Effects of antidepressants on glucocorticoid receptor binding
1. Effects of antidepressants on glucocorticoid receptor
binding and downstream gene expression
Student: Kate Jones Supervisor: Angela Bithell
I confirm that this is my own work and the use of materials from other sources has been
properly and fully acknowledged.
Signature: Date:16/4/2015
2. Abstract
Cortisol isa glucocorticoidhormone knowntoaffect the expressionof several genesassociatedwith
hippocampal neurogenesis,howeverinmanycasesit isunknownwhetherthisisthroughdirect
bindingof the glucocorticoidreceptor(GR) tothe targetgene.Inorderto investigate this,we
establishedaworkingchromatinimmunoprecipitation(ChIP)procedure tobe usedinthe analysisof
GR protein-DNA interactions.Usingthis methodologyinahumanhippocampal neural progenitorcell
line (HPC03A/07),we foundthatthe GR bindsdirectlytoat leastone suchtargetgene, Sgk1,and
that exposingcellstodexamethasoneincreasesGRbindingatthislocus whencomparedtocontrols.
Thissuggests thatup-regulationof Sgk1mayoccur in timesof stress. Previousstudiesindicate that
Sgk1 playsa role inmediatingcortisol-inducedreductionsinneurogenesis.Thus,understanding how
thisisactivatedmay helptofurtherunderstandits role andpotential asatherapeutictarget.
Throughimmunofluorescence staining,we alsoobservedthatsertraline mayreduce cortisol-
dependantreductionsinneuronal proliferationanddifferentiationinHPC03A/07 cells. Thissupports
the hypothesisthatthe therapeuticefficacyof sertraline isbasedonaGR-dependantmechanismin
a subsetof patientswithdepression.
3. Introduction
Depressionisacommonaffective disorder, prevalentin approximately14% of the global population
and contributingsignificantdetrimentaleffects topatientqualityof life,withwidersocial and
economicimplications (Mitchelletal.,2009). Whilstseveral clinicalsubtypesexist,common
symptomsinall formsof major depressive disorderinclude anhedonia, feelingsof guilt, changesin
appetite and/orsleepandpersistentlylow mood. (Cizzaetal.,2012). The exactcause of depression
isnot fullyunderstood,howeverthere are several psychological,sociological and biological theories
that attemptto addressthe disorder’sunderlying,oftenmultifactorial,pathology.
Biologically, the monoamine theoryisperhapsthe mostwell-knownpathological cause.The
underlyingprinciple isthatdepressedpatientsexhibitreducedsynaptictransmissionof dopamine,
noradrenaline and/orserotonin.Originally,itwasthoughtthatthiswascausedby reducedactivityof
the pre-synapticmonoaminergicreceptors,resultinginasubsequentmonoaminergicdeficit across
the synapses.The monoamine theoryissupportedbythe factthat all majorclassesof
antidepressantdrugs(mono-amineoxidase inhibitors,tricyclicantidepressantsandselective
serotoninre-uptakeinhibitors(SSRIs)) agonise the effectof atleastone monoamine (Morrissette
and Stahl,2015). Furthermore,monoamine antagonistssuchasreserpineare knowntocause
depressionasaside effect (Antkiewicz-Michaluketal.,2014). Low levelsof serotoninmetabolites
have alsobeencorrelatedwithdepression(BrownandLinnoila,1990).
However,antidepressantdrugshave beenshowntoincrease monoaminergiclevelsrapidly,yet
clinical improvementinpatientsisnottypicallyseenuntilafter approximately 3weeks of treatment.
To account for this,revisedversionsof the theory have been proposed.One version suggeststhat
depressedpatientsexhibitoveractive monoaminergicactivityandthatthe delayinclinical efficacyof
these antidepressantsiscausedbythe time takenformonoaminergicpost-synapticreceptorsto
become desensitised.The second,more established, hypothesissuggeststhatdepressedpatients
have more sensitive presynapticautoreceptors,whichare responsible forsendinginhibitorysignals
towardsmonoamine release.Inthistheory, antidepressantdrugs initiallytriggeradecrease in
monoamine release asthe receptorsrespondandincrease inhibitorysignalling.However,overtime,
the receptorsbecome lesssensitive andthusmonoaminergicreleaseacrossthe synapse isincreased
(Elhwuegi,2004).
The monoamine theory of depressionisreasonable butnotwithoutitsassumptions (Sapolsky,
2004). Critically,bothmodelsusedtoexplainthe delay inatreatment’sclinical effect relyonthe
conceptthat neural compensationwill occurinresponse tomonoamineneurotransmitterlevel
fluctuations. Additionally,resistance tocurrentpharmacological treatment withdrugsthatact on
thissystemissignificant.Approximately one thirdof patientsexhibit aninadequateresponse to
first-linemonotherapyandaround10% of patientsexperience prolongedstatesof depression
regardlessof multiple treatments (Soueryetal.,1999, NierenbergandAmsterdam,1990).
An emerging,alternative theorysuggeststhatinasubsetof patientswithdepression,dysfunctional
hippocampal neurogenesis withinthe granularlayerof the dentate gyrus couldbe a significant
causative factorand mightpartiallydictate individual responseto anti-depressive treatment
(Boldrini etal.,2012). Thishas beencorrelatedwithobservationsof lowergranularcell layer
volumesindepressedpatients andincreasedhippocampal neurogenesisinpatientstreatedwith
SSRIs(Kempermann,2002,KempermannandKronenberg,2003). Furthermore, the timingof onset
for therapeuticefficacyinSSRIs issimilartothe time ittakesforthe formationof neuronesfrom
4. theirrespective neural stemcells. (Ge etal.,2007, Jacobset al.,2000). Zhao etal showedthatneural
stemcellswere functionallyintegratedintothe hippocampus of mice after2-3weeksandwere
requiredforthe expression of trace memory (Zhaoetal.,2006). There isa lackof directevidence
determiningthe functionalrole of neurogenesisinhumans,howeverthe proliferation rate and
relative abundance of these neurones iscomparable tothatobservedinmice. Insuchstudies,
furthereffectsonbehaviourhave beenidentifiedandinclude the abilitytoseparate memories,a
feature lackinginsome patientswithdepressionandanxiety (Spaldingetal.,2013).
Much of the current research onthe hypothalamic-pituitary-adrenal (HPA)axis’involvementinthe
stressresponse indicatesatwo-way relationshipbetween the HPA axis regulation andhippocampal
neurogenesis.Inthe presenceof acute physiological stress,the hippocampussignalsforincreased
levelsof corticotrophinreleasinghormone (CRH),whichtriggersa cascade of events associatedwith
the HPA axisthatleadto increasedplasmalevelsof cortisol (Anacker,2014, Anackeretal.,2011a,
Holsboeretal.,1984). In mice withlowlevelsof hippocampal neurogenesis,increasedlevelsof
cortisol were observedfollowingstress,suggesting anincreasedHPA axisresponse (Schloesseretal.,
2009, Snyderetal.,2011).
Cortisol isa steroidhormone thatacts onglucocorticoid(GR) andmineralocorticoidreceptors(MR).
Both GR and MR are nuclearreceptorswithwidespread expression inthe body. Cortisol-GRbinding
causesdissociationfromthe GRinhibitorycomplex,resultinginaconformational shape change that
increasesthe availabilityof the corresponding nuclearlocalisationsequence.Subsequent
translocationof GR monomersanddimers fromthe cytoplasmtothe nucleusallowsthe GRto bind
to glucocorticoidresponseelements(GREs) andnegative glucocorticoidresponse elements(nGREs)
respectively, leadingtogene transcription viamodulationof factorsthatinfluence chromatin
configuration (Herrlich,2001, Aoyagi andArcher,2011). Otherresearchalsoshows translocationto
mitochondriawhere they influence mtDNA gene expression(Duetal.,2009). A previousstudyby
Anackeretal showedthatdexamethasone couldbe used in-vitro insteadof cortisol toelicitthe
same agonistresponse atthe GR (Anackeretal., 2011b).
Recentin-vitro studies usingahumanhippocampal neural progenitorcellsline(HPC03A/07) have
showninterestingresults relatedtohippocampal neural progenitorcell (NPC)proliferationand
differentiation whenexposedtovaryinglevelsof dexamethasone andSSRIs.Inuntreated NPCs,
there wasa high rate of proliferationbutlow differentiation. Withexposure tosertraline,
proliferationwaslowerbutdifferentiationwashigherwhileinNPCs treatedwithhigh
concentrationsof cortisol (1µm) alone,proliferation anddifferentiation remainedlow. InNPCs
exposedto highconcentrationsof cortisol andsertraline, proliferation anddifferentiation washigh
(Anackeretal.,2011b). These findingssuggested thattoincrease hippocampal neurogenesis
throughthe use of sertraline,GRagonistactivitybyhighlevelsof cortisol are required asapre-
requisite. Thiswasfurthersupportedbyresultsof decreasedneurogenesisfollowingGR-antagonism
withRU486. Froma broader perspectivethis impliesthatina subsetof patients,the therapeutic
effectof sertraline isonlyinitiatedinresponse toa cortisol-induced depressivestate.
Furthermore,exposure of progenitorcellstohigh levelsof cortisol and sertraline mayresultin
unique GRphosphorylationandGR-dependentgeneexpression. Anackeretal foundthatthe
PDE4/PKA signallingcascade regulatedthe effectsof sertraline oncell proliferationand
differentiationbyalteringthe phosphorylationstate of the GR,dependingonthe combinationof
conditionsused (Anackeretal.,2011b). Thiswas shownbydifferentpatternsof phosphorylationat
the receptor’sserine residuesS203, S211 and S226 whencortisol andsertraline concentrationswere
varied.The subsequentphosphorylationstate of the GRwas then demonstratedtomediate the
levelsof p27kip1
and p57kip2
gene expression. P27kip 1
andp57kip2
are CDK2 inhibitorswhichpromote
5. the terminationof cell divisionand increase differentiation.Levelsof p27kip1
and p57kip2
expression
were showntobe consistentwiththe proliferationanddifferentiationstatesof the cells.The
conceptthat targetgenesof the GR can be selectivelyactivatedviathe promotorregiondepending
on the specificGRphospho-isoformpresent issupportedfromanumberof otherstudies. (Galliher-
Beckleyetal.,2008, BlindandGarabedian,2008, Chenet al.,2008, Kumarand Calhoun,2008,
Websteretal.,1997, Anackeretal.,2011b).
