Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes
Fig 4
Mitochondrial autophagy evaluation by confocal and electron microscopy.
(A) Mitophagic flux for control, starved and rapamycin-treated cells. For each experimental condition, MTDR fluorescence was measured in the presence or absence of chloroquine (CQ). Each value is expressed as the ratio of MTDR mean fluorescence intensity (MFI) in the presence of CQ to that in the absence of CQ. The difference between cell lines was determined to be significant by two-way ANOVA (***P < 0.001). (B) Evidence of mitophagy type 1/2 (classic mitophagy) in confocal microscopy. (i) Single confocal optical sections (~0.8 μm thickness) showing overlay of LTG (green) and MTR (red) in wild-type and pathologic samples for control, starved and rapamycin-treated cells. White arrows represent the LTG-labeled acidic organelles colocalizing with MTR-labeled mitochondria. Bars: 10 μm. (ii) Pearson's colocalization coefficient (PCC) of LTG and MTR for control, starved and rapamycin-treated cells. The uncoupling agent CCCP was used as a positive control. Pearson's coefficients were derived from three completely independent experiments with >10 fields per experiment contributing to the cumulative result. Each value is expressed as PCC ± SD; **P < 0.01 and ***P < 0.001 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 (++P < 0.01) between tWT and tNP starved cells. (C) TEM evidence of mitophagy type 1/2 (classic mitophagy) in Niemann-Pick cells in control condition (a), after nutrient deprivation (b, c) and rapamycin administration (d, e, f). In NP samples, mitochondria were recognizable within autophagic vacuoles (black arrows). Mvb: multivesicular bodies. Bars: 0.5 μm for a, c, d, f; 0.2 μm for b, e. (D) TEM evidence of mitophagy type 3 (micromitophagy) in normal (a, i) and NP-B B lymphocytes (b-h, j) after rapamycin administration (a-h) and nutrient deprivation (i, j). Black arrows indicate mitochondria-derived vesicles (MDVs) or mitochondria closely linked to autophagic vacuoles. Several of these structures were located right next to lipid droplets (b) and multivesicular bodies (mvb, d, f-h). Bars: 0.5 μm for a, b, d, f, g, i; 0.2 μm for c, e, h, j. (E) Mitochondria-derived vesicles (MDVs) from non-pathological and pathological cells were counted in all experimental conditions. Each value is expressed as an absolute number ± SD (n = 3; 50 cells counted/experiment). *P < 0.05, **P < 0.01 and ***P < 0.001 vs respective control. The difference between cell lines was determined to be significant by two-way ANOVA (***P < 0.001). Two-way ANOVA with Bonferroni post test revealed a P value < 0.001 (+++P < 0.001) between tWT and tNP basal condition and rapamycin-treated cells.
