Spatial reference memory deficits precede motor ...

Brain, Behavior, and Immunity 33 (2013) 90–101

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Brain, Behavior, and Immunity

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Spatial reference memory deficits precede motor dysfunctionin an experimental autoimmune encephalomyelitis model:The role of kallikrein–kinin system

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⇑ Corresponding author. Address: Laboratório de Autoimunidade e Imunofar-macologia, Campus Araranguá, Rua Pedro João Pereira, 150 – Mato Alto, Univer-sidade Federal de Santa Catarina, CEP 88900-000 Araranguá, SC, Brazil. Tel.: +55 483721 6448.

E-mail addresses: [email protected], [email protected] (R.C. Dutra).

Rafael C. Dutra a,b,⇑, Eduardo L.G. Moreira b,c, Thaís B. Alberti b, Rodrigo Marcon b,Rui D. Prediger b,c, João B. Calixto b

a Laboratory of Autoimmunity and Immunopharmacology, Campus Araranguá, Universidade Federal de Santa Catarina, 88900-000 Araranguá, SC, Brazilb Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, 88049-900 Florianópolis, SC, Brazilc Neuroscience Graduate Program, Centre of Biological Sciences, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil

a r t i c l e i n f o

Article history:Received 19 February 2013Received in revised form 21 May 2013Accepted 7 June 2013Available online 15 June 2013

Keywords:Multiple sclerosisExperimental autoimmuneencephalomyelitis (EAE)Cognitive dysfunctionCholine acetyltransferase (ChAT)Genetic deletion – kinin B1R

a b s t r a c t

Multiple sclerosis (MS) is a progressive T cell-mediated autoimmune demyelinating inflammatory dis-ease of the central nervous system (CNS). Although it is recognized that cognitive deficits represent amanifestation of the disease, the underlying pathogenic mechanisms remain unknown. Here we provideevidence of spatial reference memory impairments during the pre-motor phase of experimental autoim-mune encephalomyelitis (EAE) in mice. Specifically, these cognitive deficits were accompanied by down-regulation of choline acetyltransferase (ChAT) mRNA expression on day 5 and 11 post-immunization, andup-regulation of inflammatory cytokines in the hippocampus and prefrontal cortex. Moreover, a markedincrease in B1R mRNA expression occurred selectively in the hippocampus, whereas protein level was up-regulated in both brain areas. Genetic deletion of kinin B1R attenuated cognitive deficits and cholinergicdysfunction, and blocked mRNA expression of both IL-17 and IFN-c in the prefrontal cortex, lymph nodeand spleen of mice subjected to EAE. The discovery of kinin receptors, mainly B1R, as a target for control-ling neuroinflammatory response, as well as the cognitive deficits induced by EAE may foster the thera-peutic exploitation of the kallikrein–kinin system (KKS), in particular for the treatment of autoimmunedisorders, such as MS, mainly during pre-symptomatic phase.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Multiple sclerosis (MS), a chronic inflammatory and demyelin-ating disease that affects the central nervous system (CNS) (Sospe-dra and Martin, 2005) is considered to be an autoimmunepathology in which autoaggressive Th1 and Th17 lymphocytes in-duce a response against components of myelin (Goverman, 2009;Sospedra and Martin, 2005; Steinman, 2007). Th1 and Th17 cellsare characterized by their expression of interferon-c (IFN-c) andinterleukin-17 (IL-17), respectively. Experimental autoimmuneencephalomyelitis (EAE), a CD4+ T cell-mediated disease of theCNS, is the best known animal model of MS and can be inducedin susceptible rodents and other animals by immunization withmyelin antigens such as myelin oligodendrocytes glycoprotein(MOG) (Steinman, 2007). White matter inflammation, loss of mye-

lin and consequent neuronal degeneration, the pathological hall-marks of MS and EAE, are thought to determine the diseaseseverity (Ffrench-Constant, 1994). Clinical symptoms in MS in-clude a progressive decline in motor and sensory functions andpermanent disability (Steinman, 2007).

Nonetheless, recent evidence has shown that, in MS, the neuro-nal compartment of the CNS is affected in parallel to, and evenindependently of, white matter damage, which has led to a reeval-uation of the perceived relationship between inflammation andneurodegeneration in this disease (Centonze et al., 2010; Steinman,2007). Studies using magnetic resonance imaging (MRI) revealedthat the gray matter atrophy, which occurs in cortical and deepsub-cortical brain regions (Filippi et al., 2003), begins early in thedisease, continues as the disease progresses (Lisak, 2007) and cor-relates with motor, sensory and visual disability (Magnano et al.,2006). Moreover, there is few evidence indicating that about 50–70% of MS patients experience important cognitive deficits (Rao,1995; Shi et al., 2008) which are detectable even before a definitivediagnosis of MS is made. These cognitive and emotional symptomsrelated to MS strongly affect patients’ ability to work, as well astheir quality of life (Engel et al., 2007). Although the cause of mem-

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ory dysfunction in MS is currently unknown, information-process-ing speed and working memory are the most frequently detectedcognitive deficits in MS (Rao et al., 1991). In addition, verbal andspatial learning are also affected, reflecting hippocampal dysfunc-tion, especially atrophy of the CA1 region (Sicotte et al., 2008). Inthis regard, some reports have proposed a linkage between cholin-ergic system, neuroinflammation and cognitive impairments (Fieldet al., 2012; Ruan et al., 2010; Ullrich et al., 2010). Particularly,impairment within the cholinergic system has been demonstratedin MS (Antonelli et al., 2013). For instance, Kooi et al. (2011) re-ported that in MS hippocampus, both activity and protein expres-sion of choline acetyltransferase (ChAT), the acetylcholinesynthesizing enzyme, were decreased, whereas the activity andprotein expression of acetylcholinesterase (AChE), the acetylcho-line degrading enzyme, were found to be unaltered (Kooi et al.,2011). Moreover, cholinergic projections arise from basal forebrainnuclei such as medial septum/vertical limb of the diagonal band tothe hippocampus and from nucleus basalis (of Meynert) to the cor-tex (Wevers, 2011). This anatomical split of the cholinergic systeminto neocortical and hippocampal divisions underlies cholinergicmodulation of working (Croxson et al., 2011) and spatial-referencememories (Deiana et al., 2011), respectively.

