The Selective Sphingosine-1-Phosphate Receptor 1 (S1P1) Modulator Ponesimod Enhances Murine Oligodendrocyte Precursor Cell (OPC) Differentiation and Retains OPC Migration

Abstract

Methods: Reversible, blue light (BL) stimulated, optogenetic A1 protein expression plasmids, containing wild-type A1, tagged with both the optogene Cryptochrome 2 and mCherry, were trans-fected into HEK293T cells and a differentiated neuronal cell line and used to examine the effects of RNA oligo treatment on protein dynamics and downstream cellular pathway functions in real-time. Using this in vitro optogenetic paradigm of A1 dysfunction, we gathered evidence on how RNA oligo treatment affects A1 self-association clustering and downstream neuronal morphology and viability. Results: Imitating an acute environmental cell stress using a BL stimulus followed by a steady period of recovery, our data show that RNA oligo treatment significantly decreased the kinetics of cytoplasmic A1 cluster formation [half-maximal formation time (minutes): Oligo=100; no treatment (NT)=62] and significantly decreased the number of cells with A1 clustering (percent cells w/ A1 clusters: Oligo=33%; NT=50%). We found that RNA oligo treatment stabilized the structure of A1 upon binding (avg. protein denaturation temp.: Oligo=62°C; NT=61°C), and significantly increased the quantity of A1 clusters (avg. clusters/cell: Oligo=19 ; NT=5) and decreased their size (avg. cluster size (µm2): Oligo=0.31; NT=0.95). Finally, we show that neuron morphology and viability were perturbed with A1 cluster formation. Conclusions: Using an in vitro optogenetic approach, this study presents evidence that A1 clustering negatively affects neuronal morphology, and that RNA oligo treatment attenuates MS-associated A1 dysfunction, indicating that A1 protein dys-function perturbation may affect NDG in MS. Background: Sphingosine-1-phosphate receptor (S1PR) modu-lators are clinically applied to target relapse remitting multiple sclerosis (MS) and the early phase of progressive MS when inflammation still prevails. S1P1 receptor modulation (functional antagonism) is known to prevent lymphocytes egression from lymph nodes, hence hampering neuroinflammation in MS. Recent findings suggest a potential additional role for S1P1 receptor modulation in neuroprotection and remyelination. Objectives: As the Giα-coupled S1P1 is the most prominently expressed S1P receptor in oligodendrocyte precursor cells (OPCs), we hypothesized that functional antagonism by the S1P1-monoselective modulator ponesimod induces OPC differentiation. Methods: Primary mouse OPCs were harvested via the shake-off method and treated in vitro with the S1P1-selective modulator ponesimod (3nM-3000nM), the S1P5-selective modulator A971432 (3nM-3000nM) or a phosphorylated form of the non-selective modulator fingolimod (3000nM). Migration was