Improved spatial learning and memory by perilla diet is correlated with immunoreactivities to neurofilament and α-synuclein in hilus of dentate gyrus

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1 Lee et l. Proteome Science 212, 1:72 RESEARH Open Access Improved sptil lerning nd memory y perill diet is correlted with immunorectivities to neurofilment nd α-synuclein in hilus of dentte gyrus Jinwoo Lee, Sunmin Prk, Ju-Young Lee, Yeong Keun Yeo, Jong Sng Kim nd Jinkyu Lim Astrct Bckground: Perill (Perill frutescens) oil is very rich in α-linolenic cid, n omeg-3 ftty cid. As it is widely reported tht omeg-3 ftty cid supplementtion improves cognitive function in children nd dults, feeding rts with perill diets followed y nlysis of proteomic chnges in the hippocmpus cn provide vlule informtion on the mechnism of lerning nd memory t the moleculr level. To identify proteins plying roles in lerning nd memory, differentilly expressed proteins in the hippocmpus of the 5 week old rts fed perill diets for 3 weeks or 3 months were identified y proteomic nlysis nd vlidted y immunologicl ssys. Results: The perill diet groups showed improved sptil lerning nd memory performnces in T-mze test. They lso displyed elevted level of 22:6n-3 ftty cid, n omeg-3 ftty cid (p<.5), in the rin compred to the control diet group. Quntittive proteomic nlysis using 2-D gels s well s functionl nnottion grouping with the differentilly expressed proteins in the hippocmpus showed tht those proteins involved in cytoskeleton nd trnsport were the mjor differentilly expressed proteins in the 3-week group, wheres those involved in energy metolism, neuron projection nd poptosis in ddition to cytoskeleton nd trnsport were the mjor ones in the 3 month group. Differentil protein expression in the hippocmpus ws vlidted y Western lotting using four selected proteins, known to e involved in synptic plsticity; AMPA receptor, neurofilment, α-synuclein, nd β-solule NSF ttchment protein. Brin sections from the perill-diet groups showed enhnced immunorectivities to α-synuclein nd neurofilment. Especilly, neurofilment immunorective cells mnifested longer neurite projections in the hilus of dentte gyrus of the perill-diet groups. onclusion: Improved cognitive function upon dministrtion of n-3 ftty cid-rich perill diet is ssocited with the differentil expression of hippocmpl proteins relted to cytoskeleton, energy metolism, trnsport, neuro-projection, nd poptosis. Prticulrly, the enhnced immunorectivities to α-synuclein nd neurofilment in the hilus of dentte gyrus suggest tht perill diet supplementtion promotes neuronl signling nd lters synptic plsticity for improved lerning nd memory. Keywords: Perill, n-3 ftty cid, ognition, Hippocmpus, Proteome, 2-D gel, Immunohistochemistry orrespondence: jkylim@knu.c.kr Equl contriutors Mjor in Food Biomterils, Kyungpook Ntionl University, Degu 72-71, South Kore 212 Lee et l.; licensee BioMed entrl Ltd. This is n Open Access rticle distriuted under the terms of the retive ommons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Lee et l. Proteome Science 212, 1:72 Pge 2 of 12 Bckground Memory is the process of storing informtion from lerning, which cquires informtion out experiences over time. For long term memory genertion, protein synthesis is required in the rin [1]. Further trnsient multiple protein expression s well s constitutive prolonged locl increse in protein synthesis in the rin re influenced y lerning [2]. Newly synthesized proteins re involved in the growth of new synpses, including oth pre- nd post-synptic ltertions [3]. As long term memory nd synptic plsticity require new protein synthesis, it hs een reported tht long term chnges in neurl circuits s well s long term modifiction of ehvior cn e regulted y trnsltionl control [2,4]. Mny reports hve shown tht foods cn ffect the cognitive processes in the centrl nervous system, such s, cognitive impirment y Western diet consumption [5], potentil efficcy of cognitive nd psychologicl functioning y vitmins [6], protection of vulnerle neurons y flvonoids from fruits nd erries [7], reducing the chnces of developing cognitive impirment y omeg-3 polyunsturted ftty cid (n-3 PUFA) [8], etc. It hs een known tht dietry omeg-3 ftty cids improve memory nd lerning processes, nd lso ffect gene expression in the rin [9-12]. Previous studies hve shown tht PUFA-enriched diets significntly lter the mrna expression levels of severl genes in centrl nervous tissue, nd these effects might e relted to the (n-3)/(n-6) lnce of polyenoic ftty cids in the cell memrne. Modern diets enriched in sturted ftty cids nd simple crohydrtes re often deficient in omeg-3 ftty cids, which re essentil ftty cids nd must e otined through dietry sources. Perill (P. frutescens) is good source of omeg-3 ftty cids (over 5% totl ftty cid) [13] which re essentil ftty cids tht