Background
Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS), affecting young adults. Clinically, two main forms of MS can be distinguished, the relapsing-remitting (RRMS) and the progressive, where the latter is described as the gradual progression of clinical disability in a patient either with a preceding relapsing course (secondary progressive MS (SPMS)) or without a preceding relapsing course (primary progressive MS (PPMS)) [
1].
Such phenotypic distinction seems to rely on predominance of distinct pathogenic mechanisms that appear different, though poorly understood, throughout RRMS, PPMS, and SPMS [
2]. In general, although inflammation has a recognized pivotal role in triggering the cascade of events leading to both white and gray matter (GM) damage, anti-inflammatory or immunomodulatory drugs have been found successful only in RRMS [
3], with the recent exception of ocrelizumab, which was found to reduce disability progression also in PPMS [
4]. A forthcoming challenge is therefore to identify drugs able to interfere with the mechanisms leading to neurodegeneration in PPMS and in SPMS.
GM pathology in MS is increasingly recognized to contribute to the progression of the disease. In fact, GM damage occurs early in the disease course, is independent of demyelination, and is associated with neurological and neuropsychological disability [
5]. Several studies in experimental autoimmune encephalomyelitis (EAE), the murine model of MS, have underscored an inflammation-dependent synaptopathy affecting different brain structures (striatum, hippocampus, cerebellum) and occurring independently of demyelination [
6‐
10]. In such brain areas of EAE mice, the excitatory glutamatergic transmission is potentiated, while the GABAergic inhibitory transmission is reduced: this imbalance in synaptic transmission leads to excitotoxicity and subsequently to neurodegeneration [
11]. Of note, glutamate receptor antagonists and gamma-amino butyric acid (GABA) agonists exert beneficial effects in both EAE and MS [
12‐
16]. This synaptopathy has been proposed as a valuable therapeutic target [
11].
Several drugs are under investigation for their neuroprotective effects in progressive MS. In particular, an ongoing phase-III clinical trial is currently conducted with the sphingosine-1-phosphate receptor (S1P) modulator, siponimod (BAF312) [
17]. Siponimod is a next-generation S1P modulator selective for S1P1 and S1P5 that showed good safety and tolerability in humans. This compound has recently successfully passed a phase-II clinical trial in RRMS [
18].
Compared to fingolimod, an S1P modulator selective for S1P1, S1P3, S1P4, and S1P5, the half-life of siponimod is shorter, allowing recovery of peripheral lymphocyte count to a normal range within 1 week after treatment cessation. The risk of bradycardia is mitigated using a dose titration scheme during treatment initiation [
19,
20].
Most of the effects of siponomid are attributed to S1P1 expressed on lymphocytes, thereby preventing lymphocyte trafficking into the brain, but both S1P1 and S1P5 are widely expressed in brain-resident cells, like neurons, microglia, astroglia, and oligodendrocytes [
21].
The aim of the present study was therefore to assess the central effects of siponimod in mice with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced EAE, a model of progressive MS, to provide a substrate of the putative neuroprotective effects of this drug.
Methods
EAE model
EAE was induced as described previously [
7,
20]. Mice were injected subcutaneously at the flanks with 200 μg of MOG
35-55 emulsion to induce EAE by active immunization. The emulsion was prepared under sterile conditions using MOG
35-55 (85 % purity; Espikem) in 300 μl of complete Freund’s adjuvant (CFA; Difco) containing
Mycobacterium tuberculosis (8 mg/ml, strain H37Ra; Difco) and emulsified with phosphate buffer solution (PBS). All animals were injected with 500 ng of pertussis toxin (Sigma) intravenously on the day of immunization and 2 days later. Control animals received the same treatment as EAE mice without the immunogen MOG peptide, including complete CFA and pertussis toxin (referred to as hereafter as “CFA”). Animals were scored daily for clinical symptoms of EAE according to the following scale: 0 = no clinical signs, 1 = flaccid tail, 2 = hindlimb weakness, 3 = hindlimb paresis, 4 = complete bilateral hindlimb paralysis, and 5 = death due to EAE; intermediate clinical signs were scored by adding 0.5 [
15,
22]. For each animal, the onset day was recorded as the day post-immunization (dpi) when it showed the first clinical manifestations.
Experiments were carried out in accordance with Internal Institutional Review Committee, the European Directive 2010/63/EU and the European Recommendations 526/2007, and the Italian D.Lgs26/2014. All the efforts were made to minimize the number of animals utilized and their suffering.
