The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells

21 Pages·2017·6.65 MB·English

RESEARCHARTICLE
The effect of Bacopa monnieri on gene
expression levels in SH-SY5Y human
neuroblastoma cells
How-WingLeung1☯,GabrielFoo1‡,GokulakrishnaBanumurthy1‡,XiaoranChai1‡,
SujoyGhosh1‡,ToraMitra-Ganguli2¤*,AntoniusM.J.VanDongen1☯
1 PrograminNeuroscienceandBehavioralDisorders,Duke-NUSMedicalSchool,Singapore,Singapore,
2 GlaxoSmithKlineConsumerHealthcare,Gurgaon,Haryana,India
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☯Theseauthorscontributedequallytothiswork.
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¤ Currentaddress:IndianInstituteofTechnologyTirupati,AndhraPradesh,India
a1111111111 ‡Theseauthorsalsocontributedequallytothiswork.
a1111111111 *[email protected]
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Abstract
BacopamonnieriisaplantusedasanootropicinAyurveda,a5000-year-oldsystemoftradi-
OPENACCESS
tionalIndianmedicine.Althoughbothanimalandclinicalstudiessupporteditsroleasa
Citation:LeungH-W,FooG,BanumurthyG,Chai memoryenhancer,themolecularandcellularmechanismunderlyingBacopa’snootropic
X,GhoshS,Mitra-GanguliT,etal.(2017)The
actionarenotunderstood.Inthisstudy,weuseddeepsequencing(RNA-Seq)toidentifythe
effectofBacopamonnieriongeneexpression
levelsinSH-SY5Yhumanneuroblastomacells. transcriptomechangesuponBacopatreatmentonSH-SY5Yhumanneuroblastomacells.
PLoSONE12(8):e0182984.https://doi.org/ WeidentifiedseveralgeneswhoseexpressionlevelswereregulatedbyBacopa.Biostatisti-
10.1371/journal.pone.0182984
calanalysisoftheRNA-Seqdataidentifiedbiologicalpathwaysandmolecularfunctionsthat
Editor:NatarajanAravindan,Universityof wereregulatedbyBacopa,includingregulationofmRNAtranslationandtransmembrane
OklahomaHealthSciencesCenter,UNITED
transport,responsestooxidativestressandproteinmisfolding.Pathwayanalysisusingthe
STATES
IngenuityplatformsuggestedthatBacopamayprotectagainstbraindamageandimprove
Received:February24,2017
braindevelopment.Thesenewlyidentifiedmolecularandcellulardeterminantsmaycontrib-
Accepted:July27,2017 utetothenootropicactionofBacopaandopenupanewdirectionofinvestigationintoits
Published:August23,2017 mechanismofaction.
Copyright:©2017Leungetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. Introduction
DataAvailabilityStatement:Allrelevantdataare Bacopamonnieri(Bacopa),alsoknownasBacopamonniera,Herpestismonniera,waterhyssop
withinthepaper. orBrahmi,haslongbeenusedinIndiantraditionalmedicine(Ayurveda)asabraintonicto
Funding:Theworkwassupportedbyagrantfrom enhancememoryperformance,learningandconcentration[1,2].Thesetraditionalclaims
GlaxoSmithKlinetoAV,whichwereinvolvedinthe haverecentlybeensupportedbyseveralanimalandclinicalstudies.Animalstreatedchroni-
studydesign.Thefundershadnoroleindata callywithBacopashowedbetteracquisitionandimprovedretentioninlearningtasks[3–11].
collectionandanalysis,decisiontopublish,or
Similarly,clinicalstudiesandameta-analysisofrandomizedcontroltrialsalsodemonstrated
preparationofthemanuscript.Thefunderprovided
thatchronicoraladministrationofBacopa(overaperiodofmorethan12weeks)tohealthy
supportintheformofsalariesforauthorsHL,GF
subjectsresultedinimprovementsinthesubjects’informationprocessingspeed,freerecall,
andGB,butdidnothaveanyadditionalroleinthe
studydesign,datacollectionandanalysis,decision verbalmemoryandlearning.Bacopatreatmentalsoresultedinadecreaseinanxiety,which
PLOSONE|https://doi.org/10.1371/journal.pone.0182984 August23,2017 1/21
EffectofBacopaongeneexpressioninhumanneuroblastomacells
topublish,orpreparationofthemanuscript.The improvedlearning[12–19].Withitslowtoxicityrisksandapparentbeneficialeffectsasanoot-
specificrolesoftheseauthorsarearticulatedinthe ropic[20–22],Bacopahasbeenextractedandmarketedasadietarysupplement(KeenMindor
‘authorcontributions’section.