The identification of the specific cognitive deficits observed be-fore motor symptoms in MS is of high interest because they may beuseful as temporal markers of the disease, and for testing of poten-tial therapeutic treatments even before the onset of motor impair-ments. Moreover, drugs currently used to treat MS focus mainly onthe control of autoimmune and neuroinflammatory responses.Hence, it is incumbent upon novel studies to elucidate a molecularsubstrate, with the view of discovering therapeutic interventions.In this context, we and others have previously demonstrated thatkinin receptors, mainly the B1 receptor (B1R), exert a critical rolein regulating the early development of experimental autoimmuneencephalomyelitis (EAE) progression by modulating the onset ofthe immune response and affecting the functioning of astrocytes/microglia cells (Dutra et al., 2011).

Kinins (such as bradykinin and kallidin) are the most potentautacoids involved in inflammatory, vascular and pain processes,and constitute the end-products of the so-called kallikrein–kininsystem (KKS). Kinins exert most of their biological effects by theactivation of two G-protein coupled receptors, denoted B1 and B2

receptors (Calixto et al., 2004; Marceau and Regoli, 2004). In thisregard, Wang and Wang demonstrated that injection of bradykinin(BK), a preferential B2R agonist, is able to induce learning andmemory impairment when administered into the rat hippocampus(Wang and Wang, 2002). Extending this idea, Prediger et al. (2008)showed that kinins, acting via activation of B1R and B2R in the CNS,exert a critical role in the spatial learning and memory deficits in-duced by the b-amyloid peptide (Ab) in mice (Prediger et al., 2008).In addition, up-regulation of both kinin receptors in the brain wasrelated, directly or indirectly, to cognitive processes after infusionof Ab (Viel et al., 2008). On the other hand, the linkage betweenKKS and the cholinergic system is currently not fully established.A relevant study conducted by Buccafusco and Serra (1985)showed that the intracerebroventricular (i.c.v.) injection of BK inconscious, freely moving rats evoked dose-related increases inarterial pressure, and pretreatment with hemicholinium-3, whichdeplete brain acetylcholine levels, produced a choline-reversibleblockade of the cardiovascular response to BK (Buccafusco and Ser-ra, 1985). This notion has been extended further by Barnes et al.(1998), who demonstrated that BK-induced bronchoconstrictor re-sponse in asthmatics is dependent of cholinergic and sensorynerves (Barnes et al., 1998). However, no consistent evidence forthe linkage between KKS and the cholinergic system during neur-oinflammatory response, such as MS, has to date beendocumented.

Because kinin receptors are reported to be involved in the etiol-ogy of EAE (Dutra et al., 2011; Gobel et al., 2011; Schulze-Topphoffet al., 2009), and also directly modulate cognitive impairments inexperimental models of neurodegenerative disease, such as Alzhei-mer’s disease (Prediger et al., 2008), and some kinin-induced ef-fects depends of cholinergic and sensory nerve stimulation(Abraham et al., 2006), herein we hypothesize that the KKS mayplay a key role in modulating the development of cognitive impair-ments in MS by decreasing neuroinflammation and mitigating cho-linergic system dysfunction.

2. Materials and methods

2.1. Experimental animals

Experiments were conducted using female C57BL/6 wild-type,kinin B1R-knockout (B1R�/�), kinin B2R-knockout (B2R�/�) and ki-nin B1B2R-knockout (B1B2R�/�) mice (6–10 weeks old). Deletionof the entire coding sequences of the kinin receptors was achievedaccording to previously described methods (Dutra et al., 2011; Pes-quero et al., 2000). B1R�/�-, B2R�/�- and B1B2R�/�-deficient mice onthe 129/SvJ background were backcrossed to C57BL/6 to produceF10 offspring. Following the F10 offspring the line is 99% geneticallyidentical to the recipient strain (C57BL/6) and is considered con-genic with it; therefore we maintained this linage by crossing themwith each other. The C57BL/6 animals were used as controls. Themice were kept in groups of six to nine animals per cage, main-tained under controlled temperature (22 ± 1 �C), with a 12 hlight/dark cycle (lights on at 7:00 a.m.) and were given free accessto food and water. All procedures used in the present study fol-lowed the Guide for the Care and Use of Laboratory Animals (NIHpublication No. 85-23) and were approved by the Animal EthicsCommittee of the Universidade Federal de Santa Catarina (CEUA-UFSC, protocol number 23080038266/2008-43).

2.2. EAE induction

Experimental autoimmune encephalomyelitis (EAE) was in-duced by subcutaneous (s.c.) immunization into the flanks with200 ll of an emulsion containing 200 lg MOG35–55 peptide and500 lg Mycobacterium tuberculosis extract H37Ra in incompleteFreund’s adjuvant oil, as previously described (Stromnes and Gov-erman, 2006). In addition, the animals received 300 ng of Pertussistoxin intraperitoneally (i.p.) on day 0 and day 2 post-immuniza-tion. The animals were monitored daily and neurological impair-ment was quantified using a clinical scale after day 1 post-immunization (Stromnes and Goverman, 2006): 0, no signs of dis-ease; 1, loss of tone in the tail; 2, hindlimb paresis; 3, hindlimbparalysis; 4, tetraplegia and 5, moribund and/or death.

2.3. Behavioral tests

2.3.1. Rotarod testIn order to evaluate motor coordination, the mice were placed

on a rotarod apparatus at a fixed rotational speed of 4 rpm, as pre-viously described (Dutra et al., 2011) (Fig. 1A). The maximum timefor each trial was set at 60 s. Rotarod training was performed priorto disease induction and consisted of three consecutive trials inwhich the animals became familiar with the task. After diseaseinduction, the mice were repeatedly tested at different time pointsuntil day 25 post-immunization.

2.3.2. Object location taskAfter seven days of disease induction, the spatial memory of

mice was assessed using the object location task (Fig. 1A). The task

Fig. 1. Spatial reference memory deficits precede motor dysfunction during EAE. Animals were immunized with MOG35–55 peptide/CFA and Pertussis toxin. Experimentaldesign (A), motor coordination (B), clinical score (C), neurological state (D), novel object location memory (E and F) and Morris water maze (G and H) were analyzed in controland EAE group. Motor coordination was evaluated using the rotarod apparatus over 25 days post-immunization (p.i.). Neurological severity scale scoring was performed ninedays after EAE induction; a higher score indicates greater neurological impairment up to a maximum of 10 points. The object location test was performed seven days afterimmunization and the location index (%) assessed 90 min after training sessions in mice with or without EAE. The results of water maze test are expressed as latency to findthe hidden platform during the four training trials (G) or as a delta value for Trial 1 – Trial 2 (H). Data are presented as means ± SEM (B, C, E, F, G and H) ormedians ± interquartile range (D) of 6–9 mice per group and are representative of three independent experiments. ⁄p < 0.05 versus random chance (50% of displaced objectinvestigations in test trial), and ⁄⁄p < 0.001 versus control group (Student’s t-test – B, C and F; and repeated measures ANOVA – G).

