Journal of NeurochemistryLippincott—RavenPublishers, Philadelphia© 1997 InternationalSociety forNeurochemistry

Ethanol Exposure Affects Glial Fibrillary Acidic Protein GeneExpression and Transcription During Rat Brain Development

S. Vallés, J. Pitarch,~J.Renau-Piqueras,and C. Guerri

Instituto de InvestigacionesCitológicas (FIB), and *Centro de Investigación,Hospital La Fe, Valencia, Spain

Abstract: Exposure to ethanol during fetal developmentreduces the astroglial-specific marker glial fibrillary acidicprotein (GFAP) and its mRNA levels in brains of fetalrats and in radial glia in primary culture, affecting theproliferation and differentiation of astrocytes. The objec-tives of this study were to evaluate the possible effectof ethanol on GFAP mRNA levels in astrocytes and toinvestigate the molecular mechanism(s) involved in etha-nol-induced changes in GFAP expression by analyzingthe GFAP transcription rate, GFAP mRNA stability, andGFAP DNA methylation. We show here that prenatal ex-posure to ethanol reduces significantly GFAP immunore-activity and its mRNA levels in both astrocytes in primaryculture and brains of pups from alcohol-fed mothers.Runoff experiments from nuclei of astrocytes indicate thatethanol exposure decreases GFAP transcription rate sig-nificantlyand reduces GFAP mRNA stability slightly. DNAmethylation analysis indicates that prenatal ethanol expo-sure induces a hypermethylated state of the GFAP DNAin fetal brains. Methylation-mediated repression of GFAPtranscription could be a mechanism involved in ethanol-induced reduction of GFAP expression. Ethanol-inducedalterations in GFAP expression and astroglial develop-ment may underlie the CNS dysfunctions observed afterprenatal alcohol exposure. Key Words: Prenatal ethanolexposure— Brain development—Astrocytes in culture—Glial fibrillary acidic protein—Vimentin—mRNA—Tran-scription—Runoff—mRNA stability—Methylation—Fe-tal alcohol syndrome.J. Neurochem. 69, 2484—2493 (1997).

Although the developingbrain ishighly vulnerableto the effectsof ethanol,the mechanisms underlyingthedeleterious effectsof ethanolremainto be clarified(West et al., 1994). Normal brain developmentde-pendson thecoordinatedpatternsof a seriesof com-plex processesin which neuronsandglial cells partici-pate (Rakic, 1991). It has been shown that ethanolproducesa variety ofdisruptionsin some ofthesede-velopmentalprocesses, includingdepressionof neu-rogenesis,delayed andaberrantneuronalmigration,anomalous morphologicaldevelopment (Miller, 1992),changes in theontogenyof neurotransmitter synthesis(Druse,1992), andneuronal depletionin severalbrainregions(Westet al., 1990).Moreover,studies ofboth

humans andexperimental animals provide evidencethat alterationof glial developmentmay contribute totheeffectsof alcohol on thedevelopingbrain (Phillips,1994;Guerri andRenau-Piqueras,1997).Thus, in vivostudies show that prenatal ethanol exposure toalcoholreducesthe numberof glial cells in cortex (Gressenset al., 1992; Miller,1992),altersmyelinogenesis(Lan-casteret al., 1982; Phillips,1994), inducesabnormalmorphologyof radial andBergmannglia (ShettyandPhillips, 1992; Miller andRobertson,1993; Shettyetal., 1994),anddecreasessignificantly the levels of theastrocyte-specificmarkerglial fibrillary acidicprotein(GFAP) in thebrainsof pups fromethanol-fedmothers(Sáezet al., 1991). Studies usingprimary cultures ofcorticalastrocytesalso show that in uteroand in vitroethanol exposureaffects thefunctional and structuralcharacteristicsof these cells,including their prolifera-tion anddifferentiation,the contentand distribution ofseveral cytoskeletalproteins such as GFAP, andthereleaseof growth factors (Guerri et al., 1993; GuerriandRenau-Piqueras,1997).

Glial cells are oneof the mostabundantcell typesin thevertebrate CNS,and they areessentialfornormaldevelopmentof the nervous system. Theyprovidethestructureandnutritive support fordevelopingneurons(Hattenet al., 1990; Rakic, 1991) and are involved inmany functions includingstimulation ofneuriteout-growth and guidance ofmigrating neurons,synapto-genesis and synapticplasticity, regulationof ions andtransmittersin the microenvironment,production ofseveral growth factors, andregulationof water, energy.and nutrient supportof neurons (Norenberg, 1994:Hanssonand Rönnbäck,1996).

Little is known, however, regardingthe molecularmechanisminvolved in theeffect of ethanol on glial

ReceivedApril 15, 1997; revised manuscript received July30.1997; acceptedJuly 30, 1997.

Addresscorrespondenceandreprint requeststo Dra. C. Guerri atInstituto de InvestigacionesCitologica, Amadeo de Saboya 4,46010-Valencia, Spain.

Abbreviationsused: BAL, blood alcohol level; DTT,dithiothrei-tol; GFAP, glial fibrillary acidic protein; PBS, phosphate-bufferedsaline;PEE, prenatallyethanolexposed; SDS, sodiumdodecyl sul-fate; SSC,saline—sodiumcitrate.

