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Photoreceptordamageinducedbylowintensitylight:modelofretinaldegenerationinmammals

MolVis.201319:16141625.

PMCID:PMC3724958

Publishedonline2013Jul25.

Photoreceptordamageinducedbylowintensitylight:modelofretinal
degenerationinmammals
MariaAnaContn,MilagrosM.Arietti,MaraM.Benedetto,ClaudioBussi,andMarioE.Guido
CentrodeInvestigacionesenQumicaBiolgica,(CIQUIBIC,UNCCONICET),DepartamentodeQumicaBiolgica,FacultaddeCiencias
Qumicas,UNC,HayadelaTorreyMedinaAllende,CiudadUniversitaria,X5000HUA,Crdoba,Argentina
Correspondenceto:MariaAnaContin,CIQUIBICDepartamentodeQumicaBiolgica,FacultaddeCienciasQumicas,UniversidadNacionalde
Crdoba,CiudadUniversitaria,5000Crdoba,ArgentinaPhone:543514334171/168,FAX:543514334074email:mcontin@fcq.unc.edu.ar
Received2012May18Accepted2013Jul22.
Copyright2013MolecularVision.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

ThisarticlehasbeencitedbyotherarticlesinPMC.

Abstract

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Purpose
Retinaldegenerationcausedbyadefectinthephototransductioncascadeleadstotheapoptosisofphotoreceptor
cells,althoughtheprecisemolecularmechanismisstillunknown.Inaddition,constantlowlightexposureproduces
photoreceptorcelldeaththroughtheactivationofdownstreamphototransduction.Theauthorsinvestigatedthetime
courseandmolecularmechanismsofdeathandtherhodopsinphosphorylationoccurringduringretinal
degenerationafterexposuretocontinuouslowintensitylight.
Methods
Wistarratswereexposedtoconstantcoolwhite200lxintensityLEDlight(LL)foronetotendaysandcompared
withanimalskeptinthedark(DD)orcontrolsexposedtoaregular12:12h(LD)cycle.Oneeyefromeachratwas
usedforhistologicalandquantitativeouternuclearlayer(ONL)analysisandtheotherforbiochemicalassays.
Results
ThehistologicalanalysisshowedasignificantreductionintheONLofLLexposedratsaftersevendayscompared
withLDorDDexposedrats.RetinalanalysisbyflowcytometerandtheTUNELassayrevealedanincreasein
celldeathintheONL,theinvitroenzymaticactivityassayandwesternblotanalysisshowingnocaspase3
activation.Therhodopsinanalysisdemonstratedmorephosphorylationinserine334residues(Ser334)inLL
exposedthaninLDorDDexposedrats.However,foralltimesstudied,rhodopsinwascompletely
dephosphorylatedafterfourdaysofDDtreatment.
Conclusions
ConstantlightexposureforsevendaysproducesONLreductionbyphotoreceptorcelldeaththroughacapase3
independentmechanism.IncreasesinrhodopsinphosphoSer334levelswereobserved,supportingthenotionthat
changesintheregulationofthephototransductioncascadeoccurduringretinaldegeneration.

Introduction

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Retinaldegeneration(RD)causedbydefectsinthephototransductionmechanismisgenerallycharacterizedby
photoreceptorcelldeathasaresultofgeneticmutations,vitaminAdeficiency,orprolongedlightexposure[14].
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Photoreceptordamageinducedbylowintensitylight:modelofretinaldegenerationinmammals