Furtheradvanceshave since beenmade toidentify otherspecificGRtargetgenesthatcontribute
towardshippocampal neurogenesis bothupstreamanddownstreamof the GR.Evidence todate
fromgene expressionanalysis suggests thatGR transactivationstronglyinfluences the level of
expressionof the genes p11and β-arrestin2 (whichrelate tothe serotoninreceptor); CCND1,HDM2
(cell cycle promotinggenes) andthe stress-responsive genes SGK1,FKBP5,FOXO1and GADD45B.
(Anackeretal.,2013a, Anackeretal.,2013b, Anackeretal.,2011b). However,itis unknownwhether
the expressionof the geneslistedabove isthroughdirectactivationand epigeneticmodifications
mustbe considered.Furthermore,we hypothesisethatthere are othergeneslinkedtohippocampal
neurogenesisthatare yetto be identified.
Cortisol causesincreasedexpression of SGK1and isof particularinteresttoourresearch.A previous
study by Anackeretal identifiedSGK1as a key inhibitory mediatorof the Hedgehogsignalling
pathway,whichisa major cause of cortisol-induced reductions inneurogenesis (Anackeretal.,
2013b). Furthermore, SGK1increasesGRfunctionupstreamandregulatesgenesdownstreamof the
GR thatare involvedinreducing NPCproliferation. Ithasbeenshowntopotentiate the effectsof GR
activationevenaftercortisol isremovedbyfacilitatingGRtranslocationtothe nucleus.SGK1
expressionwasfoundtobe increasedinthe hippocampusof bothpatientswithdepressionandin
mice exposed tostress. However,these studiesdonotfullyelucidate the extentof the effectsof
SSRItreatment,incombinationwithcortisol,onSGK1expressionindepressedpatients.
In thisstudy, we firstaimedtodeveloparobustmethodtoperform chromatinimmunoprecipitation
(ChIP) assaysusingDNA fromthe HPC03A/07 cell line todetermine directtargetgenesof the GR(via
directbinding).GRproteins,taggedwithcorrespondingantibodies,will be addedtothe chromatin
samples thatwere extractedfromHPC03A/07 NPCs,treatedwith varyingconcentrationsof
dexamethasone orethanol,the vehiclecontrol. The antibodieswillbe usedtorecognise the amount
of GR that isboundto specificgene loci withinthe chromatin,inthe differentconditions. We will
alsoconduct qualitativeanalysisof immunofluorescentlylabelledHPC03A/07cells todetermine the
effectsof dexamethasoneandsertralineonchangesincellulardifferentiationandproliferation.
6. Materials andMethods
Cell culture
Thissectionof the methodologywasperformedbyDrBithell.
HPC03A/07 is a multipotent,immortalised humanfoetal hippocampalprogenitorcell line usedin
these experiments. Cellswereimmortalisedusingthe c-myc-ER™ transgeneandtreated for3 days
withethanol (vehicle,EtOH),dexamethasone(Dex)and/orsertraline (Sert) inproliferative
conditions (withepidermal growthfactor(EGF),fibroblastgrowthfactor2 (FGF2) and 4-
hydroxytamoxifen(4-OHT).Theywere thendifferentiatedusingreducedmodifiedmedium without
EGF. FGF2, 4-OHT andwithremoval of othertreatments. Asanadditional control,some cellswere
leftuntreated inthe proliferative stage.Atproliferationday3and thenafter2 weeks of
differentiation,cellswerefixedandusedforimmunofluorescenceanalysis.Forchromatin,cellswere
treatedfor1hr withvehicle ordexamethasone inproliferationconditionsbefore harvestingfor
chromatin. Furtherinformationcanbe foundunder Appendix 3H:Tissue Culture Methodsfor
ReNeuron Cells (HPC03A/07).
Chromatinimmunoprecipitation (ChIP)
Thissectionof the methodologywasperformedbyDrBithell.
Cellswere fixedin1%HCHO inPBS for5 minutes thenquenchedin630 µl of 2M glycine for5
minutes toproduce a final glycine concentrationof 0.125M. Theywere thenwashed3times with
ice-coldphosphate bufferedsaline (PBS),scrapedoff dishesandcentrifugedat1300rpm for 5
minutesat4°C. The final PBSwashcontained1x protease inhibitors. Pelletswerethenre-suspended
inice-coldLysisBuffercontaining protease inhibitorandincubatedonice for30 minutes. A
microfuge wasusedtospin pelletnucleiat5000rpm for 10 minat 4°C before re-suspensioninto
shearingbufferwithproteaseinhibitorsin17ml Falcontubes,storedonice.Chromatinwas then
sonicatedincyclesof 30 secondson,30 secondsoff,until the shearedchromatinwaswithin200-
600bp (determinedbyde-crosslinking25µl of each sample in200µl usingMilliQwater,8µl 5M NaCl
and 10µg RNase A for4h at 65°C. 10µl of Proteinase Kwasthenaddedandsampleswere incubated
for 2hr at 42°C before DNA cleanupand gel electrophoresis).The sharedchromatinsampleswere
centrifugedat10,000 rpm at 4°C for 10 minutesandsupernatantchromatinremovedforuse.
Protein-Gmagneticbeads(PGMbeads) were firstpre-blockedbyrotatingat4°C 4x 10mins inmRIPA
with1x protease inhibitorsand1mg/ml Bovine SerumAlbumin(BSA),thentwice with1ml mRIPA
bufferwith1x protease inhibitors.Chromatinwaspre-clearedusing20µl of proteinG magnetic
(PGM) beadsineachchromatinsample (Dex orEtOH) dilutedinmRIPA bufferwith1x protease
inhibitorsandrotatedduringincubationfor2h at 4°C. 20µg of chromatinwasusedfor eachChIP
reaction(36.7µl of Dex or 39.22µl of EtOH chromatindilutedto100µl in mRIPA/protease inhibitors).
PGM beadswere thencapturedand10µl of sample wasretainedatthisstage for use as input
chromatin.ChIPsampleswere setupinproteinLoBindtubeswith2.5µl or 5µl (5µg) of antibody,
377.5µl or 375µl of mRIPA bufferrespectively,100µl of pre-clearedchromatin(20µg) and20µl of
25x protease inhibitors. The followingantibodieswere used:anti-REST,rabbitIgG(providedby
Millipore);rabbitIgG(negative control providedbySigma);mouse monoclonal antibodyIgG2b
(provided byDiagenode) andrabbit pAb(providedbyThermoScientificPierce).Sampleswere
incubatedat4°C on a rotator overnight.Pre-blockedPGMbeadswere addedtoeachrotating
sample at4°C for fourhours.Supernatantwasremovedusingamagneticstandto pelletthe beads.
800µl Wash Buffer1 was added2x,each for3mins ona rotator,thenthe same technique wasused
for WashBuffer2 (1 x 3mins) and2x 3minsTris-EDTA (TE). Washedbeadswere re-suspendedin
7. 100µl Elutionbuffer(alsofor10µl inputsamples). 4µl 5M NaCl and1µl RNase wasthenaddedand
sampleswere incubatedat65°C for4 hoursto de-crosslink.
At roomtemperature,2µl Proteinase K (10mg/ml) wasaddedthensampleswere incubatedat 42°C
for 2h, thenallowedtoreturntoroom temperature again.Magneticbeadswere re-captured,and
supernatant(containingDNA) removed forclean-up.DNA waspurifiedusingaQiagenQIAquickPCR
cleanup kitaccordingto the manufacturer’sinstructions.Thisinvolvedmixingsampleswith5x
volume of bindingbuffer andapplyingtoacolumn.Thiswasspun at 13000rpm for 30-60s. Flow-
throughwas discardedand0.75ml washbufferPE wasappliedtothe columnanda furtherspinat
13000rpm for 30-60s was conducted.Flow-throughwasdiscardedagain,the columnwasspunat
13,000rpm for2mins andthe columnwasplacedina new 1.5ml Eppendorf tube.The DNA was
elutedinto50µl of HPLC H2O bycentrifugationat13000rpm for1min.Sampleswere storedat -20°C
until usedinQPCR.
Quantitative Real-time polymerase chain (QPCR) reactions
Thissectionof the methodologywasperformedbyKate Jones.
ChIP DNA samples (see above) were usedinqPCRreactionsto investigate changesinGRbindingby
amplificationof boundtargetsequences. Primersdesignedtobe compatible with Dscam,Gilz,Mt2a,
Sgk1 and Slc19a (see Appendix 3A:Forward and ReversePrimerSequences) were prepared instock
solutions,storedin1.7ml Eppendorf tubes, tobe usedin20µl reactions in96-well plate assays.Each
reactioncontained0.4/1.0µl of 10µM forward primer,0.4/1.0µl of 10µM reverse primer, 10µl of 2x
SYBR® greendye (Biorad),2µl of ChIP DNA and 6.6/6.0µl of high-performance liquidchromatography
(HPLC) grade water.The reagentswere mixed inamastermix withoutChIPDNA,aliquotted,and
each ChIP DNA sample added,includingwater/negative controls.Quantitative analysiswasbasedon
standardcurves for eachgene,usingthe average value of 2 duplicates containing0.1, 0.3, 1.0, 3.0,
10.0 and 30.0ng/ml. StepOnePlusfromAppliedBiosystems wasusedtoperformthe polymerase
chainreactionover3.5 hoursand subsequentanalysiswasthrough StepOnePlus software. The
polymerase chainreactionwasranover40 cycleswitheachsteptaking30s. Denaturationtookplace
at 95°C, annealingat60°C andextensionat72°C.
