Effects of FTY720 on brain neurogenic niches in vitro and after kainic acid-induced injury

All procedures and experiments involving animals and their care were carried out according to the guidelines of the European Union Council (Directive 2010/63/EU) and Spanish regulations (Real Decreto 53/2013) on animal ethics and welfare, and under the supervision and with the approval of our internal animal ethics committee (University of the Basque Country, UPV/EHU). All possible efforts were made to minimize animal suffering and the number of animals used.

NSC cultures were prepared from 4 to 7-day-old Sprague-Dawley rat pups, as previously described [27, 28], with slight modifications. Briefly, the SVZ was isolated and minced with a McIlwain tissue chopper. SVZ tissue from three to four brains was digested for 7 min at 37 °C in 5 ml of trypsin/EDTA (Sigma). Digestion was stopped by adding an equal volume of trypsin inhibitor (Gibco) and 0.01% DNAse I (Sigma) for 5 min at room temperature. The cell suspension was centrifuged for 10 min at ×600g and the pellet mechanically dissociated 25 times in 3 ml NeuroCult^TM medium (STEMCELL Technologies) using a glass Pasteur pipette and 20 times using 1 ml pipette tips. The cells that remained in suspension were decanted for 20 min and the single cell suspension counted using the Neubauer method. Cells were seeded in proliferation medium [NeuroCult^TM medium supplemented with 10% neural stem cell factors from STEMCELL Technologies, 2 mM glutamine, penicillin/streptomycin mix, 20 ng/ml EGF/epidermal growth factor (Promega), 10 ng/ml bFGF/basic fibroblast growth factor (Promega), 10 ng/ml PEDF/pigment epithelium-derived factor (Millipore)] at a density of 10⁴cells/cm² and cultivated in suspension for 7 days at 37 °C, 5% CO₂. EGF, bFGF and PEDF were added fresh every 2–3 days. After 7 DIV (days in vitro), cells were aggregated as neurospheres. To differentiate cultures from neurons, floating neurospheres were then allowed to attach onto poly-ornithine-coated glass coverslips in 24-well plates in neuron differentiation medium (NeuroCult™ medium supplemented with differentiation factor 10× (both from STEMCELL Technologies), NGF/nerve growth factor (NGF-beta, human recombinant, #4303R-100; Biovision) and BDNF/brain-derived neurotrophic factor (BDNF, human recombinant, #4004–50; Biovision)) and differentiated for additional 7 DIV in the presence of FTY720 (reconstituted in dimethyl sulfoxide hydrochloric acid (DMSO)/50 mM HCl). Alternatively, the neurospheres were maintained for 3 days in oligodendrocyte differentiation medium, composed of DMEM supplemented with 4.5 mg/ml glucose and sodium pyruvate (Gibco), SATO (100× stock solution: 100 μg/ml BSA, 100 μg/ml transferrin, 16 μg/ml putrescine, 40 ng/ml thyroxine, 30 ng/ml tri-iodothryronine, 60 ng/ml progesterone, 40 ng/ml selenium, all of from Sigma), 6.3 mg/ml N-acetyl-cysteine (Sigma), 0.5 mg/ml insulin (Sigma), 1 μg/ml CNTF/ciliary neurotrophic factor and 10 μg/ml NT3/neurotrophin-3 (both from Peprotech). This step was considered to be the pre-commitment stage before oligodendrocyte differentiation. After 3 DIV, floating neurospheres were allowed to attach onto poly-ornithine-coated glass coverslips in 24-well plates in oligodendrocyte differentiation medium and differentiated for 4 DIV in the presence of FTY720.

At the end of the differentiation phase, on DIV 14, cells were fixed and immunostained for cell-specific markers of mature neurons or oligodendrocytes. The extent of differentiation both to neurons and to oligodendrocytes was evaluated in treatment conditions and control conditions as described below.

Following differentiation, cell cultures were fixed in 4% paraformaldehyde and permeabilized with 0.05% Triton and 5% normal goat serum in phosphate-buffered saline (PBS). Primary antibody rabbit, anti-β-III tubulin (1:300; Abcam, #Ab18207) and mouse anti-CNPase (1:500; Sigma, #C5922) were incubated overnight at 4 °C and then washed three times with 0.05% Triton in PBS. Alexa Fluor 488-conjugate secondary antibodies were incubated for 1 h in the dark at room temperature (1:500). After three washes with 0.05% Triton in PBS, cells were stained for 10 min at room temperature with propidium iodide (PI) to stain total nuclei, and further washed with PBS. Fluorescence intensity was measured using a fluorescence microplate reader equipped with appropriate excitation and emission filters to detect the fluorescent signal from Alexa 488 and PI. Differentiation was evaluated as a ratio of fluorescence intensity from β-III tubulin or CNPase-positive cells over total nuclei.