CDRI08,SohoFlordisInternational)anditsproductionandimportsaretightlyinspectedby
Competinginterests:Thisworkissupportedbya theFoodandDrugAdministration(FDA)(https://tinyurl.com/ybkmhfes).Notwithstanding
grantfromGlaxoSmithKlinetoAV.Thefunder itswideavailability,themechanismsofactionofBacopahaveyettobedelineated.Several
providedsupportintheformofsalariesas
mechanismshavebeenproposedconcerningthenootropiceffectsofBacopa.Theseincluded
indicatedintheFundingStatement.This
alterationofthelevelsofseveralneurotransmitters,includingserotonin(5-hydroxytrypta-
commercialaffiliationdoesnotalterouradherence
mine,5-HT)[6,23,24],acetylcholine[4,13,25]anddopamine[6,20,24,26].Elevationofthe
toPLOSONEpoliciesonsharingdataand
materials. neurotransmitter5-HTresultedinactivationofcAMPresponseelement-bindingprotein
(CREB)andsubsequentchangesintranscription,proteinphosphorylationandhistonemodi-
fication[8,23,27].OtherresearchgroupshavesuggestedthatBacoparegulatepre-andpost-
synapticproteins[9]andinducetheformationofnewdendrites[11,28].Theseproposed
mechanismsarenotmutuallyexclusiveandthediscrepanciescouldbeattributedtoaselective
biasinthetargetsthatwereinvestigatedbyeachresearchgroup.
InordertobetterdefinethemolecularandcellularcomponentsofBacopaaction,wehave
appliedadeepsequencingtechnique,RNA-Seq,toidentifythechangesinthetranscriptome
uponBacopatreatment.Wehaveperformedbothatranscriptlevelandgenelevelanalysisto
investigatethechangesingeneexpressioncausedbyBacopa.Theseexperimentshavesug-
gestedunderlyingmechanismsofactionforBacopa,thebiologicalpathwaysalteredbythis
plantextractandthepotentialupstreammediatorsfortheseprocesses.
Materialsandmethods
Cellculture,differentiationofSH-SY5YcellsandBacopatreatment
Thehumanneuroblastomacellline,SH-SY5Y,waspurchasedfromtheAmericanTypeCul-
tureCollection(ATCCCRL-2266).CellswereculturedinDMEM/F12(Sigma)supplemented
in10%(v/v)fetalbovineserum(FBS,Gibco)and1%(v/v)penicillin/streptomycin(P/S,JR
Scientific).ThismediumwillbereferredtoasCompleteMediumhenceforth.Subculturingwas
performedaspermanufacturer’sinstructions(ATCC).Inbrief,astheSH-SY5Ycellsgrowasa
mixtureoffloatingandadherentcells,carewastakentoensurethefloatingcellsinthe
mediumwerecollectedandrecoveredbycentrifugation.Thesecollatedfloatingcellswouldbe
combinedwithtrypsinizedadherentcellsandsubcultured.Cellswerealsopassagedlessthan
threetimestoensurethatthecellsremainedneuroblast-like[29](Fig1Aand1C).Forexperi-
mentsinvolvingundifferentiatedSH-SY5Ycells,theplatingdensitywas0.4x106cells/cm2.To
differentiateSH-SY5Ycells,theywereplatedatadensityof0.5x106/cm2onculturesurfaces
coatedwith10μg/mllaminin(Sigma)andmaintainedinCompleteMediumfor18h.After
which,theyweremaintainedinserum-freeCompleteMedium.50nMofhumaninsulin-like
growthfactor-I(IGF-1)(Sigma)wasaddedtopromotedifferentiation[30].48hafterthe
switchtoserum-freeCompleteMediumandtheadditionofIGF-1,themediumwasreplen-
ished.Bacopatreatmentwascarriedout72hafterthestartofdifferentiation.Undifferentiated
anddifferentiatedcellsweretreatedwith3μg/mlBacopafor24hor10μg/mlBacopafor4h,
orwithvehiclecontrols.Forallexperiments,weusedastandardizedextractofBacopa(CDRI-
08),containingnolessthan55%bacosideAandbacosideBasitsbioactivecomponentsthat
wasextractedbyethanolextraction(LailaImpex,Vijaywada,India)[31,32].
Hydrogenperoxide(H O )toxicityassay
2 2
UndifferentiatedordifferentiatedSH-SY5Ycellswereexposedtodifferentconcentrationsof
H O for24h.NumberoflivecellsweremeasuredusingtheLIVE/DEADassay(Invitrogen)
2 2
usinghigh-contentscreening(HCS)microscopyplatform(MetaXpress,Moleculardevices).