Immunofluorescence Staining/Immunocytochemistry
Thissectionof the methodologywasperformedbyDrBithell andKate Jones.
Mediumwas aspiratedfromthe HPC03A/07 culture mediumandexcessdebriswasremoved
throughcareful rinsingwithPBS.4%paraformaldehydewasaddedtopreserve cellsthroughcross-
linkingfor10 minutes,thenterminatedby3washeswith PBS.Cell membraneswerepermeabilized
using0.1% TX-100 (triton) inPBSfor 5 minutesatroom temperature toallow antibodiesaccessto
theirtargetintracellularepitopesandthenwashedagaininPBS.30µl of primary antibody solution
was usedpercoverslipincombinationwith10% normal seruminPBS.
The primaryantibodiesusedwere 1:1000 TuJ1 mouse IgG2a (beta-IIItubulin,fromCovance) which
recognises microtubulesin neurons;1:200 anti-GFAPmouse IgG1(Millipore) whichisspecificto
intermediate filamentsin astrocytes;1:1000 Ki67 polyclonal rabbitIgG(Abcam) whichisa
proliferationmarkerand1:60 anti-humanNestin mouse IgG1(fromR&D Systems),anNSC/NPC-
specificmarker.Sampleswere incubated withoutlightand atroomtemperature for2 hours and
washed with3x PBS to remove unbound antibody.The same stepswere alsoconductedforthe
secondaryantibodies.To examineneuronaldifferentiation, 1:1000 polyclonal goatanti-rabbitIgG
(foruse with anti-GFAP) fluorescentlytaggedwithAF-594and1:1000 goat anti-mouse monoclonal
9. Results andDiscussion
Establishmentof a functional ChIP protocol with REST ChIP
In orderto testthat the chromatinimmunoprecipitationprocedure worked,RepressorElement-1
SilencingTranscriptionFactor(REST) wasuseda positive control ChIP incombinationwiththe
primers fora bindingsite inthe Snap-25promoter(QR1primers,where RESTbinds), andfora site in
the codingregionof a gene where RESTdose notbind(QC3 primers,M4coding). Informationabout
REST’s bindingsite hasalreadybeenwell-establishedandtobe consistentwithpreviousfindings,it
was anticipatedthatthe REST ChIPs wouldshow significantrelativeenrichment atthe Snap-25
bindingsite and minimal enrichmentatthe M4coding site,toact as positive andnegative controls
respectively.
In the experimentinvolvingRESTandQR1 Snap-25,RESTconsistentlyshowedenrichmentbetween
35 and 40 fold relative toIgG.In these samples,GR showed amaximumof 3-foldenrichmentusing
eitherantibody.Comparatively,RESTshowed verylow levelsof enrichmentatthe M4coding region
(at 0.5 foldinDEX and 1-foldin EtOH ChIPs).Interestingly,minorenrichmentwasobservedin GR
ChIPs(1.5-2 in DEX samplesand2.5 to 3-foldinEtOH). Thisislikelytobe the backgroundlevel,
howeveritcouldsuggestlowlevel GRbindingwhichrequiresfurther study.
0
5
10
15
20
25
30
35
40
45
Foldenrichment
Conditions
Figure 1. REST bindsto QR1 Snap 25 significantlymore than GR in both dexamethasone and ethanol
samples.The graph showsthe average relative enrichmentof RESTand GR bindingatthe Snap-25gene
locusrelative toIgG,usingthe resultsof twobiological replicates,eachof whichusedthe average of 3
technical triplicate values. Rawvaluesforthe technical triplicatescanbe viewedin Appendix 3G:QR1
Snap 25 NumericalResults Data.
10. The highdegree of enrichmentof RESTat the RE1 site of the Snap-25gene,incombinationwithno
enrichmentof RESTat the M4coding gene,isconsistentwithwhatwasexpectedandindicatesthat
the ChIPprotocol is functional.Thissuggeststhatthe qualityof the componentsusedwithinthe
protocol,suchas the chromatinandbuffers,were suitable foruse infurtherexperimentsthat
investigatedchangesinGRbinding.
0
0.5
1
1.5
2
2.5
3
3.5
FoldEnrichment
Conditions
Figure 2. REST showsminimal bindingto the m4coding gene. The graph showsthe average relative
enrichmentof RESTand GR bindingatthe M4coding gene locusrelative toIgG,usingthe resultsof two
biological replicates,eachwhichusedthe average of 3technical triplicate values. Raw valuesforthe
technical triplicatescanbe viewedin Appendix 3F:M4coding NumericalResults Data.
11. ChIP assaysusingchromatinextractedfrom (HPC03A/07) usingdifferentantibodies againstGR(from
ThermoScientific(Thermo) andDiagenode)withrabbitIgG(rIgG) as a negative control were
performed toimmunoprecipitate andthus identifydirectGRtarget genes. Cellswere treatedwith
eitherdexamethasone (DEX) orethanol (EtOH),wheredexamethasone mimicscortisol andethanol
acts as a negative vehiclecontrol, tocompare the relative effectsof dexamethasone treatment.By
comparingbothconditions,we were able toqualitativelydefine whetherornot protein-DNA binding
occurs at a specificlocus andthenquantitativelyexaminethe relative effectdexamethasone hadon
the degree of GR binding.Thiswasmeasuredbyfoldenrichment,relativetoIgG. IgG is a non-
specificantibodyusedasanegative control ChIP todeterminethe thresholdlevel of background
noise.
Numerous primerpairs were thenusedtoamplifythe isolated,immunoprecipitatedstrandsof
genomicDNA forquantitative polymerasechainreaction (q-pcr) analysis.These include:Dscam,Gilz,
Mt2a,Sgk1 and Slc19a2. The primersequencesare available inthe Appendix3A:Forward and
Reverse Primer Sequences.
12. Optimisationof ChIPfor GR bindingin NPCs
PotentialGR binding at the Dscamnegativecontrollocus
ModifiedDscam wasusedas a negative control tomeasure the degree of non-specificDNA binding
and subsequentimmunoprecipitation,althoughintheorythisshouldnotexist.Onaverage, DEX-
treatedimmunoprecipitatedwitheitherGRantibodyshowedapproximately2.5-foldrelative
enrichment. EtOH-treatedsamples showed 3-foldand2.5-foldenrichmentforGR ThermoandGR
Diagenode respectively. Enrichmentatthe REST bindingsite waslow indicatingspecificitytowards
the GR and supportingthe conclusionthatthe ChIPprocedure wasfunctional. There isasignificant
amountof variabilitybetweenthe biological replicates thattargetthe GR bindingsite,asshownby
the standard error,particularlyforethanol containingsamples. The resultsshow thatthe GRbinds
to the Dscam gene bothin the presence andabsence of dexamethasone,althoughthe variabilityin
resultsmakesitdifficulttodeterminethe extentof agonistorinhibitoryeffectthatdexamethasone
has on GR bindinginthiscase and furtherstudiesare neededtoelucidatethis.
0
1
2
3
4
5
6
Foldenrichment
Conditions
0
0.5
1
1.5
2
2.5
3
3.5
FoldEnrichment
Conditions
A
B
13. 0
1
2
3
4
5
6FoldEnrichment
Conditions
Figure 3. GR bindsto Dscam in the presence orabsence ofdexamethasone. Graphsshow the relative
enrichmentof GRand REST bindingatthe DSCAMgene relative toIgGcontrols.Infigure 3A,y values
representthe meanaverage of correspondingyvaluesinfigures3Band3C. Infigure 3B and 3C, y values
correspondtothe meanaverage of technical triplicates,excludingresultsdeemedanomalous.Raw
valuesforthe technical triplicatescanbe viewedin Appendix 3B:DSCAMNumericalResults Data.
C
14. GR binding atthe Gilz positivecontrol locus may be inhibited by the presenceof dexamethasone
Glucocorticoid-inducedleucinezipper(Gilz) isa mediatorof GR-dependantimmunomodulation
pathways.Transactivationof the GR increases Gilztranscriptionandso GR bindingatthislocuswas
expectedwithinourexperiments. The gene therefore servesasafurtherpositive control. Ourresults
show GR enrichmentof the Gilzlocus inthe absence of dexamethasoneandinsome samples
containingdexamethasone.Onaverage,approximately2-foldenrichmentwasobservedinGR
samplescontainingdexamethasone,whichisdeemednon-significantanda4.5 foldenrichmentwas
seeninsamplescontainingethanol. Thiscouldsuggestthatdexamethasone hasaninhibitoryeffect
on GR binding. RESTwas notenrichedineitherconditions(DEXorEtOH),withlessthan1-foldin
dexamethasone andjustover1-foldinethanol,thoughthislattersample alsohadmore variation
betweenbiological replicates.
0
1
2
3
4
5
6
FoldEnrichment
Conditions
0
1
2
3
4
5
6
FoldEnrichment
Conditions
A
B
15. 0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
FoldEnrichment
Conditions
C
Figure 4. GR bindsto Gilzin the presence orabsence of dexamethasone. Graphsshow the relative
enrichmentof GRand REST bindingatthe Gilz locusrelative toIgG controls.GR bindsmore readilyin
samplescontainingethanol comparedtothose containingdexamethasone.Infigure 4A,yvalues
representthe meanaverage of correspondingyvaluesinfigures4Band4C. Infigures4B and 4C, y
valuescorrespondtothe meanaverage of technical triplicates,excludingresultsdeemedanomalous.
Raw valuesforthe technical triplicatescanbe viewedin Appendix 3C:GILZ NumericalResults Data.
