Fingolimod Phosphate Attenuates Oligomeric Amyloid β–Induced Neurotoxicity via Increased Brain-Derived Neurotrophic Factor Expression in Neurons

The neurodegenerative processes that underlie Alzheimer's disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P) receptor (S1PR) agonist fingolimod phosphate (FTY720-P)-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF) in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer's disease.


Introduction
Alzheimer's disease (AD) is the most common cause of dementia [1,2]. Senile plaques consisting of insoluble fibrillar amyloid b (Ab) are pathologic hallmarks of AD. Ab is formed after sequential cleavage of amyloid precursor protein and is secreted to the extracellular space. Ab has a strong fibrillogenic property, and soluble Ab monomers gradually convert to oligomers and ultimately to insoluble fibrils. Soluble oligomeric Ab (oAb) is considered to be more important in the pathogenesis of AD than fibrillar Ab, because oAb is more neurotoxic [3,4]. Naturally secreted oAb inhibits hippocampal long-term potentiation and disrupts of synaptic plasticity [2]. In addition, oAb induces elevation of reactive oxygen species levels in neurons, leading to neuronal death [5].
Many studies have shown that S1PRs are widely expressed in many cell types, including neurons, astrocytes, microglia, and oligodendrocytes [14]. The functions of these receptors have not been elucidated completely, however. There are five S1PR subtypes: S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 . FTY720-P binds to all S1PR subtypes except S1P 2 [15,16]. Previous studies demonstrated that FTY720-P directly induced oligodendrocytes to promote remyelination [17] and enhanced neuroprotective effects in astrocytes [18]. Moreover, we have recently showed that FTY720-P augmented microglial neuroprotective effects by downregulation of pro-inflammatory cytokines and upregulation of neurotrophic factors [19]. However, it is still uncertain whether FTY720-P directly affects neurons. S1P reportedly promotes neurogenesis from proliferation of neuronal progenitor cells [20]. S1P also contributes to the migration of neuronal stem/progenitor cells [21]. Therefore, FTY720-P may directly affect neurons. Here, by assessing the effects of FTY720-P on neurons, we show that FTY720-P directly upregulates neuronal brain-derived neurotrophic factor (BDNF) production to attenuate oAb-induced neurotoxicity.

Neuronal cultures
Protocols for animal experiments were approved by the Animal Experiment Committee of Nagoya University (permission number: 12122). Primary mouse cortical neurons were prepared as described previously [4,23]. Briefly, cerebral cortices were isolated from embryonic day 17 C57BL/6J mouse embryos, minced and treated with dissociation solution (Sumitomo Bakelite, Akita, Japan). Resulting neurons were resuspended in Nerve Culture Medium TM (Sumitomo Bakelite), plated on polyethyleniminecoated glass coverslips (Asahi Techno Glass, Chiba, Japan) at a density of 56103 cells/well in 96-well multidishes, 56104 cells/ well in 24-well multidishes or 56105 cells/well in 6-well multidishes, and incubated at 37uC in an atmosphere containing 5% CO2 at 100% humidity. The purity of the cultures was.95% based on NeuN-specific immunostaining. Neurons were used at 14 days in vitro for the following assessments.

Western blotting
To confirm the oligomerization, oAb1-42 was also dissolved in Laemmli sample buffer. Cell lysates were used to examine S1PR subtypes and BDNF production. The neurons at 14 days in vitro were treated with 5 mM oAb or 100 pM FTY720-P for 24 h. Cells were lysed in lysis buffer (50 mM Tris-HCl at pH 7.6, 1% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, and protease inhibitor mixture (Complete Mini EDTA-free, Roche, Germany)). Soluble fractions were collected following centrifugation for 5 min at 10,000 rpm and the protein concentration was determined in a bicinchoninic acid assay (Thermo Fisher Scientific, Rockford, IL, USA). The soluble fractions of the cell lysates or neuronal culture supernatant were dissolved in Laemmli sample buffer.