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is based on the spontaneous tendency of rodents, previously ex-posed to two identical objects, to later explore one of the objects– replaced in a novel location – for a longer time than they explorethe non-displaced object (Assini et al., 2009), and has been used forthe evaluation of hippocampal-dependent memories (Assini et al.,2009). The experimental apparatus used in this study was an open-field box (50 cm wide � 50 cm deep � 40 cm high) made of trans-parent PVC, placed in a dimly lit (7 lx) and sound-isolated room.Identical plastic rectangles (4 cm high � 4.5 cm wide) were usedas objects. The mice were placed in the center of the apparatuswith two identical objects for 5 min. The objects were placed7 cm away from the walls of the open field. Exploration of the ob-

jects was recorded using a stopwatch when mice sniffed, whisked,or looked at the objects from no more than 1 cm away. After thetraining phase, the mice were removed from the apparatus for90 min. After this inter-trial interval, one object was moved to anew location. The time spent by the animals exploring the objectsin new (novel) and old (familiar) locations was recorded over5 min. All locations of the objects were counterbalanced amongthe groups. In order to analyze the cognitive performance, a loca-tion index was calculated as previously described (Assini et al.,2009): (Tnovel � 100)/(Tnovel + Tfamiliar), where Tnovel is thetime spent exploring the displaced object and Tfamiliar is the timespent exploring the non-displaced object.

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2.3.3. Water maze taskThe apparatus was made of black painted fiberglass

(97 � 60 � 60 cm), and the water was maintained at 23 ± 2 �C. Fourdistant cues (55 � 55 cm) were placed 30 cm above the upper edgeof the water tank. The target platform (10 � 10 cm), made of trans-parent acrylic resin was submerged 1–1.5 cm beneath the watersurface. Starting points were marked on the outside of the poolas north (N), south (S), east (E), and west (W). These were posi-tioned with the lower edge 30 cm above the upper edge of thewater tank, and the position of a symbol marked the midpointon the perimeter of a quadrant (circle = NE quadrant, square = SEquadrant, cross = SW quadrant, and diamond = NW quadrant). If amouse did not find the platform during the trial period of 60 s, itwas gently guided to it. The water maze test was evaluated from8–11 days after disease induction, and the animals were submittedto a working memory version of the water maze using a protocolidentical to that previously described (Moreira et al., 2012). Thereis compelling evidence that the prefrontal cortex plays a criticalrole in this kind of memory (Faw, 2003). Specifically, we used aprotocol that consists of four training days (four trials per day),during which the animals were placed in the tank facing the walland were then allowed to swim freely to the submerged platform.The initial position in which the animal was placed in the tank wasone of the four vertices of the imaginary quadrants of the tank, andthis was varied among trials in a pseudo-random way. The animalwas allowed to remain on the platform for 30 s and was thenmoved to the next initial position without leaving the tank. Thisprocedure was used to ensure that the animals maintained the vis-uospatial information of the maze that was available during execu-tion of the working memory task. On each subsequent training day,the platform position was moved to the center of another quadrantof the tank in a pseudo-random way. The experiments were video-taped and the scores for latency of escape from the starting point tothe platform and swimming speed were later measured throughthe ANY-maze™ video tracking system (Stoelting Co., Wood DaleIL, USA).

2.3.4. Neurological severity scoreAfter nine days of disease induction, mice were rated on the 10-

point neurological severity scale (NSS) (Fig. 1A), a compositebehavioral scale designed to measure the general neurologicalstate, as previously described (Schwarzbold et al., 2010). The micewere assessed for the following traits: presence of paresis; inabilityto walk straight; impairment of seeking behavior; absence of a per-ceptible startle reflex; inability to exit a 30-cm-diameter circle;inability to walk on 3-, 2-, and 1-cm-wide beams; and inabilityto balance on a 0.7-cm-wide beam and a 0.5-cm-diameter roundbeam for at least 10 s. If a mouse showed impairment in one ofthese traits, a value of 1 was added to its NSS score. Higher scoreson the NSS thus indicate greater neurological impairment.

2.4. RNA extraction and real-time quantitative PCR

Total RNA from the hippocampus and prefrontal cortex (11 dayspost-immunization, Fig. 1A) was extracted using the Trizol proto-col and concentrations were determined using a NanoDrop 1100(NanoDrop Technologies, Wilmington, DE, USA). An amount of100 ng of total RNA was used for cDNA synthesis. Reverse tran-scription was performed as described in the M-MLV Reverse Trans-criptase protocol according to the manufacturer’s instructions.cDNA was amplified in duplicate using the TaqMan� UniversalPCR Master Mix Kit with specific TaqMan Gene Expression targetgenes, the 30 quencher MGB and FAM-labeled probes for mousecholine acetyltransferase (ChAT, Mm01221887_m1) and acetyl-cholinesterase (AChE, Mm00477275_m1), interleukin-17 (IL-17,Mm00439618_m1), interferon-gamma (IFN-c,

Mm99999071_m1), kinin B1R (Mm00432059_s1) and kinin B2R(Mm01339907_m1). GAPDH (NM_008084.2) was used as anendogenous control for normalization. The PCR reactions were per-formed in a 96-well Optical Reaction Plate (Applied Biosystems,Foster City, CA, USA). The thermocycler parameters were as fol-lows: 50 �C for 2 min, 95 �C for 10 min, 50 cycles of 95 �C for 15 sand 60 �C for 1 min. Expression of the target genes was calibratedagainst conditions found in control animals or cells (i.e. naïve orwild-type (WT) mice).

