Fig 2: RBC-EVs contain oligomeric a-syn species and promote a-syn aggregation. a Levels of oligomeric a-syn in CD235a containing-EVs (RBC-EVs) in plasma were isolated by immuno-capture from healthy control subjects (n = 12 pools combined from 59 subjects), patients with PD (n = 11 pools combined from 109 subjects) and reference plasma samples (n = 4; pooled plasma generated from 30 healthy controls) or IgG captured EVs reference plasma samples (n = 4) measured by ECL immunoassays (means + S.E.M; *p < 0.05 by One-way ANOVA test). b Dot blot analysis of RBC-EVs isolated from control human subjects. Blots were probed with antibodies specifically against oligomeric a-syn, and oligomerized synthetic a-syn was used as a loading control. c Schematic representation showing the chronic injection strategy. RBC-EVs obtained from the plasma from human PD patients or healthy controls were injected 2 times/week i.v. into 3 month old A53T mice for 6 weeks. d-e After chronic RBC-EVs injection, mouse brain slices were immunofluorescently stained for oligomeric a-syn. Representative images of striatum (STR) (d) and quantification of fluorescence intensity of oligomeric a-syn in various brain regions (cortex (CTX), striatum (STR), cerebellum (CERE) and midbrain (MIDB) (e) are shown . Note that A53T mice injected with RBC-EVs derived from PD subjects showed increased levels of oligomeric a-syn fluorescence intensity compared to A53T mice injected with control RBC-EVs. (Scale bar, 10 µm, n = 5 independent animals were used in each group means + S.E.M; *p < 0.05 by One-way ANOVA test). f Mouse tissues from different brain regions were also homogenated after chronic RBC-EVs injection, and the levels of oligomeric a-syn in mouse brain homogenate of cortex (CTX), striatum (STR), midbrain (MIDB) and cerebellum (CERE) were measured by an ECL immunoassay (n = 5 independent animals were used in each group; means + S.E.M; *p < 0.05 by Student’s t-test *p < 0.05 compared to control-EVs injected group)