Fingolimod Impedes Schwann Cell–Mediated Myelination:  Implications for the Treatment of Immune Neuropathies?

Figure 1. Graphs of RNA expression. A, RNA expression of all 5 known sphingosine-1-phosphate receptors (S1P₁-S1P₅receptors) in rat Schwann cells. All receptors were found to be expressed by Schwann cells (light gray). Treatment with FTY720P did not change the expression on the RNA level (dark gray). In DRG cocultures from day 1 to day 21 of culture age, S1P₁ to S1P₅ receptors were found to be expressed on the RNA level (light gray; B-F). During the course of myelination, receptor-expression levels did not change. Treatment with FTY720P at different concentrations (here 1 μM of FTY720P) did not alter expression levels of any S1P receptor studied (dark gray). RNA levels expressed as a fraction of the housekeeping gene GAPDH, expressed as ΔC_T (A) or ΔΔC_T (B-F). NS indicates not significant. The error bars indicate standard deviation.

Figure 2. Sphingosine-1-phosphate₁ (S1P₁) receptor expression on the protein level. Pure rat Schwann cells express the S1P₁ receptor in vitro (A), which colocalized to the S100 merge (B and C). In the peripheral nerve of a Lewis rat, the S1P₁ receptor could be detected (D), which localized toward S100 (E) and merge (F). In the human peripheral nervous system, the S1P₁ receptor could be localized within the endoneurium (G), where it colocalized to S100 (H) and merge (I). Arrows indicate Schwann cells expressing the S1P₁ receptor; given the ubiquitous expression of this receptor, mononuclear cells in the perineurium stained positive for the S1P₁ receptor (arrowhead). S1P₁ receptor stained with phycoerythrin and S100 with fluorescein isothiocyanate conjugated. Original magnifications ×40 (A-C), ×60 (D-F), ×20 (G-I), and ×100 (C and I inserts). DAPI indicates 4',6-diamidino-2-phenylindole.

Figure 3. Effect of FTY720P on myelination in vitro. A, Continuous treatment with FTY720P resulted in a dose-dependent decrease in the total amount of myelin per well studied. This effect was significant for a treatment with 100nM and 1 μM of FTY720P. B, The coincubation with the competitive sphingosine-1-phosphate₁ antagonist W123 in a dose of 1 μM inhibited this dose-dependent effect. + indicates with W123; −, without W123. Dorsal root ganglion coculture after 21 days of myelination without treatment (C) or with FTY720P treatment (D). The error bars indicate standard deviation. * indicates P < .05.

Figure 4. FTY720P treatment results and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) stainings. A, Treatment of cultured cells with FTY720P resulted in a significant decrease in proliferation and cellular viability, as assessed by the CellTiter-Blue assay. B, Concentrations of 100nM and 1 μM of FTY720P activated significantly cysteine aspartic acid–specific protease (caspase) 3 and caspase 7, an effect that could be abrogated by coincubation with the competitive sphingosine-1-phosphateantagonist W123. These results were corroborated by TUNEL staining. C, Densitometry revealed increased TUNEL fluorescence per area investigated in a dose-dependent fashion. This was confirmed visually. In cultures cotreated with FTY720P plus W123, apoptosis could not be seen (D), whereas 4',6-diamidino-2-phenylindole (DAPI) revealed nuclear staining (D insert). Treatment with FTY720 alone resulted in apoptosis (E and F). Original magnifications ×40 (D-F) and ×100 (D and F inserts). The error bars indicate standard deviation. * indicates P < .05.

Figure 5. Inhibition of reactive oxygen species production by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium (DPI) significantly protects Schwann cells from FTY720P-associated apoptosis, resulting in significantly better cellular viability. + indicates with DPI; −, without DPI. The error bars indicate standard deviation. * indicates P < .05. NS indicates not significant.