cn e converted to docoshexnoic cid (DHA) in the liver nd the developing rin, nd contins one of the highest contents of omeg-3 ftty cid mong edile plnt seeds [14]. In this study, 5 week old rts fed with either perill oil- or corn oil-sed diet for 3 weeks or 3 months, fter which the cognitive functions of rts nd protein profiles in the hippocmpus were determined. The rin proteome of rts with improved sptil cognition induced y perill-diet intke ws compred with tht of rts fed with regulr diet. Here, we report the significntly ltered expression of cognition-ssocited proteins in the rin upon perill diet-intke. Differentil expression of the proteins ws confirmed y Western lotting nlysis nd immunohistochemistry. Our findings indicte tht n-3 ftty cid supplementtion could lter synptic plsticity in lerning nd memory performnce y generting new hippocmpl neurl memrne structures s well s y inducing specific protein expression. Results Improvement of sptil lerning nd memory y perill diets After 3 weeks or 3 months of perill- or perill oil-diet intke, 1 rts in ech group were tested for lerning nd memory performnce y T-mze tests. Throughout the trining period, rts fed control diet took longer time to rech the gol ox thn the perill-diet groups (Figure 1). Further, reversl of the gol ox rm induced lrger time difference etween the perill-diet groups nd control group (dt not shown). The numer of trils it took to successfully rech the gol ox five consecutive times ws significntly ltered y reversl of the gol ox position. Rts fed with control diet for 3 weeks took n verge of 2.9 ±.32 trils, wheres rts fed perill- or perill oil-diet showed improved cognitive function (2.3 ±.48 or 2.4 ±.52 trils, respectively). (Figure 1). When these results were sttisticlly compred with ech other in two-tiled t-test, the perill-diet nd perill oil-diet groups showed significnt differences compred to the control group with p-vlues of.48 nd.192, respectively. Along the sme line, rts fed perill- or perill oil-diet for 3 months lso showed significnt improvements in sptil cognitive function (1.8 ±.42 or 2.1 ±.32 trils, respectively), wheres the control diet group took 2.6 ±.52 trils. These dt show significnt differences etween the control group nd perill- nd perill oil-diet groups with p-vlues of.14 nd.197, respectively. Ftty cid composition in the rin Ftty cid composition in the rin of rts ws nlyzed y gs chromtogrphy nd normlized y comprison with n externl stndrd, heptdecnoic cid (17:). The results were compred etween the control diet nd perill-diet groups (Additionl file 1). The verge percentge of 22:6n-3 ftty cids in the rin of the control group ws 9.94±.49, nd this numer incresed to 12.1±.6 or 12.43±.62 in the perill- or perill oil diet groups, respectively, fter 3 weeks (Figure 2). Similr differences were lso oserved in the 3 month feeding groups: 1.9±.5 in the control group, 11.98±.59 in the perill group nd 12.91±.64 in the perill oil group (Figure 2). Sttisticl nlysis of the results showed significnt increse in 22:6n-3 ftty cids in the rin of perill-diet fed rts fter oth 3 weeks nd 3 months. Similr results were reported for rts fed n-3 ftty cids or perill oil [1,12]. However, in our study, 18:3 ftty cid, which is the mjor ftty cid component of perill, could not e detected in either the control or perill-diet groups. Bsed on these results, we concluded tht perill-diet chnged the ftty cid composition in the

3 Lee et l. Proteome Science 212, 1:72 Pge 3 of 12 Numer of Trils A B Trining 1 Trining 2 Trining 3. Trining 1 Trining 2 Trining 3 Figure 1 Performnces in T-mze test. Ten rts in ech group fed control diet (lck r), perill seed diet (white r), or perill oil diet (gry r) for 3 weeks (A) nd 3 months (B) were trined until they rech the gol ox 6 times consecutively nd the numers of errors they mde were counted in ech trining section. Likewise, fter the three trining sessions for two weeks, ctul tests were given with reversed gol ox t the T-mze (). The numer of trils efore reching the gol ox 6 consecutive times were significntly reduced (, p<.5) in rts fed perill or perill oil diet.. rin, consequently improving cognitive functions y ltering the protein profile. Proteome profiles on 2-D gel nd imge nlysis Proteins extrcted from pooled hippocmpl tissues of rts from ech group were seprted on 2-D gels in order to quntittively compre individul protein expression levels. Differentilly expressed proteins compred to control group were identified y peptide mss fingerprinting nd re listed in Tles 1 nd 2. Among the 27 proteins selected from the 3 week groups, 33.3% of proteins (nine proteins) were clustered with cytoskeleton function with n enrichment score of 2.97 nd DHA / Totl Ftty Acids (%) Figure 2 Reltive DHA content in the rin of rts. Ftty cid contents in the rin of rts fed control diet (lck r), perill diet (white r) or perill