AlthoughthefunctionalalterationanddiseasemechanismsinvolvedinRDmaydifferdependingonthegene
affected,thecommonresultiscelldeathbyapoptosis[1,59].Retinaldamagebylightexposure,leadingtocell
deathinthevisualcortexviaaseriesofapoptoticevents,hasservedasamodelforhumanRDarisingfrom
environmentalinsult,agingandgeneticdiseases[10].Thephenomenonofretinallightdamageisavisualpigment
mediatedprocess[11]associatedwithbothlongexposuretimesandshorterwavelengthlightexposure.The
exposureofretinaltissuetoradiantenergycangeneratefreeradicals,withtheretinabeingunabletoovercomethe
protectivemechanismtorevertthisprocess(reviewedin[12]).In1966,Noelletal.[13]suggestedthatlow
intensitylightcanalsocausedamagetotheretina,andthereisevidencethatrodphotoreceptorsexposedtolow
intensitylightdiefromalightinducedconstitutivesignaltransductionmechanism[14,15].
ApoptosisismanifestedbytheappearanceofdoublestrandedDNAbreakswithintheinitialhoursoflight
exposure,whichdependsonthewavelengthandintensityoflightused[2,7,16,17].However,thereare
contradictoryresultsregardingtheapoptoticmechanismandtheroleofcaspase3inlightinducedmodels:some
authorshaveattributedacentralroletothisenzymeinphotoreceptordegeneration[1820],whereasothershave
reportedacaspase3independentmechanismassociatedwiththeroleofCa+2dependentproteasecalpainsor
cathepsinDasalternativedeathpathways[2024].Itisclearfromallthesefindingsthatphotoreceptordeathvaries
inbothseverityanditsapoptoticmechanisms,whichdependonthestrain,lightintensityandwavelengthused.
Haoetal.[25]providedevidenceoftwoapoptoticpathwaysthatareinitiatedbylightactivationofrhodopsin.
Brightlighttriggersapoptosisofphotoreceptorcellsthroughamechanismrequiringactivationofrhodopsinbutnot
ofthephototransductionmechanism,whereaslowlightintensitiesinducephotoreceptorapoptosisbyphotopigment
activationandsubsequentdownstreamsignaltransduction[25].Inalbinorats,retinalstimulationwithcontinuous
lowwhitelightcausestheprogressivedeteriorationofphotoreceptors,aneffectthatdoesnotoccurinratsexposed
tocyclicilluminationconditions[13,2631]thethresholdcycliclightintensitythatproducesdamagetotheretinas
ofalbinoratsliesaround270lx[32].Althoughlightmicroscopefindingsrevealedfragmentationanddisorientation
ofthephotoreceptoroutersegments(OS)afterthreetofivedaysofconstantexposuretolowlight,withno
photoreceptorsatallremainingafterthirtydaysofexposure[33],nosinglechangecouldbeidentifiedthatwould
inexorablyleadtocelldeath[33].Ultrastructuralchangesinphotoreceptorssuggestthatdeathoccursbecausethe
cellscannolongermaintaintheiranabolicprocesses[3436],butelucidationoftheprecisemechanismsinvolved
callsforthesystematicstudyofphotoreceptorcellfunctions.
Weconsiderthatabetterunderstandingofthemolecularmechanismstriggeredbylowlightintensityexposure
leadingtoRDcouldprovidevaluableinsightsintotheprogressionofclinicaldisordersrelatedtophototransduction
defects.Theaimofthiswork,therefore,wastoinvestigatethetimecourseofRDandthemechanismsofdeathin
Wistarratsstimulatedbyconstantexposureto200lxofdiffuse,coolwhite,LEDlight.Ourresultsshowthat
continuousilluminationproducesretinaldamage,withareductionintheONLoccurringaftersevendaysoflight
stimulationthroughacaspase3independentmechanism.Moreover,rhodopsinproteinexpressionanalysisshowed
thatrhodopsinlevelsdidnotreducebeforeONLcellsdied,butbecamemorephosphorylatedatSer334residues.

Methods

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Animals
AllanimalprocedureswereperformedinaccordancewiththeARVOstatementfortheuseofanimalsin
ophthalmicandvisionresearch,whichwasapprovedbythelocalanimalcommittee(SchoolofChemistry,UNC,
Exp.2013291).MaleadultWistarratsaged1215weekswereexposedtoa12:12hlightdark(LD)cycle,with
thelighton(lessthan10lx)fromzeitgebertime(ZT)0to12forallanimalsfromthetimetheywerebornuptothe
experiment.Foodandwaterwereavailableadlibitum.
Lightdamage
Retinaldegenerationwasinducedinatemperaturecontrolledlightstressboxequippedwith200lxofdiffuse,cool
white,LEDlampsfixedontheinneruppersurface,whichilluminatedtheinteriorwhitewalls.Theilluminationat
theleveloftheratseyeswasmeasuredas200lxwithalightmeter(model401036ExtechInstrumentsCorp.,
Waltham,MA).AllratswerekilledinaCO2chamberatZT12atonetotendaysafterconstantlightstimulation
(LL)accordingtotheexperimentaldesign.

