Fig. 4

PD-EEVs reduce tight junction levels in BMEC-like cells and induce dopaminergic neuron atrophy. (A) Representative confocal images of control BMEC-like cells immunostained for VE-cadherin (white), internalized EEVs are shown in red (red, white arrows) and DAPI nuclear stain is shown in blue. Scale bar: 20 μm. (B-C) Quantification of BMEC-like cell area (B) and frequency distribution (C) in untreated conditions or after 48 h of treatment with EEV derived from control, mild PD or severe PD. (D) Western blot-based quantification of tight junction protein levels for VE-cadherin, ZO-1, and Claudin 5 in BMEC-like cell monolayers after a 48-h incubation with EEVs. Protein levels are normalized to BMEC-like cell treated with EEV control. (E) Representative confocal images of iPSC-derived neurons labeled for the pan-neuronal marker MAP2 (white) and a marker specific for dopaminergic neurons (TH, red). Images were taken after a 72-h treatment with vehicle control (i.e. untreated), age-/sex-matched control EEVs, mild or severe PD EEVs. Scale bar: 25 μm. (F-G) Quantification of MAP2⁺/TH⁻ (F) and TH⁺/MAP2⁺ (G) neurite lengths when iPSC-derived neurons are incubated in the absence or presence of EEVs derived from PD donors diagnosed with mild or severe PD, or age-/sex-matched controls, as shown in (E). Demographics: control subjects (n = 2–4, mean age = 70±4), patients with mild (n = 2–4, UPDRS = 26±3, mean age = 60±3) or severe (n = 4, UPDRS = 90±8, mean age = 68±5) PD. Statistical analysis: Error bars represent mean + SEM. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test: Comparison vs. untreated ***p-value < 0.001, ****p-value < 0.0001; Comparison vs. control EEV ^%%p-value < 0.01, ^%%%%p-value < 0.001 (B, F-G); Kuskal-Wallis test followed by Dunn’s multiple comparisons test: Comparison vs. untreated ***p-value < 0.001, ****p-value < 0.0001; Comparison vs. PD severe EEV p-value < 0.0001 (C). Statistical analysis was performed using Wilcoxon signed rank test with theoretical median (D – Ctrl EEV) set at 1 (*p-value < 0.05). (B-C) Corresponds to the BMEC-like area (B) or EEV/BMEC-like cell (C) of two independent replicates where two different iPSC donors (line IDs 38554 and 41658) and EEVs from two different individuals per group were used (i.e. at least 300 cells/experimental group); (D) Corresponds to two different iPSC donors (line IDs 38554 and 41658) and EEVs sampled from three to four different individuals per group were used. (F-G) Corresponds to the combined neurite length values of two independent replicates where two different iPSC donors (line IDs 38554 and IM2-GC) and EEVs from two different donors per group were used (i.e. at least 150 neurites/experimental group). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; EEV, extracellular vesicles derived from erythrocytes; MAP2, microtubule-associated protein 2; ZO-1, Zonula occludens-1