oil diet (grey r) were nlyzed y gs chromtogrphy using n externl ftty cid stndrd, heptdecnoic cid (17:). The reltive rtios of DHA in the totl ftty cid were significntly incresed in the rin of rts fed perill or perill oil diet (, p<.5). p-vlue of 9.2 x 1-4, wheres 26% (seven proteins) were clustered with memrne ound vesicle or vesicle medited trnsport with n enrichment score of 1.79 nd p-vlue of 9.9 x 1-4. The remining proteins were clustered with insufficient sttisticl significnce (p>.5) (Tle 3), suggesting tht the short period (3 weeks) of perill-diet intke improved sptil cognition y chnging cytoskeletl structure nd vesiculr trnsport in the hippocmpus of rts. Functionl nnottion clustering of the 26 proteins selected from the 3 month perill-diet intke groups resulted in seven nnottion groups with enrichment scores higher thn 2.1 (p<.13) (Tle 3). Among the seven nnottion clusters, the iologicl functions of the proteins could e seprted into five clusters: energy metolism, vesiclemedited trnsport, cytoskeleton, neuron projection, nd regultion of poptosis. In ddition to the nnottion clusters of the proteins from the rins of the 3 week group, proteins from the rins of the 3 month groups could e seprted into more clusters, including energy metolism (27%), neuron projection (27%), nd regultion of poptosis (27%), suggesting tht more diverse mechnisms of lerning nd memory re required s the period of perill-diet tretment increses nd the rts ecome older. To test whether or not nnottion clusters from the differentilly expressed proteins re simple representtion of the totl proteins content in the rin, we rndomly picked 2 undnt proteins (Additionl file 2) from the hippocmpl proteins seprted y 2-D gel electrophoresis nd identified them for nnottion clustering (Tle 3). Interestingly, we found tht the functions of mjor proteins in the rin could e grouped into severl clusters. The three enriched clusters in the rin, energy metolism, neurogenesis nd cytoskeletons, re lso the mjor functionl groups in the rin ut with lower percentges nd higher

4 Lee et l. Proteome Science 212, 1:72 Pge 4 of 12 Tle 1 Differentilly expressed proteins in the hippocmpus of rts fed perill diets for 3 weeks Accession numer MOWSE score overge (%) Protein nme log(p/) log(o/) P E Protein disulfide-isomerse A3 precursor + c + O omplement component 1Q-inding protein + d P T-complex protein1 suunit lph + + P protein zet/delt + Q5U2Z Unchrcterized protein KIAA82 homolog + P E Serum lumin precursor - e - P E Elongtion fctor Tu, mitochondril precursor - - P R GDP dissocition inhiitor et + + Q99NA Isocitrte dehydrogense NAD] suunit lph + Q9Z Homer protein homolog 1 + Q6P9V Tuulin lph-1b chin + P E+7 21 Protein disulfide-isomerse A3 precursor - + Q4V8G entromere protein U - - P E+8 28 Neurofilment medium polypeptide - - Q6QLM Kinesin hevy chin isoform 5A - - P E Alph-solule NSF ttchment protein - - P Alph-synuclein + + P ytochrome c oxidse suunit 5B + P Glucose-6-phosphte1-dehydrogense + + P6318 M 1.8E Het shock cognte 71 kd protein + + P E Bet-solule NSF ttchment protein - - Q99JY9 M 2.68E Actin-relted protein P62815 M 1.22E V-type proton ATPse suunit B, rin isoform - + Q9RQ7 M Prostglndin E synthse P Serine/threonine-protein phosphtse 2A - + Q6PE4 M S-phse kinse-ssocited protein Q9WTT6 2.65E Gunine deminse - -, log vlue of the rtio of the spot intensity of perill diet group (P) over control group ()., log vlue of the rtio of the spot intensity of perill oil diet group (O) over control group (). c, + mens tht the rtio of P/ or O/ over 1.2 is recognized s incresed expression, nd the vlue of log(p/) or log(o/) is over.79. d, mens tht the rtio of P/ or O/ etween.8 nd 1.2 re recognized s unchnged, nd the vlue of log(p/) or log(o/) is. e, - mens tht the rtio of P/ or O/ lower thn.8 recognized s decresed expression, nd the log vlues re under.97. p-vlues thn the nnottion clusters of rin proteins from rts fed perill-diets. This suggests tht the nnottion clusters of the differentilly expressed proteins in the hippocmpus re not simple representtions of the mjor protein groups in the rin. Western lot nlysis To verify the results of differentil protein expressions in the rin upon perill-diet intke, Western lotting nlyses using ntiodies ginst the four selected proteins were performed. Selection of protein ws crried out ccording to the vilility nd specificity of the ntiodies, reproduciility nd fold differences of the proteomic results, nd involvement of the proteins in synptic plsticity. Since AMPA receptor suunits re essentil for hippocmpl synptic plsticity, sptil lerning nd memory, s well s long term potentition induction nd mintennce [15-17], n ntiody ginst AMPA receptor ws used to investigte improved sptil lerning nd memory in rts supplemented with perill-diets. After 3 weeks of perill-diet intke, there ws significnt increse in AMPA receptor expression, positive control for lerning nd memory improvement, in the hippocmpus (Figure 3). AMPA receptor protein ws lso differentilly expressed in the hippocmpus of rts supplemented with perill-diets for 3 months. α-synuclein protein showed good correltion with the proteomic dt from Western lotting nlysis. Protein expression in the hippocmpus significntly incresed upon oth