We used a total of 24 adult male Sprague-Dawley rats (200–250 g). Rats were kept on a 12/12 h light/dark cycle with constant ambient temperature and humidity. Food and water were available ad libitum. We used unilateral icv injection of KA as model of induced-seizure and neurodegeneration. Rats (n = 6 per experimental group) were anesthetized by ip of ketamine 80 mg/kg (Imalgene®, Merial Laboratorios SA) and xylazine 10 mg/kg (Rompun®, Bayer) and placed into a stereotaxic apparatus (David Kopf Instruments). KA (0.5 μg in 2 μl in saline; Abcam), alone or in combination with FTY720 (1 μg in 2 μl in saline), was injected into the right lateral ventricle (right side referred thereafter as ipsilateral), at the following coordinates from bregma: −1 mm anterioposterior, 2 mm mediolateral and 4 mm dorsoventral [29]. Injections were carried out over 5-min period using an infusion pump (KD Scientific), with a constant infusion rate of 0.4 μl/min. Animals were injected ip with vehicle (saline solution) or FTY720 (1 mg/kg) 24 h before icv injection and subsequent daily until sacrifice 8 days after KA application (see Fig. 2a for experimental design). For the ip injection, FTY720 was freshly prepared every day. Control animals received vehicle only. All animals received the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU, Sigma, #B5002) as ip injection (100 mg/kg, diluted in sterile saline) every 2 days starting the day after icv injection, as previously used [30]. Rats were sacrificed 24 h after the last BrdU injection.

Eight days after icv injection, the animals were deeply anesthetized with chloral hydrate 400 mg/kg (Panreac Quimica) and transcardially perfused with 4% paraformaldehyde in 0.1M PBS (pH 7.4). Brains were removed and immediately post-fixed in the same solution for 3 h. Then, they were washed and stored at +4 °C in PBS/Azide (0.02%) until sectioning. For all brains, series of 40-μm-thick coronal sections at the level of lateral ventricles and dorsal hippocampus were cut on a vibratome (HM 650V, Microm International) and used for immunohistology on free-floating sections, as hereinafter described. HCl antigen retrieval was used for BrdU immunostaining: before blocking step, sections were incubated in 2N HCl at 37 °C for 30 min to denature DNA, followed by two 10-min rinses in 0.1M sodium tetraborate pH 8.5 at room temperature to neutralize HCl and then rinsed twice with PBS. Immunoperoxidase staining was used for identification of proliferating, BrdU-positive nuclei. Briefly, after quenching of endogenous peroxidase (H₂O₂ 0.3%) and blocking/permeabilization (4% normal goat serum and 0.1% Triton X-100 in PBS), we incubated the sections overnight at 4 °C with a rat anti-BrdU antibody (AbD Serotec, #MCA2060) diluted 1:400 in blocking buffer. Subsequently, the primary antibody was detected using biotinylated goat anti-rat secondary antibodies (1:200), followed by incubation with avidin-biotin-peroxidase complex (both from Vector Laboratories). Peroxidase activity was visualized by incubation in 3,3′-diaminobenzidine (DAB) substrate (Roche). Finally, the sections were mounted in gelatin-coated slides, dehydrated through graded alcohols, cleared with xylene and coverslipped with DPX.

To analyze the cell fate of BrdU-positive cells, we performed double immunofluorescence colocalization studies with cell-specific markers. The sections were incubated with blocking and permeabilization solution (4% normal goat serum, 0.1% Triton X-100 in PBS) for 1 h at room temperature and then incubated overnight with the primary antibodies (diluted in the same solution) at 4 °C. After washing with PBS, the sections were incubated with Alexa Fluor 488- or 594-conjugate secondary antibodies (molecular probes) diluted 1:400 in the blocking solution for 1 h at room temperature. After washing with PBS, the sections were mounted on gelatine-coated slides with ProLong® Antifade Mountant (ThermoFisher Scientific). In addition to anti-BrdU antibody, we used rabbit anti-DCX (1:1000; Abcam, #Ab18723) as a marker of new neurons and mouse anti-NG2 (1:500; Chemicon, #MAb5384) as a marker of OPCs.

Negative controls in all experiments included the omission of the primary antibodies and provided no labelling, indicating the reliability and specificity of the immunostaining.