16. GR binding atthe Mt2a positivecontrollocus may be increased by the presenceof dexamethasone
Metallothionein2A (Mt2a) isa knownGR targetand thusservesas a positive control inour
experiment.Ourresultsconsistentlyshow that significantenrichmentof the GRat this locus occurs
inthe presence of dexamethasone,butthere issome variabilityinthe ethanol containingsamples.
The average of the resultsindicatesthatdexamethasone increasesenrichment between2.7- and
3.5-foldcomparedtothe control and between2-2.5-foldinthe ethanol vehicle,howeverfurther
investigationisneeded.
REST doesnot showenrichmentineitherDEXor EtOH, there isapproximately1-foldaverage
enrichmentinboth,withlowvariabilitybetweenthe biological replicates.Thisisconsistentwith
whatis expectedof the negativecontrol,asthere isnoRE1 bindingsite atthe Mt2a locus.This
supportsthe validityof the resultsof GRbinding.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Foldenrichment
Conditions
0
0.5
1
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Foldenrichment
Conditions
B
A
17. 0
0.5
1
1.5
2
2.5
3
3.5
4
Foldenrichment
Conditions
Figure 5. GR bindsto Mt2a with and without the presence ofdexamethasone. Graphsshow the relative
enrichmentof GRand REST bindingatthe Mt2a gene locusrelative toIgGcontrols. GR bindsmore
readilyinsamplescontainingdexamethasone comparedtothe ethanol control. Infigure 5A,yvalues
representthe meanaverage of correspondingyvaluesinfigures5Band5C. Infigures5B and 5C, y
valuescorrespondtothe meanaverage of technical triplicates,excludingresultsdeemedanomalous.
Raw valuesforthe technical triplicatescanbe viewedin Appendix:Mt2a NumericalResultsData.
C
18. GR binding atthe Slc19a2 positivecontrol locus may be increased by the presenceof dexamethasone
Slc19a2 was usedas a positive control,followingdataof successful GR-bindinginA549human lung
cells(Soetal.,2007). Significantaverage enrichment of GR at the Slc19a2 locuswas observedin
bothsamples butwas higheroverall inthe presence of dexamethasone,at5.5-fold,3-fold,2-fold
and 1.5-foldinthe DEX GR Thermo,DEX GR Diagenode,EtOHThermoandEtOH Diagenode samples
respectively. There isalargerdistinctioninthe resultswhencomparingDEXto EtOH inthe first
biological replicate,withveryhighrelative enrichmentvaluesforDEXsamplesandnoenrichmentat
EtOH. If this resultwasto be replicated,itcouldsuggestthatdexamethasone isrequiredforGR-
bindingatthe Slc19a2 locus.Inthe secondbiological replicate, noenrichmentisobservedexceptfor
inEtOH Thermoat approximately3.2-fold. Itislikelythatthe datafrom the firstbiological replicate
ismore reliable,asthisisconsistentwithexpectationsof apositive control,howeverfurtherstudies
are neededtoconfirmthese findings.
On average,noenrichmentof RESTat the Slc19a2 locuswasobserved,whichisasexpectedasitwas
a negative control.Thissuggeststhatdespitethe variationinresults,the ChIPprocedure usedwas
appropriate andthat erroris likelytooriginate fromthe PCRreactions.
-1
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Figure 6. GR bindsto Slc19a with and without the presence of dexamethasone. Graphsshowthe
relative enrichmentof GR and REST bindingatthe Slc19a gene locusrelative toIgGcontrols. GR binds
more readilyinsamples containingdexamethasonecomparedtothe ethanol control. Infigure 6A,y
valuesrepresentthe meanaverage of correspondingyvaluesinfigures6Band6C. Infigures6b and 6c, y
valuescorrespondtothe meanaverage of technical triplicates,excludingresultsdeemedanomalous.
Raw valuesforthe technical triplicatescanbe viewedin Appendix:Slc19a NumericalResults Data.
20. GR binding atSgk1 locus may be dependenton thepresence of dexamethasone
Serine/threonine-proteinkinase 1(Sgk1) isa GR target gene associatedwithmediationof the
Hedgehogsignallingpathway (Anackeretal.,2013b).GR bindingatthe Sgk1 locusinNPCswas
enrichedbyapproximately5-foldinDEXtreatedsamplescomparedwithapproximately2-foldin
EtOH vehicle controls,suggestingthatdexamethasoneadministrationisrequiredforGRbinding.
Furtherstudiesare neededtoconfirmthese findingshowever,asthere isa large amountof
variabilityinthe resultsfrombiological replicatesof DEXtreatedsamples. Inthe firstbiological
replicate (figure7B),DEXsamplesshowapproximately7to 8-foldenrichmentcomparedto1.5 to 2-
foldenrichmentinthe second(figure 7C),whichisnotgenerallyconsidered significantdue tobeing
relative toIgG.Minimal enrichmentisobservedinEtOHsamplesfrombothreplicates.
REST showsan average of approximately3-foldand1-foldenrichmentinDEXandEtOH treated
samples.The biological replicatesof DEXtreatedsamplesshow approximately6-fold(figureB) and
0.5-fold(figure C) enrichment.RESTwasintendedforuse asa negative control asthere isno known
RE1 bindingsite atthe Sgk1 locus,therefore the resultfromthe secondbiological replicate (figureC)
islikelytobe more accurate.Althoughthe possibilityof RESTbindingshouldnotbe excluded, itis
noteworthythatmostenrichmentvalues,includingREST,are higherinthe firstreplicate.Thiscould
implicate amore general erroneousresult,whichislikelytohave arisenfromerrorsinthe PCR
reaction.
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Figure 7. GR bindsto Sgk1 with and without the presence ofdexamethasone. Graphsshow the relative
enrichmentof GRand REST bindingatthe Mt2a gene locusrelative toIgGcontrols. GR bindsmore
readilyinsamplescontainingdexamethasone comparedtothe ethanol control. Infigure 7A,yvalues
representthe meanaverage of correspondingyvaluesinfigures7Band7C. Infigures7B and 7C, y
valuescorrespondtothe meanaverage of technical triplicates,excludingresultsdeemedanomalous.
Raw valuesforthe technical triplicatescanbe viewedin Appendix:Sgk1NumericalResultsData.
B
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22. Summary
ResultsfromChIPdata showsuccessful optimisationof the procedure.We are confidentthatthe
ChIPprocedure isfunctional due tothe Snap-25andM4coding resultsbeingconsistentwithwhatis
expectedof positive andnegative controlsforRESTbindingrespectively.
Resultsfromthe PCRdata showsthat there maybe directGR bindingatthe Sgk1 locus,whichis
dependentonthe presence of dexamethasone.The resultsof the positive controls Mt2a,Slc19a2
and Gilz improve the validityof ourexperimentsbyshowingthatthe ChIPandPCR procedures
display positive resultswhereexpected.Atthe Dscamnegative control locus, ourresultsindicated
that some GR bindingmaybe present despite theorysuggestingthatitshouldnot.Alternatively,the
perceivedGRbindingmaybe at leastpartially attributable tothe Dscamprimerdesign.Infuture
studiesitwould advisable touse adifferentGRnegative control locus if possible.
23. Immunofluorescence imagingofchangingmorphologiesinHPC03A/07 NPCs
Qualitative analysisof cellswasthenconductedusingimmunofluorescence labellingtoestablishthe
effectsof dexamethasoneand/orsertraline oncellularproliferationanddifferentiation.Similarto
our previousexperiments,ethanol wasusedasa negative vehicle control.The ethanol concentration
was doubledinsampleswhere the intentionwastocompare the effectsof samplescontainingboth
sertraline anddexamethasone. Thisactedasa control for the volumesof sertralineand
dexamethasone added,bothof whichuse EtOHas a solvent. A furthernegativecontrol wasused
whichwasuntreatedwithanyvehicle otherthanreducedmodifiedmedium(without
FGF2/EGF/4OHT).
The effectsof dexamethasoneand sertralineon neuralprogenitorcells at proliferation day 3
NPCswere growninvaryingconditionsfor3 daysand labelled withDAPI,NestinandKi67
conjugatedwithdifferentfluorochromestopermitimmunoflouresceneimaging.The conditions
were treatmentwitheitherdexamethasone1µm,sertaline 1µm, dexamethasone 1µmandsertraline
1µm, ethanol 0.1mMor ethanol 0.2mM. A furthercontrol was alsocreatedwherebythe cells
receivednotreatment.Ourresultsshow thatdexamethasone mayhave aninhibitoryeffectonNPC
proliferationthatiscounter-actedtosome extentbyco-treatmentwithsertraline.
Figures8a and 8b: Scale bar: 20µm. The top leftpanelsindicatestainingbyDAPI(blue);topright:
Nestin(green);bottomleft:Ki67(red) andthe bottomright panel combinesimagestakenfromall
filtersets. DAPIhasbeen usedtostainall cell nuclei;Ki67isa non-specificcellularproliferation
markerand Nestinstainsanintermediate filamentfoundinNSCs/NPCs. The white arrow intopleft
quadrantof figure 8Ai showsan example of anucleusina cell thatis notproliferating.The white
arrow inthe bottomleftquadrantof thisfigure showsanucleusof a proliferatingcell,asindicated
by a positive Ki67stain.
These figures show thatdexamethasoneslightlydecreases NPCproliferationatday3 comparedto
the negative vehicle control EtOH.Thisis shownby increasednumbersof Ki67-positivecellsin both
ethanol samples (figures8Bi and 8Bii) relative tothat observedinfigure 8Ai.Sertraline-treatedcells
alsoshowedincreased proliferationcomparedtodexamethasone.Allcellstreatedwithsertraline
were showntobe positive forthe Ki67marker(figure 8Aii).Cellsthatwere co-treatedwith
sertraline anddexamethasone showedanintermediate amountof proliferation comparedtoeach
stand-alone treatment,withapproximatelyhalf of the cellsstainedbeingKi67positive (figure8Aiii).