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EffectofBacopaongeneexpressioninhumanneuroblastomacells
Fig1.DifferentiationofSH-SY5YcellsusinglamininandIGF-1.SH-SY5Ycellswereplatedonlamininandgrownfor24hoursinDMEM/F12
supplementand10%FBS.Toinducedifferentiation,FBSwasremovedand50nmIGF-1wasadded;cellswereallowedtogrowfor72hours.(A)
Differentialinterferencecontrast(DIC)imageoftheundifferentiatedcontrols.Redarrowsmarkedtheneuritesinundifferentiatedcellsthatwere
characteristicforneuroblast-likecells.(B)DICimageofthedifferentiatedcells.Theincreaseinneuritelengthupondifferentiationwasmarkedout
bythegreenarrowheads.(CandD)Toquantifythechangeinthelengthoftheneurites,twodaysintothedifferentiationprotocol,cellswere
transfectedwithGFPcDNAandimagedonday3usingfluorescencemicroscopy.TransfectingwithGFPhighlightedtheneuritesamongthe
confluentcelllayers,allowingforeasyquantification.(C)Anoverviewoftheundifferentiatedcontrols.Redarrowsmarkedouttheneuriteofeach
GFPtransfectedcells.(D)Differentiatedcellsdisplayedlongneuritesasoutlinedbygreenarrowheads.(E)Theincreaseinthelengthofthe
neuritesupondifferentiationwasstatisticallysignificant(unpairedt-test,****indicatesP-val<0.0001).
https://doi.org/10.1371/journal.pone.0182984.g001
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EffectofBacopaongeneexpressioninhumanneuroblastomacells
High(10μg/ml)andlowconcentration(1μg/ml)ofBacopaweresupplementedduringthe
additionofH O forinvestigationofneuroprotectiveeffectsofBacopa.
2 2
RNAsamplepreparation,libraryconstructionandRNAsequencing
RNAwasharvestedfromtreatedSH-SY5YcellsusingtheNucleoSpinRNA/Proteinkit
(Macherey-NagelGmbH).LibraryconstructionandRNAsequencingwereperformedbythe
Duke-NUSGenomeBiologyFacility(DGBF).Briefly,2.2μgofRNAwassentforlibrarycon-
struction.QualityofRNAwasanalyzedwiththeAgilentBioanalyzerandRNAwithRIN>9
wasused.Followingpoly-Aenrichment,recoveredRNAwasprocessedusingtheIllumina
TruSeqRNASamplePreparationKitv2protocol(non-stranded)togenerateadaptor-ligated
libraries.Atotalofsixsamplesweresequenced,obtainedfromundifferentiatedanddifferenti-
atedSH-SY5Ycellstreatedwith(i)vehicle-treatedcontrol,(ii)3μg/mlBacopafor24h,and
(iii)10μg/mlBacopafor4h.SamplesweresequencedusingtwolanesofanIllumina
HiSeq2000sequencerusing76-bppaired-endreads.
ComputationalanalysesofRNA-sequencingdata
RNA-SeqdatawasmappedtothehumangenomeusingPartekFlow(version4.0.15.0406)and
PartekGenomicsSuite(version6.15.0327).Aftertheadaptorsequencesweretrimmedaway,
readsweremappedtotheHomosapiensgenome(hg38)withTopHat2(version2.0.8).Local
alignmentwasperformedontheunalignedreadsfromTopHat2tothehumangenome(hg38)
withBowtie2(version2.1.0).AlignedreadsfromtheTopHat2andBowtie2alignmentwere
combinedinPartekFlow.Post-alignmentQA/QCwasperformedaftereachalignmentstep
andalignedreadshadanaveragequalityPhredscoreabove30.Theuniquepairedreadswere
usedforgeneexpressionquantification.Readswereassignedtoindividualtranscriptsofa
genebasedontheExpectation/Maximization(E/M)algorithm[33].InthePartekGenomics
Suitesoftware,theE/Malgorithmwasmodifiedtoacceptpaired-endreads,junctionaligned
reads,andmultiplealignedreadsifthesearepresentinthedata.RNAexpressionwascalcu-
latedasfragmentsperkilobaseoftranscriptpermillionmappedreads(FPKM)valuesofthe
humanRefSeqgenesforpaired-endsequencing.Toidentifydifferentiallyexpressedgenes,
Partek’sGeneSpecificAnalysis(GSA)algorithmwasused.Readcountsbetweensampleswere
normalizedwiththeUpperQuantilemethodandanalysiswasperformedatthetranscript
level.AcutoffvalueofmultimodalP<0.05andfoldchange>2or<-2wereset.Agene
ontologyanalysiswasconductedusingPartekGenomicsSuite.
