Fig 1.
Evaluation of cell death by Propidium Iodide (PI), Annexin (Anx) and transmission electron microscopy.
(A) Dot plots PI vs SSC from Niemann-Pick (tNP) and wild-type (tWT) B lymphocytes for control (CTRL), starved (STARV), rapamycin (RM), nocodazole (NZ) and wortmannin (WM) treated cells. Rectangular regions show events with low PI uptake (PI+ in R3) and high PI uptake (PI++ in R2), pointing to apoptosis and necrosis, respectively. (B) The statistical histogram shows the total PI uptake percentage (events in R2 and R3) in non-pathological and pathological lymphocytes. Each value is expressed as a percentage ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 and ***P < 0.001 vs respective control. (C) Dot plots Annexin-FITC vs FSC from Niemann-Pick (tNP) and wild-type (tWT) B lymphocytes from control (CTRL) and starved (STARV) cells. Rectangular regions (populations in green) show Anx+ early apoptotic events. (D) The statistical histogram shows Annexin (Anx) positivity percentage in non-pathological and pathological lymphocytes. Each value is expressed as a percentage ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 and ***P < 0.001 vs respective control. (E) TEM images of tWT (a) and tNP (b-e) cells from different experimental conditions: in control condition (a, b), after nutrient deprivation (c), rapamycin (d) and nocodazole (e) administration. In c, d and e autophagic cell death in vacuolated cells is recognizable (arrows: autophagic vacuoles, AVs). Bars: 5 μm for a, b, e; 2 μm for c, d.
Fig 2.
Evaluation of mitochondrial dysfunctional features by flow cytometry and transmission electron microscopy.
(A) Statistical histogram of MFI variation of NAO in tWT and tNP cells for each experimental condition. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 vs respective control. The difference between cell lines was significant as shown by two-way ANOVA (***P < 0.001). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition. (B) Flow cytometry histogram overlay depicting NAO MFI values of tWT and tNP basal condition. (C) Statistical histogram of MFI variation of TMRE in tWT and tNP cells for control, starved and rapamycin-treated cells. Mean values were converted to arbitrary units (A.U.) setting control values from wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 vs tWT control. The difference between cell lines was significant as shown by two-way ANOVA (*P < 0.05). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition. (D) Electron microscopy analyses of normal (a) and NP-B B lymphocytes (b-g) in control condition (a, b), and after starvation (c), rapamycin (e), nocodazole (f, g) and wortmannin (d) administration. In Niemann-Pick cells, dysfunctional mitochondria are recognizable (arrows): altered morphology is characterized by dilated and distorted cristae (c, d, e) and by stress-damaged features (b, f, g), absent in non-pathological cells (a). Bars: 1 μm for a, b, c, d, f, g; 0.5 μm for e. (E) Mitochondria from non-pathological and pathological cells were counted and classified according to their morphology—mitochondria with standard cristae (i), mitochondria with unusual cristae (ii) and oxidative stress-damaged mitochondria (iii)–in control, starved and rapamycin-treated cells. Each value is expressed as a percentage ± SD (n = 3; 50 cells counted/experiment); *P < 0.05 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 and < 0.05 for standard and unusual cristae numbers respectively (++P < 0.01 for standard cristae and +P < 0.05 for unusual cristae) between tWT and tNP in basal condition. (F) ROS detection by CM-H2DCFDA in flow cytometry. (i) Statistical histogram of MFI expression of CM-H2DCFDA in tWT and tNP cells. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 and **P < 0.01 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition and after nocodazole treatment; NS: not significant. (ii) CM-H2DCFDA flow cytometric histograms are overlaid to show the comparison between MFI values of tWT and tNP basal condition. (G) Statistical histogram of MFI expression of MitoSOX in tWT and tNP cells. Mean values were converted to arbitrary units (A.U.) setting as 100 control MFI values of wild-type cells. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition and after nocodazole treatment, and a P value < 0.01 (++P < 0.01) between tWT and tNP after starvation and wortmannin treatments; NS: not significant.
Fig 3.
Autophagic Vacuole (AV) detection and endocytic compartment evaluation.