Control samplesandthose treated withethanol alone appearedtobe greaterincell numberand
showsimilarrelativeamountsof proliferationcompared tothose co-treatedwithdexamethasone
and sertraline.
Morphological changes asa resultof the differentconditionsare minimal.Inthe untreatedsample,
there appearsto be a highercell countwithincreasedclustering,howeverthe significance of thisis
unknown.
26. The effectsof dexamethasoneand sertralineon neuronaldifferentiation atday 14
Followingtreatmentinproliferativeconditions,NPCswere allowedtodifferentiateinthe presence
of eitherdexamethasone1µm,sertaline 1µm, dexamethasone 1µmandsertraline 1µm, ethanol
0.1mM or ethanol 0.2mM. As before,anadditional untreatedcontrol wasalsocreated.Ourresults
suggestthatdexamethasonehasaninhibitoryeffectonneuronal differentiation.Thiscouldeither
be a consequence of cell deathoranincrease inthe proportionof cellsthatdifferentiate into
astrocytes. Treatmentwithsertraline increasedthe extentof neuronaldifferentiationbothinthe
presence andabsence of dexamethasone.
Figures9a and 9b: Scale bar: 20µm. The top leftpanelsindicatestainingbyDAPI(blue);topright:
TuJ1 (green);bottomleft: Anti-GFAP(red) and the bottomrightpanel combinesimagestakenfrom
all filtersets.DAPIhasbeenusedtostainall cell nuclei;TuJ1isstainsmicrotubulescontainedin
neuronal cells andAnti-GFAPstainsanintermediatefilamentfoundin astrocytes. Figures9Ai and
9Bi, 9Bii and 9Biii showthat differentiationintoneuronsisreducedinthe presence of
dexamethasone comparedtonegativecontrols,asshownbylowerTuJ1staining.Incellstreated
withsertraline,neuronal differentiationisincreasedcomparedtocontrols (see figure 9Aii in
comparisontofigures 9Bi, 9Bii and9Biii).Co-treatementwithsertraline anddexamethasone (figure
9Aii) showsan intermediateamountof neuronsinrelationtoastrocytes.
Furthermore,in 9Ai an example of anasteron(a hybridcell thatcontainspropertiesof both
astrocytesandneurons) ishighlighted (Laywell etal.,2005). The nucleusisshownbythe white
arrow and can be foundinthe same positioninall quadrantsof the figure, indicatingthatitisboth
TuJ1 and Anti-GFAP positive. The presenceof asteronswasnotobservedinothersamplesanditis
therefore difficulttopredictthe significance of this.
It isdifficulttoassessthe effectsof dexamethasoneonneuronal morphologydue tothe low number
present(figure 9Ai).Those thatdoexistappeartobe smallerbutwithmore dendriticregions.This
effectseemstobe consistentwhensubsequentlycomparingcellstreatedwithsertralinealone
(figure 9Aii) tothose that receivedcotreatmentwithsertraline anddexamethasone (figure9Aiii),
wherebyinthe lattersample there isalsogenerallyahighernumberof dendriticregionsobservedin
neurons.
29. Discussion
Establishinga working ChIP protocol
The firstaim of these experimentswastoensure thatthe procedure usedforchromatin
immunoprecipitationwasfully-functional.Totestthis, repressorelement-1silencingtranscription
factor (REST) bindingwasfirstvalidatedbymeasuringthe relative enrichmentof RESTat a gene
where itwasknownto bindas well asone where itwasnot. Asexpected,RESTshowedsignificant
enrichmentatthe knowntargetgenes,averagingatbetween 35- to40-foldenrichmentin
dexamethasone andethanol-treatedHPC03A/07NPCs.Thisshowsthat the procedure usedforChIP
inour experimentswassuccessfulandthatthe chromatinand REST antibodieswereappropriate.
Therefore,errorsinexperimentsthatinvolve GRbindingare likelytoresultfromproblemswiththe
GR antibodiesand/orthe quantitative polymerase chainreaction(qpcr).
Both GR antibodies(fromThermoScientificPierce andDiagenode) showedarelativelylow but
significant3-foldand2.5-foldenrichmentatthe M4coding locusinEtOH treatedsamplesfromone
biological replicate.GRwas notexpectedtobindinthisregion,whichsuggeststhatthese antibodies
exhibitednon-specificbindingtosome extent.However,the GRbindinginthe otherbiological
replicate wasasexpected.ItislikelythaterrorinvolvingGRtargetgenesmayalsobe predominantly
technical andassociatedwiththe qpcrprocedure.Thisisdiscussedfurtherinthe section: Errorand
furtherconsiderationsforexperimentoptimisation.
Overall,we achievedourfirstaimof successfullyoptimisingthe ChIPprotocol foruse in
interrogatingputativeGRtargets.
Establishingthe extentof GR-bindingat target genes
PreviousworkbyAnackeretal…Knowntargetgenesof the GR were showntohave higherrelative
enrichmentvaluesrelative tointernal controlsandthe negative control gene, Dscam. Furthermore,
we were able todetermine the extentof inhibitoryand stimulatoryeffectsthatdexamethasonehad
on GR-bindingatthe targetgenesandour resultsare largelyconsistentwithpreviousfindings,
howeverthere issignificant variabilityinsome results andfurtherstudiesshouldbe conductedto
confirmthese.
Of the twovalidatedand GR specificantibodies used,samplesthatcontainedGR-Thermo,arabbit
polyclonal antibody,typicallyshowedhigherenrichmentvalues forGR.Our resultscouldindicate
that GR-Thermoantibodymaybe more effectiveatbindingtothe GR in ourChIP conditions
comparedto the monoclonal GR-Diagenode antibody.Monoclonal antibodiesare oftenfavoured
due to theirhigherspecificitytowardsasingle epitopeandthussubsequentlylowerbackground
noise,howeverinourexperimentsthe use of apolyclonal antibodyhasbeenadvantageous.This
couldbe due to the fact thatit can recognise multiple epitopesonthe same GR proteinandthusbe
slightly more resilientagainstanymaskingthatcanoccur due to areas of heterochromatin
formation. (Lipmanetal.,2005). Regardless,bothantibodieswereadequateandshowedconsistent
enrichment.Thus,ourfindingsshowthatwe have a workingprotocol todetermine GRbindingusing
commercial GR antibodies,whichcanbe usedinthe future forstudiesthatinvestigateothergene
loci that may be directlyactivatedbythe GR.
PossibleGR binding atDscam negativecontrollocus
The Dscam gene isresponsible forthe formationof Downsyndrome celladhesionmoleculesandis
not knownto be a GR target.Modifiedversionsof previouslypublishedhuman Dscamprimerswere
30. usedinour experiments(see Appendix 3A:Forward and ReversePrimer Sequences) withthe
intention of improvingtheirefficiencyinthe PCRto act as a negative control region,basedonpoor
resultswithpreviousprimers(AB,personal communication).Regardless,the resultsindicate possible
GR bindingatthe Dscamlocus,whichthe presence of dexamethasone doesnotappearto
significantlyinfluence.Thiscouldsuggestpotential non-specificbindingwithinourexperiments,
whichcan resultinbackgroundenrichment,slightlydecreasingthe validityof relativequantitative
results.Alternatively,GRmayin fact bindtothisregionand thusinfuture experiments,abetter
negative regioncontrol wouldneedtobe selected. Toaccountfor this,a range of othercontrols
have alsobeenused incombination andenrichmenthasbeenmeasuredrelative toIgGinsteadof
throughabsolute quantification.
Gilz is an effectivepositivecontrol locusfor GR binding thatmay be inhibited by the presenceof
dexamethasone
The glucocorticoid-inducedleucine zipper(Gilz) geneiswell-knownasa mediatorof glucocorticoid-
dependantimmunomodulationpathways,yetitswidespreadexpressionsuggeststhatitmayalso
have more basic roles (AyroldiandRiccardi,2009, Yachi etal.,2007). Murine water-immersion
restraintstresstestsstronglyincreasedGILZmRNA expressionandsubsequentproteinexpression.
Furthermore,itwasobservedthatinmice withoutadrenal glands,up-regulationof GILZwasceased,
suggestingthatthe increase inexpressionwasdependantonHPA axisstimulation andsubsequent
cortisol release.(Yachi etal.,2007) Up-regulationof the gene wasobservedinseveral areasof the
brainassociatedwithstress,includingthe hippocampus,inanunevendistributionthatsuggested
specificfunctionalroles.Howeverthe exactnature of these roles stillremainsunclear,particularlyin
relationtohippocampal neurogenesis.PreviousresearchbyYachi etal observedincreased
expressionspecificallyinpyramidal neuronsof Ammon’shornandgranule neuronsfromthe dentate
gyrusas a response toincreasedHPA activation, butdidnotexaminethe effectsondifferentiation
or the formation of these cellsfrom respective precursors (Yachi etal.,2007).
Furthermore,whilstitisknownthatglucocorticoidsincreasetranscriptionof Gilzviatransactivation
of the glucocorticoidreceptor, the extenttowhichindividual syntheticglucocorticoidsinfluence
gene expressionisunknown.Whilstrecognisedasadexamethasone-induciblegene,there exists
contradictoryresultsarisingfromdexamethasone suppressiontests (Smitetal.,2005, Yachi et al.,
2007). Interestingly,othersyntheticglucocorticoidssuchashydrocortisone andbudesonide alsohad
varyingeffectson GILZ mRNA levels (Smitetal.,2005).
Our results suggestthatdexamethasonehasinhibitoryeffectson GRbindingatthe Gilz loci
comparedto the ethanol control. If these findingscanbe replicatedinthe future,itcouldsuggest
that inpatientswithcortisol-induceddepression,there isalowerextentof GRbinding inNPCs.
