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.
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.
We utilized primary mouse astrocyte cultures, scintillation counting of labeled glutamate, Taqman gene expression analyses, and Seahorse analyses.
Mouse primary astrocytes express S1P1,2,3 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.
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., S1P1-3 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).