers of both clathrin and caveolin-mediated endocytosis as well as micropinocytosis (Delenclos et al 2017). Clearly, the resolution of such discrepancies between methodological approaches will be required in the future.…”