To analyze cell fate of newly, BrdU⁺ cells, double-immunolabelled sections were imaged with a Fluoview FV500 confocal laser scanning microscope (Olympus) equipped with Fluoviewer software (Olympus). Positive cells were counted using confocal acquired images, and the percentage of BrdU⁺ cells colabelling with cell fate markers was determined using Z-stack maximal projection of 5–10 confocal layers spaced 2.5 μm in coordination with three-dimensional orthogonal reconstruction of confocal layers (ImageJ software, http://​rsbweb.​nih.​gov/​ij/​download.​html). DCX and BrdU colocalization was determined over the total ipsilateral SGZ of DG in two slices per animal corresponding to two different levels of the dorsal hippocampus, and data is presented as mean positive cells per section in the case of total BrdU⁺ cells and as percentage of DCX/BrdU double-positive cells on total BrdU⁺ cells per section. NG2 and BrdU colocalization was determined at the level of lateral ventricle and at the level of the dorsal hippocampus: the measured areas selected five fields of the corpus callosum (both at ventricle and hippocampal levels), three fields of hilus and two fields of fimbria of the dorsal hippocampus (see Figs. 4a and 5d). Data are illustrated as the average of positive cells per square millimeter (mm²) in the case of total BrdU⁺ cells and percentage of NG2/BrdU double-positive cells on total BrdU⁺ cells per mm².

Data are expressed as mean ± SEM. Statistical analyses were performed using Prism version 5.0 (GraphPad Software, USA). Comparisons between the two groups were analyzed using two-tailed Student’s t test. Comparisons among multiple groups were analyzed by one-way or two-way analysis of variance (ANOVA), as appropriate, followed by Bonferroni post hoc test. p ≤ 0.05 or p ≤ 0.01 was defined as significant or highly significant, respectively.

We examined the neurogenic potential of FTY720 in vitro, on differentiation of NSC derived from young rat SVZ [27]. NSCs in culture proliferate and aggregate in neurospheres with multipotent stem cells properties which depending on local signals can differentiate into neurons or glial cells. In Fig. 1a, a schematic diagram of the experimental conditions is reported (see ‘Methods’ for further details). After a common phase where neurospheres proliferate as suspension cultures, they were split into two groups: neuron-oriented and oligodendrocyte-oriented differentiation cultures that were exposed to specific mitogen growth factors. At that stage, FTY720 was added in the medium at different concentrations (10 and 100 nM) to verify the ability of the drug, as compared to control conditions, to promote differentiation toward a specific cellular type. We measured the differentiation to neurons and to oligodendrocytes using immunofluorescence for β-III tubulin or CNPase, as markers of neurons or oligodendrocytes, respectively. FTY720 at either tested concentrations significantly increased NSC differentiation both into neurons (Fig. 1b–d) and oligodendrocytes (Fig. 1e–g), as compared to control conditions.

Previous studies demonstrated that neurospheres in culture express all five subtypes of S1P receptors [16], and the treatment with FTY720 was able to activate intracellular signalling cascades, indicating receptor binding and activation [16, 19, 20]. FTY720 enhanced survival, proliferation and migration of NSCs in vitro [18‐21]; however, effect on differentiation remains controversial. Our results, demonstrating a positive effect of FTY720 on differentiation of postnatal NSCs, both to neurons and oligodendrocytes, indicate that FTY720 is able to accelerate neurogenesis and oligodendrogenesis from neural precursors and suggest a potential neurogenic role also in vivo. To verify that the observed effect is due to involvement of S1PRs pathways, we performed the same differentiation experiment using FTY720-P instead of FTY720, obtaining similar results (see Additional file 1: Figure S1).

