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Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype

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Version 2 2020-01-20, 11:49
Version 1 2017-12-04, 08:26
journal contribution
posted on 2020-01-20, 11:49 authored by Georgia Minakaki, Stefanie Menges, Agnes Kittel, Evangelia Emmanouilidou, Iris Schaeffner, Katalin Barkovits, Anna Bergmann, Edward Rockenstein, Anthony Adame, Franz Marxreiter, Brit Mollenhauer, Douglas Galasko, Edit Irén Buzás, Ursula Schlötzer-Schrehardt, Katrin Marcus, Wei Xiang, Dieter Chichung Lie, Kostas Vekrellis, Eliezer Masliah, Jürgen Winkler, Jochen Klucken

The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct “autophagosome-exosome-like” profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.

Funding

This study was supported by the Interdisciplinary Center of Clinical Research (IZKF) of the University Hospital Erlangen, Germany (IZKF no. E21 and E11) and the Bavarian State Ministry of Education and Culture, Science and Arts in the framework of the Bavarian Research Network for Induced Pluripotent Stem Cells (ForIPS). In addition, it was supported by the deutsche Forschungsgemeinschaft/German Research Foundation (DFG, no. KL1395/8-1), and the Research Foundation Medicine, University Hospital Erlangen (Parkinson and Nutrition) WX was supported by the Johannes und Frieda Marohn-Stiftung (Alpha-synuclein). KB and KM were supported by P.U.R.E., Protein Unit for Research in Europe, North Rhine-Westphalia and the H2020 project NISCI, GA no. 681094. EM and DG were supported by National Institutes of Health (NIH) grants NIH AGO5131 and NIH AG18440.

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