Fig 1.
Microvesiculation of myelin in the Twitcher mouse.
Electron microscopy micrographs of myelinated axons from peripheral nervous system shows myelin damage and vesicle association (arrows) demonstrated at sciatic nerves (Sc. Nv.) in presymptomatic P12 Twitcher (B) and late stage P40 Twitcher (C, top inset: large vesicle expanded. Bottom inset: myelin protruding from lamella) as compared to healthy wild-type (A). The ultrastructure of the Node of Ranvier in sciatic nerve of a healthy wild-type (D) vs Twitcher (E) presenting vesicular disruption of myelin lamellae (E, arrows) and region specific paranodal normalcy with myelin rupture at one side of the paranode region (white arrowhead) whereas other regions of the paranode were intact (dark arrowhead). D, E are composite images from multiple photographs. Bar = 1 μm, applies to all panels.
Fig 2.
Psychosine destabilizes submicrometric lipid domains in red blood cells.
A) Red blood cells (RBCs) from Twitcher mice and healthy littermates collected at different days of age (P14-P40) were analyzed for psychosine content. Psychosine in Twitcher RBCs was significantly higher at all ages as compared to healthy control, with psychosine levels increasing with age (n = 3 in 4 independent experiments). B) Wild-type RBCs incubated with 0–10 μM of psychosine, washed and processed for psychosine extraction and quantification. Data from 6 independent experiments. C) Hemolysis was measured after incubation with 0–20 μM psychosine. D, E) Confocal images of P40 RBCs labeled with BODIPY-SM showed a decrease of BODIPY-SM domains in Twitcher (D) as compared to wild-type (E). Scale bars, 2 μm. F) Isolated-RBCs collected from P14-P40 were dyed with BODIPY-SM, -PC and -GM1 and labeled sub-micrometric domains were counted by confocal microscopy. There was no difference in domains of Twitcher vs wild-type RBCs from mice below 30 days of age, while domains were significantly decreased in Twitcher RBCs at P30 and P40 (N = 109–924 in 3–6 independent experiments). G) Domains were counted in P40 wild-type hemi-RBCs after exposure to 0–5 μM of psychosine and labeled with BODIPY-SM, -PC or -GM1. H) RBCs were incubated with various concentrations of psychosine before labeling with BODIPY-SM and membrane fluorescence determined. I) RBCs were incubated with various concentration of psychosine. Sphingomyelin (SM) and phosphatidylcholine (PC) were extracted and separated on thin layer chromatography and quantified by densitometry. All aforementioned psychosine incubations were 30 minutes at 37°C. The results are mean ± SEM. NS, not significant (>0.05); *, p <0.05; **, p <0.01; ***, p <0.001.
Fig 3.
Psychosine reduces sphingomyelin lateral mobility.
A-C) Delayed time to recover from photobleaching by BODIPY-SM (A), BODIPY-PC (B) and BODIPY-GM1 (C) (N = 61–648 in 1–4 independent experiments). D-F) The mobile fraction of BODIPY-SM (D) but not that of BODIPY-PC (E) and BODIPY-GM1 (F) in Twitcher and wild-type RBCs treated with psychosine were significantly lower than wild-type RBCs. G-I) Similarly, the half-life of FRAP signal was increased for BODIPY-SM (G) but not for BODIPY-PC (H) and BODIPY-GM1 (I) in Twitcher and wild-type RBCs treated with psychosine, evidencing an effect of psychosine in the plasma membrane and an interaction with sphingomyelin. All aforementioned psychosine incubations were 30 minutes at 37°C Results are mean ± SEM. NS, not significant (>0.05); *, p <0.05; **, p <0.01; ***, p <0.001.
Table 1.
Psychosine reduces sphingomyelin and cholesterol lateral mobility.
Fig 4.
Psychosine increases rigidity in focalized areas of the RBC plasma membrane.
A-C) Overall fluidity in RBCs (A,B) and purified P40 brain myelin (C) measured by TMA-DPH is not affected by psychosine or in Twitcher mice. Wild-type, Twitcher, and wild-type RBCs preincubated with increasing concentrations of psychosine were analyzed by TMA-DPH anisotropy. D-H) Multiphoton excitation microscopy imaging of Laurdan in P40 wild-type (D), Twitcher (E), and wild-type RBCs treated with 2 (F), 5 (G) or 10 μM of psychosine for 30 minutes at 37°C. (H). GP images are in pseudo-colors with the range indicated by the color bar going from fluid (blue/green) to rigid (yellow/red). Scale bar, 10 μm. I) GP value from the center of RBCs were analyzed as represented inside the white dashed circles in (D) and plotted as % of change from wild-type values. J) Distribution of domains of high GP and high-rigidity. Results are mean ± SEM of samples N = 68–257 in 3–4 independent experiments. NS, not significant (>0.05); *, p <0.05; **, p <0.01; ***, p <0.001.
Fig 5.
Psychosine affects cell surface area by vesiculation.
A) RBCs from P40 TWI or WT pre-incubated with psychosine for 30 minutes at 37°C were labeled with BODIPY-SM, plated on poly-lysine coated slides and their surface area was measured by confocal microscopy. Twitcher cell surface area was significantly decreased, an effect also elicited in wild-type cells after exposure to 2–10 μM of psychosine (N = 28–44 in 3 independent experiments). B) RBCs labeled with BODIPY-SM were exposed to psychosine during a 60 min on stage incubation. Vesicles appeared at the surface following different stages of budding, from membrane outward bending (#1, #2), to small (1–2 μm, #3, #4) and large (2–4 μm #5 to #7) vesicle budding. C-E) Flow cytometry graphs plotting side (SSC) and forward scatter (FSC) of events with a gate selected for size 1–4 μm (microvesicles, mv) and 6–7 μm (RBCs) (C) showed a significant increase in Twitcher with respect to wild-type cells (D). Analysis of RBC CD235a confirmed the erythrocyte origin of the vesicles (expressed as erythrocyte-derived particles, EdP in E). Results in D and E were normalized by total RBCs counted in the 6–7 μm region from 3 independent experiments. Results are mean ± SEM. NS, not significant (>0.05); *, p <0.05; **, p <0.01; ***, p <0.001.
Fig 6.
Membrane rigidification and vesiculation of Twitcher oligodendrocytes and myelin.
A, B) Flow cytometry quantification of vesicles (0.5–3 μm) from enriched cortical oligodendrocytes from Twitcher or wild-type mice incubated with 2–5 μM of psychosine overnight at 37°C (A) and from CG-4 cell cultures in proliferating or differentiating conditions (B). Incubation with lactosyl-sphingosine (LacS) did not significantly promote the release of vesicles in brain-purified oligodendrocytes (A) and CG-4 cells (B). C-H) Psychosine content in myelin extracted from P40 wild-type and Twitcher mice (C). Brain myelin extracted from P40 wild-type (D), Twitcher (E) and wild-type treated with 5 (F) or 10 μM of psychosine for 90 minutes at 37°C (G) were incubated with Laurdan and analyzed by multiphoton microscopy (H). I) Vesicles were analyzed by flow cytometry from purified wild-type myelin after incubation with 5–20 μM of psychosine for 90 minutes at 37°C. Results are expressed as percentage from control levels (i.e. myelin incubated with vehicle) of 3–7 independent experiments and presented as the mean ± SEM. NS, not significant (>0.05); *, p <0.05; **, p <0.01; ***, p <0.001.