2.5. Western blot analysis

The hippocampus and prefrontal cortex tissues were collectedfive days after EAE induction and homogenized in complete radioimmunoprecipitation lysis buffer (RIPA). Equal amounts of proteinfor each sample (30 lg) were loaded per lane and electrophoreti-cally separated using 10% denaturing polyacrylamide gel electro-phoresis (SDS–PAGE). After that, the proteins were transferred tonitrocellulose membranes using a Mini Trans-Blot Cell System(Bio-Rad Laboratories Inc., Hercules, CA, USA). Western blot analy-sis was carried out using polyclonal rabbit anti-B1R (1:1000) (Alo-mone Labs, Jerusalem, Israel) incubated overnight. After washing,membrane was incubated with secondary antibodies conjugatedto horseradish peroxidase (1:25,000, Cell Signaling Technology,Danvers, MA, USA). The immunocomplexes were visualized usingSuperSignal West Femto Chemiluminescent Substrate detectionsystem (Thermo Fischer Scientific, Rockford, IL, USA) and densito-metric values were normalized using monoclonal mouse b-actinantibody (1:500, Cell Signaling Technology, Danvers, MA, USA).

2.6. Drugs and reagents

Pertussis toxin, phosphate buffered saline (PBS) and incompleteFreund’s adjuvant oil were all purchased from Sigma Chemical Co.(St. Louis, MO, USA). The MOG35–55 peptide (MEVGWYRSPFSRVVH-LYRNGK) was obtained from EZBiolab (Carmel, IN, USA) and the M.tuberculosis extract H37Ra from Difco Laboratories (Detroit, MI,USA). The primers and probes for mouse ChAT, AChE, IL-17, IFN-c, B1R, B2R and GAPDH were purchased from Applied Biosystems(Warrington, UK). The polyclonal rabbit anti-B1R was purchasedfrom Alomone Labs (Jerusalem, Israel). Other reagents used wereall of analytical grade and obtained from different commercialsources.

2.7. Statistical analysis

Data are presented as means ± SEM. The object location taskwas analyzed by one-sample t-test to determine whether the loca-tion index was different from random chance (50%). Other statisti-cal analyses were carried out by using a Student’s t-test or by one-way analysis of variance (ANOVA), with repeated measures whenappropriate. Following significant ANOVAs, multiple post-hoccomparisons were performed using Bonferroni or Newman–Keulstests, depending on the experimental protocol. p-Values of lessthan 0.05 (p < 0.05) were considered significant. The statisticalanalyses were performed using GraphPad Prism 4 software(GraphPad Software Inc., San Diego, CA, USA).

3. Results

3.1. Cognitive deficits associated with cholinergic dysfunction precedemotor impairments in EAE

As previously described (Dutra et al., 2011), C57BL/6 miceimmunized with MOG35–55 antigen developed EAE starting 13 days

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following immunization, and the clinical severity increased tomaximum by day 20 (Fig. 1B and C). However, recent reportshowed that 50–70% of MS patients have some cognitive deficitsduring the course of the disease (Rao, 1995; Rao et al., 1991; Shiet al., 2008). Thus, here we assessed whether learning and memoryimpairments could onset during the pre-motor phase of EAE (days0–11 post-immunization, Fig. 1A). Firstly, we evaluated short-termspatial memory of mice using an object location task. One weekafter EAE induction, object location memory performance was sig-nificantly impaired, as no difference between the time spentexploring displaced and non-displaced objects could be observed(t = 1.43; p = 0.20; Fig. 1F). In contrast, the control group, treatedwith saline one week before behavioral testing, explored the objectreplaced in a novel location for a longer time than the non-dis-placed object, as indicated by a significant increase in location in-dex in comparison to random chance (t = 9.88; p < 0.0005; Fig. 1F).It should be noted that no innate preference for object position wasexhibited in either control (t = 0.64; p = 0.55) or EAE (t = 0.38;p = 0.38) mice, as indicated by the similar amounts of time spentsniffing both objects during the 5-min familiarization (training)session (Fig. 1E). Moreover, at this time, the animals (regardlessof group) did not present any gross motor alterations, as assessedby rotarod test (Fig. 1B), clinical score (Fig. 1C) and neurologicalseverity scores (Fig. 1D).

Next, we assessed the spatial working and reference memory ofmice over days 8–11 post-immunization using the water mazetask. The performance of the EAE-group in the water maze testwas significantly impaired compared with control animals, as illus-trated by the mean latency to escape to the hidden platform(Fig. 1G). The latency to escape to the hidden platform decreasedover the training trials for both groups (main effect of repetition,

Fig. 2. EAE-induced spatial reference memory deficits associated with cholinergic regulatpeptide/CFA and Pertussis toxin. On days 5 and 11 post-immunization, total RNA was extrEAE groups and processed for choline acetyltransferase (ChAT) (A, B, E and F) and acetylthe relative amounts of mRNA. Data are presented as means ± SEM of 6–9 mice per grouimmunized control group (Student’s t-test).

F(3, 174) = 11.19, p < 0.00005), however, treatment differenceswere observed (main effect of EAE, F(1, 58) = 13.32, p < 0.0005).Specifically, escape latencies significantly differed across treat-ments on training trials 3 and 4 (p < 0.05; Fig. 1G), indicating spa-tial reference memory impairments. Moreover, we calculated adelta value by subtracting the value for trial one from trial two,as an index of working memory. As showed in Fig. 1H we observedno differences between groups (p = 0.86), indicating that EAEimmunization did not disrupted working memory of mice. It isworth mentioning that the learning and memory deficits wereindependent from motor deficits as mice from each group had sim-ilar swimming speeds (data not shown).