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ETHANOL AFFECTSGFAP GENE EXPRESSION 2485

cells.We recently showedthat ratradial glia expressGFAP (Sancho-Telloet al., 1995) and thatethanolexposure duringembryogenesisdelays theappearanceof this protein and itsmRNA, as demonstratedin invivo and in vitro studies (Vallés et al., 1996). Al -thoughGFAP appearsto play a critical role in themorphogenesisof the CNS(Liedtkeet al., 1996),littleis known regarding themolecularmechanisminvolvedin the effect of ethanolon GFAP expression.A de-creasein the GFAPmRNA levels inducedby ethanolcould bemediatedby eithera decreasein its transcrip-tion rate oran increasein GFAP mRNA instability, orboth. In addition, theextentof DNA methylationcanalso influenceGFAP expression(Michalowsky andJones,1989).Indeed,methylationof cytosine residuesis thoughtto play animportantrole in the regulationof mammaliangeneexpression,especiallyduring em-bryogenesisanddifferentiation,and thushypermethyl-ated regionsof DNA are generally not transcribedwhereashypomethylationis associatedwith geneex-pression(Edenand Cedar, 1994;Razinand Shemer,1995). Thus, methylation changesin tissue-specificgenes duringembryogenesisare likely to lead to aderangementof normalfetal geneexpressionand fetaldevelopment.

Thepresentstudy wasundertakento furtherinvesti-gate themolecular mechanismsby which ethanol in-duces adecreasein GFAP expression,by analyzing(1) the possibleeffect of ethanol on theGFAP tran-scription rate (nuclear runoff assay), and (2) theGFAP mRNA stability inastrocytesin primarycultureobtainedfrom control or alcohol exposedfetuses. Inaddition,because changesin DNA methylationduringdevelopmentare closely relatedto regulationof tran-scription andgene expression(Edenand Cedar,1994;Teter etal., 1996), we also analyzedthe methylationstatus of GFAPDNA in control and ethanol-exposedfetal brains. Anunderstandingof themolecularmecha-nisms controlling GFAP expressionmay provide in-sight into thepotentialrole of this glial-specificproteinin variousneurological disorders,including the patho-geneticrole of fetal alcohol syndrome.

MATERIALS AND METHODS

Animal treatmentFemaleWistarratsweighing 200—250 g wereused. Rats

were fed the Lieber—DeCarli diet (Lieber and DeCarli,1994) eithercontaining5% (wt/vol) ethanolor isocalori-cally balancedwith dextrin—maltosefor pair-fed controls.Femalerats receivedthe liquid diet (ethanolor control)fora minimum of 30 daysbeforeexposureto males.After mat-ing, the dams were place in separatecages andkept onethanolor control liquid diets duringgestation.Mean bloodalcohol levels (BALs) reachedby pregnantrats andtheirfetuses were105 ±45 and 106 ±23 mg/dl, respectively(Guerri and Sanchis,1985; Sanchiset al., 1986).Pregnantratswere killed bydecapitation,fetuseswereremoved,andwhole brainswere frozenquickly in liquid nitrogenandthenstoredat —70°Cuntil used.In the postnatalstage,animalsat

differentdays weredecapitatedandbrainsremovedquickly,frozenin liquid nitrogen,and storedat —70°Cuntil usedforprotein or RNA extraction.

All experimentsusing rats wereperformedin strict com-pliancewith the EuropeanCommunity Guidefor the Careand Use of Laboratory Animals.

Astrocyte culturesControl and prenatally ethanol-exposed(PEE) primary

culturesof astrocytesfrom21-day-oldfetuseswere preparedfrom brain hemispheresas describedby Renau-Piquerasetal. (1989). In brief, fetuseswereobtained understerilecon-ditionsfrom both controlandchronicalcoholicrats.Thecellsuspensionwasvortex mixedat maximumspeed(I mm) todestroythe neuronspresent. Cellswere plated on 60-mmComing plastic tissue-culturedishes (2.5 x l0~’cells perdish).Themediumwaschangedevery3 days.Culturesweregrownin a humidified atmosphereof 5% CO2 at 37°C.Thepurity of astrocyte cultureswasroutinely assessedby immu-nofluorescenceusingamouseanti-GFAP.Thepossiblecon-taminationby neuronswasassessedby using ananti-neuro-filament 68 kDa monoclonalantibody (BoehringerMann-heim).

Immunofluorescence microscopyAstrocyte cells growing on 16-mm glass coverslipswere

used for double-immunofluorescencestudies following theproceduresdescribedby Shez et al.(1991).Astrocyteswereincubatedsimultaneouslywith anti-neurofilament68 kDamonoclonal antibody (Boehringer Mannheim) and anti-GFAP polyclonal antibody (Sigma). The incubation wasperformedat 37°Cfor 60 mm. After washingseveral timesin bovine serumalbumin—Tris-bufferedsaline, cells wereincubatedfor 60 mm at 37°Cwith phosphate-bufferedsaline(PBS) containing anti-mouselgG—tetramethylrhodamineisothiocyanateand anti-rabbit IgG—fluoresceinisothiocya-nate. All the dilutions were 1:100.