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Controlexperimentswereperformedbyexposingallanimalsto200lxundera12:12hLDcycle,withlightonat
ZT0andoffatZT12.ToverifythattheLDcycledidnotinducedegenerativedamageduringthelightstimulias
observedinconstantlight(LL),otheranimalsweresubjectedtoconstantdarkness(DD)duringthesametimesas
animalsexposedtotheLDcycleandunderidenticalconditionsinthetemperaturecontrolledstressbox.
Histologicalandquantitativeouternuclearlayeranalysis
Themethodsemployedforfixation,embedment,sectioningandhistologicalanalysisofeyeswereaspreviously
described[37].Briefly,rateyesatdifferentdaysafterlightorcontroltreatmentwerefixedin4%(W/V)
paraformaldehydein100mMsodiumphosphatebuffer(PBS,pH7.3)overnightat4C,beforebeing
cryoprotectedinsucroseandmountedinanoptimalcountingtemperaturecompound(OCTTissueTekSakura).
Retinalsections10mthickwerethencutalongthehorizontalmeridian(nasaltemporal)usingthecryostat.The
sectionswerelightlystainedwith1%(W/V)propidiumiodide(PI)fortwominutesandphotographedusinga
confocalmicroscope(OlympusFV300,Japan)at40magnification.ThenumberofONLnucleiwascountedon
thephotographofeachofthefourdesignatedretinalareasleft,middleleft,middlerightandrightforten
differentanimalspertreatment.QuantitativeONLanalysiswasperformedtoquantifyphotoreceptorsurvivalonthe
entireONL(widthandlengthofthemicrograph),usingthesoftwareImageJ(v.1.45),andthepluginAutomatic
NucleiCounter[38].
FACScanflowcytometer
Retinasweredissected,dissociatedin0.25%(W/V)trypsin(Lifetechnology,Inc.)andprocessedasdescribed
previously[20]withmodifications.Briefly,cellswerewashedtwiceandthenresuspendedwithcoldPBS(100
mMsodiumphosphate).Aliquotsof100lwereseparatedandloadedintoaNeubauerchamberforcounting.
Afterlabeling1104cellsbyadding5lofFITCAnnexinV(PEAnnexinVDetectionKitI,559763,BD
Biosciences)and10lofpropidiumiodide(PI0.05mg/mlstocksolution,Sigma,P48864),thesolutionwasgently
vortexed,incubatedfor15minatroomtemperature(25C)inthedarkandanalyzedbyflowcytometryona
BectonDickinsonFACSflowcytometerwithinthefirsthour.Thecelldataofsixretinaspertreatmentfromthree
independentexperimentswereanalyzedbytheFlowJosoftware.
TUNELstaining
TheterminaldeoxynucleotidyltransferasedUTPnickendlabeling(TUNEL)assaywasperformedfollowingthe
proceduresprovidedwiththekitfromRocheDiagnosticsCorp.(Indianapolis).Briefly,frozensectionsofratretinas
werecutonacryostatbeforebeingpostfixedwith4%paraformaldehydeandpermeabilizedinPBS,0.1%(W/V)
sodiumcitrate,0.1%(V/V)TritonX100.Thereactionmixture(50lTUNEL)wasaddedtoeachsampleandthe
slideswereincubatedinahumidifiedatmospherefor60minat37Cinthedark.Then,sectionswerelightly
stainedwith1%(W/V)propidiumiodide(PI)fortwominutes.Fornegativecontrols,theTdTenzymewas
replacedbylabelsolution,andthesamplesfromthreedifferentanimalspertreatmentwereanalyzedbyaconfocal
microscope(OlympusFV300,Tokyo,Japan).
Outersegmentpreparation
Animalswerekilledinthedarkunderinfraredillumination.TheeyeswerewashedoncewithPBSbufferandthe
retinasremovedandmaintainedin20%(W/V)sucroseinthedark.TheOSwerepurifiedbysucrosegradient
purificationasdescribedpreviouslywithsomemodifications[39].Briefly,theretinaswereputoniceinaglass
tubecontaininganisolationmediumcomposedof20%sucrosePBSinthepresenceofproteaseinhibitorcocktail
P33360(Invitrogen)andphosphataseinhibitorcocktailtablets(PhosphoSTOP,Roche),andwerehomogenized
withfiveupanddownpassesinaDouncehomogenizer.Theretinalhomogenate(500l)waslayeredontopofa
discontinuoussucrosegradientatconcentrationsof20%,27%,and50%(W/V)sucrose,beforebeingcentrifugedat
141,000gfor2hat4CinaBeckmanultracentrifuge,50.2TIrotor(WSBECKOPTXL90).After
ultracentrifugation,therodOSsedimentingbetweenthe27and50%sucrosephasewerecollected,dilutedinPBS,
andcentrifugedat7700gfor10minat4CbeforebeingsuspendedinPBSbuffer.Thesedimentcollectedfrom
theultracentrifugationat141,000ginthesucrosegradientwasresuspendedinPBSandcentrifugedat800gfor
10min,withtheprocedurerepeatedthreetimes.Thispreparationwasnamedpelletpreparation(P).
Westernblot
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Caspase3 Homogenatesofwholeratandembryonicchickretina(embryonicstage11,E11)resuspendedin