Functionalclassscoringusinggene-setenrichmentandover-
representationanalysis
Toidentifybiologicalfunctionsaffectedbydifferentialgeneexpression,evidenceforfunc-
tionalclassenrichmentinvolvingbiologicalpathwaysorgene-setswassoughtviatwoindepen-
dentmethods.First,pathwayenrichmentanalysiswasconductedviatheGeneSetEnrichment
Analysistoolusingthe“pre-ranked”option[34].Forthisanalysis,atotalof10744geneswith
aFPKM(cid:21)5inatleast1samplewereincluded,andrankedbytheirfold-changeinexpression
betweenthecontrolandBacopatreatedsamples.Therankedgene-listwasusedtoquerypath-
waysfromGeneOntology-BiologicalProcessandGeneOntology-MolecularFunction,
(downloadedfromtheMolecularSignaturesDatabase,MSigDB,[35],aswellascustompath-
ways(Reactomepathwaysplususer-definedgene-setsforbrain-specificfunctions)forstatisti-
callysignificantenrichmentofhigherorlowerrankedgenes.Significanceestimateswere
adjustedformultipletestingviathefalsediscoveryrate(FDR).
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EffectofBacopaongeneexpressioninhumanneuroblastomacells
Over-representationanalysis(ORA)ofbiologicalfunctionsandputativeupstreamregula-
torswascarriedoutbysubjectingapre-filteredlistof576differentiallyexpressedgenes
(FPKM(cid:20)5inatleast1sampleandabsolutefold-change(cid:21)1.5)toQIAGEN’sIngenuityPath-
wayAnalysistool(IPA,QIAGENRedwoodCity,https://www.qiagenbioinformatics.com/
product-login/).First,referencegene-setscorrespondingto‘biologicalfunctions’(asdefined
intheIngenuityKnowledgeBase),wereanalyzedviaFisher’sexacttestforstatisticallysignifi-
cantover-representationinthelistofdifferentiallyexpressedgenes.Additionally,predictions
ofchangesintheactivitystatusof‘upstreamregulators’(specifically,transcriptionfactors),
thatcouldputativelyexplaintheobservedgeneexpressionchangesduetoBacopatreatments,
wasalsocarriedout.AnORAwasfirstperformedtodeterminewhetheranupstreamregulator
wasenrichedfordifferentialexpressionofitstargetgenes(thelistofregulatorsandtheirtarget
geneswere,again,obtainedfromtheIngenuityKnowledgeBase).Theoverallactivationor
inhibitionstatusoftheregulatorwastheninferredfromthedegreeofconsistency(up-or
down-regulation)intheexpressionpatternsofitstargetgenes,expressedasaz-score.Regula-
torswithz(cid:21)2orz(cid:21)−2wereconsideredtobeactivatedorinhibited,respectively.
cDNAsynthesisandquantitativeReal-TimePolymeraseChainReaction
(qRT-PCR)
RNAwasharvestedusingtheNucleoSpinRNA/Proteinkit(Macherey-NagelGmbH).The
amountofRNAwasmeasuredspectrophotometricallyusingaNanodropND-1000Spectro-
photometer.cDNAwasgeneratedfrom2μgofRNAusingtheiScriptcDNAsynthesiskit(Bio-
RadLaboratories).Randomhexanucleotideswereannealedfor5minat25˚C.cDNAsynthesis
wasperformedfor30minat42˚C,followedbyanenzymeinactivationstepfor5minat85˚C.
cDNAwasstoredat-20˚Cuntiluse.1μlofthecDNAreactionmixwasusedforqRT-PCR,
whichwasperformedusingiQSYBRgreenreagents(Bio-RadLaboratories)ontheiQ5Multi-
colorReal-TimePCRDetectionSystem(Bio-RadLaboratories)withthefollowingPCRprofile:
95˚Cfor3min,40cyclesof95˚Cfor10sand55˚Cfor30s.AfterthecompletionofthePCR,
meltcurveanalysiswasperformedusingthefollowingparadigm:95˚Cfor1min,55˚Cfor1
minfollowedbyrampingupthetemperaturefrom55˚Cto95˚C.RPL19wasusedasacontrol.