Preparation of astrocytes and immunoblottingCulture plates were rinsedtwo timeswith PBS. Mono-

layerswere resuspendedin sodiumdodecyl sulfate (SDS)samplebuffer [0.125M Tris-HCI, pH 6.8, 2% SDS,2 mMEDTA, 2 mM phenylmethylsulfonylfluoride, 5% (wt/vol)2-mercaptoethanol,10% glycerol, and0.025%bromophenolblue]. Before adding2-mercaptoethanolandbromophenolblue, aliquotswere withdrawnto determineprotein (Lowryetal., 1951). Sampleswerethen boiledfor 5 mm andstoredat —70°C(Sancho-Telloet al., 1995; Vallés et al., 1996).

Proteinswere separatedin SDS-polyacrylamideslab gels,following thediscontinuousgel andbuffer systemof Laem-mli (1970),using10% (wt/vol) polyacrylamidein thesepa-ration gel. After electrophoresis,proteins were transferredto nitrocellulose membraneusing a SemidryElectroblotter(Millipore) apparatus. The nitrocellulosemembranewas in-cubatedfor 2 h at room temperaturewith monoclonalanti-bodies to GFAP or vimentin (both at 1:500 dilution),washed, andthen incubatedfor 1 h with goat anti-mouseIgG conjugatedwith alkalinephosphatase.After 5—10 mmof color development,the nitrocellulosepi~mbraneswerewashedandphotographed.The intensity of the bandswasmeasuredusinga laserdensitometer(Ultrascan,LKB). Inall the experiments,the protein concentrationused was inthe linear rangeof the densitometer.

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RNA extraction and northern blot analysisThe following cDNA probeswere used: mouseGFAP

from Dr. N.Cowan, mousevimentinfromDr. E.Reichmann,andrat cyclophilin from Dr. J. G. Sutcliffe.

ThecDNA probeswerelabeledwith [a-32P]dATP (3,000Ci/mmol, Amersham)by randomprimer (FeinbergandVo-gelstein, 1984)to a specific activity of —1 x io~cpnV~.tg.

Total RNA wasextractedfrom 60-mm culture dishesbythe methodof Chirgwinet al. (1979)aspreviouslydescribed(Valléset al., 1996). For northernblots, total cellular RNAwasseparatedon a denaturingagarosegel containing2.2 Mformaldehyde,transferredto a nylon membrane(Zeta-Probeblotting membranes,Bio-Rad), andhybridized with the la-beledeDNA probesovernightat 65°C.Themembraneswerewashedtwo times (30 mm each,65°C)in 1 mM EDTA,40 mM Na

2HPO4, pH 7.2, and1% SDS. After washing,membranesweresetup for autoradiographyat —70°CwithAmershamHyperfilm-MP anda CronexLightning-Plusin-tensifying screen. The autoradiogramswere quantified bydensitometricscanningwith a laserdensitometer(Ultrascan,LKB) (Vallés et al., 1996).

To verify that equal amountsof RNA were loaded perlane, cyclophilin eDNA wasused. Blotspreviouslyhybrid-ized with GFAP or vimentin were washedtwo times (20mm each)in a largevolume of 0.1% saline—sodiumcitrate(SSC),0.5% SDS at95°C,andthen hybridized overnightwith [ct-

32P]cyclophilin eDNA.

Nuclear runoff assaysNuclei were isolatedby sucrosegradient centrifugation

(Ausubel et al., 1994), andtranscriptionrate analysiswasconductedas describedpreviously(Colottaet al., 1992). Inbrief, on day 14 of cultureastrocytes(40—SO X 10”) wereresuspendedin Nonidet P-40 lysisbuffer [0.32 M sucrose,3 mM CaCI

2, 2 mM magnesiumacetate,0.1 mM EDTA,10 mM Tris-HC1, pH 8, 1 mM dithiothreitol (DTT), 0.5%NonidetP-40], gently homogenizedin aDouncehomoge-nizer, storedon ice for 5 mm, and examinedby phase-contrastmicroscopyfor theefficiencyof cell lysis. Thenthenuclei suspensions were diluted withsucrosebuffer (2 Msucrose,5 mM magnesiumacetate,0.1 mM EDTA, 10 mMTris-HC1,pH 8, and 1 mM DTT). Nuclei freeof membranesandcytoplasmic componentswerecollectedby ultracentrifu-gation at 30,000g for 45 mm, througha sucrose cushion(Ausubelet al., 1994). For transcriptionrate analysis, 230~l of nuclei suspensionwasincubatedwith 100 1.,Ci of [a-2P]UTP (Amersham)and 60 1.51 of 5x runoff buffer (25mM Tris-HC1, pH 8, 12.5 mM MgC1

2, 750 mM KCI, 1.25mM each of ATP, CTP, andGTP) for 30 mm at 30°C.Elongatedtranscriptswere then isolated using guanidine/cesium(Chirgwin et al., 1979),with 50 ~.tgof yeasttRNAaddedas carrier.The RNA pelletwasresuspendedin 1801.51 of ice-coldTNE(0.5 mMTris-HC1,pH 8,1.5 MNaC1).RNA was thenprecipitatedby ethanol andthe pellet wasresuspendedin hybridization solution [10 mM N-tris[hy-droxymethyl]methyl-2-ammnoethanesulfonic acid (TES),2% SDS, 10 mM EDTA, and 300 mM NaCI]. The

32P-labeled RNA was then hybridized at 65°Cfor 48 h withcDNAs for GFAP, vimentin, and cyclophilin, which hadbeenimmobilized on nitrocellulosefilters in a slot-blotappa-ratus. Filters werethenwashedwith severalchangesof 0.2xSSC at65°Cfor 30 mm andincubatedat 37°Cin 0.2x SSCwith 1 ~.eg/mlRNaseA for 30 mm. Filterswerethenexposedfor autoradiographyandquantifiedby densitometricanalysisas describedabove.