200lPBSbuffercontainingtheproteaseinhibitorcocktailwerelysedbyrepeatedcyclesofultrasonicationand
thetotalproteincontentwasdeterminedbytheBradfordmethod[40].Chickretinaservedasapositivecontrolof
caspase3expression[41].Then,thehomogenatesofwholeratandembryonicchickretinaswereresuspendedin
samplebuffer(SB:62.5mMTrisHClpH6.82%(W/V)SDS10%(V/V)glycerol50mMDTT0.1%(W/V)
bromophenolblue)andheatedat90Cfor5min.Theproteins(20g)wereseparatedbySDSgelelectrophoresis
on12%polyacrylamidegels,transferredontonitrocellulosemembranes,blockedfor1hatroomtemperaturewith
5%(W/V)skimmilkinPBSandthenincubatedovernightat4Cwithantibodyagainstcaspase3(HPA002643
Sigma,StLouis,MO)intheincubationbuffer(2.5%(W/V)skimmilkand0.1%(V/V)Tween20detergentin
PBS).Membraneswerewashedthreetimes(15mineachwash)inwashingbuffer(PBScontaining0.1%Tween
20Sigma,P1379),andthenincubatedwiththecorrespondingsecondaryantibody(goatantirabbitIRDye
OdysseyLICOR)intheincubationbufferfor1hatroomtemperature,followedbythreewashes(15mineach
wash)withwashingbuffer.MembraneswerescannedusinganOdysseyIRImager(LICORBiosciences)before
beingstrippedwith0.5MNaOHandincubatedfor1hatroomtemperaturewithtubulin(SigmaT6199)
antibody.Theywerethenwashedthreetimes(15mineachwash)withwashingbufferandincubatedwith
secondaryantibody(goatantimouseIRDye800CW)intheincubationbufferfor1hatroomtemperaturebefore
carryingoutthreefurtherwashesof15mineachwithwashingbuffer.MembraneswerescannedusinganOdyssey
IRImagerandtworetinasfromfourindependentexperimentswereanalyzedforeachtimepoint.
Rhodopsin Homogenatesofwholeratretinas,resuspendedin200lPBSbuffercontainingtheproteaseinhibitor

cocktail,werelysedbyrepeatedcyclesofultrasonication.Asthedegeneratedretinas(LL)containedlessprotein
thancontrolretinas(LD),andaswewishedtocomparetheratioofrhodopsininLLtothatinLD,ratherthanthe
absoluteproteinquantity,thesameamountofsamplesperretina(nottotalprotein)wereloaded.Homogenates
(200l)wereresuspendedinsamplebufferand40lwereseparatedbySDSgelelectrophoresison15%
polyacrylamidegels.Thegelwasthentransferredontonitrocellulosemembranes,blockedfor1hatroom
temperaturewith5%skimmilkinPBSandthenincubatedovernightat4Cwithrhodopsinantibodies(RetP1
Sigma)intheincubationbuffer.Themembranesweresubsequentlywashed,incubatedwiththesecondaryantibody
goatantimouseIRDye800CWandscannedintheOdysseyIRImagerasindicatedinthecaspase3westernassay.
Sixretinasfromfourindependentexperimentswereanalyzedforeachtimepoint.Densitometryofwesternblots
wasperformedusingtheImageJsoftwareandtheratiofromthequantitativeanalysisofthreeoligomerbandoptical
densitiesinLLrelativetoLDwasexpressedasapercentageofchange.
Rhodopsinphosphorylation ThetotalproteinsoftheOSandPpreparationwerequantifiedbytheBradford

method[40]and40gwereseparatedbySDSgelelectrophoresison15%polyacrylamidegels.Thegelwasthen
transferredontonitrocellulosemembranes,blockedandincubatedovernightat4Cwithrhodopsinphosphorylated
antibodies(PhosphoSer334assayBiotech)andsecondaryantibodygoatantimouseIRDye800CW,and
scannedintheOdysseyIRImagerasindicatedinthecaspase3westernassay.Themembranesweresubsequently
strippedwith0.5MNaOHandincubatedovernightat4Cwithtubulin(SigmaT6199)antibodyandscanned
usinganOdysseyIRImager.Fourretinasfromthreeindependentexperimentswereanalyzedforeachtimepoint.
DensitometryofwesternblotswasperformedusingtheImageJsoftwareandtheratiofromthequantitativeanalysis
ofthreeoligomerbanddensitiesofrhodopsintotubulininOSsampleswasexpressedastherelativeopticaldensity.
Caspase3activity
Retinalcaspase3activitywasmeasuredwithacommercialcolorimetrickit(EnzChekCaspase3AssayKit#1
MolecularProbesInc.Eugene,OR)accordingtothemanufacturersinstructions.Briefly,equalaliquotsofretinal
homogenatewereincubatedat37Cfor30minwithcaspase3specificsubstrateZDEVDAMCinthekit
reactionbuffer.Theabsorbanceofeachsamplewasreadat441nmandcaspase3activitylevelsweredirectly
proportionaltothecolorreaction.Chickretinas(embryonicstage11)wereusedaspositivecontrolofcaspase3
activity.ResultsofeachLLtreatmentwereexpressedasthepercentagechangeintheabsorbancemeasuredinLL
withrespecttoLD.
Statisticalanalysis
Dataareexpressedasmeanstandardderivation(SD).Statisticalcomparisonsweremadeusingaoneway
ANOVA(ANOVA)pvalue<0.05wasconsideredstatisticallysignificant.Toprovethenormalityand
homogeneityofvarianceassumption,weusedShapiroWilksandLevenetests.Inallcaseswithsignificance,a
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Duncantestwasapplied.Apvalue<0.05wasconsideredstatisticallysignificant.Whenweworkedwith
percentagedataweappliedalogarithmtransformationandwhendatadidnotpresentnormality,anonparametric
KruskalWallistestwasconducted.

