Siponimod (BAF312) prevents synaptic neurodegeneration in experimental multiple sclerosis

Abstract

Background: Data from multiple sclerosis (MS) and the MS rodent model, experimental autoimmune encephalomyelitis (EAE), highlighted an inflammation-dependent synaptopathy at the basis of the neurodegenerative damage causing irreversible disability in these disorders. This synaptopathy is characterized by an imbalance between glutamatergic and GABAergic transmission and has been proposed to be a potential therapeutic target. Siponimod (BAF312), a selective sphingosine 1-phosphate1,5 receptor modulator, is currently under investigation in a clinical trial in secondary progressive MS patients. We investigated whether siponimod, in addition to its peripheral immune modulation, may exert direct neuroprotective effects in the central nervous system (CNS) of mice with chronic progressive EAE.

Methods: Minipumps allowing continuous intracerebroventricular (icv) infusion of siponimod for 4 weeks were implanted into C57BL/6 mice subjected to MOG35-55-induced EAE. Electrophysiology, immunohistochemistry, western blot, qPCR experiments, and peripheral lymphocyte counts were performed. In addition, the effect of siponimod on activated microglia was assessed in vitro to confirm the direct effect of the drug on CNS-resident immune cells.

Results: Siponimod administration (0.45 μg/day) induced a significant beneficial effect on EAE clinical scores with minimal effect on peripheral lymphocyte counts. Siponimod rescued defective GABAergic transmission in the striatum of EAE, without correcting the EAE-induced alterations of glutamatergic transmission. We observed a significant attenuation of astrogliosis and microgliosis together with reduced lymphocyte infiltration in the striatum of EAE mice treated with siponimod. Interestingly, siponimod reduced the release of IL-6 and RANTES from activated microglial cells in vitro, which might explain the reduced lymphocyte infiltration. Furthermore, the loss of parvalbumin-positive (PV+) GABAergic interneurons typical of EAE brains was rescued by siponimod treatment, providing a plausible explanation of the selective effects of this drug on inhibitory synaptic transmission.

Conclusions: Altogether, our results show that siponimod has neuroprotective effects in the CNS of EAE mice, which are likely independent of its peripheral immune effect, suggesting that this drug could be effective in limiting neurodegenerative pathological processes in MS.

Keywords: Experimental autoimmune encephalomyelitis; GABA; Neurodegeneration; Parvalbumin neuron; Striatum; Synaptic transmission.

Figures

Fig. 1

Intracerebroventricular administration of 0.45 μg/day siponimod attenuates EAE motor deficits without inducing peripheral lymphocyte depletion. a Schematic representation of the experimental design used in the study. Minipump implantation and siponimod/vehicle release preceded the induction of EAE by 1 week. All examinations were performed on animals during the peak of the symptomatic phase of the disease 18–24 dpi, both in the peripheral blood samples and in the striatum of the animals. b CD3+ lymphocytes were counted in the peripheral blood of EAE mice receiving vehicle or different siponimod dosages: significant reduction was observed for the 4.5 μg/day dosage. Siponimod 4.5 μg/day vs vehicle ##p < 0.01 unpaired T test; siponimod 0.45 μg/day vs vehicle p > 0.05 unpaired T test; siponimod 0.225 μg/day vs vehicle p > 0.05 unpaired T test. c Siponimod concentrations were determined in peripheral blood samples of EAE mice receiving intracerebroventricular release of different siponimod dosages: the highest concentration (4.5 μg/day) used was significantly higher compared to both 0.450 μg/day and 0.225 μg/day, while there were no statistically differences between the other two dosages. Tukey’s post hoc ***p < 0.001. d Representative clinical course of EAE mice treated with three different siponimod dosages: EAE disease progression is strongly affected by 4.5 μg/day dosage during the symptomatic phase of the disease and significantly attenuated by 0.45 μg/day concentration in the dpi range of 18–24. Daily statistical significance was evaluated by non-parametric Mann-Whitney test