DNA extraction, methylation-sensitive restrictionenzyme digestion, and Southern blotting analysis

GenomicDNA was extracted from 21-day fetalbrain (con-trol and prenatallyethanolexposed)andadult liver, usingtheproceduredescribedby Sambrooket al.(1989).DNA samples(SO ~.tg)were digestedwith HpaII or MspI (6 U/fig DNA)at 37°Cfor 16 h. Digested DNA samples wereelectrophoresedin 1% agarose gels and transferredonto nylon membranesaccording totheprocedure of Southern(1975).Hybridizationwas performed as described above. The GFAPfragmentEcoRI—Hindill (Dr. N. Cowan) was used as probe and3Plabeled by the random primingmethod. Specificactivity of10” cpmJ~.cgDNA was routinely obtained.

Statistical analysisResultsare reported as mean± SD values. Datawere

analyzedusing two-wayANOVA. A p value of <0.05wasconsideredstatisticallysignificant.Linearregressionanalysiswas used todeterminekD and thehalf-life wascalculatedbyt112 = In

2IkD.

RESULTS

Purity of astrocyte culturesIn our experimentalconditions, the proportion of

GFAP-positivecells in thecultureranged,as alreadyreported (Renau-Piqueraset al., 1989), from 90 to95%. A small proportionof oligodendrocytes(2—4%)was also observed.Lack of staining with anti-neuro-filament 68 kDa indicatesthe absenceof neuronsintheculture.

Cells obtained from PEE fetusesshowed, as pre-viously described (Renau-Piqueraset al., 1989; Sáezet al., 1991), alterations ofintermediatefilamentorga-nization pattern anddelayedmorphologicalastrocytedifferentiation.

Western blot analysis of the GFAP and vimentincontent in astrocytes in culture

GFAP and vimentin are two intermediatefilamentcomponentsof astroglial cells, andbothproteins canbe expressed simultaneouslyin’ some astroglial cellsin vivo and inprimary culture(Schnitzeret al., 1981;Fedoroffet al., 1983;Abd-el-Bassetet al., 1992;San-cho-Tello et al., 1995).

The relative contentof GFAP andvimentin in con-trol andPEEastrocytesat days7 and 15 wasanalyzedby westernblotting. As shownin Fig. 1, immunoblotsusing anti-GFAP showeda single band of ‘~51kDa,which correspondsto the molecularmass of GFAP.The intensityof the stainingincreasedthroughouttheentire cultureperiod and was I .5-fold higheron day15 than on day7 of the culture.WhenPEEastrocyteswere analyzed,the relative absorbancein thesecellswassignificantly lower (p < 0.01)than in thecontrols(Fig. IA).

With respectto vimentin, onebandof ~57 kDa wasobservedvvhenamonoclonal antibodyagainstvimentinwasused (Fig.1 B). Although prenatalexposureto etha-nol appearsto decreasethevimentin levels, the differ-encesbetweenthe two groups (control and PEE)werenot statistically significant(p < 0.08) (Fig. 1B).

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ETHANOL AFFECTSGFAP GENE EXPRESSION 2487

FIG. 1. Densitometric analysis of immunoblots demonstratingGFAP (A) and vimentin (B) levels from control (C) and ethanol-treated (E) astrocyte cells at different days of culture. A constantprotein concentration (10 1.sg/lane in A and 15 ©g/lane in B) wasused. Representative immunoblots are shown. As a control ofthe immunostaining, adult brain (Ad) was used. Data are mean±SD values of three independent astrocyte cultures, each froma different litter. *p < 0.01 vs. control for treatment effects bytwo-way ANOVA.

Influence of ethanol on GFAP expression duringpostnatal brain development

To determinewhetherthe effect of ethanol on theGFAP expressionobservedin astrocyteswasalso re-flected in vivo, weanalyzedthe levels of both GFAPand its mRNA in brain from control andalcohol-ex-posedpups at different stagesof postnataldevelop-ment. Immunoblotsusing anti-GFAPrevealedthat theGFAPproteinincreasedstrikingly throughoutthepost-natalperiod,which may reflectastroglialcell differen-tiation (Fig. 3). This increasein GFAP was also re-flectedby aparallel enhancementof theGFAP mRNAlevels, but in this casea progressiveincreasewasob-serveduntil postnatal day 14, after which the levelremainedconstant.Alcohol exposuresignificantly de-creasedthe brainlevelsof bothGFAP and itsmRNAon all thepostnatal daysanalyzed(Fig. 3). Thesere-sults are consistentwith thedataobtainedin primarycultureof astrocytesandsuggest,as previouslydemon-strated (Guerri et al., 1993; Guerri and Renau-Pi-queras,1997),thecongruity of the astrocyte response

Northern blot analysis of GFAP and vimentinmRNA

To investigate further the molecular changesinGFAP andvimentin expressionduring astrocytedif-ferentiationin primaryculture, northern blot analysiswas used toexaminethe possiblechangesin theirencodingmRNAs at different times of culture(days7, 14, and21). Asshownin Fig. 2A, theGFAP probeidentifieda 2.7-kbmRNA species.TheGFAP mRNAlevel increased throughoutthe entire cultureperiod.In PEE cells, the levelsof GFAP mRNA also in-creasedbetweendays7 and21 (Fig. 2A), but theselevelswere significantly lower (p < 0.001) than thecontrol values.Analysis of vimentin expressionindi-cates that ethanol reduces slightly the vimentinmRNA levels, althoughthis reductionwas not statis-tically significant comparedwith control values(p< 0.09) (Fig. 2B).