Results

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Histologicalandquantitativeouternuclearlayeranalyses
AsdescribedintheMethodssection,retinasofanimalsexposedtocoolwhite200lxlight(LL)wereprocessedby
quantitativeONLanalysistoassessretinallayerthinning.Photoreceptordamageoccurredinthelightexposedeyes
andresultedinthelossofaround50%oftheONLphotoreceptornucleiattendaysofLLstimulation(Figure1B)
comparedwithanimalsrearedinanLDcycle(Figure1AandAppendix1).Therewasasignificantlylower
numberofnucleiintheONLofLLexposedgroupsaftersevendaysoflighttreatment(535.25nuclei142.99
Figure1C)comparedwiththeLDCycle(1003.22nuclei95.32)orDD(1058.23nuclei79.31).
Figure1
Effectofconstantlowlightexposureonthenumberofouternuclearlayer
nuclei.A:Retinasrearedina12:12hlight(200lx)darkcycle(LDCycle).
PhotoreceptorsinONLweremorphologicallynormal.B:Retinasfromrats
exposedtotendays...
Analysisofdeath
FACScanflowcytometer Biochemically,apoptosisischaracterizedbythephospholipidphosphatidylserine(PS)

becomingenrichedintheouterleafletoftheplasmamembrane.AnnexinVisa3536kDaCa2+dependent
phospholipidbindingproteinthathasahighaffinityforPS[42,43]andisusedtodifferentiateapoptoticfrom
necroticcells[42].Earlyoninapoptoticcells,membranePSistranslocatedfromtheinnertotheouterleafletofthe
plasmamembrane.Whenapoptosisismeasuredovertime,earlyAnnexinVlabelingispositiveandPInegative,
withthemembranestillretainingitsintegrity.However,whenbothAnnexinVandPIarepositive,thisindicates
thatthemembranehaslostitsintegrityandsignalstheendstageofapoptosisanddeath.
Figure2A,BpresentsthetemporalquantificationofAnnexinVandPIstainingbyflowcytometerstudies.
Figure2BshowsthatafterfivedaysofLLexposure,PIandAnnexinVincreasedinretinalcells(LL5D,
PI:2.430.41%PI/AnnexinV:2.750.54%andAnnexinV:0.220.17%)comparedwithcontrolcells(LDCycle:
PI:0.190.42%PI/Annexin0.130.14%,AnnexinV0.040.03%).Atsevendays,PIincreasedandPI/Annexin
decreasedwithrespecttoLL5D(LL7D:PI:3.980.64%PI/Annexin:2.810.33%andAnnexinV:0.490.33%).
TheseresultsindicatethatexposureofWistarratstoLLproducesretinalcelldeath.
Figure2
AnnexinV,PIandTUNELassayinratretinaexposedtolowintensitylight.
A:Imageshowingarepresentativeanalysisofretinafromratsexposedtoa
regularLDcycle200lx(L12hD12h)andforfiveandsevendaysof
constantlight(LL).B:...
TodemonstratethatthecelldeathsoccurredintheONLwherethephotoreceptornucleireside,weanalyzedDNA
fragmentationusingtheTUNELassayintheretinalhistologicalsection.TheseresultsshowedpositiveTUNEL
staining(green)localizedattheONLintheretinasofratsexposedtofourdaysofLL(Figure2E,F),whereasno
stainingwasdetectedafterfourdaysinretinasintheLDCycle(Figure2C,D).
Caspase3analysis Theexpressionofcaspase3proteinanditsactivitywasinvestigatedduringphotoreceptor

celldeathusingtwodifferentanalyticaltechniques(Figure3).Theimmunoblotanalysisofretinasfromanimals
exposedtoLLstimulationrevealedprocaspase3protein(32kDa)tobepresentatalltimesstudied,althoughno
activeformoftheprotein(17kDa)wasobservedunderanyconditiontested(Figure3A).However,afractionof
cleavedcaspase3immunoreactivitywasdetectedinthehomogenatesofcontroltissue(chickenretinaatembryonic
stage11).

