The sphingosine 1-phosphate receptor agonist FTY720 is neuroprotective after cuprizone-induced CNS demyelination

Abstract

Background and purpose: Modulation of the sphingosine 1-phosphate receptor is an approved treatment for relapsing multiple sclerosis because of its anti-inflammatory effect of retaining lymphocytes within the lymph nodes. Here, we evaluated the potential of an agonist at this receptor, FTY720 (fingolimod), to activate the promyelinating pathways within the brain to encourage remyelination and neuroprotection.

Experimental approach: In this study, we used the cuprizone model in male C57BL/6 mice and tested the promyelinating and neuroprotective effects of FTY720 after acute and chronic toxin-induced experimental demyelination. We used histological, immunohistochemical and gene expression methods.

Key results: The midline of the corpus callosum was severely demyelinated after acute and chronic cuprizone-induced demyelination. Robust endogenous remyelination was evident after acute, but impaired after chronic, demyelination. FTY720 treatment modestly accelerated myelin recovery after acute but not chronic cuprizone exposure. Markers of gliosis (astrocyte and microglia activation) were not affected by FTY720 treatment. Remarkably, the accumulation of amyloid precursor protein-positive spheroids in axons was less distinct in FTY720-treated animals, indicating that this compound alleviated ongoing axonal damage.

Conclusions and implications: We show that even during endogenous remyelination, axonal degeneration continued at a low level, accumulating over time. This continuous neurodegenerative process was ameliorated by FTY720 treatment. FTY720 preserved CNS integrity by direct interaction with brain resident cells, the actions of which are still to be defined.

Figure 1

Daily FTY720 treatment results in stable phosphorylated FTY720 (FTY720-P) blood levels. (A) Blood levels of phosphorylated and non-phosphorylated FTY720. (B) Expression levels of Sphk2, Ppap2b and Sgpp1 in liver tissues. (C) Expression levels of Sphk2, Ppap2b and Sgpp1 in CNS tissues after acute demyelination. Note constant expression of the two key FTY720-metabolizing enzymes Sphk2 and Sgpp1, paralleled by increased FTY720 and FTY720-P levels in FTY720-treated animals. **P < 0.01, significantly different from controls.

Figure 2

Remyelination parameters are not affected by FTY720 after acute cuprizone-induced demyelination. (A) Effects of FTY720 on myelin recovery after acute cuprizone-induced demyelination (5 weeks cuprizone exposure) demonstrated by anti-PLP (first and second row) and LFB/PAS stains (third row) in the CCm and cortex (Cx). The box in (A) indicates the CCm, the arrow in (A) indicates the somatosensory cortex. (B) Oligodendrocyte numbers estimated by anti-OLIG2 IHC in the CCm. Note that neither myelination nor oligodendrocyte numbers are different in FTY720- and vehicle-treated groups (scale bar A:100 μm; B: 30 μm).

Figure 3

FTY720 ameliorates acute axonal damage after acute cuprizone-induced demyelination. (A/B) Glial response determined by evaluating anti-GFAP-stained sections for astrocytosis (A) and anti-IBA-1-stained sections for microgliosis (B) respectively. (C) Acute axonal damage determined by evaluating anti-APP-stained sections. Note that acute axonal damage is significantly lower in animals treated with FTY720 for 11 days after acute cuprizone-induced demyelination. Further note that APP⁺ spheroids do not colocalize to cell bodies but are flanked by axonal neurofilaments (red arrows in the lower part of C) (black scale bar: 100 μm; white scale bar: 10 μm). *P < 0.05; ** P < 0.01; ***P < 0.001, significantly different as indicated.

Figure 4

FTY720 ameliorates acute axonal damage and axonal loss after chronic cuprizone-induced demyelination. (A) Effects of FTY720 on myelin recovery after chronic cuprizone-induced demyelination (12 weeks cuprizone exposure) demonstrated by anti-PLP and LFB/PAS stains. (B) Oligodendrocyte numbers estimated by anti-OLIG2 IHC in the CCm. (C) Acute axonal damage determined by evaluating anti-APP-stained sections. (D) Effects of FTY720 on axonal density after chronic cuprizone-induced demyelination demonstrated by anti-NF200 stains. Arrowhead indicates the CCm. Note that acute axonal damage is significantly lower and that axonal density is preserved in FTY720-treated animals after chronic cuprizone exposure (scale bar A and C: 100 μm; scale bar B: 30 μm; scale bar D: 200 μm). *P < 0.05, significant effect of FTY720.