As a control for determiningRNA loading levels,blots were strippedand hybridized with cyclophilineDNA. As shown in Fig. 2, a single band of 1.0-kb mRNA specieswas expressedat constant levels(monitored densitometrically)throughout the timeof culture.

FIG. 2. Northern blot analysis of GFAP (A) and vimentin (B)from control and ethanol-treated astrocyte cells at different daysofculture. A constant RNAconcentration (10 ©g/lane) was used.Representative autoradiograms of a northern blot are shown.Data are mean ±SD values of four independent cultures, eachfrom a different litter. Cyclophilin mRNA is shown as a controlfor the loading and integrity of RNA. *p < 0.001 vs. control fortreatment effect by two-way ANOVA. On all days studied, vimen-tin mRNA was reducei, but not significantly (p < 0.09 vs. controlfor treatment effects by two-way ANOVA).

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FIG. 3. Western blot (A) and northern blot (B) densitometricanalysis of GFAP and its mRNA levels from control and ethanol-exposed rats during postnatal brain development. A constantprotein (20 ©g/lane) or mRNA (15 ©g/lane) concentration wasused. Representative western blot and northern blot are shown.Data are mean ±SD values of three to five different samples,each from a different litter. °p< 0.001 (A) and °p< 0.01 (B),for treatment effect by two-way ANOVA. Cyclophilin mRNA isshown as a control for the loading and integrity of RNA. P, post-natal day.

to prenatal exposureto ethanolin in vivo and invitrosystems.

The levels of vimentin and its mRNA were alsoanalyzedin control andPEEbrains,but no significantvariationsbetweenthetwo groups wereobserved(datanot shown).

Influence of ethanol on the transcription anddegradation of GFAP mRNA and vimentinmRNA in astrocytes

The alcohol-induced reductionin the levels ofGFAPmRNA could resultfrom a decreasein the rate ofGFAP mRNA transcription,an increasein therate ofGFAP mRNA degradation,or a combinationof thetwo mechanisms.To distinguishbetweenthesepossi-bilities, nuclearrunoff assayswere conductedin con-trol and PEE astrocytes.A representativeblot from anuclearrunoff experimentis shownin the insetof Fig.4, and the datafrom threeindependent experimentsare averaged in thebargraphof Fig. 4. As showninthis figure, no consistent differenceswere found in the

ability of nuclei to synthesizevimentin or cyclophilinmRNAs when control andPEEcells werecompared.However, a significantreductionin the GFAP tran-scription rates was observed inPEE astrocytes.Thisfinding suggeststhat prenatalexposureto ethanolspe-cifically altersthe rateof GFAP mRNA transcriptionin isolated nucleiof astrocytes.

To determinethe stability of theGFAPmRNA tran-scripts, control andPEEastrocytesat day14 of culturewere treatedwith 10 ,uM er-amanitin, an inhibitor ofRNA polymeraseII, and GFAP mRNA levels weremeasuredat varying timeperiods(3, 6. 9, and15 h).As shown in Fig. 5A, prenatal exposureto ethanolincreasedslightly the degradation of GFAP. Thus.whereasin control cellsthehalf-life of GFAP mRNAwas 5.48 ± 0.56 h (kD = —0.1263 ± 0.014, r= 0.979), in PEE cellsthehalf-life decreasedto 4.11±0.45 h (k~= —0.1688±0.017,r2 = 0.979).Analy-sis of vimentin mRNA levelsafter treatmentrevealedthat the stability of the vimentin mRNA transcriptswas not affected by prenatal ethanol exposure(Fig. 5B).

Changes in DNA methylation of GFAP gene incontrol and prenatally ethanol-exposed fetalbrains

Becauseduring embryogenesis,genetranscriptionand gene expressionappearto be controlled by themethylation state of the cytosine intissue-specificgenes (Eden and Cedar, 1994; Razin andShemer,1995),we investigatedthepossibleinfluence ofprena-tal ethanolexposure onGFAP genemethylation.Forthis purpose,genomicDNA wasextractedfrom fetalbraintissue(controland alcoholexposed)anddigestedwith the methylation-sensitive restrictionenzymeHpaII or its methylation-insensitiveisoschizomerMspI (Waalwijk and Flavell,1978). Methylated CpGsequenceswere detected by Southern blot probedwitha 2.7-kbEcoRI—HindIIIfragmentof themouseGFAP

FIG. 4. Transcription rate analysis of GFAP and vimentin fromcontrol (C) and ethanol-treated (E) astrocytes in primary culture.Runoff analysis was conducted as described in Materials andMethods. A constant cDNA concentration (5 gig) was used. Datafor GFAP and vimentin transcription (means ±SD) were ob-tained from three independent astrocyte cultures, each from adifferent littcr. Data were analyzed by normalization tocyclophilinmRNA. °p< 0.05 vs. control values.