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Figure3
Caspase3analysesofratretinasexposedtolowintensitylight.A.Awesternblotshowing
caspase3inretinasafterexposuretoaregularLDcycleof200lx(LDCycle12hL:12hD)or
to12hortoone,two,fiveortendaysofconstantlight...
Caspase3activitywasfurtherassessedbymeasuringthecleavageofthecolorimetricsubstrateDEVDpNA(
Figure3B).TheresultsinFigure3BshowtheaverageabsorbancemeasuredineachLLandDDtreatment
comparedwithLDcontrols(%).Inagreementwiththeresultsoftheimmunoblotanalysis,nosignificantactivation
ofcaspase3wasobservedintheretinasatanyoftheexaminedtimescomparedwiththecontrolgroup(chicken
embryonicretinasdatanotshown),stronglysuggestingthatthecelldeathmechanisminphotoreceptorswas
caspase3independent.
Rhodopsinanalysis
Rhodopsinexpressionandphosphorylation,knowntobeinvolvedinthecriticalquenchingstepofthe
phototransductioncascade,wereevaluatedtoinvestigatewhetherthephotopigmentofthephototransduction
cascadewasaffectedbylowlightstimulationwithinthephotoreceptorcells.AsshowninFigure4,westernblotof
retinasimmunolabeledwithrhodopsinantibodyshowedthepredictedisoformsofthisprotein[44].Afterseven
daysofLLstimulation,proteinlevelswerelowerthanthoseinLDcontrolsandDD,withthehighestlevelsof
rhodopsinimmunoreactivitybeingrecordedinthelatter(Figure4A).Densitometricanalysisoftherhodopsinlevels
showedasignificantreductionaftersevendaysofLL,coincidingwithareductioninONL(Figure4B)and
suggestingthatconstantlowlightdidnotaffectrhodopsinexpressionbeforeapoptoticphotoreceptorcelldeath.
Theeffectsoflowlightstimulationstressonrhodopsinphosphorylation(Ser334residue)wasstudiednextinthe
OSandPpreparationbywesternblotmethodsinvolvingaspecificantibodyagainstrhodopsinphosphoSer334,as
describedintheMethodssection.InFigure5A,arepresentativewesternblotofthepositiveimmunoreactivityof
rhodopsinSer334,isshownattwo,fourandsevendaysofLLexposure.Ascanbeseen,therhodopsinphospho
Ser334washigherinLLthaninLDcontrolsandwaslocalizedmainlyintheOSofretinaphotoreceptorsexposed
totwodaysofLLstimulation.HoweveratLL4DandLL7D,itwasalsofoundinthePelletfraction.A
quantitativeanalysisoftheOSfractionshowedaslightincreaseinrhodopsinphosphoSer334betweenfourto
fortyeighthoursofLL(Figure5B)incontrast,atlongertimesofLL(LL4DorLL7D)therhodopsinphosphor
Ser334washigherandstatisticallysignificantaftersevendaysofLL(Figure5C).Moreover,therhodopsinwas
completelydephosphorylatedaftersevendaysinDD(Figure5A).Theseresultssuggestthathigherlevelsof
phosphorylationordelayeddephosphorylationactivityoccurredwithconstantlowlightstimulation.
Figure4
Analysisofrhodopsinexpressioninratretinaexposedtolowintensitylight.
A:Awesternblotimmunolabeledrhodopsinwithaspecificantibody
showingthepredictedisoformbands.Themarginshowstheoligomerization
statesoftherhodopsinfragment...

Figure5
AnalysisofrhodopsinphosphorylatedinSer334intheoutersegment(OS)
andpellet(P)ofratretinasexposedtolowintensitylight.A:Awesternblot
ofrhodopsin(phosphoSer334)inretinasafterexposuretoaregularlight
darkcycle(LDCycle12...