5 Lee et l. Proteome Science 212, 1:72 Pge 5 of 12 Tle 2 Differentilly expressed proteins in the hippocmpus of rts fed perill-diets for 3 months Accession numer MOWSE score overge (%) Identified protein log(p/) log(o/) P E protein epsilon + + P E Het shock cognte 71 kd protein + + P Alph-synuclein + + P E Gmm-enolse - - P E ATP synthse suunit et, mitochondril - - P E Alph-internexin + + P E kd glucose-regulted protein - - P E Neurofilment medium polypeptide + + P E Bet-solule NSF ttchment protein + + P E+7 22 Het shock cognte 71 kd protein + P E Trnsitionl endoplsmic reticulum ATPse + P E Tropomodulin P Pltelet-ctivting fctor cetylhydrolse IB suunit et - O Mlte dehydrogense, cytoplsmic - P E Actin-relted protein P E Alph-enolse + Q E Nucleoside diphosphte kinse A - - P E Protein disulfide-isomerse A3 - P E Serum lumin - + P Hypoxnthine-gunine phosphoriosyltrnsferse - + Q9JJM9 4.31E Septin P E Dihydropyrimidinse-relted protein P E Glycogen phosphorylse, rin form + P E Hexokinse-1 + P E Dynmin-1 + Q9QUL6 1.49E Vesicle-fusing ATPse - perill- nd perill oil-diet supplementtion for 3 weeks or 3 months. Neurofilment ws down-regulted in oth the perill-diet nd perill oil-diet groups fter 3 weeks, ut it ws up-regulted in the 3 month perill oil-diet group. β-solule NSF protein showed slight downregultion in the 3 week smples of perill-diet group ut up-regultion in the 3 month smples of oth perillnd perill oil-diet groups. Overll, the fold chnges of expression level in AMPA receptor, α-synuclein, neurofilment, nd β-solule NSF protein were correlted with the proteomic dt nd were lwys greter in the perill oil-diet group compred to perill-diet group in Western lotting nlysis. Improved sptil lerning nd memory re ssocited with incresed immunorectivity to α-synuclein nd neurofilment in dentte gyrus As AMPA receptor, neurofilment nd α-synuclein were ll significntly up-regulted in the hippocmpus of rts perill-diet intke, we questioned whether or not the upregultion of protein expression hd ny histologicl effects on the hippocmpus. To test this, we used primry ntiody ginst AMPA receptor s positive control for improved lerning nd memory function long with ntiodies ginst α-synuclein, nd neurofilment. We lso performed immunohistochemistry stining on the sgittl microtome sections of rt rins (Figure 4). Tissue sections were prepred fter completion of the ehviorl studies. Anti-AMPA receptor ntiody decorted the dentte gyrus of rts with significntly higher intensities following perill-diet intke for 3 weeks or 3 months (Figure 5A) in comprison with control rts, suggesting tht the rts indeed experienced improved sptil cognitive function y perill-diets. Likewise, ntiody ginst α-synuclein showed higher immunorectivity in the dentte gyrus of rtsfed perill- or perill- oil-diet for 3 weeks or 3 months (Figure 5B). However, no significnt differences were oserved in the

6 Lee et l. Proteome Science 212, 1:72 Pge 6 of 12 Tle 3 Functionl nnottion clustering of the proteins differentilly expressed in the hippocmpus of rts fed perill-diets Annottion cluster Numer of proteins Enrichment score p-vlue 3 week-feeding cytoskeleton E-4 memrne-ounded vesicle E-4 vesicle-medited trnsport E-4 3 month-feeding vesicle-medited trnsport E-5 cytoskeleton E-4 neuron projection E-4 regultion of poptosis E-3 2 mjor proteins neurofilment cytoskeleton E-5 orgniztion glycolysis E-3 regultion of xonogenesis E-3 A1 to 3 regions in the hippocmpus or in the cortex (dt not shown). ontrry to the results of the proteomic (Tle 1) nd the Western lotting nlyses (Figure 3) of the hippocmpus, no significnt differences in neurofilment M were detected etween the two groups in terms of immunostining intensitied (quntified s intensity of immunostined cells/mm 2 dentte gyrus: control=12.961; perill=12.286; perill oil= , Figure 4) in the dentte gyrus. However, cells stined y the ntineurofilment M ntiody showed different cell morphologies. Immunorective cells in the hilus of dentte gyrus of the control group showed less neurite projections compred to cells of the perill-diet groups (Figure 4). To oserve the projections, high mgnifiction photomicrogrphs (representtive imges shown in (Figure 4)) were tken, nd the projections re indicted with rrows. The reltive length of the projections mesured s descried in the Methods (Figure 5) significntly incresed in the perill-diet groups, suggesting enhnced neuron projections in the dentte gyrus upon supplementtion of perill- or perill