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ETHANOL AFFECTSGFAP GENEEXPRESSION 2489

more extensivelythan in 21-dayfetal brain expressingGFAP. Moreover,when21-day fetalbrains fromcon-trols andPEEwerecompared(Fig. 6A), a relativelyintensivehybridization signalat the4.5-kDafragmentappeared in 80% of thePEEfetal brains,but this frag-ment wasabsentor veryweak in theDNA from controlbrains. These resultssuggestthat whereasin controlbrain a demethylationof GFAP DNA takesplacebe-tweendays 15 and21 as previously reported(Teteretal., 1996), in PEE a reduction in the demethylationprocessof GFAP DNA occurs in21-day fetal brains,and this is consistentwith the reduction in GFAPmRNA observedin treatedfetal brains (Vallés et al.,1996).

DISCUSSION

FIG. 5. Influence of ethanol on the stability of GFAP (A) andvimentin (B) mRNA5 in astrocytes. RNA was extracted from cul-tures treated for 0, 3, 6, 9, and 15 h with 10 ©M a-amanitin (apowerful inhibitor of RNA polymerase II) and northern blot analy-sis was conducted, as described in Materials and Methods, fol-lowed by densitometric analysis. Representative autoradiogramsare shown. Data are mean ±SD values of five or six experimentsfrom different astrocyte cultures (each from a different litter).Note that some reduction (p <0.06 vs. control) of GFAP mRNAwas observed in the PEE cultures.

eDNA (Lewis etal., 1984).BothenzymesrecognizedandcutunmethylatedCCGG sequences dependingonthe methylation of the internal cytosine, and thismethodthereforeallowed us to detectreliablediffer-encesin themethylation level.

An example of the Southernblot autoradiogramsobtainedwith DNA extracted from 15- and 21-dayfetal brains(control and PEE) and adult liver afterMspI and F/pall digestion is shown in Fig. 6. DNAfrom adultliver wasusedasanegativecontrol,becausethis tissuedoesnot expressdetectablelevelsof GFAP.As shownin Fig. 6, after completedigestion withthemethylation-insensitiveisoschizomerMspI, two mainhybridizationfragmentsof ~.‘2.1 and 1.2 kb wereob-tained from both liver and fetal brain DNAs. WhenHpaII digestion, which cleaves the sequenceCCGGonly if the internal cytosineis unmethylated,wasused,however,four fragmentsof ‘—~5.5,4.5, 2.1, and 1.2 kbwerevisualized for adult liver (Fig. 6A) and 15-dayfetal brains (Fig. 6B), but only two main fragmentsof ‘—~5.5and2.1 kb were observedin 21-day controlfetal brain (Fig. 6A). Thesedatasuggestthat in liverand 15-dayfetalbrain, wheretheexpression of GFAPis absent or very low,the GFAP geneis methylated

Astrocytesare believedto be crucialfor the nutri-tional andstructuralsupport ofneuronsand theregula-tion of local concentrationsof neurotransmittersandions (Glowinski et al., 1994). During nervoussystemdevelopment,astrocyteshave beenshown to play acritical role in neuronal migration, positioning, andmaintenanceof a functionalenvironmentfor neurons(Rakic, 1981, 1991;Privat et al., 1995).

GFAP isan intermediate-filamentprotein expressedalmostexclusively in astrocytesin the CNS, anditsexpressionis thoughtto be thehallmark of astrocytedifferentiation (Bignami et al., 1972). This astrogliaprotein not only provides structuralsupport for theneuronsbut also appears to be critical during themor-phogenesisof CNS. GFAP isrequiredfor the forma-tion of stable astrocytic processesin responseto neu-rons (Weinstein et al., 1991;Chen andLiem, 1994),

FIG. 6. Changes in GFAP DNA methylation pattern in control(C) and alcohol-exposed (A) fetal brains. Genomic DNA ob-tained from controi and ethanol-ex~osedfetal rat brain was di-gested by the methylation-sensitive isoschizomers Hpall andMsp I restriction enzymes, and DNA fragments were electropho-resed on agarose gels, transferred to nylon membranes, andhybridized with cDNA probe as described in Materials and Meth-ods. A: Twenty-one-day-old fetal rat brain and adult liver (L). B:Fifteen-day-old fetal rat brain. Molecular weights are expressedin kilobases (kb). F, fetal day.

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and in micecarryinga null mutation,GFAP geneex-pressionwas shownto be essentialfor normal whitematter architecture,vascularization,blood—brainbar-rier integrity (Liedtke et al., 1996), cerebellarlong-term depression(Shibuki et al., 1996),andhippocam-pal long-term potentiation (McCall et al., 1996).Moreover,a recent reportrelates a novelGFAPmRNAisotype with memory retention(Huang and Lee,1997).Therefore, alterationsin GFAP geneexpressionarea particularly attractivemechanismfor explainingthe deleterious effectof prenatal exposureto ethanolon astrocytesand,consequently,on different aspectsofCNS development,including neuronal migrationandmyelination (Liedtkeet al., 1996).