(A) Acidic vesicular organelle (AVOs) detection by LysoTracker Green (LTG) in flow cytometry. (i) Statistical histogram of MFI variation of LTG in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments). 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 < 0.01 (++P < 0.01) between tWT and tNP starved cells (ii) Flow cytometry histogram overlay showing lysotracker green MFI values of tWT and tNP basal condition. (iii) Fold increase related to histogram in (i) starved and rapamycin-treated cells in both cell lines, versus respective control. (B) Autophagic vacuole detection by Monodansylcadaverine (MDC) in fluorescence microscopy and flow cytometry. (i) Microscopy images showing monodansylcadaverine fluorescence from control and starved tWT and tNP cells. White arrows indicate cells rich in AV. Bars: 10μm. (ii) Statistical histogram depicting MFI variation of monodansylcadaverine in tWT and tNP cells for control, starved and rapamycin-treated cells. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments). Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP in basal condition and rapamycin treated and a P value < 0.01 (++P < 0.01) between tWT and tNP after starvation treatment (iii) Monodansylcadaverine flow cytometric histograms are overlaid to show the comparison among MFI values of basal condition, starved and rapamycin-treated samples in tWT (upper panel) and tNP (lower panel) lymphocytes. (C) TEM autophagic vacuole detection in control condition of tWT (a, b) and tNP (c-g) cells. Black arrows indicate AVs. Bars: 2 μm for a, b, c, d, f, g; 0.5 μm for e. (D) TEM autophagic vacuole detection in normal (a, e) and NP-B B lymphocytes (b, c, d, f), after nutrient deprivation (e, f) and rapamycin administration (a-d). Black arrows indicate AVs. Black arrowhead indicates a lipid droplet inside AV. Mvb: multivesicular bodies. Bars: 1 μm for a, f; 2 μm for b, c, d, e. (E) Double- and single- membrane autophagic vacuoles from tWT and tNP lymphocytes were counted in control, starvation and rapamycin conditions. Each value is expressed as an absolute number (70 cells counted/experiment). Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 between tWT and tNP basal condition and a P value < 0.05 between tWT and tNP rapamycin treated.
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.
Fig 5.
Evaluation of intracellular lipid content by Nile Red (NR).
(A) Single confocal optical sections (~0.8 μm thickness) showing overlay of yellow (FL2) and red (FL3) NR fluorescence from all experimental conditions in tWT and tNP cells. Bars: 10 μm. (B) Statistical histogram of FL2 MFI variation of intracellular NR in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 vs respective control.
Fig 6.
Evaluation of intracellular lipid content and lipophagy by BODIPY® 665/676 (BP).
(A) Single confocal optical sections (~0.8 μm thickness) showing overlay of LTG (green) and BP (red) and the relative merge with bright field (BF), from control, nutrient deprivation and rapamycin- treated tWT and tNP cells. Insets show lipid droplets bound to the outer cell surface, probably newly extruded from the cell, or just below the outer cell membrane, most likely about to be exocytosed by the cell. Bars: 10 μm; 5 μm for insets. (B) Pearson's colocalization coefficient (PCC) for LTG and BP in control and pathologic cells for control, nutrient deprivation and rapamycin administration. 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 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 < 0.01 (++P < 0.01) between tWT and tNP basal condition and rapamycin-treated cells. (C) Statistical histogram of MFI variation of intracellular BP in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 vs respective control.
Fig 7.
Detection of microvesicles and lipid particles in the extracellular environment and analysis of lysosomal exocytosis.
(A) Statistical histogram showing lipid particle counting by NR-FL2 in the extracellular environment for each experimental condition. Each value is expressed as an absolute number ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP starved cells. (B) Statistical histogram showing CD63+ events counted in the extracellular environment for each experimental condition. Each value is expressed as an absolute number ± SD (Results from n ≥ 3 independent experiments). (C) Extracellular microparticle/microvesicle detection by FC. (a) Dot plot FSC vs SSC in logarithmic visualization. P1: viable cells; P2: Dako CytoCount counting beads 5.2 μm diameter; P3: beads 1 μm diameter; P4: beads 0.5 μm diameter; (b) Density plot FSC vs FL1 of the negative sample; (c) Density plot FSC vs FL1-CD63 of starved and (d) rapamycin-treated tWT cells. (e) Density plot FSC vs FL1-CD63 of starved and (f) rapamycin-treated tNP cells. (D) Statistical histogram of surface Lamp-1/CD107a detection on fresh cells: MFI values shown are for each experimental condition and from both cell lines. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *P < 0.05 and **P < 0.01 vs respective control. Two-way ANOVA with Bonferroni post test revealed a P value < 0.05 (+P < 0.05) between tWT and tNP basal condition and a P value < 0.001 (+++P < 0.001) between tWT and tNP starved cells.
Fig 8.
A proposed model for the pathogenesis of Niemann-Pick disease type B.
Defective ASMase and lysosomal storage could lead to a reduced ability of lysosomes to fuse with autophagosomes. This in turn could result in a partial block of autophagy maturation and defective degradation, accompanied by accumulation of autophagic vacuoles, peroxidized lipid droplets and aberrant mitochondria (autophagic stress). Additional evidence and hypotheses are more fully explained in the text. Dotted lines: proposed mechanism; solid lines: proven mechanism.