Extracellular vesicle-associated α-synuclein disrupts retrograde signalling-endosome trafficking in recipient neurons

<title>Abstract</title> <p> α-Synuclein (α-syn) transfer between neurons is increasingly implicated in the spread of synucleinopathies, yet its functional impact on recipient neurons remains poorly defined. Here, using microfluidic chamber technology, we show that conditioned medium from α-syn ^WT -GFP-expressing donor hippocampal neurons, but not Parkinson’s disease mutant α-syn ^A30P or EGFP controls, selectively reduced the flux of retrogradely transported cholera toxin B-positive carriers in recipient axons. A similar reduction was observed with conditioned medium from neurosecretory PC12 cells co-expressing α-syn ^WT -GFP and the chaperone HSP90 ^WT , but not the HSP90 ^MD7 mutant that disrupts multivesicular body fusion. This effect was prevented by inhibiting dynamin-dependent endocytosis, but not by immunodepletion of soluble α-syn, consistent with uptake of extracellular vesicle (EV)-associated α-syn. Accordingly, isolated EVs from α-syn ^WT -GFP-expressing PC12 cells contained α-syn. Single-molecule imaging further detected EV-transmitted α-syn ^WT in recipient axons, where it exhibited reduced mobility and periodic nanoscale clustering relative to α-syn ^A30P . Together, these findings identify EV-associated α-syn ^WT as a modulator of retrograde axonal trafficking. </p>