Results
CharacterizationofSH-SY5Ycells
ThegoaloftheseexperimentswastocharacterizetheeffectsofBacopaonfunctionalproper-
tiesandgeneexpressionprofilesofSH-SY5Yhumanneuroblastomacells.Inthepresenceof
serum,undifferentiatedSH-SY5Ycellscanbepropagatedindefinitely,whiletheycanbemade
toterminallydifferentiatebywithdrawingserum,platingonaproperlycoatedsurfaceand
additionofdifferentiatingfactors.SH-SY5Ycells(passagenumber27,www.atcc.org)weredif-
ferentiatedbyplatingthemonlaminin-coatedcoverslips,usingaprotocoloptimizedby
Dwaneetal[30],whichconsistedofremovingfetalbovineserum(FBS)andadding50nM
InsulinGrowthFactor1(IGF-1).Cellsshowedadifferentiatedneuronalphenotype(forma-
tionofextensiveneurites)after3–5days(Fig1B).Inordertoobtainabettervisualizationof
theindividualcells,wetransfectedthecultureswithacDNAencodingGreenFluorescentPro-
tein(GFP)andusedfluorescencemicroscopytodocumentthedifferentiation(Fig1Dand
1E).Fig1illustratestheresultsofdifferentiationonthemorphologyofSH-SY5Ycells.Upon
differentiation,therewasasignificant3.4foldincreaseintheneuritelengthintheSH-SY5Y
cells(undifferentiatedcells:19.4μm;differentiatedcells:66.2μm,studentt-test,P-
val=0.0001)(Fig1E).
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EffectofBacopaongeneexpressioninhumanneuroblastomacells
RNA-Seqanalysis
InordertounderstandthechangesingeneexpressionlevelscausedbyBacopa,weperformed
acomprehensiveRNA-SeqanalysisforbothundifferentiatedanddifferentiatedSH-SY5Yneu-
roblastomacells.Twodifferentconcentrationsandtreatmenttimeswereused:10μg/mlfor4
hoursand3μg/mlfor24hours.Theseconcentrationsweredeterminedinpilotexperimentsto
bethehighestconcentrationsthatwerenotdeleterioustothecells.Vehicle(DMSO)wasused
asacontrolfortheBacopaeffect.Inaddition,weevaluatedtheeffectofdifferentiationitself.
Foreachcondition,thesequenceof50millionmRNAfragments(“reads”)wasobtained
whichwerethenmappedtothehumangenome,asdescribedintheMethodssection,resulting
inarelativeabundanceforallmRNAsexpressedatareasonablelevel.Foreach“treatment”,a
listofdifferentiallyexpressedmRNAswasgenerated,usingaP-valuesmallerthan0.05andan
absolutefold-changeofatleast2.Table1summarizestheresults.
Effectofdifferentiation
DifferentiationofSH-SY5Ycellswasaccomplishedbygrowingthemonlamininandreplacing
serumwithIGF-1.Cellsshowedadifferentiatedphenotype(formationofextensiveneurites)
after3–5days(Fig1B,1Dand1E).TheRNA-Seqanalysisdetected31,500differenttranscripts
inSH-SY5Ycells.Duetoalternativesplicing,thenumberofdistinctmRNAsinacellistypi-
callylargerthanthenumberofgenes(~23,000)inthehumangenome.Thedifferentiationpro-
tocolresultedinachangeof502mRNAlevels,ofwhich78couldbeclassifiedastranscribed
from‘neuronal’genes(S1Table).Theresultsindicatedthat(1)ourdifferentiationprotocol
waseffectiveinalteringthegeneexpressionprofiletowardsamoreneuronalphenotype,and
(2)theRNA-SeqapproachcanbeusedtoidentifychangesinmRNAlevelsinanun-biased
mannerandatagenomewidescale.
EffectofBacopatreatment:Individualtranscriptlevelanalysis
Table1summarizestheeffectoffourdifferentBacopatreatmentprotocols(2concentrations/
durationsinundifferentiatedanddifferentiatedSH-SY5Ycells)onthemRNAprofileofthe
neuroblastomacells.The4-hourtreatmentwithBacopaalteredtheexpressionofmore
mRNAsthanthe24-hourBacopatreatment:57and66vs.37and29alteredmRNAsinundif-
ferentiatedanddifferentiatedcells,respectively(Table1).Thisindicatedthattheeffectof
Bacopaongenetranscriptionseenafter4hourssubsidesafteronedayformanyoftheaffected
transcripts.4hoursofBacopaondifferentiatedcellswasmosteffective,with66mRNAsbeing
alteredatleast2-fold(Table1).Thisdatasetwasthereforeselectedforfurtheranalysis.Agene
ontologyanalysiswasconductedusingPartekGenomicsSuite.Fig2illustratestheresults,
Table1. NumberofmRNAsalteredbydifferentiationandBacopatreatment.