Siponimod (Novartis Pharma AG) was dissolved in a solution containing 10 % Solutol/Kolliphor HS15 (BASF Pharma Solutions)—final pH range between 6 and 7—at a final concentration of 2 mg/ml. This preparation allowed stability of the drug for up to 6 weeks at 37 °C.
One week before immunization, mice were implanted with subcutaneous osmotic minipumps allowing continuous intracerebroventricular (icv) infusion of either vehicle or siponimod for 4 weeks (three sets of immunizations) [
8,
22]. Different siponimod dosages were tested: 4.5, 0.45, and 0.225 μg/day.
T cell absolute count
T cell absolute count was performed on blood samples kept from the mandibular vein of the mouse.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
Determination of siponimod in mouse blood by LC-MS
For quantitative determination, 10 spiked samples from 0.5 up to 10,000 ng/ml were prepared in the same matrix. Proteins were removed by protein precipitation by adding an organic solvent mixture. The organic layer was evaporated to dryness, and the residue was re-dissolved in HPLC buffer B, containing 5 mM ammonium formate.
Aliquots of 2 μl were directly injected on a Agilent Eclipse Plus, RRHD 2.1 × 50 mm reversed-phase column with 1.8-μm particles, and kept at 40 °C.
For separation, a linear gradient from 50 to 100 % B within 1.7 min was used. Solvent A was 0.2 % formic acid in water and solvent B 0.2 % formic acid in acetonitrile. The flow was kept at 500 μl/min during the whole cycle.
For detection, the column effluent was guided directly to the electrospray source of the Agilent 6490 triple quadrupole MS with parameters optimized for siponimod. Compound and a structure-related internal standard were detected as their [MH]+ ions with the multiple reaction monitoring (MRM) transition 517.3 → 159.0, 416.1 for siponimod. For data processing, the compound to internal standard ratio of the extracted ion chromatograms was used.
The calculation was based on a second order fitted and 1/x weighted calibration curve (r
2 = 0.9987). The accuracy of the 10 individually prepared calibration samples was better than 15 %. The recovery of the compounds was 94.3 % (RSD = 1.3 %). The precision of the control samples (n = 3), distributed over the whole series, was better than RSD = 1.8 %. The LOQ for siponimod was found to be 0.5 ng/ml, based on the lowest calibration sample.
Electrophysiology
Mice were killed by cervical dislocation, and corticostriatal coronal slices (200 μm) were prepared from fresh tissue blocks of the brain with the use of a vibratome [
6,
15]. Single slices were then transferred to a recording chamber and submerged in a continuously flowing oxygenated artificial cerebrospinal fluid (ACSF) (34 °C, 2–3 ml/min) gassed with 95 % O
2–5 % CO
2. The composition of the control ACSF was (in mM) as follows: 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, 25 NaHCO3. Whole-cell patch clamp recordings were made with borosilicate glass pipettes (1.8 mm o.d.; 4–8 MΩ), in voltage-clamp mode, at the holding potential (HP) of −80 mV. Spontaneous excitatory and inhibitory post-synaptic currents (EPSCs, IPSCs) were recorded from medium spiny neurons (MSNs) as in Centonze et al. [
15] and Rossi et al. [
6]. Siponimod (1 μM) was added to the bath solution for 1 h, before recording. Synaptic events were stored by using P-CLAMP 9.2 (Axon Instruments) and analyzed offline on a personal computer with Mini Analysis 5.1 (Synaptosoft, Leonia, NJ, USA) software. Offline analysis was performed on spontaneous synaptic events recorded during fixed time epochs (1–2 min, three to five samplings), sampled every 5 or 10 min. One to six cells per animal were recorded, and four animals were sacrificed for each experimental group.
Western blot
Animals were killed by cervical dislocation at 24 dpi. Striata were quickly removed, snap frozen in dry ice, and homogenized as in Mandolesi et al. [
8] and Gentile et al. [
22]. Soon after blocking with 5 % milk in Tris buffered solution (TBS), the membrane was incubated for 1 h with mouse anti-β-actin primary antibody (1:20,000; Sigma-Aldrich) followed by anti-mouse IgG HRP (1:10,000, GE Healthcare, formerly Amersham Biosciences) secondary antibody. Immunodetection was performed by ECL reagent (Amersham) and membrane was exposed to film (Amersham). Next, a mouse anti-glial fibrillary amino acid protein (GFAP, 1:2000, Immunological Science, over night) primary antibody was used in combination with anti-mouse IgG HRP (1:4000 for GFAP; GE Healthcare, formerly Amersham Biosciences) secondary antibody. Immunodetection was performed as for β-actin. Densitometric analysis of bands was performed by NIH ImageJ software (
http://rsb.info.nih.gov/ij/). Western blot (WB) results are presented as data normalized to control CFA values.