Discussion
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Lightinducedstressontheretinacausestheapoptoticcelldeathofphotoreceptors[29,45,46],withitsseverity
dependingonthelightintensity,exposuredurationandwavelength[10,11,47].However,themolecular
mechanismsgivingrisetothisresponsehavenotbeenfullyelucidatedtodate.
Inalbinorats,retinalexposuretolowwhitelightleadstoseveredegenerativechanges,withreducedthicknessof
theONLandultrastructuralalterationssuchasfragmentation,disorientationoftheOSandchangesinthe
amplitudeoftheelectroretinogrambwave[13,2629].Thesephenomena,however,arenotfoundinratsexposed
tocycliclowintensitylight(lessthan270lx)lowerthanthethresholdofcycliclightintensitycapableofproducing
damagetotheretina[32].Ourdatashowthatconstantlight,butnotcyclicexposureto200lx,produced
photoreceptordeathinalbinoWistarrats(Figure1).Nevertheless,wecannotruleoutthepossibilityof
ultrastructuralalterationshavingoccurredunderbothLLandLDexposure.RetinalONLreductionwasstatistically
significantafteronlysevendaysoflowlightexposurecomparedwithanimalskeptinDDoratregularLDcycles
of200lxintensity,indicatingthatcelldamageprocessesmaybeslowerthanbrightlightdamageandcorrespondto
adifferent,phototransductiondependentcelldeathmechanism.Inthisconnectionitisknownthatphotoreceptor
apoptosisinducedbyconstantlightexposureatlowerintensitiesrequirestheactivationofphotopigmentsand
consequentdownstreamsignaltransduction[25],suggestingthatimpairmentofthephototransductionmechanism
couldberesponsibleforcelldeath.
FlowcytometryandTUNELassaysrevealedcelldeathinretinasexposedtoLLafterfourdays,withretinalcell
deathandanincreaseinAnnexinVbeingobservedafterfivedaysoflightstimulationwithaconcomitant
incrementinPI(Figure2A,B).TUNELanalysisshowedpositivestainingintheONLarea(seeFigure2B),
demonstratingthatallcelldeathswereofphotoreceptors.
Caspase3isrecognizedasbeingoneofthekeyexecutionersofapoptosis.However,theapoptoticpathwayand
theroleofcaspase3inlightinducedmodelsdependonmultiplefactorssuchasanimalstrain,lightintensityand
thewavelengthused.Someauthorshaveattributedacentralroletothisenzymeinphotoreceptordegeneration[18
20,48],whileothershavereportedacaspase3independentmechanisminvolvingCa+2dependentprotease
calpainsorcathepsinDasalternativedeathpathways[2024].Totrytoelucidatethetypeofmechanismsinvolved
inthisphenomenon,wesystematicallystudiedcaspase3proteinexpressionandenzymaticactivationapplying
antibodiesagainstactivatedandnonactivatedisoformsusingacommercialcolorimetricactivityassaykit.As
showninFigure3,neithermethodwasabletodetectcaspase3activityatanytimeofthelightexposuresstudied.
Althoughtheapoptosismechanismisknowntobeinvolvedafterretinaldamage,otherpathwayssuchas
necroptosismightalsoplayarole.Inthissense,Trichonasetal.[49]demonstratedthatreceptorinteractingprotein
(RIP)kinasemediatednecroptosisdeathwasimplicatedintheretinaldetachmentinducedbysubretinalinjectionof
sodiumhyaluronate,indicatingthatRIPmaybeacomplementarymechanismofphotoreceptorcelldeath.Here,we
showthatphotoreceptorcelldeathinretinasexposedtoLLoccurredthroughacaspase3independentmechanism,
suggestingthatphotoreceptorcelldeathisanapoptoticcaspase3independentmechanism.Nevertheless,the
participationofanecroptosismechanismcannotberuledout.Furtherstudiesarenecessarytodeterminewhether
Ca+2dependentproteasecalpainsorcathepsinDapoptoticprocesses,necroptosisorindeedbothmechanismsare
involvedinphotoreceptorcelldeath.
QuantitativeanalysisofrhodopsinexpressionrevealedasignificantreductionintheproteinaftersevendaysofLL,
coincidingwithareductioninthenumberofphotoreceptorcells(Figure4)andthussuggestingthatconstantlow
lightstimulationdoesnotaffectrhodopsinexpressionbeforephotoreceptorcelldeath.However,westernblot
analysisrevealedanincreaseinthepositiveimmunoreactivityofrhodopsinphosphoSer334atdifferenttimesofLL
exposure(Figure5).AlthoughtheseincrementswerenotstatisticallysignificantatshorttimesofLL(betweenLL4
htoLL48h),atalongertime(LL7D)therhodopsinphosphoSer334washigherandsignificant.
Dependingonthemammalspecies,sixorsevenpotentialphosphorylationsitesintheCterminusofrhodopsin[50]
Ser334andSer338havebeenidentifiedasmajorsitesforthephosphorylationofrhodopsininvivo[51],soasto
obtainmaximalhighaffinitybindingofarrestin1[52].Phosphorylationoccursinasequentialmanner,correlating
withthedegreeofillumination[53].Thus,underhighlevelsofillumination,rhodopsinisphosphorylatedat
relativelyhigherlevelsforthemono,di,tri,andtetraphosphorylatedspeciesthanforthepentaand
hexaphosphorylatedspecies[54],resultinginmaximumarrestinbindingandreceptordesensitization[55].
Studiesontheretinalphotoreceptordegenerativemodelofcancerassociatedretinopathy(CAR)haverevealed
significantlyhighlevelsofrhodopsinphosphorylation[5658].Rhodopsinmutantscausingautosomaldominant
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retinitispigmentosaarealsophosphorylatedatsignificantlyhigherlevelsthaninwildtypecells[59],andrhodopsin
dephosphorylationkineticsintheretinaldegenerationofP23Hratshavebeenshowntobeextremelyprolonged,
especiallyinSer344[60].Furthermore,micewithamissensemutation(K296E)thatproducesanopsinwithanon
chromophorebindingsite(spontaneouslyactivated)presentphosphorylatedrhodopsintightlyboundtoarrestinin
vivo[61].Inthissense,Allowayetal.[62]demonstratedthatincertaingeneticbackgroundsinDrosophila,the
lightdependentformationofstablephosphorylatedrhodopsinarrestincomplexes,whichmostlyaccumulateinthe
innersegmentofphotoreceptorcells,mayconstituteamolecularmechanismfortheinitiationofretinal
degeneration.
Ithasbeenshownthatthelevelandmultiplicityofrhodopsinphosphorylationwerereducedasafunctionof
exposuredurationinretinasofSpragueDawley(SD)ratssubjectedtolightstressintheformofexposuretointense
greenlightforuptoeighthours[54].Theseauthorshypothesizethatasregeneratedrhodopsinisconstantlyre
activatedbylight,thesupplyof11cisretinaldeclinesgraduallyandreducesthelevelofrhodopsin
phosphorylation.However,SDandRoyalCollegeofSurgeons(RCS)ratsexposedtowhitelightfor24hshowed
asignificantdelayinthekineticsofrhodopsindephosphorylationinRhodopsinSer334andSer338,withhighlevels
ofphosphorylatedrhodopsinbeingdemonstratedbyimmunofluorescencelabeling[57].Thisdiscrepancymaybe
duetotherhodopsinphosphorylationresiduestudiedoraresultofthelighttreatment,withalowerintensityof
greenlightorthefullspectrumofwhitelightperhapsbeingnecessarytoproducetheeffect.Takentogether,all
theseresultssuggestthatdefectsintheregulationofthephototransductioncascadebyrhodopsinphosphorylation
andtheconsequentinternalizationoftherhodopsinarrestincomplexmayconstituteakeystepinphotoreceptor
degenerationbymutationsinkeycomponentsofphototransductionorlightstress.Ourstudiessuggestthat
rhodopsinphosphorylationmaybeaffectedbyprolongedphototransductionmechanismactivityandgeneratesa
riseinphosphorylationand/ordelayeddephosphorylationofrhodopsin.Furtherstudieswillberequiredto
investigatethephosphorylationstateinotherresiduesofrhodopsinandinternalizationtotheinnersegmentinLL
bylowlightandtoexaminewhetheralterationsinRKandPKCactivityaredependentonthedurationofthelight
stimulus.
Basedonourresultsdiscussedhere,weconcludethatconstantilluminationoflowwhitelightintensity(200lx)
producedretinaldegenerationthroughacaspase3independentmechanism,wherebytheONLwassignificantly
reducedaftersevendaysofconstantstimulation.Oneofthekeymechanismsfortheinitiationofthisprocesscould
berhodopsinhyperphosphorylationasaconsequenceofimpairmentoftheenzymesrelatedtothe
phosphorylation/dephosphorylationprocesses,andinternalizationtothephotoreceptorcellsoma.Inthissense,
constantlowlightinducedphotoreceptorcelldeathcouldbeausefulmodelforstudyingthemechanismsinvolved
inphototransductiondefectsatthemolecularlevel.Asthemostcommonformofretinaldegenerationresultsfroma
primarydefectinrodswithsecondaryconeloss[5,63],theuseofsuchamodelinthecomprehensivestudyof
retinaldegenerationmaythusprovideabasisforfuturetherapiestopreventoratleastdelayvisualcellloss.