Fig. 2

CNS-directed delivery of siponimod reduces astrogliosis in the EAE striatum. a Confocal microscopy images depict the striatum of EAE-vehicle and siponimod mice stained for the astrocyte marker GFAP (green fluorescence, counterstained with DAPI, gray). Expression of GFAP is markedly reduced in EAE-siponimod striatum compared to EAE-vehicle striatum (scale bar: 100 μm). The images are representative of stained sections from three animals per groups from two immunizations. The panel in b shows western blot comparing EAE-siponimod and EAE-vehicle striatal extracts probed for anti-GFAP antibody. Densitometric analysis of the bands in b’ reveals reduced GFAP content, relative to β-actin, in EAE-siponimod lysates compared to EAE-vehicle. WB data are normalized to EAE-vehicle values. Unpaired T test was used for two-group analysis. Values are means ± SEM, *p < 0.05

Fig. 3

Attenuated microgliosis and reduced lymphocyte infiltration characterizes the striatum of EAE mice treated with siponimod. a Double immunostaining of coronal striatal sections (in gray DAPI nuclei) showing expression of Iba1-positive microglia/macrophage cells (red) and of CD3 (green) in EAE-siponimod and EAE-vehicle mice. Only few lymphocytes could be detected in the striatum of mice treated with siponimod, and microgliosis is visibly reduced in the same animals. Scale bar: 100 μm. b The levels of IBA1 and CD3 mRNA were quantified by qRT-PCR in the striatum of EAE-siponimod versus EAE-vehicle mice using β-actin as internal control. The histogram shows that both IBA1 and CD3 mRNA were significantly reduced in the striatum of siponimod-treated mice. Statistical significance was evaluated by unpaired T test: *p < 0.05

Fig. 4

In vitro siponimod application to activated BV2 microglial cell reduces IL-6 and RANTES release. BV2 cells were pre-treated with siponimod prior to stimulation with TNF. The levels of secreted IL-6 (a) and RANTES (b) were measured by Luminex assay on collected media. Application of siponimod to non-activated cells does not modulate the secretion of both molecules. Data were expressed as picograms per milliliter (pg/ml) and were analyzed by one-way ANOVA, followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Siponimod treatment corrects GABAergic defects in EAE striatum. a The glutamatergic transmission was recorded from MSNs of EAE-vehicle and EAE-siponimod mice. The analysis of the increased kinetic properties (rise time, decay time, and half width) of sEPSCs showed that there were no differences between the two groups (unpaired T test, p > 0.05). a’ The frequencies of the glutamatergic currents, increased in EAE striatum, were not corrected by siponimod (unpaired T test, *p > 0.05). a” Electrophysiological traces representative of glutamatergic currents recorded from MSNs of EAE-vehicle and EAE-siponimod mice. b Siponimod restores normal GABAergic frequencies in the striatum of acute phase EAE mice (unpaired T test,*p < 0.05). b’ Electrophysiological traces representative of GABAergic currents recorded from MSNs of EAE-vehicle and EAE-siponimod mice. c The frequencies of the GABAergic currents, reduced in EAE striatum, were completely rescued after 1 h of siponimod incubation in EAE slices (unpaired T test, **p < 0.01)

Fig. 6

Brain infusion of siponimod promotes the survival of PV+ GABAergic interneurons during the course of EAE. a Low magnification images (×5 objective) of coronal striatal sections from control CFA-vehicle, EAE-vehicle, and EAE-siponimod showing area selected for PV counting on serial sections (dorsal striatum) and DAB-immunolabeled interneurons (scale bar: 200 μm): at high magnification, the density of PV+ cells is visibly reduced in EAE-vehicle compared to that in CFA-vehicle with a partial recovery in EAE-siponimod slices. Yellow arrows indicate PV-labeled cells (scale bar: 20 μm). b Histogram represents the quantitative stereological counts of PV+ cells performed on striatal slices: the intracerebroventricular treatment with siponimod significantly increases the total number of PV+ cells in the dorsal left striatum of EAE mice. Data are expressed as mean ± S.E.M. Statistical comparisons showed in the graph were calculated with Newman-Keuls post hoc: CFA-vehicle vs EAE-vehicle **p < 0.01; CFA-vehicle vs EAE-siponimod p > 0.05; EAE-vehicle vs EAE-siponimod*p < 0.05