oil-diet. Discussion As synpses re lile nd plstic structures, ny microenvironmentl chnges in the rin cn lter the numer nd structurl configurtions of existing synpses. hnges in neurologicl signling y ltertions of the gene expression profiles nd lipid composition in the synptic memrne cn modulte cognitive function in the rin [18,19]. Thus, the gols of this study were to nlyze proteomic chnges in the rin upon supplementtion of α-linolenic cid, n n-3 ftty cid, rich perill, or perill oil s well s to identify proteins closely relted to the cognitive function. Short term (for 3 weeks) feeding of 18:3n-3 ftty cidrich perill-diets to rts significntly improved sptil lerning memory (Figure 1), chnged the level of 22:6n-3 ftty cid, DHA, in the rin (Figure 2 nd Additionl file 1), nd ltered proteome profiles compred to the corn-oil sed control diet group (Tles 1 nd 2). These chnges were lso oserved in the rt groups fed perilldiets long term (for 3 months). From the proteomic nlysis, we found tht the differentilly expressed proteins in the hippocmpus of rts fed perill-diets for 3 weeks were minly nnotted into severl functionl groups, including cytoskeleton nd trnsport, wheres the mjor functionl groups of those proteins from the 3 month groups were energy metolism, neuron projection nd poptosis in ddition to trnsport nd cytoskeleton (Tle 3). The nnottion grouping in this study shows tht the chnges in ftty cid composition in the memrne of the rin of rts fed n-3 ftty cid rich perilldiets ltered the expression profiles of hippocmpl proteins, including cytoskeleton nd energy metolism proteins, herey ffecting neurogenesis nd synptic plsticity. Further, chnges in the protein expression profile grew more complex s the nimls ecme older, suggesting tht there re common nd specific pthwys for cognition in response to vrious stimuli, nd the pthwys involved in cognitive function were much more complicted for the mture rin thn for the young one. Previously, we demonstrted tht estrogen deprvtion in rts y ovriectomy results in llevited sptil lerning nd memory in T-mze test, nd we oserved significnt proteomic chnges in energy metolism, cytoskeleton, nd nti-oxidtion [2]. These results indicte tht cytoskeleton s common ground in conjunction with other proteins from different functionl groups, improves cognition through vrious chnges in synptic plsticity. To understnd how the differentilly expressed proteins re involved in synptic plsticity, nd sed on the fold differences in 2-D gel electrophoresis, we selected four proteins for verifiction nd functionl nlysis of the proteomic dt. Antiodies ginst AMPA receptor, neurofilment, -synuclein, nd β-solule NSF ttchment protein were used for Western lotting nd immunohistochemistry nlysis. Firstly, Western lotting nlysis using ntiodies ginst the selected proteins were performed (Figure 3). Although AMPA receptor could not e detected y 2-D gel electrophoresis nd its differentil expression in the hippocmpus ws not cler, it ws selected s positive control since the receptor suunits re essentil for long

7 Lee et l. Proteome Science 212, 1:72 Pge 7 of 12 A AMPAR PS PO PS PO α-syn NeuroF M β-nsf GAPDH B Expression Level Expression Level AMPA P P O O α-syn P O P O NeuroF M P O P O β-nsf P O P O Figure 3 Western lotting nlysis on the hippocmpus of rts fed perill diets for 3 weeks () nd 3 months (). A. The nds of hippocmpl proteins seprted on SDS-PAGE nd trnsferred to PVDF memrne were incuted with ntiodies ginst AMPA receptor (AMPAR), α-synuclein (α-syn), neurofilment M (NeuroF M), nd β-solule NSF ttchment protein (β-nsf) nd decorted with chemiluminescence. B. The intensities of the nds were cptured with densitometer nd normlized ccording to tht of GAPDH. The normlized nd intensities were compred etween control () nd perill (P) or perill oil (O) diet groups, nd presented s reltive expressions (%) with the control group s 1%. Letters nd on the r grphs designte differences etween the expression levels in ech group with different letters for significnt differences (p <.5). term potentition induction nd mintennce [16] nd ply importnt roles in sptil lerning nd memory [15]. Using nti-ampa ntiody, rts fed perill-diets for 3 weeks or 3 months showed elevted AMPA receptor expression in the hippocmpus, indicting tht, together with the results from the T-mze test, n-3 ftty cid rich perill-diets improved cognitive functions. Among the selected differentilly expressed proteins in Tles 1 nd 2, α-synuclein nd neurofilment showed significntly incresed expression, wheres β-solule NFS ttchment protein underwent slight chnges in expression etween the control group nd perill-diet groups in Western lotting nlysis (Figure 3B). Further, the up-regulted proteins in the 2-D gel nlysis showed higher expression in the Western lotting nlysis. Interestingly, the perill-diet group lwys showed lower protein expression thn the perill oil-diet group. This could e due to the higher vilility of n-3 ftty cids