Acknowledgments

Goto:

TheauthorsaregratefultoDr.CeciliaSampedro,Mrs.SusanaDezaandGabrielaSchachnerfortheirexcellent
technicalsupport.ThisworkwassupportedbygrantsfromtheAgenciaNacionaldePromocinCientficay
Tcnica(FONCyT,PICT2005No31,971,PICT2006No898andPICTbicentenarioNo647),ConsejoNacional
deInvestigacionesCientficasyTecnolgicasdelaRepblicaArgentina(CONICETPIP20092011),Secretara
deCienciayTecnologadelaUniversidadNacionaldeCrdoba(SeCyTUNC)andMinistryofSciencesand
TechnologyofCrdoba.

Appendix1.Effectofconstantlowlightexposureoncentralretinalthickness. Goto:
Toaccessthedata,clickorselectthewordsAppendix1.AC:Retinasrearedina12:12hlight(200lx):dark
cycle(LDCycle).DF:Retinasexposedtoconstantlowlightfortwodays(LL2D).GI:Retinasexposedto
constantlowlightfortendays(LL10D),A,D,G:RetinaslabeledwithPIB,E,H:Transmittanceimage.Scalebar
indicates30mC,F,I:Transmittanceimage.Scalebarindicates15m.ONL,outernuclearlayerINL,inner
nuclearlayerGCL,ganglioncelllayerOS,outersegmentIS,innersegment.

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