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Behavioural Brain Research 268 (2014) 88–93

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

ingolimod increases brain-derived neurotrophic factor levels andmeliorates amyloid �-induced memory impairment

azuya Fukumotoa, Hiroyuki Mizoguchia,∗, Hideyuki Takeuchib, Hiroshi Horiuchib,un Kawanokuchib, Shijie Jinb, Tetsuya Mizunob, Akio Suzumurab

Futuristic Environmental Simulation Center, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku,agoya 464-8601, JapanDepartment of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

i g h l i g h t s

Fingolimod ameliorated memory and learning impairments in amyloid �-injected mice.Fingolimod restored normal levels of BDNF in cerebrum of amyloid �-injected mice.Fingolimod may be a promising therapeutic agent for Alzheimer’s disease.

r t i c l e i n f o

rticle history:eceived 7 January 2014eceived in revised form 26 March 2014ccepted 31 March 2014vailable online 5 April 2014

a b s t r a c t

Alzheimer’s disease is a progressive neurodegenerative disorder. Amyloid �, a neurotoxic protein, causesdisruption of hippocampal synaptic plasticity, and induces cognitive impairment in Alzheimer’s disease.We previously revealed that fingolimod, a new oral immunosuppressant used to treat multiple sclerosis,ameliorates oligomeric amyloid �-induced neuronal damage via up-regulation of neuronal brain-derived

eywords:ingolimodlzheimer’s diseasemyloid �

neurotrophic factor (BDNF). Here, we showed that oral administration of fingolimod ameliorated theimpairment in object recognition memory and associative learning in mice injected with amyloid �. Thiseffect was associated with restoration of normal BDNF expression levels in the cerebral cortices andhippocampi, suggesting that neuroprotection was mediated by up-regulation of neuronal BDNF levels.Therefore, fingolimod may provide therapeutic effects in patients with Alzheimer’s disease.

© 2014 Elsevier B.V. All rights reserved.

rain-derived neurotrophic factor

. Introduction

Alzheimer’s disease, the most common age-related neurode-enerative disorder in humans, is characterized by deteriorationf cognitive and mental functions. The formation of extracellu-ar deposits of amyloid � (A�) peptide, leading to the formationf neuritic plaques and neurofibrillary tangles in the cortex andippocampus, is a prominent pathological feature of Alzheimer’sisease [1,2]. A� is produced from amyloid precursor protein

hrough sequential cleavages and secreted to the extracellularpace. A� is strongly fibrillogenic: soluble oligomeric A� (oA�)onomers gradually convert to oligomers and ultimately to

Abbreviations: oA�, oligomeric amyloid �; BDNF, brain-derived neurotrophicactor; NORT, novel-object recognition test; S1P, sphingosine-1-phosphate; S1PR,phingosine-1-phosphate receptor; Trk, tropomyosin-related kinase.∗ Corresponding author. Tel.: +81 52 789 3929; fax: +81 52 789 3999.

E-mail address: h-mizo@riem.nagoya-u.ac.jp (H. Mizoguchi).

ttp://dx.doi.org/10.1016/j.bbr.2014.03.046166-4328/© 2014 Elsevier B.V. All rights reserved.

insoluble fibrils. Recent studies have shown that oA� is moreneurotoxic than fibrillar A� [3,4]. A�-mediated neurotoxicity hasbeen extensively demonstrated both in vivo and in vitro studies.oA� induces neuronal oxidative stress by activating N-methyl-d-aspartate (NMDA) receptors, leading to neuronal death [5]. We alsohave demonstrated that the intracerebroventricular (i.c.v.) injec-tion of A� induces cognitive deficits [6] as well as impairmentof the cholinergic system and down-regulation of brain-derivedneurotrophic factor (BDNF); these changes play important rolesin cognitive and morphological deficits associated with aging andneurodegenerative diseases [7,8].

Fingolimod is an oral immunosuppressant, which has beenrecently approved for treatment of multiple sclerosis [9–12]. Inrats, orally administered fingolimod localizes to white matter in thecentral nervous system (CNS), preferentially along myelin sheathes

[13]. The drug was synthesized by modifying myriocin, which isderived from Isaria sinclairii [14]. Fingolimod is phosphorylatedin vivo by sphingosine kinase to form the active metabolite. Afterbinding to sphingosine-1-phosphate (S1P) receptor 1 (S1P1) on the

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urface of lymphocytes, phosphorylated fingolimod induces S1P1nternalization; as a result, the lymphocytes can no longer egressrom lymphoid tissues. Thus, fingolimod acts as a functional antag-nist S1P1 and reduces the migration of immune cells into the CNS,hereby providing therapeutic effects against subsequent neuroin-ammation [15–17].