8 Lee et l. Proteome Science 212, 1:72 Pge 8 of 12 Figure 4 Immunohistochemicl nlysis of the dentte gyrus of rts with improved cognitive function fed perill diets. Representtive DAPI-leled sections contining the dentte gyrus of rts fed perill (P) or perill oil (O) diets showed incresed immunorectivities y ntiodies ginst AMPA receptor (A) nd α-synuclein (B) compred to control (ont) diet group. Wheres no significnt chnges in the density of immunorectivity ws oserved in the sections stined y nti-neurofilment ntiody (). However, the immunorective cells y nti-neurofilment ntiody showed longer neurites (indicted with rrows) in the hilus of dentte gyrus of rts fed perill- or perill-oil diets. Scle rs in the first left column represent 1 μm nd re the sme for other sections. Otherwise, scle r ws designted with size for higher mgnifiction (lst rw in ). in the digestive trct in the perill oil group compred to perill group (Figure 2). Oils in perill seeds should e less ville to the digestive enzyme system nd thus less miscile nd less sorptive in the intestine compred to the extrcted perill oil. Secondly, using rin sections nd ntiodies whose trget proteins were the most differentilly expressed in the Western lotting nlysis, we performed immunohistochemistry to exmine where the proteins were expressed in the hippocmpus s well s ny chnges in cell morphology. In ddition, the specificity nd rectivity of the ville ntiodies to the prffin sections were considered. α-synuclein gene, which encodes 14-mino-cid protein, is key plyer in neurodegenertive diseses. The firillr β-sheet ggregtion of α-synuclein is the mjor constituents of Lewy odies nd Lewy neurites in the pthogenesis of Prkinson disese [21]. However, in comprison to its identified roles in neuropthogenesis, the norml physiologicl function of α-synuclein in neurogenesis is not well understood. As presynptic protein, α-synuclein, is locted in the memrne, intercts with tuulins, is enriched in presynptic termini, nd is involved in microtuule trnsport nd neurotrnsmitter relese [22-24]. Mice lcking α-synuclein gene show norml long-term potentition (LPT) of

9 Lee et l. Proteome Science 212, 1:72 Pge 9 of 12 A Stining Intensity Figure 5 Sttisticl presenttion of the immunohistochemicl stining of rin sections of rts. Expression levels of AMPA receptor (A) nd α-synuclein (B) represented y immunorectivities in the sections were quntitted y softwre, isolution, nd sttisticlly compred (, p<.5, nd, p<.1). The sections of rins of rts fed perill (white r) or perill oil (grey r) diet for 3 weeks () or 3 months () were more immunorective to the ntiodies ginst AMPA receptor nd α-synuclein thn those of rts fed control (lck r) diet. Although nti-neurofilment ntiody did not show significnt immunorectivity (Figure 4) etween control nd perill groups in the sections, the cells stined y the nti-neurofilment ntiody hve significntly longer neurite projections (, p<.5, nd, p<.1) (). B Stining Intensity Neurite Length (µm) glutmtergic synpses in the hippocmpus, suggesting tht α-synuclein is not essentil for LTP in the hippocmpus [22]. Nevertherless, the modultion of α-synuclein gene expression in trnsgenic mice lters hippocmpl neurogenesis nd synptic plsticity in the dentte gyrus, suggesting tht the protein might hve reltionship with lerning nd memory [25,26]. Interestingly, it ws reported tht n incresing concentrtion of DHA in the memrne of the rin induces ggregtion nd chemicl modifiction of α-synuclein in vitro [27]. Regrding the fct tht the perill-diet groups showed elevted DHA levels in the rin (Figure 2 nd Additionl file 1), n-3 ftty cid converted to DHA could modulte the redistriution of α-synuclein in the hippocmpl presynpse for synptic plsticity nd improved cognitive function. This result supports the previous finding tht the mrna level of α-synuclein ws elevted upon n-3 ftty cid tretment to rts for 3 weeks [9]. Neurofilment medium moleculr weight protein (NF-M) is neuron-specific intermedite filment protein tht heteropolymerizes with other immunologiclly distinct neurofilments, NF-L nd NF-H, nd constitutes mjor prt of the neuronl cytoskeleton [28]. The three different NF proteins form filments t different stoichiometries under different physiologicl conditions, nd the resulting siderm structures of the -termini of the NF proteins long the core fier cn interct with different cytoskeletl components to estlish xonl cytorchitecture [29]. Structures of the siderms, which cn e ltered y NF stoichiometry, influence xonl trnsport nd determines xonl clier, which ffects the conduction velocity of neurons. Therefore, ltertion in the NF stoichiometry cn contriute to neurodegenertive diseses [3] s well s other neurologicl functions. Up-regulted NF-M protein in the hippocmpus of rts fed perill-diets (Figure 3) cn lter the neuronl cell morphology in the hilus of dentte gyrus, neurogenic re (Figure 4 nd 5), therey fcilitting the conduction velocity of neurons to improve lerning nd memory. Moreover, the up-regultion of neurofilments in the hilus of dentte gyrus fcilitted neurite outgrowth

