Background

The sphingosine 1-phosphate (S1P) receptor modulators, siponimod and fingolimod, have overlapping yet also distinct mechanisms underlying their efficacy for treating multiple sclerosis (MS). Siponimod directly binds to S1P receptors (S1P_1,5), whereas fingolimod is a prodrug that requires sphingosine kinases (SPHK1/2) to generate active fingolimod-phosphate (engaging S1P_1,3,4,5). Both drugs functionally antagonize S1P₁ on immune and central nervous system (CNS) cells. Siponimod showed activity in clinical trials of progressive MS (SPMS), whereas fingolimod (trialed in PPMS) did not. However, the mechanistic explanation for the clinical differences between these two S1P receptor modulators is unclear.

Objectives

Single-cell transcriptome profiles of SPMS and RRMS brains might identify differential gene pathways in relevant cell types that may explain the clinical differences between siponimod vs. fingolimod.

Methods

We applied single-nucleus RNA sequencing (snRNA-seq) using 10x Genomics on nuclei derived from region-matched prefrontal cortices of SPMS vs. RRMS-affected brains.

Results

The snRNA-seq yielded 33,197 high quality nuclei that were clustered into major CNS cell types. SPMS brains, as compared to RRMS brains, showed perturbation in sphingolipid pathway genes, and most notably, a downregulation of SPHK1 in astrocytes. Functional proof-of-concept was obtained in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), which resulted in diminished fingolimod efficacy in astrocyte-specific Sphk1/2 double knockout mice.

Conclusions

SPMS brains show reduced SPHK1/2 expression, particularly SPHK1 in astrocytes of SPMS brains. Although it is not clear whether or not this results in reduced local exposure to active fingolimod-P in the MS brain, animal EAE studies are consistent with its functional reduction. These data further support changes in the metabolism of sphingolipids during MS progression that highlight a major metabolic difference for fingolimod vs. siponimod, which could have significant clinical relevance particularly since siponimod does not require SPHK1/2 for receptor activation.

We thank Dr. Shaun R. Coughlin (UCSF) for providing the Sphk1/2^flox/flox mice. This work was supported by a grant from Novartis Pharma AG (JC) and the NIH R01NS103940 (YK).