S1P receptors (S1PRs) are widely expressed in various CNSell types, including neurons, astrocytes, microglia, and oligoden-rocytes [18]. Phosphorylated fingolimod can bind to all S1PRubtypes (S1P1, S1P3, S1P4, and S1P5) but S1P2 [19,20]. Previ-us studies demonstrated that phosphorylated fingolimod directlyromotes remyelination mediated by oligodendrocyte progenitorells [21] and enhances neuroprotective effects through astrocyte1P1 modulation [22]. Recently, we showed that phosphorylatedngolimod binds to S1P1 receptors, resulting in down-regulationf pro-inflammatory cytokine production by activated microglia,p-regulation of microglial production of neurotrophic factors,nd augmentation of the neuroprotective effects of microglia [23].oreover, we also showed that phosphorylated fingolimod directly

ffects neurons and prevents oA�-induced neuronal damage viap-regulation of neuronal BDNF production [24].

In this study, we investigated the efficacy of fingolimod onognitive impairment in a mouse model of Alzheimer’s diseasenduced by i.c.v. injection of oA�. Fingolimod significantly ame-iorated cognitive impairments, and this effect was accompaniedy restoration of normal BDNF production in the brain. Our find-

ngs suggest that fingolimod may have therapeutic potential forreatment of Alzheimer’s disease.

. Materials and methods

.1. Animals

Male C57BL/6J mice (7–8 weeks old; weighing 40 ± 5 g at theeginning of the experiments) were obtained from Japan SLCHamamatsu, Japan). The animals were housed in plastic cages andept in a regulated environment (23 ± 1 ◦C; 50 ± 5% humidity) with

12-h light − dark cycle (lights on at 9:00 am). Food (CE-2, CLEAapan) and tap water were available ad libitum. All experiments

ere performed in accordance with the Guidelines for Animalxperiments of Nagoya University; the Guiding Principles for theare and Use of Laboratory Animals approved by the Japanese Phar-acological Society; and the United States National Institutes ofealth Guide for the Care and Use of Laboratory Animals.

.2. Peptides and drugs

Soluble oligomeric A�1-42 (oA�1-42) was prepared asescribed previously [4,25]. Briefly, synthetic human A�1-42Peptide Institute, Osaka, Japan) was dissolved in 100% 1,1,1,3,3,3-exafluoro-2-propanol and dried to completely remove theolvent. The obtained film was resuspended in dimethyl sulfoxidend diluted with Dulbecco’s Modified Eagle Medium/F12 (Invitro-en, Carlsbad, CA, USA) at a concentration of 100 �M. This solutionas incubated at 4 ◦C for 24 h to obtain oA�. A final concentration

f 300 �M oA�1-42 was used for all experiments. Oligomerizationf oA� was characterized by Western blotting.

All peptides were injected as described previously [26] andummarized as follows. A microsyringe with a 28-gauge stainless-teel needle was used for all experiments. Mice were anesthetizedsing 50 mg/kg sodium pentobarbital i.p. before stereotaxic

mplantation of a microinjection cannula into the right lateralentricle (anteroposterior +0.3 mm, mediolateral +1.0 mm fromhe bregma, and dorsoventral −2.5 mm from the skull) accordingo the method of Franklin and Paxinos (1997). oA�1-42 (300 pmol)

n Research 268 (2014) 88–93 89

was i.c.v. injected in a volume of 3 �L for 3 min. The same volumeof oA�42-1 (a peptide with identical amino-acid composition tooA�1-42, but with the reverse sequence) was injected as a control.Mice exhibited normal behavior within 1 min after the injection.Behavioral assessments were performed starting at 5 days afterinjection (day 5, Fig. 1A).

Fingolimod, kindly provided by Novartis Pharma AG (Basel,Switzerland), was dissolved in distilled water. Fingolimod or vehi-cle was administered at a dose of 1 mg/kg by oral gavage needle,once a day for 14 days after oA� injection, as previously described(Fig. 1A) [22].