Combiningthiswithresearchdiscussedpreviously,thiscouldindicate that increased GR-bindingat
thisloci has an inhibitoryeffectonGILZ expression andthatsubsequently,cortisol-induced
depressionmayresult inlowerGILZexpression.However,thisassumesthat GR-bindinghasthe
same effectsongene expressioninbothNPCsandfullydifferentiatedneurons.Furtherstudiesare
neededtoconfirm ourfindingsof the effectsof dexamethasone onGR-bindingatthe Gilz locusand
then,throughparallel gene expressionanalysis,how GR-bindingatthe Gilz locusaffectsGILZ gene
expression.
The primary purpose of usingGilz in our experimentswasasa positive control toaidinthe
validationof ourresults.Thishasbeenachieved,asshownby consistentenrichmentof GR binding
at the Gilz locus. Additionally,noenrichmentof RESTbindingatthe Gilz locuswas observed,whichis
31. alsoas expectedasthere isnoRE1 bindingsite.Thus,the use of a REST antibodyinall GR binding
experimentsisasa furthernegative control.
Mt2a is an effectivepositivecontrollocus forGR binding thatmay be stimulated by dexamethasone
Metallothionein2A (Mt2a) isa gene whichfunctionsbybindingtoheavymetalsandplaysarole in
diseases suchascerebral lymphoma(Yangetal.,2007). There islittle evidence linkingMt2a
expressiontocortisol-mediateddepression,althoughone small studyshowedthat increased Mt2A
expressionwithinthe anteriorcingulate cortex inpatientswhohadcommittedsuicide (Sequeiraet
al.,2012). GR isknownto bindto MT2A locus andMT2A is consideredadexamethasoneinducible
gene,thusthisexperimentserves asapositive control.However, bindingof GR to the Mt2A locus
may be cell specific(Soetal.,2007). This couldbe due to differentchromatinstatesindifferentcells
and the subsequentextentof epigeneticmasking. ItwasunknownwhetherGRbindstothese gene
locusin HPC03A/07 cellsspecifically. TOur resultsshowedenrichmentinthe presence andabsence
of dexamethasone,butwithahigherenrichmentindexamethasone-treatedsamples,suggesting
that dexamethasone hasastimulatoryeffectonGR-MT2A binding. However,the variabilityinthe
ethanol containingsamplessuggeststhat technical sourcesof errorare likelytobe presentandthus
experimentsneedtobe repeated. Regardless,MT2A was a successful positive control inour
experimentsandGRbindingtothe gene loci wasas predicted.
Slc19a2 is an effectivepositive controllocus forGR binding thatmay be dependenton thepresence
of dexamethasone
Slc19a2 isa known,dexamethasone-responsive, humanGRtarget gene thatcodesfor a thiamine
transporterprotein (Soetal.,2007, Aoyagi and Archer,2011). The GR bindingsite ispositioned
upstreamof the gene,136 base pairs fromthe transcriptionstartsite (Soet al.,2007). In our
experiment,itwasintendedasapositive control,however previous dataonGR-bindingspecificityin
HPC03A/07 cellsislacking.Ourresultsindicate thatGRdoesinfact bindto Slc19a2 inHPC03A/07
cells,howeverthere issignificantvariationinresultswhencomparingthe effects of dexamethasone
to the samplescontainingethanolalone. NoenrichmentwasobservedforRESTbindingatthe locus,
confirmingthatthe ChIPprocedure usedwasaccurate.Variationinresultsislikelytobe a
consequence of technical errorassociatedwith the PCR.
On average,dexamethasone appearsto be a pre-requisite forGRbindingtothe Slc19a2 locus.
Furtherstudiesare neededtoquantifythe effectsof dexamethasone andconfirmthese findings.
Dexamethasoneincreases GR binding atthe Sgk1locus, which may directly upregulateSGK1 gene
expression
There isa significantamountof literature thatsuggeststhatSgk1,a GR targetgene,isinvolvedinthe
body’sresponse tostress (Lucaetal., 2009). The glucocorticoidbindingregionispositioned
upstreamof the gene (Soet al.,2007). The hedgehogpathwayisresponsibleforthe transmissionof
informationresponsibleforneural stemcell developmentwithinbothembryosandadultstemcells.
Accordingto researchbyAnackeret al,SGK1 is necessaryforcortisol induced,neurogenic, hedgehog
pathwayinhibitionandthussubsequentdecreasesinneurogenesis (Anackeretal.,2013a, Anacker
et al.,2013b). SGK1 alsoincreasedcortisol-dependantactivationof the GR byincreasingGR
translocationtothe nucleusandphosphorylation (Anackeretal.,2013b). Previousresultsfrom
Anackeretal showedthatSGK1 expressionisincreasedbyactivationof the GRreceptor.However,
directbindingof GR to SGK1 followingactivationwithcortisol ordexamethasonehadnotbeen
previouslyproven.
32. Our resultsshowsignificantbindingof GRat the Sgk1 locususingbothGR-targetedantibodies
tested.Ourresultsalsoindicate thatGR maytherefore up-regulateSGK1directly,inkeepingwith
the findingsfromAnackeretal., (Anackeretal.,2013b). Our resultsalsoshowedalowerbut
significantamountof bindinginethanol containingsamples,suggestingalow degree of GRbinding
and thuspossiblythatSGK1 expressionisnotdependentondexamethasone activation.
However,there isasignificantdegreeof variabilitybetweenthe biological replicates,whichislikely
due to technical errorassociatedwiththe PCRreaction. Notably,the differenceinthe extentof
enrichmentof GRbindingindexamethasone treatedsamples,betweenthe biological replicates, was
considerablyhigh.Furthermore,RESTbindingwasalsoobservedatthe Sgk1locusinone biological
replicate despite noknownRE1bindingsite and the use of an optimisedChIPprotocol. Therefore,
more studiesare neededtoconfirmourfindings.
If our findingscanbe replicatedandGR does upregulate SGK1directly,thismayhave important
implicationsforthe waysinwhichthisgene isutilisedasatherapeutictargettotreat stress-induced
depression.
Qualitative observationsof dexamethasone andsertraline’seffectsonneural progenitorcell
proliferationand neuronal differentiation
Our resultsare consistentwithfindingsbyAnackeretal (Anacker,2014, Anackeretal.,2013a)
suggestingthatsertraline mediatesbothproliferationanddifferentiation of hippocampalprogenitor
cells.
We firstexaminedthe effectsof sertralineanddexamethasone onNPCproliferation andobserved
that dexamethasone mayinhibitthis. Incellstreatedwithbothdexamethasoneandsertraline,this
inhibitoryeffectwasreduced,suggestingthatsertralineantagonisesthe effectsof dexamethasone.
We thenobservedthatincellsallowedtodifferentiate,those thatwere treatedwith
dexamethasone exhibitedlowerdifferentiationintoneurons.Thissuggestedthathighlevelsof
dexamethasone wereinhibitorytowardsneuronaldifferentiation,possiblybyencouragingthe
formationof astrocytesinstead. Similarly,the presence of sertraline counter-actedthiseffect.
Interestingly,hybridasteronswereobservedinsome samples,althoughthe relationshipbetween
differentiationintothesecellsandlevelsof dexamethasone and/orsertralineremainsunclear.
Asteronscanbe definedascellsthatshare the same morphologyasbothastrocytesandneurons.
Theyare hypothesisedtobe anintermediatestepbetweenthe differentiationof neuronsinto
astrocytes(Laywell etal.,2005).
Furtherstudiesare neededtoconfirmourfindingsandquantifythe effectsdescribedabove.These
shouldbe conductedona largerscale usingtechnical triplicatesandappropriatenumbersof
biological replicates.Quantificationcanthenbe achievedthroughstandardcell countingmethods.
From a broaderperspective, ourresultssupport the conceptthatsertraline mayexhibitanti-
depressanteffectsoutside of those describedinthe monoaminergichypothesis.Aspreviously
discussed,the combinationof highlevelsof dexamethasone andthe presence of sertralinemay
conferunique phosphorylationstatesatthe GR viaPKA signallingmechanismsatserine residues
S203, S211 and S226 (Anackeretal.,2011b). This mayhave significantconsequencesonsubsequent
GR-dependentgene expression,asourfindingssuggestthatleastone stress-relatedgene (SGK1) is
upregulateddirectlyasaresultof GR binding.The phosphorylationstate of the GR mayinfluence the
extentof GR binding,forexample throughmodificationof itscompatibilitywithvaryingtarget
chromatinstates. Future research testingthe effectsof sertraline anddexamethasone onGRbinding
33. at the SGK1 locus, usingouroptimised ChIPprotocol,maytherefore be useful,particularlyif ranin
parallel togene expressionanalysis.Itwouldalsobe goodtoassesswhetherornot GR bindsdirectly
to othergene loci usingourChIPprotocol such as P27kip 1
andp57kip2
where the phosphorylation
state of the GR hasalreadybeenestablishedtoinfluence expressionandwherebythe geneshave
alreadybeenlinkedtothe terminationof cell divisionandincreasesinneuronal differentiation.
(Galliher-Beckleyetal.,2008, BlindandGarabedian,2008, Chenetal.,2008, Kumarand Calhoun,
2008, Websteretal.,1997, Anackeretal.,2011b).
Potential errors and further considerationsfor experimentoptimisation
As previouslydiscussed,itislikelythatthe majorsourcesof error inGR-bindinganalysesare due to
technical mistakesassociatedwiththe q-pcrprocedure.Thisincludeshumanerror,suchas variation
inpipettingtechnique.The percentage errorarisingfromsuchvariablesislikelytobe highdue to
the small volumesof solutionsused,particularlygiventhatthe standardcurveswere alsosubjectto
thiserror. Anothersource of human errorin the q-pcr reactioncouldinclude incomplete thawingof
reagentsbefore use,resulting inunevenlydistributedconcentrations.