There is extensive literature indicating that specific neuralstructures such as the hippocampus and prefrontal cortex plays acritical roles in different types of memory such as spatial reference(Burgess et al., 2002) and working memory (Faw, 2003; Wanget al., 2011), respectively. Moreover, the close relationship be-tween the disruption of the cholinergic system and cognitive defi-cits, such as working (Croxson et al., 2011) and spatial referencememories impairments (Deiana et al., 2011) is well established(Van Beek and Claassen, 2011). Herein, in order to evaluate thepossible molecular mechanisms underlying cognitive deficits in-duced by EAE model, we next investigate whether the spatial ref-erence memory impairments were also linked to down-regulation of cholinergic basal forebrain system. Thus, we mea-sured ChAT mRNA expression in the hippocampus and prefrontalcortex during the motor pre-symptomatic phase of EAE. At 11 dayspost-immunization, the expression of ChAT mRNA was markedlyreduced in the hippocampus (Fig. 2A) and prefrontal cortex(Fig. 2B) of EAE mice, which might be the underlying cause ofthe spatial reference deficits. Accordingly to previous reports (Tu

ion during the pre-motor phase of disease. Animals were immunized with MOG35–55

acted from the hippocampus and prefrontal cortex of non-immunized (control) andcholinesterase (AChE) (C and D), respectively. GAPDH mRNA was used to normalize

p and are representative of two independent experiments. ⁄⁄p < 0.001 versus non-

Fig. 3. EAE-induced up-regulation of B1R mRNA and protein levels in the hippocampus and prefrontal cortex during the pre-motor phase of disease. Animals were immunizedwith MOG35–55 peptide/CFA and Pertussis toxin. On days 5 and 11 post-immunization, total mRNA was extracted from the hippocampus and prefrontal cortex of non-immunized (control) and EAE groups. The mRNA levels of B1R (A and B) and B2R (C and D) were measured by RT-PCR, on day 11 post-EAE induction. The housekeeping geneGAPDH mRNA was used to normalize the relative amounts of mRNA. The protein expression levels of B1R (E and F), in the hippocampus and prefrontal cortex, was determinedby Western blot analysis on day 5 post-immunization. The level of protein was expressed as ratio of signal intensity for the target protein relative to that for b-actin, and theirvalues was considered arbitrary units that represented the relative values among all samples. Data are presented as means ± SEM of 6–9 mice per group and arerepresentative of two independent experiments. #p < 0.05 and ⁄⁄p < 0.001 versus non-immunized control group (Student’s t-test).

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et al., 2009), the expression of AChE mRNA (the key enzymeresponsible for acetylcholine degradation) remained unaltered inthe hippocampus and prefrontal cortex after EAE induction(Fig. 2C and D). In another set of experiments, we investigatedwhether ChAT mRNA is down-regulated in the hippocampus andprefrontal cortex in the earlier phase of EAE. Interestingly, on the5th day after EAE, the expression of ChAT mRNA was significantlyreduced in the hippocampus (Fig. 2E) and prefrontal cortex(Fig. 2F), when compared to the control group, suggesting that cho-linergic down-regulation occur in the CNS before memory deficits.

3.2. B1R is over-expressed in the hippocampus during the pre-motorphase of EAE

Recent evidence points to the involvement of the KKS in MS andEAE models (Dos Santos et al., 2008; Dutra et al., 2011; Gobel et al.,2011; Schulze-Topphoff et al., 2009). Thus, we next investigatewhether the spatial reference memory impairments seen herewere also linked to the up-regulation of kinin B1 and B2 receptorsin specific brain areas during the motor pre-symptomatic phaseof EAE (on day 11 post-immunization). Basal expression of B1R(Fig. 3A and B) and B2R (Fig. 3C and D) were detected in the hippo-campus and prefrontal cortex of non-immunized mice. On theother hand, during pre-motor phase of EAE, levels of kinin B1Rtranscript were increased a hundred-fold in the hippocampus

(Fig. 3A), but not in the prefrontal cortex (Fig. 3B). Moreover, nosignificant difference in kinin B2R mRNA levels was observed be-tween non-immunized and EAE-groups in the hippocampus andprefrontal cortex, respectively (Fig. 3C and D). Next, we investi-gated whether B1R protein levels was up-regulated before spatialreference memory impairments induced by EAE. As shown inFig. 3, only low protein levels of B1R were detected in the non-immunized mouse hippocampus (Fig. 3E) and prefrontal cortex(Fig. 3F) tissues, respectively, but these values were markedly in-creased in EAE-group at 5 days post-immunization, suggesting thatKKS may contribute to the appearance of cognitive deficits inducedby EAE during pre-motor phase.

3.3. Genetic deletion of kinin B1R prevents cognitive impairments andcholinergic dysfunction in EAE

Accordingly, the genetic deletion of B1R (Fig. 4A) drastically re-duced the clinical score of EAE and blocked the development of thedisease (Fig. 4B and C). In marked contrast, for the genetic deletionof B2R, or in mice that lack the genes encoding both kinin B1R andB2R (B1B2R�/�) no differences were found when compared to thewild-type EAE-group (Fig. 4B and C). This indicates that B1R has adominant role in influencing the development of the disease, aspreviously demonstrated (Dutra et al., 2011). Presently, there isevidence indicating that kinin receptors exert a critical role in

Fig. 4. Kinin B1R deletion prevented spatial reference memory impairments during the pre-motor phase of EAE. Animals were immunized with MOG35–55 peptide/CFA andPertussis toxin. Experimental design (A), motor coordination (rotarod test) (B), clinical score (C) and water maze test (D and E) were analyzed in the C57BL/6 non-immunizedgroup, EAE group, and in B1R�/� knockout, B2R�/� knockout and B1B2R�/� knockout mice. The motor coordination (B) and clinical score (C) were evaluated for 25 days post-immunization (p.i.), whereas water maze test (D and E) was performed during days 8–11 p.i. (pre-motor phase of EAE). The results of water maze test are expressed as latencyto find the hidden platform during the four training trials (D) or as the delta value for Trial 1 – Trial 2 (E). The data are presented as means ± SEM of 6–9 mice/group and arerepresentative of two independent experiments. #p < 0.05, ⁄p < 0.05 and ⁄⁄p < 0.001 versus EAE-control group (one-way ANOVA with Newman–Keuls post hoc test). NS = notsignificant.

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the spatial learning and memory deficits observed with aging andin experimental models of Alzheimer’s disease (Lemos et al., 2010;Prediger et al., 2008; Wang and Wang, 2002). Thus, in another setof experiments, we investigated whether the genetic deletion of ki-nin receptors could prevent the spatial reference memory deficitsinduced by EAE in mice. In previous studies, we performed ade-quate inter-strain comparisons between wild-type and B1R�/�,B2R�/� and B1B2R�/�mice, and these findings indicated that the ge-netic deletion of both B1R and B2R did not significantly disrupt spa-tial learning and memory processes in mice evaluated in the watermaze task (Prediger et al., 2008). Herein we showed that geneticdeletion of B1R significantly mitigated the spatial reference mem-

ory deficits of EAE mice in the water maze task, since the escapelatencies significantly differed when compared with C57BL/6EAE-induced group on training trials 3 and 4 (p < 0.05; Fig. 4D).On the other hand, genetic deletion of B2R alone or both B1R andB2R (B1B2R�/�) did not significantly prevent the cognitive impair-ments induced by EAE in mice (Fig. 4D). Moreover, one-way ANO-VA indicated no differences between groups regarding the deltavalue of Trial 1 – Trial 2 (F(4, 130) = 0,85, p = 0,49; Fig. 4E), rein-forcing the suggestion of the absence of working memoryalterations.

