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).

Background

Excitotoxicity is the accumulation of extracellular glutamate, an excitatory neurotransmitter, leading to excessive activation of neurons and eventually, their death. It is an underlying condition associated with several neurodegenerative diseases, including multiple sclerosis (MS). Sphingosine 1-phosphate (S1P) is a bioactive lipid with activities that are mediated through five G-protein coupled receptors (GPCRs) – S1P₁-S1P₅. Since several sphingosine 1-phosphate (S1P) receptor modulators (e.g., fingolimod, siponimod) are being used as disease-modifying therapies (DMTs) for treating MS, an understanding of how S1P affects glutamate uptake is warranted.

Objectives

Astrocytes are the major brain cell type responsible for the uptake of glutamate upon neurotransmission. The objectives of this study were: 1) to determine how S1P modulates astrocytic glutamate uptake, 2) to determine whether any of the presently employed DMTs affect the ability of astrocytes to take up excess glutamate, and 3) to identify which S1P receptor mediates this function.

Methods

We utilized primary mouse astrocyte cultures, scintillation counting of labeled glutamate, Taqman gene expression analyses, and Seahorse analyses.

Results

Mouse primary astrocytes express S1P_1,2,3 receptors. S1P inhibited astrocytic glutamate uptake in a dose-dependent manner. Fingolimod and siponimod did not inhibit astrocytic glutamate uptake, indicating that astrocytic S1P₁ and/or S1P₃ are not likely to be involved. Notably, when primary astrocytes from GFAP Cre S1P₁^-/- and S1P₃^-/- were exposed to S1P, glutamate uptake inhibition was comparable to controls. However, preliminary data on the effects of pharmacological inhibitors on glutamate uptake by primary astrocytes indicated the involvement of S1P₂ that was confirmed by comparing S1P₂^-/-vs. WT astrocytes. This S1P₂-dependent inhibition of astrocytic glutamate uptake also altered mitochondrial respiration.

Conclusions

S1P inhibits glutamate uptake by astrocytes primarily through S1P₂. Fingolimod, siponimod, and other current S1P receptor modulators do not target S1P₂ and thus do not increase excitotoxic glutamate, contrasting with S1P itself, which increases in MS lesions. S1P receptor tone involving multiple S1P receptors (e.g., S1P_1-3 on astrocytes) could additionally affect other endpoints including vascular permeability, which would be differentially affected by fingolimod vs. siponimod, with both avoiding deleterious glutamate-related metabolism.

Supported by a grant from Novartis Pharma AG (JC) and the NIH R01NS103940 (YK).