Enzyme-linked immunosorbent assay (ELISA)
BDNF was measured using an ELISA kit according to the manufacture's protocol (Emax ImmunoAssay Systems; Promega, Madison, WI, USA). Neurons at 14 days in vitro were treated with 5 mM oAb or 100 pM FTY720-P for 24 h. Cell lysates were obtained with lysis buffer and soluble fractions were collected following centrifugation for 5 min at 10,0006g. Assays were carried out in three independent trials.

Assessments of neuronal survival
WST-8 assay was carried out in six independent trials using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the Figure 1. Neurons express all S1PR subtypes. (A) Representative RT-PCR data for S1PRs. Neurons constitutively expressed all S1PR subtypes regardless of oAb-stimulation. (B) Immunostaining data for S1PRs in untreated neurons. Neurons constitutively expressed all S1PR subtypes. Scale bar, 20 mm. (C) Western blotting data for S1PRs. Arrowheads indicate the bands for S1PRs. Neurons constitutively expressed all S1PR subtypes regardless of oAb-stimulation. NT, no treatment; oAb . 5 mM oAb1-42 treatment. The positive control samples used for each S1PR were as follows; brain for S1P 1 and S1P 3 ; heart for S1P 2 ; spleen for S1P 4

Statistical analysis
Statistical significance was analyzed with Student's t-test or oneway analysis of variance followed by post-hoc Tukey's test using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA).

Neurons express S1PRs
First, we examined S1PR expression on primary cortical neurons. RT-PCR analysis revealed that cortical neurons constitutively expressed all S1PR subtypes regardless of stimulation with 5 mM oAb1-42 ( Figure 1A). Immunostaining for S1PRs depicted that cortical neurons expressed all S1PR subtypes ( Figure 1B). Western blotting analysis also showed that cortical neurons constitutively expressed all S1PR subtypes ( Figure 1C, arrowheads), which is consistent with the RT-PCR data, although multiple non-specific bands were also detected. Treatment with 5 mM oAb1-42 did not alter the expression levels of S1PRs.

Discussion
This is the first report showing that FTY720-P is protective against oAb1-42-induced neurotoxicity. We have demonstrated that FTY720-P enhances the production of BDNF, which activates TrkB and ERK1/2 signaling.
Although Ab-stimulated mononuclear cells reportedly show increased mRNA expression levels of S1P 2 and S1P 5 [28], whether Ab alters the expression profiles of neuronal S1PRs has not been reported. We demonstrated that neurons constitutively express all S1PR subtypes regardless of Ab-stimulation. These results suggested that FTY720-P directly affects neurons via S1PRs. We had the technical limitation of evaluation for mouse S1PRs protein expression levels because well-characterized antibodies against mouse S1PRs are not commercially available at this time. Moreover, because we used primary cortical neurons in this study, additional experiments are needed to confirm whether S1PR expression profiles change in response to aging or various pathologic conditions. BDNF and its receptor TrkB are widely expressed in the CNS [29][30][31]. These molecules play important neuroprotective roles, including contributions to cell survival, axon growth, neuronal transmission, and synaptic plasticity [32][33][34]. Several reports have documented that BDNF and TrkB contribute to long-term potentiation and memory formation [35][36][37][38]. Decreased BDNF-TrkB signaling induces deficiencies in spatial memory [39], whereas overexpression of full-length TrkB can enhance learning and memory [40]. Moreover, BDNF expression levels are lower in patients with AD [41,42]. Thus, BDNF-TrkB signaling may play an important role in the pathology of AD. In this study, we demonstrated that BDNF-TrkB signaling is critical for the neuroprotective effects of FTY720-P against oAb-induced neurotoxicity. We believe that treatment with FTY720 and resulting increases in BDNF expression are a promising therapeutic strategy for AD. Recently, FTY720-P was shown to improve spinal cord injuries via nonimmunologic mechanisms [43]. Furthermore, FTY720-P injections induced BDNF production and improved disease symptoms in mouse models of Rett syndrome [44]. These two papers corroborate the potential therapeutic utility of our approach.
In conclusion, FTY720 appears to be a promising therapeutic agent against not only multiple sclerosis but also various neurodegenerative diseases, including AD.