Sphingosine kinase 2 is essential for remyelination following cuprizone intoxication

Abstract

Therapeutics that promote oligodendrocyte survival and remyelination are needed to restore neurological function in demyelinating diseases. Sphingosine 1-phosphate (S1P) is an essential lipid metabolite that signals through five G-protein coupled receptors. S1P receptor agonists such as Fingolimod are valuable immunosuppressants used to treat multiple sclerosis, and promote oligodendrocyte survival. However, the role for endogenous S1P, synthesized by the enzyme sphingosine kinase 2 (SphK2), in oligodendrocyte survival and myelination has not been established. This study investigated the requirement for SphK2 in oligodendrocyte survival and remyelination using the cuprizone mouse model of acute demyelination, followed by spontaneous remyelination. Oligodendrocyte density did not differ between untreated wild-type (WT) and SphK2 knockout (SphK2 À/À ) mice. However, cuprizone treatment caused significantly greater loss of mature oligodendrocytes in SphK2 À/À compared to WT mice. Following cuprizone withdrawal, spontaneous remyelination occurred in WT but not SphK2 À/À mice, even though progenitor and mature oligodendrocyte density increased in both genotypes. Levels of cytotoxic sphingosine and ceramide were higher in the corpus callosum of SphK2 À/À mice, and in contrast to WT mice, did not decline following cuprizone withdrawal in SphK2 À/À mice. We also observed a significant reduction in myelin thickness with aging in SphK2 À/À compared to WT mice. These results provide the first evidence that SphK2, the dominant enzyme catalyzing S1P synthesis in the adult brain, is essential for remyelination following a demyelinating insult and myelin maintenance with aging. We propose that persistently high levels of sphingosine and ceramide, a direct consequence of SphK2 deficiency, may block remyelination.

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes

“…We next sought to determine if loss of myelin in CerS2 mice is accompanied by loss of mature oligodendrocytes, by immunofluorescent labelling for the mature oligodendrocyte marker aspartoacylase (ASPA) (Madhavarao et al, 2004; Song et al, 2021; Zhang et al, 2014). At 6 weeks of age, there was no significant difference in the density of mature oligodendrocytes in CerS2 ΔO/ΔO compared to CerS2 fl/fl mice (Fig.…”

“…Six distinct fields of view per mouse were imaged at 15,000x magnification and captured as 3×3 tile scans using the JEOL integrated software and a high-sensitivity sCMOS camera (JEOL Matataki Flash). G-ratios were calculated as the diameter of the axon lumen divided by the diameter of the lumen plus myelin sheath (Song et al, 2021). These measurements were taken from 3 mice per genotype, and 220 - 500 axons per mouse, using ImageJ software.…”

“…Targeted lipidomic analysis and GalCer/GluCer isomer separation Lipids were extracted from ~50 µg isolated myelin protein or ~500 µg liver homogenate and analysed by multiple reaction monitoring on a TSQ Altis triple quadrupole mass spectrometer, as described (Song et al, 2021). An internal standard for each lipid class was included, as described above.…”

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes

“…No discernible loss of myelin (Figure 4m) or myelin protein markers (Figure 4n,o) was observed in 16-week-old mice heterozygous for CerS2 deletion in oligodendrocytes (CerS2 ΔO/+ ). These mice did not exhibit any deficits in the rotarod test or any change in grip (Madhavarao et al, 2004;Song et al, 2021;Zhang et al, 2014). At 6 weeks of age, there was no significant difference in the density of mature oligodendrocytes in CerS2 ΔO/ΔO compared to CerS2 fl/fl mice (Figure 6a,b).…”

Disrupted myelin lipid metabolism differentiates frontotemporal dementia caused by GRN and C9orf72 gene mutations

“…Sections were dehydrated in two changes of 100% ethanol, cleared in two changes of xylene, and coverslipped using DPX mounting medium. Myelination scores from 0 to 3 were assigned by two blinded observers, where 0 is complete absence of myelin and 3 is dense myelin [ 33 ].…”