There may alsobe casesof systematicerror. The nature of polymerase chainreactionsrequires
everyindividual reactiontobe optimised.The mainstepsof PCRare denaturation,annealingand
extension.Eachmaybe a potential source of errorif the time givenforeachis notcarefully
considered.Forexample,notenoughtimegiventothe denaturingstepcanresultinincomplete
strand separation,hinderingthe abilitytobindtospecificprimersinthe annealingstage. Inthe
annealingstage,toolowof a temperature permitsnon-specificbinding. The time giventoeachstep
and the numberof cyclesperformedwasbasedupon the numberof base pairsinthe target
sequence.The efficiencyof the reactionswere thenobservedbasedandprimerswere then
optimisedaccordingly.
The frequencyof calibrationchecksof the qpcrmachine wasunknown. Additionally,onanalysisof
the meltcurvesof some samples,the peakindicatingprimerdimerizationwaspresentinsome
samples. Itmayalsobe possible thatsome primersboundtonon-targetregionsof DNA,forexample
due to repeatsequences. M4codingandSnap25 primerswere designedindividuallyusingPrimer3
to checkfor possibilityof secondarystructure formationandthusreduce errorfromDNA hairpins.
However,otherprimersthatwere usedoriginatedfromexistingpublicationstocompare resultsto
knowndata andit may be possible tofurtheroptimise theseinfuture experiments.
From the ChIPprocedure, itisunlikelythatthe degree of errorwasas significant.Howeverone
potential source of errorinthis is slightvariationinchromatinsize due totimingsof eachstage,
whichcan affectPCRkinetics.
In future experiments, itmaybe more cost-effective toconductgenome wide ChIPsequencing
analysisof GR binding, if analysingalarge numberof genes. Itmay alsointerestingtorungenome-
wide gene expressioninparallel tothis,tofurtherelucidate the directeffectsof GR-bindingongene
expression. If budgetpermits,there is alsothe potential forerrorinthe q-pcrto be reducedthrough
the use of sequence specificfluorescentDNA probes.These are more selectivetowardsthe target
DNA to be amplified,thusdecreasinglevelsof backgroundnoise.
Withrespectto the immunofluorescence stainingexperiments,the time-frameinwhich they were
carriedout made quantitative analysisthroughcellcountingunfeasible. The imagesobtainedfrom
these experimentsmayalso be subjecttosamplingbias. Leakage of fluorescence signal into
differentfilterssetsmayalsohave beenaproblem, forexampleredlight sometimesleakedinto
imagesviewedunderthe greenfilterset.Inideal circumstances,cellcounting wouldallow usto
34. furtherelucidate informationregardingthe effectsof dexamethasone and/orsertraline on
hippocampal cell proliferationanddifferentiation. If thiswere tohappen,more biological replicates
wouldbe neededtoensure reliable resultsandsubsequentstatistical analysis. Use of a confocal
microscope may alsobe preferable due tothe abilitytocontrol depthof fieldandthusreduce the
amountof backgroundinformation,particularlyinanalysisof differentiationpatternswhere
clusteringof cellsalongthe zplane wascommon,howeverthisisunlikelytohave hada significant
impacton our findings.
35. Conclusions and future work
We have establishedanoptimal chromatinimmunoprecipitationmethodforuse inthe analysisof
GR-bindinginHPC03A/07 cells.The gene of primaryinterestinourexperiment,Sgk1,wasfoundto
bindto the GR directly anddexamethasonehasastimulatoryeffecton the extentof this.Low levels
of Sgk1-GRbindingwere observedinsamplesonlycontainingethanol. Due totechnical variability,
furtherconclusive studiesare needed,howeverouraverage results extendupon findingsfrom
Anacker(Anackeretal.,2013b). We recommendfurtherstudiesusingouroptimisedChIPprotocol
to investigateGRbindingatmore gene loci inthe presence andabsence of dexamethasone and
antidepressanttreatment. Qualitativeanalysisof immunofluorescentlystained HPC03A/07showed
that dexamethasone reducesproliferationandmayinhibitdifferentiationintoneurons,possiblyby
directingdevelopmentintoastrocytesinstead.Sertralinedoes notsignificantlyincrease proliferation
or differentiationof cellscomparedtothe negativecontrol,butdoesappeartocounteractthe
effectsof dexamethasone,suggestingthatcortisol-induceddepressionisrequiredforatherapeutic
effectonhippocampal neurogenesis.Furtherstudiesare neededtoquantifythe effectsof
dexamethasone andsertraline oncellulardevelopment.Itwouldalsobe interestingtoanalyse
changesinmorphology,suchas the numberandsize of dendrites onneuronal cells.Some samples
were alsofoundtocontainastroneurons,howevertheirrole indepression,if itdoesexist,is
currentlyelusiveandtheirrelationshiptodexamethasone and/orsertralinelevelsisunknown.
39. Appendix2: Response to referee’scomments
Abstract
Typically don’t reference within the abstract.
Make clearwhat yourcell line isearlieron.
Referenceswereremovedandcell lineisnow
specifiedatthe earliestpointcellsare
mentioned
Introduction
Full stopsdon’tcome before areference Reference citationswere amendedsothatfull
stopscame afterwards
Thirdparagraph needsreferencing Thirdparagraph was updatedwiththe
appropriate reference
Paragraph 8 is confusing –needto clarify
resultsfromAnacker2011 study
Wordingof this paragraphhas beenimproved
Whensay evidence suggestsstrong
involvementof genesincluding Sgk1,Fkbp5etc.
needtoelaborate onwhatthe evidenceis
Evidence hasbeenclarified(fromgene
expressionanalysis) andelaboratedon
Be careful notto confuse DNA withchromatin Thishas beenrectifiedthroughoutthe text
Final ‘aims’ paragraph needs some re-wording
to make clearer the main aims, including to
establish a working GR ChIP and to look at
changes in GR binding to putative target genes.
Thisisthe central ideaof the projectandhasnot
beendone inthismodel sofar
Thishas beenre-wordedforclarificationand
the aimshave beenaddedin
Materialsand Methods
Incell culture,needtostate thiswasdonebyAB
and more precisely when growth factors were
in/out and for how long cells were in
proliferation/dex/sertetc.thendifferentiation.
I have listedatthe top of each relevantsection
whoconductedthe work.Growth factor
conditionswere made more specific.
ChIPprotocol needstoincludehowpre-blocking
and pre-clearing were done precisely and to
include how de-crosslinked ChIP DNA was
purifiedatthe endbefore PCR
I have addedthisinformationassuggestedin
line withothercorrespondence.
qPCR needs conditions for PCR programme and
greaterdetail
Greaterdetail hasbeenaddedusing
information fromthe original protocol.
ICC/immunofluorescenceneedsfull detailsof all
primaryand secondaryantibodiesusedandthe
dilution factors as well as explanation of why
each markerwasused.
Greaterdetail hasbeenaddedwithhelpfrom
furthercorrespondence andconsultingthe
original protocol andnotes.
Results
Titles for sub-sections need to say what the
resultisfor thatsection
Titleshave beenamended.
40. In all figure legends ensure you say what fold
enrichmentisrelativeto(i.e. IgG)
Thishas now beenspecified.
‘SI’ should be included as Appendices for the
thesis
Make sure it is clear that B and C are individual
biological replicates and A is the average of the
2 inPCR figures
SI has beenchangedtoappendices(allare
containedwithinAppendix 3)
Make clear why each gene locus was chosen to
interrogate
Thishas now beenmade clearthrough
specifyingwhichwere positive/negative
controlsect.
Ensure yousummarise the meaningof the result
at the end of each sub-section. E.g. if GR
enrichmentisatSgk1inDEXand notEtOH andit
is significantly (over 2-fold) above IgG then it
tellsyouthat the ChIP isworking,that GR binds
inresponse toDEXandisnotboundinuntreated
samples. Also, explain more carefully what you
meanwhendiscussingpossible sourcesof error
A small summaryhasnow beengivenineach
sub-section,followedbyalargersummaryat
the endof the results.Sourcesof errorhave
beenclarified.
Summarise all the PCR data briefly at the end
before making a sub-heading for the Immuno
experiments
A brief sub-sectionhasnow beenusedasa
summary.
Immunobitneedswork –needstobe expanded
to better describe the experiment done, why
and the results. You need to say which markers
were used on which cells and why. Then what
yousee andwhatthismeans(caninclude how it
fitswithpublisheddataisyoulike)
Thishas now beenextensivelyaddedto
Immuno part – all figures need legends to
explain what panels show including scale bars.
Some annotation would help e.g. arrows to
indicate things described in the text. (e.g.
asterons,anexampleof whichisshowninFigure
8A,white arrow).Alsoneedtosaywhatasterons
are.
Scale bars have now beenspecifiedwithinthe
mainfigure legendandexplanationaddedof
whatis withineachimage. Eachquadrant has
beengivenasubtype (I,ii,iii) andreferredto
withinthe textina logical order.Arrowshave
beenaddedtohighlightaproliferatingcell and
an asteron.
Discussion
First aim was to check ChIP was working – e.g.
chromatin good, buffers good etc. thus used
REST. Say whatREST isand reference.Thenalso
say next was to determine if this protocol was
suitable forGRChIPusing2antibodiesthatwere
beingtested
Thishas beenclarifiedanddiscussedmore
Do not always say our results support previous
findings – in places we are extending those
findings toinclude datathey didnot have – e.g.
Anacker et al., shoed gene expression changes
downstream of dex/GR activation but did not
show direct binding of GR to those genes (e.g.
Thishas now beenstressed.
41. Sgk1). You have done that and need to stress
that as it is a major finding and novelty of the
work
Again need better titles for sub-heading to
describe the importance of the findings
Be careful againnot to confuse gene expression
with GR binding/enrichment. You did not do
gene expressionanalysis
New titleshave now beengivenforsub-
sectionsandI have editedareaswhere the
termgene expressionanalysiswasused
inappropriately
Put Sgk1 findings last in the list of loci analysed
– it is the most significant. Also, be sure to
include what would be your next step in these
experiments now that you have a working GR
ChIP in the cells (think about drug treatment,
loci to analyse etc.)