2.3. Novel-object recognition test (NORT)

The NORT was carried out as described previously [27,28]. Theexperimental apparatus consisted of a Plexiglas open-field box(l × w × h: 30 cm × 30 cm × 35 cm), with a sawdust-covered floor.The apparatus was located in a sound-attenuated room and illumi-nated with a 20 W bulb.

The NORT procedure consisted of three sessions: habituation,training, and retention. Each mouse was individually habituatedto the box with 10 min of exploration in the absence of objectsfor 3 consecutive days (habituation sessions, days 5–7). During thetraining session, two novel objects were symmetrically fixed to thefloor of the box, 8 cm from the walls, and each animal was allowedto explore in the box for 10 min (day 8). The objects were con-structed from a golf ball, a wooden column, and a wooden trianglepole, which were different in shape and color but similar in size.An animal was considered to be exploring the object when its headwas facing the object or it was touching or sniffing the object. Thetime spent exploring each object was recorded. After training, micewere immediately returned to their home cages. During the reten-tion sessions, the animals were placed back into the same box 24 hafter the training session (day 9), but one of the familiar objects usedduring training had been replaced with a novel object. The animalswere then allowed to explore freely for 5 min, and the time spentexploring each object was recorded. Throughout the experiments,novel and familiar objects were of similar physical complexity andemotional valence. In the retention session, the preference indexwas defined as the ratio of the amount of time spent exploring thenovel object to the total time spent exploring both objects; thismetric was used to measure cognitive function. In the training ses-sion, the preference index was defined as the ratio of the time spentexploring the object that was replaced by the novel object in theretention session to the total exploration time.

2.4. Conditioned-fear memory test

Mice were divided into two groups according to experimentalschedule (Fig. 1A). Fear conditioning test for group 1 was performedon day 12–13, and the test for group 2 was performed on day 13–14.Fear conditioning tests were performed as previously described[26,29,30] with minor modifications. Freezing behavior, duringwhich the mouse’s head, arms, and legs did not move, was timedusing a stopwatch. To measure the basal freezing response (precon-ditioning phase), mice were individually placed in a transparentPlexiglas conditioning cage (30 cm × 30 cm × 35 cm) for 2 min. Fortraining (conditioning phase, day 12 for group 1 or day 13 forgroup 2), mice were placed in the conditioning cage and subjectedto the conditioned stimulus (a 15-s tone at 80 dB). During the last5 s of the tone stimulus, a 0.6-mA foot shock was delivered as theunconditioned stimulus via a shock generator (BrainScience Idea

Co., Osaka, Japan). This procedure was repeated six times at 15-sintervals, and mice were left in place for an additional 1 min afterthe final unconditioned stimulus. Contextual and cued tests werecarried out 1 day after fear conditioning (day 13 for group 1 or day

90 K. Fukumoto et al. / Behavioural Brain Research 268 (2014) 88–93

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Fig. 1. Oral administration of fingolimod ameliorates oA�-induced memory impairment.(A) Experimental scheme of behavioral assessments. (B, C) Effect of fingolimod treatment on object recognition memory of oA�1-42-injected mice in the novel objectrecognition test. Retention session was carried out 24 h after training. Values are means ± SE (n = 9–10). *, p < 0.05 vs. oA�42-1/vehicle. †, p < 0.05 vs. oA�1-42/vehicle. (D,E micet asureo

1t2mm

) Effect of treatment with fingolimod on associative learning of oA�1-42-injectedraining. Context-dependent (D) and tone-dependent (E) freezing times were meA�1-42/vehicle.

4 for group 2). For the contextual test, mice were placed back into

he conditioning cage, and the freezing response was measured for

min in the absence of the conditioned stimulus. For the cued test,ice were placed in the neutral cage and the freezing response waseasured for 1 min in the presence of the conditioned stimulus.

in the conditioned-fear learning test. Retention session was carried out 24 h afterd. Values are means ± SE (n = 9–10). *, p < 0.05 vs. oA�42-1/vehicle. †, p < 0.05 vs.