We have previously shown that whereasGFAPmRNA is first detectedin rats on fetal day 15 andits contentincreaseson fetal day 21, in utero alcoholexposure delays theappearanceof GFAP mRNA anddecreases significantly theGFAP expressionin fetalbrain and inprimary culture of radial glia (Vallésetal., 1996).Thepresentstudyextendstheseresults anddemonstratesthat chronic ethanol exposureduring fe-tal developmentdecreasesthe GFAPexpressionin thebrainsof alcohol-exposedpups duringpostnataldevel-opment andin astroglialcells in primary culture. Ourresults alsoshowthat theethanol-induced decreaseinGFAP expressionis mainly due to a reductionin theGFAP transcriptionrate, which appearsto be associ-atedwith ahypermethylationstateof theGFAP DNA.

Regulationof the GFAP geneappearsto be quitecomplex, withmultiple interactingDNA elements,butthe regulatorypathwaysthat operateduring develop-ment, aging, and inresponseto a varietyof physicalandchemicalinsults arenotyet completelyunderstood(Brenner,1994).Transcriptionalcontrol of the GFAPgeneappearsto occurprincipally atan early stageofastrocytic developmentby stimulating itstranscriptionrate andduring the transition from theproliferation tothe differentiation phaseof astrocytes(Riol et al.,1992). Ethanol mayinterfere with the transcriptionprocessor interactwith someof the factors involved,directly or indirectly, in the transcriptional regulationof theGFAP gene.In fact, cyclicAMP-dependentpro-tein kinaseand proteinkinaseC have beenfound tobe involvedin theregulationof theGFAP geneexpres-sion (Shafit-Zagardoet al., 1988; Brenner, 1994;Ka-neko et al., 1994), and bothof thesekinasesare af-fectedby ethanol (Pennington,1988; Messing etal.,1991;Slateret al., 1993).Exposureto ethanolduringfetal developmentalsoinducespronounced alterationsin growth factors andhormones,including glucocorti-coids andsex steroids(Weinberg,1989, 1994),whichhave beenshown to modulateGFAP expressionbyinterferingwithAP-1-mediatedtranscriptionactivation(Laping et al., I 994a,b). Therefore,ethanol-inducedalterations in some of theGFAPtranscriptionalmodu-latory elementsduring criticalperiodsof brain devel-opment(e.g., theonsetof GFAP expression)may in-fluence the transcriptionprocess,thus leading to an

alterationin this processand areductionin its expres-sion.

Another mechanism thatcould affect GFAP tran-scription and expressionis theextentof GFAP DNAmethylation.DNA methylationis essentialfor normalembryonic development,in which mostof the tissue-specific genesare almostfully methylatedand undergoprogrammedactive demethylationat the momentofactivation and transcription (Edenand Cedar,1994;Razin and Shemer,1995). In agreementwith thesedata, our resultsshowthat betweenfetal days 15 and21, the GFAP DNA goes from a highly methylatedstate to apartiallymethylatedcondition.Thesechangesin methylationare concomitantwith the onset andin-creasein GFAP expressionandwith thetransformationof radial glia into astrocytes(Sancho-Tello et al.,1995).Theseresultsagreewith the findings of a recentstudy demonstratingthat betweenembryonicday 14andpostnatalday 10 the GFAP geneis demethylatedprogressively(80 to 15%) (Teteret al., 1996).How-ever, theseauthors found that GFAPDNA suffers apartial remethylationat later postnatal ages whenGFAP mRNA remainsprevalent,which suggeststhatotherregulatoryfactors may be involved in thecontrolof GFAPgeneexpressionduring brain development.

Our results also suggest ahypermethylatedstate inthe GFAP DNA from alcohol-exposedbrains. Al -though methyl-sensitivenucleasetechniquesonly de-tect large differencesin the methylation level, whencontrol and PEE fetal brainswere comparedwe ob-served somechangesin the extent of GFAP DNAmethylation.Thus, ourresultsshowthattherestrictionprofiles obtainedfrom PEEgenomicDNA aresimilarat fetaldays 15 and21, andthis suggeststhatexposureto alcohol during fetal development induceda reduc-tion in thedemethylationprocessmanifestedby hyper-methylation stateof the GFAP DNA in 21-day fetalbrain. These resultsare consistentwith the delay intheappearanceof GFAP expressionobservedin fetalbrainsandin primary cultureof radial glia(Vallés etal., 1996). Moreover,becauseit has been suggestedthat DNA methylationregulates the GFAPtranscrip-tion process(Michalowsky andJones,1989), changesin the GFAP DNA methylation inducedby ethanolcould also explain thedecreasedGFAP transcriptionrateobservedin PEEastrocytes.

In contrast,Garro et al.(1991)reportedfetal DNAhypomethylation associatedwith acuteethanoladmin-istration.Thus,ethanol may alter themethylationpro-cessin differentways dependingon doses andalcohollevels attained. With acuteadministration,a highetha-nol level is achieved for a short period, whereaschronic administrationresults in sustained,generallylower, ethanol levels. In addition, alcoholmay onlyaffect the methylationpatternof sometissue-specificgenessuch as GFAP. Becausethegeneof vimentin,which is closely relatedto GFAP in astrocytes, is notaltered after ethanolexposure (Fletcherand Sham,1993), our results indicate that ethanol changesthe

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geneexpressionof a limited numberof specific genesand does not cause a change inoverall capacity fortranscription.