Thishas now beenswappedwithSlc19a2
Sectionon immune/proliferation/differentiation
is far too brief and needs to be expanded
including bringing in the relevant findings from
previous literature and how your data fit with
those and the next step (consider also if might
look at other cells not just NPCs – what about
whentheydifferentiate intoneurons?)
Thishas now been expandedon
Conclusions/future work
Remove bitonasteronsor make clearer Thishas beenclarified
Include what would be future work now you
have aChIPprotocol orGR (see earliercomment
above)
Thishas now beenmentionedbutdiscussed
and rationalisedinmore detail indiscussion
Other comments
Human gene namesare usuallyincapital letters
italicized (whilst e.g. mouse are usually lower
case exceptthe firstletter).Whentalkingabout
expression they are not italicized and when
talking about the protein they are capital, not
italicized. E.g. human Snap25 would be SNAP25
for the gene and SNAP25 for the mRNA or
protein.
These have now beenchanged
Ensure youmentionwhere experimentsorparts
thereof were performedbyAB,you or together
where applicabletobe clear
Thishas now beenclarified
56. H. Tissue Culture Methods for ReNeuronCells(HPC03A/07)
Human Cells
Reduced ModifiedMedium (RMM)
Remove 10 ml of media from a 500 ml bottle of DMEM:F12 with 15 mM HEPES and sodium bicarbonate but
without L-glutamine (Sigma: D6421) and add:
0.75 ml Human serumalbumin solution (0.03%final concentration)(20%stock,PAA: C11-096)
1.0 ml Human apo-transferrin (100 µgml-1 final concentration)(50 mgml-1 stock, SCIPAC:
T100-5)
1.0 ml Putrescinedihydrochloride(16.2 gml-1 final concentration)(8.1mgml-1 stock,Sigma:
P5780)
0.25 ml Human recombinant insulin (5 µgml-1 final concentration)(10 mgml-1 stock, Sigma:
I9278)
1.0 ml Progesterone (60 ngml-1 final concentration)(30 µgml-1 stock,Sigma:P6149)
5.0 ml L-glutamine (2 mM final concentration)(200 mMstock, Sigma: G7513)
1.0 ml Sodium selenite (40 ngml-1 final concentration)(20 gml-1 stock,Sigma: S9133)
5.0 ml Penicillin/Streptomycin ()
For proliferation,thefollowingcomponents should also beadded to make RMM+++:
0.5 ml Human FGF-basic (10 ngml-1 final concentration)(10 gml-1 stock,PeproTech:100-18B)
100 l Human EGF (20 ngml-1 final concentration)(100 gml-1 stock,PeproTech: AF-100-15)
50 l 4-OHT (100 nM final concentration)(1 mM stock, Sigma: H7904)
Filter the medium with all additional components through a 0.2 M filter unit and store for no longer than 2
weeks at 4C.
NB: Bithell lab there is no requirement to filter as all components are sterile. Make up RMM and only add
FGF2/EGF and 4OHT when required
DifferentiationMedium (DM)
Remove 10 ml of media from a 500 ml bottle of Neurobasal Medium (Invitrogen: 21103-049) and add:
0.75 ml Human serumalbumin solution (0.03%final concentration)(20% stock,PAA:C11-096)
1.0 ml Human apo-transferrin (100 µgml-1 final concentration)(50 mgml-1 stock, SCIPAC:
T100-5)
1.0 ml Putrescinedihydrochloride(16.2 gml-1 final concentration)(8.1mgml-1 stock,Sigma:
P5780)
0.25 ml Human recombinant insulin (5 µgml-1 final concentration)(10 mgml-1 stock, Sigma:
I9278)
1.0 ml Progesterone (60 ngml-1 final concentration)(30 µgml-1 stock,Sigma:P6149)
5.0 ml L-glutamine (2 mM final concentration)(200 mMstock, Sigma: G7513)
1.0 ml Sodium selenite (40 ngml-1 final concentration)(20 gml-1 stock,Sigma: S9133)
10 ml B27 Supplement (1x final concentration)(50x stock,Invitrogen: 17504-044)
Alternative Differentiation Medium 1 – ‘RMM’
Simply use RMM without FGF2, EGF or 4OHT
Alternative Differentiation Medium 2 – ‘NB:B27’
To Neurobasal medium add 2mM glutamine, 1x Pen/strep and 1x B27 (as used for iPSC-derived hNPCs,
11530536/17504-044,Invitrogen)
Alternative Differentiation Medium 3 – ‘N2:B27’
57. Make a 50:50 mix of ‘N2 medium’ used for iPSC-derived hNPCs (DMEM:F12:Glutamax (31331-093 Invitrogen),
1x N2 (11520536/17502-048, Invitrogen), 1xNEAA (11140-050, Invitrogen), 1mM glutamine, 1x penstrep) and
‘B27 medium’ (same as NB:B27 above)
Filter the media with all additional components through a 0.2 M filter unitand storefor no longer than 2 weeks
at 4C.
NB: Bithell lab there is no requirement to filter as all components are sterile. Make up RMM and only add
FGF2/EGF and 4OHT when required
Cell culture Plastic and Substrate Preparation
Cells grow on Nunc plasticwarecoated with laminin at1 µgcm-2. Cells grown on coverslipscoated with PDL and
laminin.Laminin stock is 1mg/ml (Sigma L2020). Stock vials should bethawed at 4oC (NOT room temperature)
and then stored at 4oC and used within 1 month. To laminin coat, dilute laminin in DMEM:F12 or HBSS in an
appropriate volume with the appropriate amount of laminin added for the surface area to be coated. For
example, 75µl of laminin in 6 ml of DMEM:F12 or HBSS for a T75 flask,10µl in 1ml for a well of a 6-well plateor
2µl in 0.5ml for a well of a 24-well plate. Ensure it covers the surface. Incubate at 37oC for a MINIMUM of 2hrs,
ideally overnight. Followingincubation,wash 3x with 1xPBS or HBSS and do notallowany laminin-coated surface
to dry before plating cells onto it (i.e. leave on the last PBS wash until you are ready to plate the cells – the
plasticwarecan happily sitin PBS atroomtemp or at37oC until required).For PDL/laminin coating(when plating
onto glass), first coat with PDL and then laminin. To PDL coat, thaw a stock vial of PDL (1mg/ml) at room
temperature and dilute1:50 (so to 20µg/ml) in dH2O.Cover the surfaceand incubateat37oC for 1- 2hrs (can do
overnight but not necessary).Ensure that coverslipsdo not float(push down if necessary).Wash 2-3x 1xPBS or
dH2O before proceeding to laminin coating(as before).
Growth and Passage of ReNeuron Cells
For proliferativecells,splitwhen 70-80%confluent and don’t splitmore than 1:4. Feed the cells every other day
with 10 ml of fresh medium (for a T75 flask,RMM+++). To passagethe cells,remove all medium, rinsein 1xPBS
then add 2-3ml Accutase (Sigma) at 37oC for ~3 minutes until cells detach. Dilute with 7 ml of DMEM:F12 or
HBSS. Spin the cells at 1000 rpm for 5 minutes, remove the supernatant (except for a small volume), flick to
resuspent before fully resuspending in RMM + FGF2/EGF/4OHT. Split into newly prepared flasks/plates as
required.
Freezing and Thawing ReNeuron Cells
To freeze cells,accutasecells off theplastic(e.g.T75 flask) and pelletby centrifugation as above.Remove almost
all the supernatant and resuspend by flickingin the small volume remaining(as above). Resuspend in 1.8ml of
warm RMM+++ and add 10% DMSO (0.2ml, D2650, Invitrogen) slowly and with agitation.Fully mix and aliquot
into cryovials, 1ml of cells per vial (i.e. 1 T75 will generate 2x 1ml vials for freezing). Transfer to a Mr Frosty
freezing vessel at room temperature and placeinto a -80oC freezer overnight. The followingday,transfer frozen
vials to liquid nitrogen for long-term storage.
To thaw a vial of cells ensurethat you have prepared a T75 flask with laminin coating(includingbeingwashed)
and that you have a 15ml tube with 11ml of pre-warmed RMM+++. Placethe vial into the 37oC waterbath until
the vial thaws except for a small pieceof ice. Transfer into the hood and take <1ml of the 11ml of pre-warmed
RMM+++. Add this dropwise into the vial of cells and then remove cells/RMM+++ into the remaining 10ml of
RMM+++. You should nowhave 12ml in total. Transfer this to the prepared T75 flask and placein theincubator.
Allow ~1hr for the cells to settle and gently remove and replace the medium with a fresh 10ml of RMM+++ to
remove the medium with DMSO. Alternatively, upon thawing, transfer the cells into 9ml of pre-warmed
DMEM:F12 and immediately centrifuge at 1000rpmfor 5mins and discard the supernatant(to remove DMSO).
Resuspend the pellet as above (for passaging) and fully resuspend into 10ml of RMM++ before transferringinto
the prepared T75.
58. ReNeuron Cell Differentiation
For differentiation, grow the cells to approximately 80% confluence before washing the cells twice with plain
DMEM:F12 or HBSS then replace with differentiation medium (see DM or alternatives above). Change half of
the medium every 2-3 days.
For Treatment with Depression Study Drugs
Cells were plated, treated in proliferative conditions and then differentiated (RMM without FGF2/EGF/4OHT)
essentially as described in Anacker et al., with: Dexamethasone (1µM, dlluted 1:10,000 from stock in EtOH),
Sertraline(1µM, dlluted 1:10,000 from stock in EtOH), Dexamethasone + Sertraline(1µM each as above) or the
appropriate amount of vehicle (EtOH, 1:10,000 for single drug conditions or 1:5,000 for dual drug conditions).
Some cells were left in untreated conditions.Some cells fromeach condition were fixed atDay 3 of proliferation
before differentiation and some following 2 weeks of differentiation and processed for immunofluorescence
analysis.