Effectof #mRNAswithfoldchange>2 #neuronalmRNAs
Differentiation(laminin,IFG-1) 502 78
10μg/mlBacopa4hours(Undifferentiated) 57 1
3μg/mlBacopa24hours(Undifferentiated) 37 4
10μg/mlBacopa4hours(Differentiated) 66 20
3μg/mlBacopa24hours(Differentiated) 29 4
WeconsideredmRNAlevelstobealterediftheP-valuewassmallerthan0.05andtheabsolutevalueofthefoldchangewaslargerthan2.Fourhoursof
Bacopaondifferentiatedcellswasmosteffective(highlightedinboldanditalics).
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Fig2.ResultsofthegeneontologyanalysisbyPartekGenomicsSuite.Thebargraphsindicatethepercentageoftranscripts
thatbelongtoBiologicalProcesses(blue),CellularComponents(red)andMolecularFunctions(green)thatweremostaffectedby
Bacopatreatment.
https://doi.org/10.1371/journal.pone.0182984.g002
listingthebiologicalprocesses,cellularcomponentsandmolecularfunctionsaffectedby
Bacopatreatment.Manyoftheaffectedbiologicalprocessesreferredtofunctionsthatwereof
criticalimportanceinthecentralnervoussystem.Table2summarizesthemRNAsalteredby
Bacopatreatmentencodedbygeneswithaneuronalfunction.Themoststrikingfindingwas
theNeuroplastingene(NPTN),becauseasinglenucleotidepolymorphism(SNP)intheNeu-
roplastinlocusassociateswithcorticalthicknessandintellectualabilityinadolescents[36].
Neuroplastinisasynapticglycoproteininvolvedinlong-termpotentiation(LTP)inhippo-
campalCA1synapsesthatmodulatesneuritogenesisandneuronalplasticity[37,38].
EffectofBacopatreatment:Genelevelanalysis
Theanalysisdescribedabovewasbasedonmappingthereadstoindividualtranscriptsofthe
humangenome(seeMaterialsandmethods).Becauseoftheextensivealternativesplicingseen
formanygenes,aprocessthatismostprominentinthebrain,mappingthereadsisadifficult
processandboundtobeerror-prone.Wehavethereforeusedanadditionalanalysis,whichis
morerobust,inwhichthereadsmappedtoallexonsbelongingtoagenewerecombinedto
provideagenelevelstatistic.SeveraladditionalgenesregulatedbyBacopawereidentifiedthis
way,assummarizedinTable3.
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Table2. SummaryofneuronalgeneswhosemRNAswerealteredbyBacopatreatmentofSH-SY5Ycells.
Gene Genename Fold Function
Symbol change
HNRNPC HeterogeneousnuclearribonucleoproteinC 19.7 PromotesAPPtranslationbycompetingwithFMRPforAPPmRNA
recruitmenttoPbodies
CIB2 Calciumandintegrinbindingfamilymember2 6.7 RoleinCa2+homeostasisandCa2+regulationinthemechano-transduction
process;Mutationscausedeafness
NPTN Neuroplastin 6.6 Regulationoflong-termneuronalsynapticplasticity,cytosolicCa2+ion
concentration,neuronprojectiondevelopment;SNPsassociatedwith
cognitiveabilitiesinadolescents
COMMD6 COMMDomainContaining6 6.4 NF-KappaBbinding;Regulatestranscriptionfactoractivity,geneexpression
NDUFA5 NADHDehydrogenase1AlphaSubcomplex5 (4.7) Mitochondrialtransport
CHAC1 ChaCGlutathione-SpecificGamma- 4.0 NegativeregulatorofNotchsignalingpathwayinvolvedinembryonic
Glutamylcyclotransferase1 neurogenesis;Promotesneurogenesisinembryos
AP2S1 Adaptor-RelatedProteinComplex2,Sigma1 3.8 Synaptictransmission;RegulatesEGFR,TRKreceptor,ephrinreceptor
Subunit pathway
KCNMA1 KChannel,CaActivatedLargeConductanceM (3.3) Regulationofmembranepotential;Synaptictransmission
Alpha1
NGFR NerveGrowthFactorReceptor 3 Mediatescellsurvivalandcelldeathofneuralcells;Necessaryforcircadian
oscillationinsuprachiasmaticnucleus
STRN3 Striatin-3 2.6 GlutamateregulationofdopamineD1Areceptorsignaling
PRKACB ProteinKinase,CAMP-Dependent,Catalytic, (2.6) MediatessignalingthroughcAMP;Involvedinneuronalstructureand