10 Lee et l. Proteome Science 212, 1:72 Pge 1 of 12 in the perill-diet groups, suggesting enhnced neurogenesis ssocited with improved sptil memory performnce [31]. onclusion This study shows tht improved sptil lerning nd memory upon supplementtion of n-3 ftty cid-rich perilldiets is ssocited with differentil expression of proteins in the hippocmpus of rts. The proteins cn e grouped into severl functions, including cytoskeleton, energy metolism, trnsport, neurogenesis, nd poptosis. Functionl vlidtion of the proteomic chnges y immunohistochemicl study using ntiodies ginst neurofilment nd α-synuclein showed differentil immunorectivities in the hilus of dentte gyrus, neurogenesis center of the rin. These results indicte tht improved cognitive function y supplementtion with dietry α-linolenic cid from perill induces chnges in memrne ftty cid composition, especilly, DHA, s well s in the immunorectivity in neurogenesis region of the hippocmpus. Together, these chnges culminte in ltertion of synptic plsticity. Methods Animls Thirty 4-week old Sprgue Dwley mle rts (Hyochng Science, Degu, Kore), weighing g were housed t 23 nd 6% humidity with d liitum ccess to food nd wter under 12/12-h light/drk cycle. After 1 week, the rts were divided rndomly nd eqully into three groups nd fed diets djusted with the lipid contents of 7% with corn oil, perill oil nd powdered perill seed, respectively, sed on the AIN-93G formultion (Additionl file 3). The rts were grown for 3 weeks or 3 months until the ehviorl tests nd rin proteome nlyses. All niml procedures were in complince with guidelines of Kyungpook Ntionl University (KNU) Animl re nd Use ommittee. Behviorl test The T-mze ws constructed ccording to the mesurements provided y Gerli [32]. After vrile inter-tril intervl in the gol ox (15 sec on verge), ech rt ws plced in the strt ox for the next tril. The pretrining session continued until the rt mde its first correct voidnce response. For 2 weeks, the rts were trined until they mde five correct voidnce responses in six consecutive trining trils (cquisition session). On the lst dy of the second week, the ctul test ws conducted, fter which reversl session ws performed with the correct gol ox locted opposite to the one used during cquisition. The mesures included the ltency to rech the correct gol ox nd the numer of trils prior to correct voidnce. Protein smple preprtion nd 2-DE Brin tissues from 7 individul rins per group were extrcted fter perfusion with ice-cold PBS nd immeditely stored t 8. For protein preprtion, tissues were ground in liquid nitrogen nd homogenized in lysis uffer contining 7 M ure, 2 M thioure, 4% HAPS, 1 M DTT nd mixture of protese inhiitors (omplete-mini EDTA-Free, Roche, Indinpolis, USA), followed y soniction 2 times. The protein smples in lysis uffer were directly pplied to immoilized ph grdient (IPG) strips (ph 3 1 nd 4 7, 17 cm; Bio-Rd, Herculus, A USA), nd then seprted on second dimensionl SDS-PAGE. The gels were stined using BB R-25 method for visuliztion. The protein profiles on the gels were digitized y reflector mode scnning using n Epson Perfection V7 scnner (Ngno, Jpn). Imge nlysis softwre, PDQuest v7.3 (BioRd), ws used to compre the imges nd detect protein spots whose expression levels were significntly incresed or decresed compred to control. Mss spectrometry nlysis, protein identifiction, nd ioinformtics Protein spots of interest on gels were mnully excised, wshed with deionized wter, nd destined using 5 mm mmonium icronte/cetonitrile (6:4, v/v) with vigorous shking. Digestion ws crried out y dding sequencing grde modified trypsin (Promeg, Mdison, WI, USA) onto the dried gel pieces, followed y incution overnight t 37. The peptides were then extrcted with extrction uffer (6% AN in wter nd.1% TFA), nd dried with the id of vcuum drier. From the concentrted peptide extrct, 1 μl ws tken nd mixed with 1 μl of mtrix solution (1% HA in 5% methnol nd.1% TFA contining externl stndrds such s rdykinin, ngiotensin nd neurotensin) on trget MALDI plte. The cquired mss spectr were nlyzed y Mscot from Mtrix Science ( nd MS- Fit from Protein Prospector ( Functionl nlysis of the differentilly expressed proteins ws crried out y serching the dtses using the Uniprot KB ( serching tool, nd the identified proteins were functionlly nnotted using n nnottion serch engine, DAVID Bioinformtics Resources 6.7 (Ntionl Institute of Allergy nd Infectious Diseses (NIAID), NIH n integrted knowledge se nd nlytic tools. Immunologicl nlysis nd stining Proteins extrcted from rin tissues were seprted y SDS-PAGE nd trnsferred to PVDF memrne (Millipore, Billeric, MA, USA) for Western lotting nlysis. After locking the PVDF memrne with BSA, specific monoclonl primry ntiodies (ell Signling,