2.5. Quantitative analysis of BDNF protein

Mice were euthanized after the behavioral experiments (days 14and 15). Contralateral left-brain hemispheres, including the cere-bral cortices and hippocampi but excluding the striatum, were

al Brain Research 268 (2014) 88–93 91

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Fig. 2. Fingolimod treatment increased BDNF production in the cerebrum of oA�1-42-injected mice. Mice were euthanized after behavioral assessments, and BDNF

K. Fukumoto et al. / Behaviour

mmediately dissected after perfusion of PBS to rule out directffects of injury induced by the injection needle. Collected sam-les were immediately frozen and stored at −80 ◦C until the assay.ll dissections were performed using brain matrix (BrainScience

dea, Osaka, Japan) and based on an anatomic atlas [31].Samples were homogenized in 10 volumes of lysis buffer

137 mM NaCl, 20 mM Tris–HCl [pH 8.0], 1% NP-40, 10% glycerol)ith protease inhibitor mixture (Complete, Mini, EDTA-free, Roche)sing ultrasonication. After centrifugation at 10,000 × g for 15 mint 4 ◦C, supernatants were assessed using the BDNF ELISA kit (EmaxmmunoAssay Systems; Promega, Madison, WI, USA) as describedreviously [24]. Assays were carried out in nine or ten independentrials.

.6. Statistical analysis

All data are expressed as means ± SE. Statistical significance wasetermined using one-way analysis of variance (ANOVA) for multi-roup comparisons. Fisher’s LSD was used for post hoc comparisonhen the F-value was significant (p < 0.05).

. Results

.1. Oral administration of fingolimod ameliorates oAˇ-inducedemory impairment

Firstly, we investigated the efficacy of fingolimod on oA�1-2-induced impairment of recognition memory in the NORT. Ashown in Fig. 1C, i.c.v. injection of oA�1-42 significantly reducedxploratory preference for the novel object in the retention sessionF(3,35) = 5.43, p < 0.05), without affecting total exploration time inhe training and retention sessions (Fig. 1B, training: F(3,35) = 0.52,

> 0.05; retention: F(3,35) = 1.26, p > 0.05), indicating that recog-ition memory was impaired in oA�1-42-injected mice. Oraldministration of fingolimod (1 mg/kg) in oA�1-42-injected miceignificantly improved exploratory preference in the retention ses-ion (Fig. 1C, F(3,35) = 5.43, p < 0.05), without affecting exploratoryreference in the training session (Fig. 1C, F(3,35) = 0.20, p > 0.05) orotal exploration time in the training or retention session (Fig. 1B,raining: F(3,35) = 0.52, p > 0.05; retention: F(3,35) = 1.26, p > 0.05).

Fingolimod treatment per se had no effect on exploratory pref-rence or total exploration time in oA�42-1-injected control miceFig. 1B and C).

Next, we evaluated associative learning in a conditioned-fearearning test. In the preconditioning phase (training), all groupsf mice exhibited very little freezing (data not shown). oA�1-2-injected mice exhibited less freezing than control mice inoth contextual and cued tests (Fig. 1D, context: F(3,35) = 14.92,

< 0.05; Fig. 1E, tone: F(3,35) = 16.79, p < 0.05), indicating thatssociative learning was impaired in oA�1-42-injected mice. Fin-olimod treatment ameliorated the oA�1-42-induced decrease inhe context-dependent freezing response (Fig. 1D: F(3,35) = 14.92,

< 0.05), but not in the tone-dependent freezing response (Fig. 1E: > 0.05). No alterations of the nociceptive response were found inny group: there was no significant difference between the groupsn the minimal current required to elicit flinching/running, jump-ng, or vocalization (data not shown).

Fingolimod treatment per se had no effect on associative learn-ng in oA�42-1-injected control mice (Fig. 1D and E).

.2. Fingolimod restored BDNF production in the brain of

Aˇ1-42-injected mice.

Finally, we investigated whether fingolimod exerts its neuro-rotective effects by up-regulating neuronal BDNF [24].

levels in homogenates of cerebral cortices and hippocampi were measured by ELISA.Values are means ± SE (n = 9–10). *, p < 0.05 vs. oA�42-1/vehicle. †, p < 0.05 vs. oA�1-42/vehicle.

BDNF levels were reduced in the cerebrum of oA�1-42-injectedmice relative to those in oA�42-1-injected control mice (Fig. 2,F(3,35) = 3.91, p < 0.05). Fingolimod treatment restored normal lev-els of BDNF production in the cerebrum of oA�1-42-injected mice(p < 0.05).