Beta signaling
LDHA LactateDehydrogenaseA (2.5) Substantianigradevelopment
MTMR2 MyotubularinRelatedProtein2 2.3 MutationsresultinCharcot-MarieToothdiseasetype4B,anautosomal
recessivedemyelinatingneuropathy
VCL Vinculin 2.3 Involvedinregulationofactincytoskeleton,axonandneuronprojection
extension;Hasanegativeregulationoncellmigration
WDR1 WDRepeatDomain1 2.3 Involvedinsensoryperceptionofsound,regulationofoligodendrocyte
differentiationorgliogenesisandneurogenesis
DBI DiazepamBindingInhibitor(GABAReceptor (2.3) ModulatessignaltransductionatGABA receptors;Displacesdiazepam
A
Modulator,Acyl-CoABindingProtein) fromthebenzodiazepinerecognitionsiteinGABA receptor
A
EFNB2 Ephrin-B2 (2.3) Mediatedevelopmentofthenervoussystem;Crucialformigration,repulsion
andadhesionduringneuronaldevelopment
ACTB Actin,Beta 2.2 Involvedinaxonogenesis,axonguidance,neuronprojection
morphogenesis,substantianigradevelopment,ATP-dependentchromatin
remodeling,ephrinreceptorsignalingpathway
ACTG1 ActinGamma1 2.2 AssociatedwithDFNA2-/26,asubtypeofautosomaldominantnon-
syndromicsensorineuralprogressivehearingloss;InvolvedinRassignaling
pathway,axonalguidance
SLC38A1 SoluteCarrierFamily38,Member1 2.2 Glutaminetransporter,precursorforthesynaptictransmitter,glutamateand
GABA;Involvedinsynaptictransmissionandneurotransmitterreuptake
STMN4 Stathmin-Like4 (2.2) Involvedinneuronprojectiondevelopment,microtubuledepolymerization,
neuronalplasticity,rapidlyinducedafterseizureorLTP
CALR Calreticulin 2.1 MajorCa2+-bindingproteininthelumenoftheER;Essentialforintegrin-
mediatedsignalingandcelladhesion
SLC1A4 SoluteCarrierFamily1(Glutamate/NeutralAmino 2.1 AssociatedwithHartnupdisorder;Chloridechannelactivity;Transporterfor
AcidTransporter),Member4 alanine,serine,cysteineandthreonine,sodiumdependent
KLHL24 Kelch-LikeFamilyMember24 (2.1) Reduceskainatereceptor-mediatedcurrentsinhippocampalneurons
ATF4 ActivatingTranscriptionFactor4 2 Transcriptionalactivator;ProtectsagainstneuronaldeathinParkinson’s
disease;Involvedinneurodegeneration;Mayconstrainlong-termsynaptic
changesandmemoryformation
NRCAM NeuronalCellAdhesionMolecule (2.0) Involvedinneuron-neuronadhesion;Promotesdirectionalsignalingduring
axonalconegrowth
STMN1 Stathmin1 (2.0) Requiredforaxonformationduringneurogenesis;Involvedinthecontrolof
learnedandinnatefear
(Continued)
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EffectofBacopaongeneexpressioninhumanneuroblastomacells
Table2. (Continued)
Gene Genename Fold Function
Symbol change
XRCC6 X-RayRepairComplementingDefectiveRepairin (2.0) Involvedinbraindevelopment;Positiveregulationofneurogenesis
ChineseHamsterCells6
https://doi.org/10.1371/journal.pone.0182984.t002
ValidationoftheRNA-sequencingresults:qRT-PCR
Next,wevalidatedtheRNA-SequencingresultsusingqRT-PCRexperimentsforasubsetof
genes.Fig3summarizestheresultsbyshowingtheabsolutevalueofthefoldchangeproduced
byBacopatreatment.Inthecaseofgeneswithalternativelysplicedtranscripts,PCRprimer
pairsweredesignedthatwereuniqueforthemRNAisoformthatwasalteredbyBacopa.In
somecases,thisprovedtobehard,sincetheexonthatwasuniqueforthealteredtranscript
wasveryshort.AcaseinpointisNeuroplastin,whichhasfourmRNAvariants,oneofwhich
isaffectedbyBacopa(NPTN_D).Thedistinguishingfeatureoftheaffectedvariantisthe
absenceofexon2andthatexon7isshortenedby12nucleotides.Wewereabletovalidatethe
Table3. GenesregulatedbyBacopaidentifiedbygene-levelanalysisoftheRNA-Seqdata.