Confident with our results in vitro, we decided to explore the effect of FTY720 in adult neurogenesis in vivo, under neurodegenerative and neuroinflammatory conditions. For this aim, we investigated the effects of FTY720 in adult rat hippocampal neurogenesis following KA-induced acute neuronal death. All animals treated with KA experienced seizures in the first 2–3 h after icv, and we previously demonstrated that FTY720 reduced CA3 excitotoxic neuronal death and microgliosis associated with KA-induced status epilepticus [32]. We speculated whether modulation of adult neurogenesis by FTY720 could contribute to tissue repair in addition to the observed neuroprotection. To test that possibility, KA was injected in the right lateral ventricle (0.5 μg/2 μl), alone or in combination with FTY720 (1 μg/2 μl); in parallel, animals also received ip injection of FTY720 (1 mg/kg) or vehicle, from day −1 every day until 24 h before sacrifice (Fig. 2a). Animals were split into a total of four experimental groups: control (animals receiving vehicle icv and ip), FTY720 (animals receiving FTY720 icv and ip), KA (animals receiving KA icv and vehicle ip) and KA + FTY720 (animals receiving KA and FTY720 icv and FTY720 ip). Also, all animals were treated with BrdU (100 mg/kg, ip), starting from post-injection day 1 and every second days until 24 h before sacrifice (Fig. 2a), to label all proliferative cells at the S phase of cell cycle. Eight days after icv injection of KA, animals were sacrificed and immunohistochemistry for BrdU performed on 40-μm-thick brain slices at the level of dorsal hippocampus. The number of BrdU⁺ cells was counted in the SGZ of the DG, to estimate the effect of FTY720 on the proliferation of NSCs in adult rats in basal and neurodegenerative conditions. Positive cells were considered to be within SGZ if they were within two cell body diameters on the border between the GCL and the hilus (Fig. 2b). Figure 2d shows representative photomicrographs of BrdU staining in ipsilateral and contralateral DG (at dorsal hippocampus level), whereby GCL and SGZ are indicated by arrows. Quantitative analysis of the number of BrdU⁺ cells in SGZ revealed that KA induced a significantly increase in proliferation both in ipsilateral and contralateral hippocampus (Fig. 2c), as reported in previous studies [2, 31]. FTY720, alone or in combination with KA, slightly enhanced the number of positive cells, but the increase was not statistically significant (Fig. 2c). No significant differences were observed between the ipsilateral and contralateral hemispheres. Subsequent analyses were then performed only in the ipsilateral hemisphere. BrdU⁺ cells in GCL were also counted, but no significant differences were observed between groups (data not shown). Data obtained with BrdU immunolabelling were further confirmed by immunohistochemistry for the proliferation marker Ki67 (a nuclear protein expressed in active dividing cells) (Additional file 2: Figure S2).

To establish the nature of newborn cells in SGZ, we carried out double immunofluorescence for BrdU and DCX, a microtubule-associated protein expressed by neuronal precursor cells and immature neurons (Fig. 3a, b) and quantified BrdU and BrdU/DCX double-positive cells in the ipsilateral SGZ. Consistent with the results described above by immunohistochemistry (Fig. 2c), no difference was observed between KA and KA + FTY720-treated group (Fig. 3c) in terms of total BrdU⁺ cells, whereas FTY720 significantly increased the percentage of BrdU cells coexpressing DCX marker, both in basal (FTY720 alone) and in after injury (KA + FTY720) conditions (Fig. 3d), indicating a role for this drug in promoting new neuroblasts formation in the hippocampus. The same results were obtained when FTY720 was applied ip only, as reported in Additional file 3: Figure S3.

FTY720 exerts direct effects on oligodendrocytic lineage via S1P receptors modulation, promoting survival, differentiation and remyelination [11, 12, 14, 33]. We tested the hypothesis that FTY720 could enhance oligodendrogenesis in vivo, promoting OPC proliferation and differentiation into mature oligodendrocytes after a brain injury caused by icv injection of KA, as oligodendrocytes are also vulnerable to excitotoxic insults [34]. Thus, we quantified newborn BrdU⁺ cells with an oligodendrocytic fate using the OPC marker NG2, at two different brain levels in order to cover a wider brain area: the ipsilateral corpus callosum (CC), fimbria and hilus at the level of the dorsal hippocampus and the ipsilateral CC at level of the lateral ventricles (Fig. 4a and 5d). Figure 4 summarizes the results obtained at the level of the dorsal hippocampus in term of total BrdU⁺ cells per mm² (Fig. 4c–f), and percentage of NG2/BrdU double-positive cells over total BrdU⁺ cells per mm² (Fig. 4g–j), in hilus, fimbria and CC, separately and all together. Percentage of newborn NG2⁺ cells has a tendency to decrease in KA-treated animals (significantly in the hilus, fimbria and in all regions pool together), compared to control, whereas significant effect between KA-treated and (KA + FTY720)-treated group was found only in the corpus callosum and in all regions plotted together. The number of analyzed cells and P values resulting from all statistical analyses are showed in Tables 1 and 2, respectively.

However, we observed no differences in the number of newly formed NG2⁺ cells at the level of lateral ventricle (Fig. 5). We also quantified in the same brain regions the number of mature oligodendrocytes using as a marker APC antibodies (directed against the protein adenomatous polyposis coli), but no significant differences were found (data not shown) between the experimental groups.