Investigating the Mitoprotective Effects of S1P Receptor Modulators Ex Vivo Using a Novel Semi-Automated Live Imaging Set-Up

Abstract

In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) +/- 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.