Fig. 4

The level and activity of MAO-B are altered in Ts2 mitovesicles. (A) Expected results after the incubation of intact mitovesicles with proteinase K (PK). Outer membrane-bound proteins (e.g., MAOs) facing the outside of the EV are cleaved. Proteins that face the inside of the EV and matricial proteins (e.g., MDH2), are shielded by one or two membranes and are not digested. The solubilization of both membranes with a detergent (SDS) causes the degradation of all proteins, regardless of the localization. (B, C) Representative Western blots (B) and densitometric quantifications (C) of MAO-A and MAO-B protein levels (normalized to MDH2 levels) in 2N mitovesicles (n = 3 mice) upon incubation with PK at increasing concentrations. MDH2: loading control. Last lane: mitovesicles lysed with SDS before PK incubation (positive digestion control). PK effect P = 0.0026 for MAO-A and P < 0.0001 for MAO-B. For MAO-A: untreated vs. 10 µg/mL PK P = 0.1202; untreated vs. 20 µg/mL PK P = 0.0122; untreated vs. 40 µg/mL PK P = 0.0013. For MAO-B: untreated vs. 10 µg/mL PK P = 0.0005; untreated vs. 20 µg/mL PK P = 0.0002; untreated vs. 40 µg/mL PK P < 0.0001. Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test. In (C-F), all bars are mean ± SEM. (D, E) Representative Western blots (D) and densitometric quantifications (E) of MAO-B protein levels (normalized to NDUFB8 levels) in equal numbers (quantified by NTA) of mitovesicles isolated from the brain of 2N and Ts2 mice (n = 4). NDUFB8 is the loading control to prove similar amount of EVs within each pair. 2N vs. Ts2 mitovesicles P = 0.0132. Two-tailed, unpaired Student’s t-test. (F) MAO-B enzymatic activity (expressed as U/L) in 1 µg intact mitovesicles isolated from the brain of 2N and Ts2 mice (n = 7), or from human DS frontal cortices (n = 3) as compared to their respective 2N controls. P = 0.0382 and P = 0.0150 for murine and human samples, respectively. Two-tailed, unpaired Student’s t-test (different test for each species). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001