4. Discussion

In this study, we demonstrated that treatment with fingolimodprotects against oA�1-42-induced cognitive impairment, and thatthis neuroprotection is accompanied by BDNF production.

oA� injection induced impairment of object-recognition mem-ory (Fig. 1B and C) and associated learning (Fig. 1D and E) in mice.These results are consistent with our previous study revealing themechanism of A� neurotoxicity in this animal model [6]. Oral treat-ment with fingolimod ameliorated oA�1-42-induced impairmentof recognition memory, and also restored the oA�1-42-induceddecrease in the context-dependent freezing response (Fig. 1D),but did not affect the tone-dependent freezing response (Fig. 1E).In general, context-dependent learning depends on hippocampusfunction, whereas tone-dependent learning mainly depends onamygdala function. In this animal model, we have shown that cor-tical and hippocampal extracellular macromolecules are involvedin A�-induced cognitive impairment and neurotoxicity [6,7,32].Moreover, it is possible that the expression levels of BDNF andits receptor TrkB are much higher in the hippocampus than inthe amygdala [33,34]. Thus, fingolimod may exert its protectiveeffects via inhibition of extracellular macromolecules that induceapoptotic and/or necrotic cell death, or via up-regulation of fac-tors that promote the cell survival and synaptic plasticity in thecortico-hippocampus. Consistent with these findings, fingolimodtreatment inhibited neuronal damage in the CA1 or dentate gyrusin rats that received intrahippocampal or intracortical A� injection,respectively [35,36].

BDNF/TrkB signaling impacts neuronal survival, axon growth,neuronal transmission, and synaptic plasticity [37–39]. Reductionin the BDNF level leads to disruption of memory formation andmaintenance [40,41], whereas overexpression of full-length TrkB

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an facilitate spatial memory and associative learning [42]. More-ver, in Alzheimer’s disease, the BDNF expression level is severelyeduced in the hippocampus and some cortical areas [43,44], andlasma BDNF level might be useful as a behavioral state marker

n Alzheimer’s disease patients [45,46]. We have also demon-trated that BDNF expression is reduced in the hippocampus in annimal model of mild cognitive impairment [8]. Thus, BDNF sig-aling is a pivotal modulator of memory formation, and may be

nvolved in the pathology of Alzheimer’s disease, which is associ-ted with A� production, accumulation, and aggregation [41]. Inhis study, we showed that repeated treatment with fingolimodestored normal levels of BDNF protein in the cortico-hippocampusf oA�1-42-injected mice (Fig. 2). Taken together with the results ofur previous study showing that BDNF/TrkB signaling is critical forhe neuroprotective effects of phosphorylated fingolimod againstA�-induced neurotoxicity [24], these findings demonstrate thatherapeutic effect of fingolimod in Alzheimer’s disease is mediatedy an increase in neuronal BDNF. Two recent reports have corrobo-ated the potential therapeutic utility of our approach: in one study,ngolimod improved functional recovery after spinal cord injuryia nonimmunologic mechanisms [47]; in the other, fingolimodounteracted NMDA-induced neuronal death in a BDNF-dependentanner in a mouse model of Rett syndrome [48]. In addition, S1P

tself exerts neuroprotective effects against soluble oA�-inducedell death, via inactivation of acid sphingomyelinase [49], andngolimod also reduces neuronal A� production via signaling path-ays independent of S1PR, although the details remain to be

lucidated [50]. Thus, fingolimod may exert neuroprotective effectsgainst oA�-induced neurotoxicity via multiple pathways.

. Conclusions

This study demonstrates that fingolimod exerts a striking ther-peutic effect in oA�-induced memory impairment. Thus, S1Peceptors and associated signaling pathways are a potential tar-et for the treatment of Alzheimer’s disease via up-regulation ofDNF production.

cknowledgments

This study was supported in part by Novartis Pharm KK; a Grant-n-aid for Scientific Research (No. 25460094) from the Japan Societyor the Promotion of Science; a grant from the Takeda Scienceoundation; and a grant from the Advanced Research for Medicalroducts Mining Program of the National Institute of Biomedicalnnovation (NIBIO). The funding sources had no involvement in theonduct of the research or preparation of the manuscript.

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