Fingolimod induces neurogenesis in adult mouse hippocampus and improves contextual fear memory

Abstract

Fingolimod (FTY720) was the first per os administered disease-modifying agent approved for the treatment of relapsing-remitting multiple sclerosis. It is thought that fingolimod modulates the immune response by activating sphingosine-1 phosphate receptor type 1 (S1P1) on lymphocytes following its in vivo phosphorylation. In addition to its immune-related effects, there is evidence that fingolimod exerts several other effects in the central nervous system, including regulation of the proliferation, survival and differentiation of various cell types and their precursors. In the present study, we have investigated the effect of fingolimod on the production of new neurons in the adult mouse hippocampus and the association of this effect with the ability for pattern separation, an established adult neurogenesis-dependent memory function. Immunofluorescence analysis after chronic administration of a physiologic dose of fingolimod (0.3 mg kg(-1)) revealed a significant increase in both the proliferation and the survival of neural progenitors in the area of dentate gyrus of hippocampus, compared with control animals. These effects were replicated in vitro, in cultures of murine hippocampal neural stem/precursor cells that express S1P1 receptor, suggesting cell-autonomous actions. The effects of fingolimod on neurogenesis were correlated to enhanced ability for context discrimination after fear conditioning. Since impairment of adult hippocampal neurogenesis and memory is a common feature of many neuropsychiatric conditions, fingolimod treatment may be beneficial in therapeutic armamentarium of these disorders.

Figure 1

(a) Illustrative graph that shows the exact time of administration of BrdU and fingolimod in animals for the NSCs proliferation experiments. (b) Coronal section of dorsal DG from 3-month-old C57/BL6 mice injected with fingolimod or vehicle daily for 14 days, co-injected with BrdU for the last 5 days. Sections were immunostained for BrdU (green) and counterstained with DAPI. (c) Quantification of BrdU+ cells in SGZ in Fingolimod or vehicle-treated mice of different ages. (d) Coronal section of dorsal DG from 3-month-old C57/BL6 mice injected with Fingolimod or vehicle daily for 14 days. Image depicts DcX (red) immunostained immature neurons. (e) Quantification of DcX cells in C57/bl6 mice of different ages treated with fingolimod or vehicle for 14 days. Scale bar, 150 μm (mean±s.d.; n=6; *P<0.05 vs vehicle). BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; DcX, doublecortin; DG, dentate gyrus; NSC, neural stem cells; SGZ, subgranular zone.

Figure 2

(a) Illustrative graph that shows the exact time of administration of BrdU and fingolimod in animals for the NSCs survival experiments. (b) Coronal section of dorsal DG from 3-month-old C57/BL6 mice injected with fingolimod or vehicle daily for 21 days, co-injected with BrdU for the first 5 days. Sections were immunostained for BrdU (green) and NeuN (red). (c) Higher magnification of a confocal image of BrdU- and NeuN-stained cells in SGZ. (d) Quantification of BrdU+ cells in SGZ and GCL in fingolimod or vehicle-treated 3-month-old mice (mean±s.d.; n=6; *P<0.05 vs vehicle). (e) Percentage of BrdU+/NeuN+ cells in the DG of fingolimod or vehicle-treated mice 21 days after first BrdU administration. BrdU, 5-bromo-2′-deoxyuridine; DG, dentate gyrus; NSC, neural stem cells; SGZ, subgranular zone.

Figure 3

(a) Western blot analysis of S1P1 expression in neurospheres derived from the respective area as indicated and at specific ages (Ctx, cortical; Hip, Hippocampal; E, embryonic; P, postnatal). (b) BrdU staining of hippocampal neural stem cells after they have been treated for 24 h as indicated in the absence of growth factors. Scale bar, 100 μM and (c) percentage of BrdU+/DAPI+ (mean±s.d.; n=3; *P<0.05 vs vehicle-treated cells). (d) Phosphorylation of ERK1/2 after 10-min exposure of NS/PCs culture in growth factor free medium in the presence or absence of 1 μM fingolimod-p after they have been pre-incubated or not with PTX. Western blot analysis was performed and relative optical density (OD) of phosphorylated ERK1/2 (pERK) to this of total ERK1/2 (tERK) was estimated and normalized to control values (mean±s.d.; n=3; *P<0.05 vs vehicle-treated cells). BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; PTX, pertussis toxin.

Figure 4

(a) TUNEL staining of hippocampal neural stem cells after 72 h treated as indicated in the absence of growth factors. (b) BDNF protein levels quantified by ELISA in lysates of hippocampal NSC cultures treated with 1 μM fingolimod-p or not in the presence or absence of U0126 or pertussis toxin for 24 h (mean±s.d.; n=3; *P<0.05 vs vehicle-treated cells). (c) Percentage of TUNEL+/DAPI+ (mean±s.d.; n=3; *P<0.05, vs vehicle-treated cells). Scale bar, 50 μM. DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 5

(a) Illustrative graph that shows the exact time periods for drug or vehicle administration, the training day and the testing days of the behavioral tasks. (b) Percent of time freezing in context A (black bars) and context B (gray bars) 1 day after fear conditioning, for mice that were treated with either vehicle or fingolimod for 14 days. (mean±s.e.m.; n=13; *P<0.05 vs freezing in context, A) (c) Percentage of freezing in context A and B for mice that have not received shock during the training day. (d) Discrimination ratio, which represent (freezing in context A−freezing in context B) to (freezing in context A+freezing in context B) (mean±s.e.m.; n=13; **P<0.01 vs vehicle).