Gene Genename Fold Function
Symbol Change
HBA2 Hemoglobin,Alpha2 405 Oxygen-transportmetalloproteinintheredbloodcells
HBB Hemoglobin,Beta 370 Oxygen-transportmetalloproteinintheredbloodcells
HBA1 Hemoglobin,Alpha1 292 Oxygen-transportmetalloproteinintheredbloodcells
ANKRD1 AnkyrinRepeatDomain1 21.7 Transcriptionfactorinvolvedindevelopmentandunderconditionsof
stress
SLC7A11 SoluteCarrierFamily7Member11 7.5 Transporterthatantiportsglutamateforcysteine
SERPINE1 SerpinPeptidaseInhibitor,CladeE 6.8 Serineproteaseinhibitorthatfunctionsastheprincipalinhibitoroftissue
plasminogenactivatorandurokinase
WNT8B Wingless-TypeMMTVIntegrationSiteFamily, 6.8 Wntisoformspecificforthedevelopingbrain
Member8B
HIST1H4K HistoneCluster1,H4k 5.3 HistoneH4isoform
HIST1H4J HistoneCluster1,H4j 5.2 HistoneH4isoform
CCL2 Chemokine(C-CMotif)Ligand2 4.8 Recruitsmonocytes,memoryTcells,anddendriticcellstositesof
inflammationproducedbyinjuryorinfection
CHAC1 ChaCGlutathione-SpecificGamma- 4.5 Proapoptoticcomponentoftheunfoldedproteinresponse;Downstreamof
Glutamylcyclotransferase1 theATF4-ATF3-CHOPcascade
NTS Neurotensin 4.4 Neuropeptideimplicatedintheregulationofhormonerelease;Has
interactionwiththedopaminergicsystem
ANGPTL4 Angiopoietin-LikeProtein4 4.0 Inducedunderhypoxicconditions;Serumhormonedirectlyinvolvedin
regulatinglipidmetabolism
PTPRH ProteinTyrosinePhosphatase,ReceptorType, (3.8) Ubiquitouslyexpressed;Upregulatedingastrointestinalcancers
H
TXNIP ThioredoxinInteractingProtein (3.8) Glucocorticoid-regulatedprimaryresponsegeneinvolvedinmediating
glucocorticoid-inducedapoptosis
YPEL4 Yippee-Like4(Drosophila) (3.6) Nuclearprotein;ActivatesElk-1intheMAPKsignalingpathway;Possible
functionincelldivision
CNN2 Calponin2 3.5 Actin-bindingproteinimplicatedincytoskeletalorganization
RAPGEF4 Rapguaninenucleotideexchangefactor4 (3.0) EPAC2;Mayregulatesynapticplasticity
STON1 Stonin1 (3.0) Componentoftheendocyticmachinery
FMN1 Formin1 (3.0) Roleintheformationofadherensjunctionandthepolymerizationoflinear
actincables
https://doi.org/10.1371/journal.pone.0182984.t003
PLOSONE|https://doi.org/10.1371/journal.pone.0182984 August23,2017 9/21
EffectofBacopaongeneexpressioninhumanneuroblastomacells
Fig3.AbsolutevaluesofFoldChange(absFC)causedbyBacopatreatment.RT-PCRwasperformedonundifferentiatedcellsand
differentiatedcellswhichweretreatedwithvehicle(DMSO)orwithBacopaforeither4h(blue)or24h(orange).Thegrayareaindicatedan
absFCvaluesmallerthan1.Genesmarkedwith*wereresultsfromthetreatmentonundifferentiatedcells.(1)NPTN_Aand(2)NPTN_A
wereresultsgeneratedfrom2setsofprimersprimingforNPTNtranscriptA.
https://doi.org/10.1371/journal.pone.0182984.g003
effectsofBacopaonthetranscriptsidentifiedbythegene-levelanalysis(ANKRD1,SLC7A11,
CHAC1,TXNIP,YPEL4andSTON1)(Table3).However,forthemRNAsforwhichonlyone
orafewofthealternativelysplicedvariantswereaffectedbyBacopa,thevalidationwasless
successful:theeffectswereeithermuchsmallerthanthoseseenintheRNA-Seqanalysis
(HBA1andHBA2)(Table3)ortherewasnomeasurableeffect(NPTN_D)(Table2).
PLOSONE|https://doi.org/10.1371/journal.pone.0182984 August23,2017 10/21