Abstract
BackgroundFTY720 (fingolimod, Gilenya™), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection.MethodsPrimary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 μg/2 μl) in Sprague–Dawley rats. FTY720 was applied icv (1 μg/2 μl), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs).ResultsFTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation.ConclusionsThese data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0308-6) contains supplementary material, which is available to authorized users.
Highlights
FTY720, a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R)
In a first set of experiments, we applied a single dose of FTY720, icv (1 μg/2 μl) together with kainic acid (KA) (Figure 2A) and found no significant differences between KA- and KA+FTY720-treated animals, neither in seizure-like score (Figure 2B) or in neuronal death in CA3 region, as assessed by quantification of Nissl’s stained sections were pre-treated with increased concentration of FTY720 (10 to 1,000 nM) for 24 h and stimulated with N-methyl-D-aspartic acid (NMDA) (25 to 100 μM) in the same conditioned medium
The damage was detected by PI uptake 24 h after treatment with NMDA and propidium iodide (PI) intensity was measured as corrected total fluorescence (CTF)
Summary
FTY720 (fingolimod, GilenyaTM), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R). The high lipophilic FTY720 crosses blood brain barrier (BBB) and accumulates in the brain and spinal cord [12], and endogenous SphK2 [13] and all the S1P receptors, with the exception of S1P4, are abundantly expressed in the brain [14], further suggesting that action of FTY720 might involve direct effects in the central nervous system (CNS) and implying a therapeutic potential of FTY720 in other neuropathologies. In vitro studies confirm direct action of FTY720 on microglial cells, reducing the production of pro-inflammatory cytokines from LPS-stimulated microglia [27] or in a model of transient demyelination in rat CNS reaggregate spheroid cell culture [28]. In cultured neurons, FTY720 counteracts N-methyl-D-aspartic acid (NMDA) or oligomeric amyloid β (Aβ)-induced neuronal death [21,29,30] increasing the neuronal production of BDNF [21,29]
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