11 Lee et l. Proteome Science 212, 1:72 Pge 11 of 12 Dnvers, MA, USA) ginst the differentilly expressed proteins selected from the imge nlysis were properly diluted in 3% BSA with.1% Tween 2 in PBS nd incuted with the memrne for 4 hr t 4. Following this, nti-mouse secondry ntiody conjugted to horse rdish peroxidse (Sigm-Aldrich, St. Louis, MO, USA) ws diluted in.1% Tween 2 in PBS nd incuted with the memrne for 1 hr t RT. Decorted nds were scnned using the LAS-3 scnner (Fujifilm, Tokyo, Jpn) fter extensive wshing with.1% Tween 2 in PBS nd chemiluminescence stining (SuperSignl Pico-chemiluminescent Sustrte, Pierce, IN). Immunohistochemicl stining of seril sgittl sections (4 μm) of prffin-emedded rin tissues ws performed using Vectsttin AB kit with the iotin/ vidin/peroxidse system, including 3,3 -diminoenzidine (DAB) s chromogen nd Hemtoxylin s counter stin (Vector, Burlingme, A) y following the suggested mnufcturer s protocol. Specificlly, prformldehydefixed nd prffin-emedded slide-mount tissue sections using 3 rins from ech group were heted in oiling citrte-sed ntigen unmsking solution for 1 min for etter ntigen retrievl y the primry ntiodies. Section slices were then incuted overnight with primry ntiody in PBS contining 2% BSA nd 2% norml got serum followed y secondry ntiodies coupled to horse rdish peroxidse. The numers of stined cells in the 4 well stined high mgnifiction fields (4x), which were tken y using H55L microscope nd DS-Fi1c digitl cmer (Nikon, Tokyo, Jpn), were counted using i-solution (ver. 7.5; IMT i-solution, oquitlm, B, nd), n imge nlysis softwre. Sections stined with nti-neurofilment ntiody were used to mesure the length of neurite projections using ImgeJ softwre (NIH). Length dt were sttisticlly compred y Student s t-test. omposition nd concentrtion of ftty cids in the rin nd perill Ftty cid nlyses in the rin, perill seeds, nd perill oil were performed on the smples (1 mg wet weight) treted with 4 ml of.5 M methnolic sodium hydroxide, nd heted t 8 for 5 min. After cooling, smples were methylted y ddition of 3 ml of oron trifluoride in methnol nd heted in n 8 wter th for 5 min. The smple tue ws then llowed to cool, fter which 2 ml of hexne nd 8 ml of distilled wter were dded. After vortexing for 1 min, the hexne frction ws removed from the tue for gs chromtogrphic nlysis. Ftty cid methyl esters (FAMEs) of lipids from the smples were nlyzed using n AME 61 gs chromtogrphy system (YL Instrument, Anyng, Kore) equipped with column (3 m in length, I.D..32 mm wide ore, film thickness of.25 μm (J&W Scientific, Folsom, A)). Ftty cid peks were determined sed on the retention times of the FAME stndrds. The re of externlly dded 17: stndrd ws used to clculte the concentrtion of ftty cids y proportionl comprison of the pek res. All smples were run five times, nd the vlues were verged for ll susequent nlyses. Additionl files Additionl file 1: Tle S1. Ftty cid composition in the rin of rts fed control or perill diets for 3 weeks or 3 months. Additionl file 2: Tle S2. 2 mjor proteins in the rin of rts fed perill diets. Additionl file 3: Tle S3. Nutrient compositions of the diets. ompeting interests The uthors declre tht they hve no competing interests. Authors contriutions JL nd SP contriuted to the overll conception nd design of the project, crried out ll the experiments, nlysis nd interprettion of the dt, performed mss spectrometry nlysis, nd preprtion of the mnuscript. JYL crried out niml ehviorl tests, mss spectrometric nlysis nd interpreting mss spectrometry dt. YKY contriuted with criticl discussions on ftty cid nlysis, ftty cid metolism in rin nd dt interprettion in ftty cids. JSK contriuted to the niml diet formultion, design of niml ehviorl test, dt nlysis nd interprettion. The corresponding uthor JL conceived the study, nd prticipted in its design nd coordintion, dt nlysis nd interprettion, nd preprtion of the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements This reserch ws supported y the grnt (# ) from Ntionl Reserch foundtion of Kore (NRF). The uthors re indeted to Dr. Kilsoo Kim for his dvice in rt rin ntomy. Received: 17 August 212 Accepted: 29 Novemer 212 Pulished: 5 Decemer 212 References 1. Goelet P, stellucci VF, Schcher S, Kndel ER: The long nd the short of long-term memory moleculr frmework. Nture 1986, 322: ost-mttioli M, Sossin WS, Klnn E, Sonenerg N: Trnsltionl control of long-lsting synptic plsticity nd memory. Neuron 29, 61: Jin I, Kndel ER, Hwkins RD: Wheres short-term fcilittion is presynptic, intermedite-term fcilittion involves oth presynptic nd postsynptic protein kinses nd protein synthesis. 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