RNA extraction and quantitative real-time PCR
Striata were dissected in RNAse-free conditions. Total RNA was extracted according to the standard miRNeasy Micro kit protocol (Qiagen). Next, 350 ng of total RNA was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem), and 10 ng of complementary DNA (cDNA) was amplified with SensiMix SYBR Hi-Rox Kit (Bioline; Meridian Life Science) in triplicate using the Applied Biosystem 7900HT Fast Real-Time PCR system. Messenger RNA (mRNA) relative quantification was performed using the comparative cycle threshold (2−ΔΔCt) method. β-actin was used as endogenous control.
Primer sequences:
Ionized calcium binding adaptor protein-1 (Iba-1, NM_019467): 5′-GACAGACTGCCAGCCTAAGACAA-3′ (sense), 5′-CATTCGCTTCAAGGACATAATATCG-3′ (antisense);
β-ACTIN (NM_007393): 5′-CCTAGCACCATGAAGATCAAGATCA-3′ (sense), 5′-AAGCCATGCCAATGTTGTCTCT-3′ (antisense).
For CD3+ mRNA detection, 17.5 ng of the same cDNA was amplified with SensiMix II Probe (Bioline; Meridian Life Science) by using TaqMan gene expression assays (ID Mm00599684_g1 and ID Mm00607939; Applied Biosystem).
Immunofluorescence and confocal microscopy
Mice from two different immunization experiments were deeply anesthetized and intracardially perfused with ice-cold 4 % paraformaldehyde. Brains were post-fixed for 2 h and equilibrated with 30 % sucrose at least one overnight. Immunofluorescence was performed as in Mandolesi et al. [
8] and Gentile et al. [
22]. The following primary antibodies were used: rabbit anti-Iba1 (1:750; Wako Chemicals, USA), rabbit anti-GFAP (1:500; DAKO), rat anti-CD3 (1:300; AbD Serotec). Secondary antibodies used are as follows: Alexa Fluor-488 (1:200; Life Technologies) and Cy3-conjugated donkey anti-rabbit or anti-rat (1:200; Jackson ImmunoResearch). 4′,6-diamidino-2-phenylindole (DAPI; 0.01 mg/ml) was used to visualize nuclei. All images were acquired using a LSM7 Zeiss confocal laser-scanner microscope (Zeiss, Göttingen, Germany) and processed by NHI ImageJ software [
8,
22].
Immunohistochemistry
A section every third (for a total of eight sections per animal) throughout the rostro-caudal extent of the striatum was selected for staining and examination. Freshly cut 30-μm serial sections, after blocking of nonspecific staining with 10 % normal donkey serum, were incubated overnight with a rabbit polyclonal antibody against parvalbumin (PV) (1:1000; Immunological Science), then with a biotinylated rabbit secondary antibody (1:500; Vector Laboratories, Burlingame, CA, USA) for 1 h, and finally with Extravidin (1:1000; SIGMA) for 1 h. Reaction was developed in a freshly prepared diaminobenzidine (DAB)/H2O2 (DAB tablet, SIGMA). Sections were mounted on glass slides and coverslipped under Eukitt.
Stereology
Quantitative observations were limited to the dorsal striatum of the left hemisphere. Using the Stereo Investigator System (MicroBrightField Europe e.K., Magdeburg, Germany) composed of a Zeiss Axioimager.M2 microscope and MicroBrightField’s Stereo Investigator software package, an optical fractionator, stereological design was applied to obtain unbiased estimates of total PV+ cells. Sampling grids and magnifications were adjusted to obtain a relatively constant number of cells sampled and a coefficient of error (CE Gunderson) of ≤0.1. A tri-dimensional optical dissector counting probe (x, y, z dimension of 200 × 200 × 25 μm, respectively) was applied. Counts were performed using a ×20 objective.
Total number was estimated according to the following formula:
$$ \mathrm{N}=\sum \mathrm{Q}\times 1/\mathrm{s}\mathrm{s}\mathrm{f}\times 1/\mathrm{a}\mathrm{s}\mathrm{f}\times 1/\mathrm{t}\mathrm{s}\mathrm{f}. $$
where ΣQ represents the total number of neurons counted in all optically sampled fields of the dorsal striatum, ssf is the section sampling fraction, asf is the area sampling fraction, and tsf is the thickness sampling fraction.