We next investigated whether B1R could modulate spatial refer-ence memory by affecting the expression levels of ChAT mRNA in

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the hippocampus and prefrontal cortex during the pre-motorphase of EAE. As mentioned previously, in the EAE-group, a signif-icant decrease in the expression of ChAT mRNA was observed inthe hippocampus (Fig. 5A) and prefrontal cortex (Fig. 5B). Notably,in B1R�/� mice the down-regulation of ChAT was completely abol-ished in the hippocampus and prefrontal cortex of mice during pre-motor phase of EAE, respectively (Fig. 5A and B). However, the ge-netic deletion of B2R failed to inhibit the decrease in ChAT mRNA inthe hippocampus (Fig. 5A) and prefrontal cortex (Fig. 5B) after EAEinduction. Moreover, in B1B2R�/� mice this effect was only ob-served in the hippocampus (Fig. 5A). Once again, the levels of AChEmRNA in the hippocampus (Fig. 5C) and prefrontal cortex (Fig. 5D)remained unchanged after EAE induction in all groups.

3.4. Genetic deletion of B1R decreases the inflammatory response in thecerebral and peripheral tissues during the pre-motor phase of EAE

Earlier studies have shown that autoaggressive Th17 and Th1cells, directed against antigens that are derived from the CNS,can induce tissue damage and neuroinflammation, and thus havea crucial role during MS development (Moldovan et al., 2003;Stromnes et al., 2008; Tzartos et al., 2008). It is now well-estab-lished that different proinflammatory mediators are produced bothby infiltrating immune cells and by CNS-resident glial cells, mainlyastrocytes and microglial cells, orchestrating a pathogenic cascadeleading to neuroinflammation and axonal damage during MS (Ger-ard and Rollins, 2001; Ibrahim et al., 2001; Lock et al., 2002). Wetherefore investigated whether B1R could modulate spatial refer-ence memory by affecting CNS inflammation during the pre-motorphase of EAE. In the EAE-group, significant increases in the expres-sion of IL-17 (Fig. 6A and C) and IFN-c (Fig. 6B and D) mRNAs wereobserved in the hippocampus and prefrontal cortex, respectively.Surprisingly, in both B1R�/� and B1B2R�/� mice, the up-regulationof IL-17 (Fig. 6C) and IFN-c (Fig. 6D) was completely abolished inthe cerebral prefrontal cortex at 11 days post-immunization, butthis effect was not observed in the hippocampus (Fig. 6A and B).

Fig. 5. Kinin B1R deletion blocked ChAT mRNA levels down-regulation in the hippocampday, brain tissues were collected and processed for choline acetyltransferase (ChAT) andhippocampus and prefrontal cortexes of mice in the C57BL/6 non-immunized group, theChAT (A and B) and AChE (C and D) were measured in the hippocampus and prefrontalmRNA. The data are presented as means ± SEM of 6–9 mice/group and are representativegroup and ⁄p < 0.05 versus EAE-control group (one-way ANOVA with Newman–Keuls po

Furthermore, the deletion of B2R did not give rise to significant de-creases in IL-17 and IFN-c expression in either the hippocampus orprefrontal cortex when assessed during pre-symptomatic phase ofEAE (Fig. 6A–D). In another set of experiments, we investigatedwhether IL-17 and IFN-c mRNA are up-regulated in the peripherallymphoid tissue, such as lymph node and spleen, after EAE induc-tion. As shown in Fig. 6(E–H), in EAE-group, a significant up-regu-lation of IL-17 (Fig. 6E and G) and IFN-c (Fig. 6F and H) mRNAs wasobserved in the lymph node and spleen, respectively. Of great rel-evance, genetic deletion of B1R resulted in a marked reduction ofIL-17 and IFN-c mRNA in the lymph node (Fig. 6E and F) and spleen(Fig. 6G and H), respectively, at 11 days post-immunization, sug-gesting that B1R modulates inflammatory response in the cerebraland peripheral tissues during the pre-motor phase of EAE.

4. Discussion

Recent evidence has shown that the neuronal compartment ofthe CNS is affected in parallel to, and even independently of, whitematter damage in MS (Centonze et al., 2010; Steinman, 2001).Accordingly, cognitive impairment occurs in up to 65% of peoplewith MS (Rao, 1995; Rao et al., 1991; Shi et al., 2008). Althoughthere is evidence that memory impairment among MS subjectsare a consequence of inadequate initial learning and not a functionof impaired retrieval (DeLuca et al., 1994), the majority studies re-port that most patients have some difficulty in remembering infor-mation learned in the past (Beatty et al., 1996; Brissart et al., 2012).For instance, verbal episodic memory impairment is frequent inpatients with MS (Brissart et al., 2012; Rao et al., 1991). Episodicmemory refers to the conscious recollection of a unique past expe-rience in terms of ‘‘what’’ happened, ‘‘where’’ and ‘‘when’’ it hap-pened (Tulving, 1983, 2007). In this regard, herein we showed animpaired object location memory in the EAE-induced group. Theobject location task allows to measure the memory for locations,where objects were initially explored, by presenting two equaland familiar objects (‘‘what’’) during the test trial, with one of

us and prefrontal cortex during the pre-motor phase of EAE. At the end of the 11thacetylcholinesterase (AChE) mRNA expression. Total RNA was extracted from the

EAE group, and B1R�/�, B2R�/� and B1B2R�/� mice on day 11 p.i. The mRNA levels ofcortex, respectively. GAPDH mRNA was used to normalize the relative amounts ofof two independent experiments. #p < 0.05 and ##p < 0.001 versus wild-type naïve

st hoc test).

