Transmitted alpha-synuclein extracellular vesicles downregulate axonal flux of retrograde carriers in recipient neurons
Alpha-synuclein (alpha-syn) is a cytosolic protein located in nerve terminals and is involved in several neurodegenerative diseases such as Parkinson's disease. Recent studies have demonstrated that alpha-syn can be transmitted from neuron to neuron via exosomal release, thereby contributing to the propagation of alpha-syn pathology. However, the mechanism by which alpha-syn-containing exosomes perturb the function of recipient neurons is currently unknown. Retrograde axonal transport of carriers emanating from the presynapse is essential for neuronal survival. To determine the effect of transmitted alpha-syn on neuronal retrograde trafficking, we used conditioned medium from alpha-syn transfected donor hippocampal neurons, applied to naive (non-transfected) recipient neurons cultured in microfluidic chambers. Time-lapse imaging of retrograde carriers containing cholera toxin B subunit (CTB) was then performed in these recipient neurons. Here, we show that conditioned medium from alpha-synWT-GFP transfected donor neurons significantly downregulated the frequency of retrograde CTB carriers when applied to recipient neurons. This effect was abolished by (i) inhibiting endocytosis in recipient neurons, (ii) blocking exosomal release from donor cells via an Hsp90-dependent mechanism, or (iii) by using conditioned medium from neurons transfected with a Parkinson's mutant (alpha-synA30P-GFP). Whilst transmitted, alpha-synWT-mEos2, alpha-synA30P-mEos2, and mEos2 alone were detected in recipient neurons using single-molecule imaging. Interestingly, alpha-synWT-mEos2 exhibited lower mobility and periodic (190 nm) immobilizations along the axon. Our data suggest that the downregulation of vesicular trafficking by transmitted alpha-syn does not rely on the specific release nor uptake of exosomal alpha-syn but probably depends on its interaction with endogenous alpha-syn following endocytosis in recipient neurons. The transmitted alpha-syn-containing extracellular vesicles therefore control essential axonal trafficking in recipient neurons, an effect lost with the Parkinson's disease mutant alpha-synA30P.