Cell culture supernatant Luminex assay
The BV2 immortalized murine microglial cell line was pre-treated for 1 h with siponimod 0.1 μM in dimethyl sulfoxide (DMSO), before incubation with tumor necrosis factor (TNF; 200 U/ml; Milteny Biotec); control cells received equal volume of DMSO (n = 3 per condition). The 24-h conditioned media was assayed for IL-6 and RANTES by Luminex assay, according to the manufacturer instructions (R&D systems). The plate was read on a Luminex-200 instrument (Luminex Corp., Austin, TX). Concentrations were calculated by using a standard 5P-logistic weighted curve generated for each target and expressed as picograms per milliliter (pg/ml).
Statistical analysis
For each type of experiment, at least three mice per group were employed. Throughout the text, “n” refers to the number of animals, except for electrophysiology, where it means the number of cells. Data were presented as the mean ± S.E.M. The significance level was established at p < 0.05. Statistical analysis was performed using unpaired Student’s T test for comparisons between two groups and non-parametric Mann-Whitney test, where needed. Multiple comparisons were analyzed by one-way ANOVA for independent measures followed by Tukey’s HSD or Newman-Keuls.
Discussion
Discovering drugs effective in progressive forms of MS is a hard challenge for both researchers and physicians working in the field of MS. Promoting neuroprotection is a promising therapeutic strategy in the light of the relevant contribution of neurodegeneration to MS pathogenesis and progression [
2]. GM damage, including neuron and synaptic loss, is currently considered as the major contributor to both cognitive impairment and motor disability in MS patients [
35,
36]. Recently, it has been highlighted that inflammation-driven synaptic abnormalities are pathological hallmarks of both EAE and MS brains: the sustained imbalance between glutamatergic and GABAergic transmission, occurring independently of demyelination and axonal loss, is supposed to induce excitotoxic neurodegeneration [
11]. Preventing such inflammatory neurodegenerative mechanisms is a novel and poorly explored therapeutic strategy.
In the present study, we investigated whether siponimod could be neuroprotective in EAE mice, by limiting striatal synaptopathy. Major findings of this study are that the GABAergic transmission alterations typical of the EAE striatum are rescued by siponimod treatment, likely due to reduced local inflammatory reaction and to increased survival of PV+ interneurons, which are the main GABAergic input to MSNs. In an attempt to discriminate the peripheral from the central effects of siponimod, we used a direct and preventive CNS administration of the drug, choosing the concentration of siponimod able to ameliorate the disease severity, but with minimal impact on blood lymphocyte count.
Adjusting the administration dose was our primary goal to avoid that the observed beneficial effects could be secondary to its peripheral immunomodulatory action. In fact, by virtue of its lipophilic nature, siponimod easily crosses the BBB reaching peripheral tissues. Not surprisingly, the highest dosage (4.5 μg/day) tested in this study resulted in significant blood exposure of siponimod and a strong reduction of T cells in the blood, likely as a result of retention in lymph nodes, and completely prevented EAE development. The lower dosages (0.45 and 0.225 μg/day) had both a minimal impact on peripheral lymphocyte count with siponimod blood levels reaching only low nanomolar range. Since the 0.225 μg/day dose had no effect on the clinical score, we chose the 0.45 μg/day dosage due to its beneficial effect on disease severity, even if this was limited to the peak of the acute phase (20–24 dpi). However, since the clinical symptoms are mainly due to lesions in the spinal cord and since we expect a gradient of the compound between the site of administration and the site of the spinal cord lesions, a lack of strong clinical efficacy does not rule out more potent effects at the proximity of the administration site. Moreover, although we cannot exclude any peripheral effect of siponimod, we might assume that the observed recovery in clinical score could be linked to central effects of this drug. Accordingly, this treatment induced attenuation of microgliosis and astrogliosis with reduced number of infiltrating CD3+ lymphocytes in the striatum of EAE mice during the symptomatic phase of the disease. A possible explanation for the low number of infiltrating lymphocytes could be a reduced inflammatory reaction in the brain, caused by the preventive and central treatment with siponimod, likely promoting a different tissue distribution of T cells compared to EAE untreated mice.