Fig. 6. Genetic deletion of B1R inhibits mRNA expression of autoimmune cytokines in the cerebral and peripheral tissues during the pre-motor phase of EAE. At the end of the11th day, hippocampus, prefrontal cortex, inguinal lymph node and spleen tissues were collected and processed for IL-17 and IFN-c mRNA expression. Total RNA wasextracted from the hippocampus, prefrontal cortex, inguinal lymph node and spleen of mice in the non-immunized group, the EAE group, B1R�/�, B2R�/� and B1B2R�/�mice onday 11 p.i. The mRNA levels of IL-17 (A, C, E and G) and IFN-c (B, D, F and H) were measured in the hippocampus, prefrontal cortex, inguinal lymph node and spleenrespectively. GAPDH mRNA was used to normalize the relative amounts of mRNA. The data are presented as means ± SEM of 6–9 mice/group and are representative of twoindependent experiments. #p < 0.05 versus non-immunized group, ⁄p < 0.05 versus EAE-control group (one-way ANOVA with Newman–Keuls post hoc test).

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the objects shifted to a novel location (‘‘where’’). Although twocomponents of an episode experienced by animals were assessedconjointly, it is worth mentioning that it does not make up an epi-sodic memory test (Ennaceur, 2010), nevertheless, at least in part,we corroborated clinical data linking episodic memory impairmentwith MS. Afterwards, we showed that the performance of the EAE-group in the Water maze task was significantly impaired whencompared with control mice. Morris water maze performance in-volves different components, including concept formation (learn-ing the general rules of the task), attention, working memory,and reference memory (Anisman and McIntyre, 2002). Our exper-imental protocol allowed distinguishing between working and ref-erence memories. In this regard, we presented evidence for specificreference memory impairments in the EAE group. Particularly, ver-bal and spatial learning are affected in MS subjects, reflecting hip-pocampal dysfunction (Sicotte et al., 2008).

Accumulated evidence now suggests that in EAE and MS pathol-ogy, T cell activation in the periphery against myelin antigens, andtheir subsequent invasion of the CNS, are considered as the primummovens of both the experimental and human diseases (Sospedraand Martin, 2005). These T cells are capable of (1) producinginflammatory cytokines that may lead to differentiation into Th1(IFN-c and TNF-a) or Th17 cells (IL-17, IL-21 and IL-22) (McFarlandand Martin, 2007), and (2) up-regulating integrins such as VLA-4,that have the ability to enter the subarachnoid space by crossingthe blood-cerebrospinal fluid (CSF) barrier in either the choroidplexus or the meningeal venules (Goverman, 2009; Steinman,2007). Through the permeabilized BBB, and attracted by chemo-kine release, other immune cells including CD8+ T cells, B cellsand monocytes/macrophages migrate into the CNS (Fletcheret al., 2010). Moreover, inside the CNS, the T cells are re-activatedby MHC class II-expressing macrophage/microglia and dendriticcells (DCs), such as astrocytes, which express myelin epitopes(Goverman, 2009). Despite the low abundance of DCs in these com-partments, experiments that limited MHC class II expression toDCs showed that these cells were sufficient to support the develop-

ment of EAE (Greter et al., 2005), and that myeloid DCs within theCNS activated naïve myelin-specific T cells, which were recruitedto the inflamed tissue, and facilitated their differentiation intoTh17 cells (Bailey et al., 2007).

In this context, several lines of evidence suggest that T-cell CNSinvasion might be considered as the first step in the chain of eventsleading to brain inflammation and the secondary neurodegenera-tion that occurs in MS patients (Centonze et al., 2010; Steinman,2007). However, only a limited number of studies have been de-voted to understanding the precise mechanisms underlying the on-set of cognitive deficits in this scenario. Here, we demonstratedthat not only the white matter, but also gray matter structuressuch as the hippocampus and prefrontal cortex, are infiltrated byautoaggressive Th17 and Th1 cells during the early phase of EAE.In agreement with our data, it was recently found that inflamma-tory infiltrates in the striatum and the cortex are mainly composedof CD3+ T cells during the pre-symptomatic and symptomaticphases of EAE (Centonze et al., 2010; Steinman, 2007). Moreover,a relevant study demonstrated learning and memory deficits fol-lowing recovery from motor symptoms (90 days post-immuniza-tion) which were associated with decreases in ChAT activity andnerve growth factor (NGF) mRNA levels in the supraspinal struc-tures (D’Intino et al., 2005). This has been extended by Sicotteet al. (2008), who demonstrated that MS patients and EAE miceshowed hippocampal neurodegeneration, selective decrease inCA1 volume and loss of GABA-ergic interneurons. Nonetheless,these cognitive deficits were evaluated at a later stage of the dis-ease. Moreover, the relationship between the cholinergic systemand cognitive impairments is well established (Field et al., 2012;Ruan et al., 2010; Ullrich et al., 2010). For instance, modulationof prefrontal and hippocampal function by forebrain cholinergicsystems plays a critical role in the performance of spatial memorytasks in rodents, since both the prefrontal cortex and hippocampusreceive substantial innervation from cholinergic cell bodies locatedin the basal forebrain. Indeed, loss of such cholinergic neurons hasbeen correlated with memory deficits in a number of neurodegen-

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erative and inflammatory disorders (Mufson et al., 2002; Pizzoet al., 2002). Our data confirm and largely extend these previousdata, by demonstrating that EAE induces spatial reference memoryimpairments during the pre-symptomatic phase of disease, andthat this is associated with ChAT down-regulation in the hippo-campus and prefrontal cortex.

In subsequent phases, autoaggressive T cells enter the paren-chyma and, together with activated macrophage/microglial cellsand astrocytes, over-express pro-inflammatory cytokines and ni-tric oxide (NO) that trigger demyelination, consequently activatingneighboring immune or neural cells, and attracting further inflam-matory cells into the CNS (Goverman, 2009). Emerging evidencesuggests that neuroinflammation could be regulated by neuropep-tides, such as kinins (Dutra et al., 2011; Prediger et al., 2008; Schu-lze-Topphoff et al., 2009). In fact, there are, so far, several reasonsfor pointing B1R kinin as a potential therapeutic approach for themanagement of inflammatory diseases, such as MS. Initially, theinduction of B1R has been associated with the production of pro-inflammatory mediators, stimulation of inflammatory cells, and, fi-nally, activation of several intracellular signaling pathways (Calixtoet al., 2004). Moreover, high levels of kinin components, namelydes-Arg9-bradykinin (DABK, selective agonist for B1R), bradykinin,kallikrein-1, kallikrein-6 and low-molecular-weight kininogens(KNGL), have been found in CNS tissue and cerebrospinal fluid fromMS patients (Schulze-Topphoff et al., 2009). In addition, the B1Rhas been found to be up-regulated not only in brain endothelialcells (Prat et al., 2000), but also in the peripheral T lymphocytes(Prat et al., 2005) of these patients. More recently, our group andothers have reported that B1R exert a critical role in regulatingthe development and progression of EAE, as well as is up-regulate