The results on ethanol-inducedalterationsin GFAPDNA methylationand thedelayin GFAP expressionin fetal brain suggest thatastrocyteprogenitorcells,suchas radialglia, are one of the maintargetsof etha-nol toxicity in the developing brain. In fact,ethanolexposure reduces thenumberof GFAP-positiveBerg-mann glial(radial glia in cerebellum)fibers, the somasize, and thepercentageof maturefibers (ShettyandPhillips, 1992; Shetty etal., 1994;Pérez-Torreroet al.,1997), suggestingthat altered developmentof Berg-mannglia could be oneof the factorscausing delayedmigration of granular neurons(Shetty and Phillips,1992; Shetty et al.,1994). Prenatal alcohol exposurealso inducesalterationsin corticalradial glia includinga decreasein the number of cells (Gressenset al.,1992) andalterationsin their morphologyand in thetiming of the phenotypictransitionof these cells intomore typical GFAP-positive astrocytes(Miller andRobertson,1993). The difference betweenthe latterresults and our data on thedelayin GFAP expressionin fetal brain could be due to thedifferentparadigmofethanol treatment. Thus,whereasMiller and Robertson(1993) usedpostnatalrats exposedto ethanolduringgestation days8—21, we haveusedfetuses(or astro-glial cells) fromrats fedwith ethanolbeforeand duringgestation(Guerri et al., 1984). In addition, thepres-ence of certain growth factors(Kentroti and Verna-dakis, 1997)and/orhormoneshasbeensuggestedtobe involved in thetransformationof radial glia intoastrocytesand in themodulationof GFAP expression(Laping et al., l994a). Therefore,changesin thesemodulatory factors duringcritical periodsof brain de-velopmentmight explain the different effectsof alco-hol. In our studies, theoffspring of chronicethanol-fedmothers showedseveralhormonal alterationsduringdevelopment (Esquifinoet al., 1986; Portoléset al.,1988). Usingthis animal model, wehavereproducedin the rat (Guerri et al., 1984; Sanchis etal., 1986,1987;Guerri, 1987;Renau-Piqueraset al., 1997) manyalterationsobservedin childrenwith fetalalcohol syn-drome (Clarren and Smith, 1978) who were born ofalcoholicmothers.

In contrast,brief postnatal(days4—9) exposuretohighlevels of alcohol(BAL5, 175—300mg/dl) causesa transientastrogliosismanifestedby an increaseinboth immunoreactive GFAP(Goodlett et al., 1993)and GFAP mRNA levels in rat cerebralcortex andin confluent corticalastrocytesin culture, but not inhippocampus,cerebellum,andbrainstem(FletcherandSham, 1993).Vasculardisruption that occursafterhigh blood alcohol concentrationshasbeenimplicatedasa potential trigger for thereactive gliosis(Goodlettet al., 1993). These resultssuggest that the effectsof ethanol onastrocytes,and specifically on GFAPexpression,dependon the levelsof alcohol, duration,and timing of exposurerelative to the stage of glial

maturation(glial progenitorcells andproliferation ordifferentiation of astrocytes)(Kennedy and Mukerji,1986a,b; Renau-Piqueraset al., 1989; Guerri et al.,1990) andlevelsof growthfactors andhormones(Lap-ing et al., 1994a;Kentroti and Vernadakis,1997). Inaddition, the brainregional differences in glial re-sponse toethanolwith respectto the expressionofGFAPmay reflecteitherstage-dependentvulnerabilityof astrocytes (differentbrain regionsperformspecificdevelopmentalfunctionsasynchronously)or heteroge-neity of astrocytes withinthedifferent brain regions.

In summary,the presentfindings and previousre-suits (Vallés et al., 1996) demonstratethat chronicethanol exposureduring fetal developmentalters thedifferentiation ofradial glial cells andtheir transforma-tion into astrocytesandthat thesechangesareassoci-ated withimportantalterationsin the gene expression,organization,andcontentof GFAP in astroglial cells.Ethanol-inducedchangesin GFAP gene expressionwould probably haveprofound effects on astrocytesand, therefore,on many astrocyte-mediateddevelop-mental eventsin theCNS, including boundaryforma-tion during neural morphogenesis,central compart-mentalization, neuralproliferation andmigration, axonoutgrowth andguidance,availability of trophic supportmolecules, and myelination. Moreover,ethanol canalso disturb the astroglial functions involved in neu-ronal physiology andsurvival suchas control extracel-lular ion concentrationand uptake andinactivationofexcitatory amino acids and otherneurotransmitters.Therefore,thepresentdatasupport thehypothesisthatethanol-inducedGFAP geneexpressionandastroglialdamagecould be a potentially important mechanisminvolved in theneurologicalandneurobehavioraldys-functions associatedwith fetal alcohol syndromeandwith prenatalalcohol exposure.

Acknowledgment: We thankM. March for herexcellenttechnicalassistance. This studywassupportedby theCICYT(SAF 94-0065-C02-0I; SAF 96-0185-C02-02),GeneralitatValenciana(GV-D-VS-20-l26-96),and FundaciónAreces.

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