Indeed, CNS-resident cells, such as microglia, astroglia, oligodendrocytes, and neurons, express sphingosine receptors, although with some differences in the receptor subunit composition [
21]. Most of the anti-inflammatory effects of S1PR agonists, like fingolimod and siponimod, in the CNS have been associated to their action on astroglia and microglia [
37,
38]. Microglia activation occurs early in the EAE brain and spinal cord, before the appearance of motor deficits [
25,
39,
40]. By secreting a number of pro-inflammatory cytokines and chemotactic factors, microglia are involved in the amplification of the inflammatory reaction and the recruitment of monocyte-blood-born cells and T cells into the brain. In our experimental paradigm of preventive and central treatment with siponimod, siponimod may prevent the chain of noxious events triggered by microglia in the EAE brain, reducing MOG-specific lymphocyte recruitment. To prove this hypothesis, we used an in vitro simplification of the in vivo experimental approach: we pre-treated microglial cells with siponimod prior to activation with TNF. We found that siponimod reduced microglial release of IL-6 and RANTES, some of the most relevant cytokines/chemokines involved in EAE/MS pathogenesis [
28‐
32]. Indeed, although IL-6 levels in the CSF of MS patients have reported to be unaltered, likely indicating that IL-6 is not a specific signature of the disease [
41], it can be hypothesized that this cytokine has a local role in the modulation of the inflammatory reaction, since its expression was detected in glial cells of MS lesions [
42]. Conversely, it is worth noting that RANTES levels have been found increased in the CSF of MS patients [
43] and, most notably, they correlated with inflammation and synaptic excitability [
44].
We have recently demonstrated that inflammation causes an imbalance between the glutamatergic and the GABAergic transmission in the EAE striatum [
6,
15]. Notably, such alterations are putative therapeutic targets, and some drugs currently used in MS treatment have already been found able to interfere with the chain of events leading to such dysregulation [
45‐
47]. Of note, fingolimod, given systemically and starting the day of immunization, was shown to strongly ameliorate clinical deficits and to correct the glutamatergic transmission alterations of EAE striatum [
45]. Here, we demonstrated that siponimod rescued GABAergic transmission defects, without improving the glutamatergic transmission in MSNs. Such differences between the results obtained with the two S1P modulators probably stems from the way of administration of the drug and the dose/drug exposure. The oral delivery of fingolimod likely prevented T lymphocytes from migrating into the brain, thus explaining the marked effect on disease progression and the excitatory currents. Notably, even if in a small number, infiltrating T cells, which have been highly involved in glutamatergic alteration in the striatum of EAE mice [
15] may be able to affect excitatory transmission.
Furthermore, an acute exposure (1 h) of siponimod to slices of EAE mice reproduced the same effect of the in vivo treatment on GABA transmission, reinforcing the idea that the compound may improve the inhibitory tone in the striatum of EAE mice by interacting with resident immune cells.
In the present study, we observed that icv treatment with siponimod recovered the frequencies of the sIPSCs in EAE striatum. This specific impairment of the GABAergic neurotransmission has been linked to inflammation, although a selective cytokine or chemokine has not been recognized, and to the reduced number of GABAergic interneurons contacting MSNs in the EAE striatum [
6]. The death of PV+ interneurons is a pathological hallmark of both MS and EAE brains [
6,
48], and the mechanisms leading to such degeneration are still unknown. Inflammation is thought to play a role in this neuronal death, which has been observed in other brain inflammatory conditions [
49] and in rodent chronic stress paradigms [
50].
In the striatum of EAE mice, GABAergic defects occur throughout the disease course [
6]. In our study, we found that siponimod promotes the survival of these neurons, thus explaining the recovery of GABAergic neurotransmission, in spite of the potentiated glutamatergic transmission. Further studies are needed to dissect the mechanisms of neuroprotection exerted by siponimod on PV+ interneurons: other mechanisms involving neuroprotective factors, like the brain-derived neurotrophic factor (BDNF), may be implied [
51].
It is worth noting that reduced GABA concentrations in the hippocampus and sensory-motor cortex have recently been correlated with physical disability in progressive MS patients [
52]. This finding strongly corroborates the role of the inflammatory synaptopathy in MS progression. Interestingly, in our study, the attenuation of the motor symptoms of EAE mice treated with siponimod was observed in the symptomatic phase of the disease in connection to the improved tone of the GABAergic transmission in the striatum, a subcortical brain area involved in motor control [
53] and compromised in MS [
54].
Acknowledgements
The authors thank Vladimiro Batocchi for helpful technical assistance.