Fig. 7. Potential mechanism by which B1R controls activation of CD4+ T cells into the CNduring EAE. CD4+ T lymphocytes are primed in the peripheral lymphoid organs by dendrinto CNS by crossing the blood–brain barrier (BBB), mainly, in the choroid plexus (2),expressing myelin epitopes (3). Finally, reactivated T cells, astrocytes and microglial celdemyelination (5), dead of cholinergic neurons (6), and consequently cognitive deficits.which is directly associated with a decrease in ChAT expression in the brain, and apautoaggressive Th17 and Th1 cells to activate and release pro-inflammatory cytokines indamage and cognitive impairments. Altogether, the modulation of this receptor may rephelper lymphocyte; DABK: des-Arg9-BK; MOG: myelin oligodendrocytes glycoprotein;histocompatibility complex; CXCL1/KC: keratinocyte-derived chemokine, TNF-a: tumcyclooxygenase-2; IFN-c: interferon-gamma.

in MS patients (Dos Santos et al., 2008; Dutra et al., 2011; Gobelet al., 2011; Prat et al., 2005; Schulze-Topphoff et al., 2009), sug-gesting that B1R inhibition may offer a novel anti-inflammatorystrategy for the treatment of MS. Moreover, a report suggest thatBK can modulate the cholinergic system, mainly in the airways(Barnes et al., 1998). Herein, we demonstrated that deletion ofB1R significantly prevented spatial reference memory impair-ments, reduction of ChAT mRNA levels in the hippocampus andprefrontal cortex, and blocked the increase of IL-17 and IFN-cmRNAs induced by EAE in the prefrontal cortex, lymph node andspleen.

Although B2R is commonly classified as a mediator of acuteinflammatory responses, while B1R is thought to be involved inchronic inflammation (Costa-Neto et al., 2008), one cannot ruleout short-term and acute effects for B1R. The induction of B1R isassociated with several processes, including: (i) production ofinflammatory mediators, such as IL-1b, TNF-a, IL-2, and growthfactors; (ii) stimulation of inflammatory cells; and (iii) activationof several intracellular signaling pathways, including NF-jB activa-tion (Santos et al., 1999). In addition, selective NF-jB blockers havebeen shown to be able to prevent B1R up-regulation, both in vitro(Sabourin et al., 2002) and in vivo (Passos et al., 2004). Therefore,in cases of acute inflammation, over-production of pro-inflamma-tory cytokines may sustain the presence of active B2R as well asB1R. These data clearly demonstrate the relevance of different clas-ses of proteins in the modulation of B1R up-regulation, and supportthe short-term effects of B1R. We have also reported that up-regu-lation of B1R in the hippocampus and prefrontal cortex, seems di-rectly regulates neuroinflammatory responses and cognitiveimpairments in the motor pre-symptomatic phase of EAE. Thus,

S, leading to neuroinflammation, loss of cholinergic neurons and cognitive deficitsitic cells (DCs) presenting myelin (MOG35–55) epitopes (1). After, CD4+ T cells enterand within the subarachnoid space, Th17 and Th1 cells are re-activated by APCs

ls (Dutra et al., 2013) (4) secrete neuroinflammatory soluble mediators that triggerSpatial reference memory impairments occur before the onset of motor symptoms,pear to be mediated by kinin B1R. Moreover, activation of B1R could stimulatethe peripheral tissue, as well as within the CNS, consequently inducing cholinergic

resent a novel therapeutic approach for MS. APC: antigen-presenting cell; Th cell: TB1R: kinin B1 receptor; B2R: kinin B2 receptor; TCR: T cell receptor; MHC: majoror necrosis factor-alpha; IL: interleukin; NOS2: nitric oxide synthase 2; COX-2:

100 R.C. Dutra et al. / Brain, Behavior, and Immunity 33 (2013) 90–101

one could suppose that, after myelin antigen presentation to naïvelymphocytes T CD4+, occurs B1R up-regulation, an effect that seemsassociate with differentiation into Th1 and Th17 cells, which mi-grate and accumulate within the gray/white matter structures,leading to neuroinflammation and consequently the loss of cholin-ergic neurons that is responsible for the onset of cognitive impair-ments (see scheme proposed in Fig. 7).

Understanding of the mechanisms leading to cumulative cogni-tive deficits in patients with MS, and the further development ofeffective therapeutic strategies aimed at reducing disease progres-sion, are major goals in MS research. Therefore, the characteriza-tion of early symptoms, plus identification of peptides/receptorsassociated with neuroinflammation, during the pre-symptomaticphase of MS, could give rise to temporal markers of the diseaseas well as open up fresh perspectives in the development of ther-apeutic approaches for MS. In summary, we described that spatialreference memory impairments occur before the onset of motorsymptoms, and these effects are directly associated with a decreasein ChAT expression in the brain areas (hippocampus and prefrontalcortex) involved in acetylcholine control of memory acquisitionand maintenance (Fig. 7). Our data also show that the cognitivedeficits, and cholinergic down-regulation, that appear in the miceprior to any overt motor deficits are strongly mediated by kininreceptors, mainly of the B1R subtype. Moreover, we demonstratedthat the mechanisms involved in kinin receptor-mediated cogni-tive impairment during the pre-motor phase of EAE appear to beprimarily associated with the ability of these peptides to stimulateautoaggressive Th17 and Th1 cell activation, proliferation andmigration to the brain (Dutra et al., 2011), release pro-inflamma-tory cytokines within the CNS, and consequently induced choliner-gic damage and cognitive impairments (Fig. 7). Therefore, themodulation of this receptor may represent a novel therapeutic ap-proach for the management of MS.

Acknowledgments

This work was supported by grants from the Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq), the Coorde-nação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),the Programa de Apoio aos Núcleos de Excelência (PRONEX), andthe Fundação de Apoio a Pesquisa do Estado de Santa Catarina(FAPESC), all of Brazil. The authors would like to thanks AllissonFreire Bento for help in schematic figure.

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