Ž .Developmental Brain Research 114 1999 201–206

Research report

Forskolin-induced expression of tyrosine hydroxylase in human foetalbrain cortex

F. Bernardo Pliego Rivero a,1, Wendy J. McCormack a, Eric Jauniaux b, Gerald M. Stern a,Henry F. Bradford a,)

a Imperial College of Science, Technology and Medicine, Department of Biochemistry, South Kensington, London SW7 2AY, UKb Department of Obstetrics and Gynaecology, UniÕersity College Hospitals, 86-96 Chenies Mews, London WC1E 6HX, UK

Accepted 23 February 1999

Abstract

Ž .Brain-derived neurotrophic factor BDNF has previously been shown by this and other laboratories to work in concert with dopamineŽ .DA to induce the dopaminergic phenotype in foetal rat and human cerebral cortex during specified sensitive developmental stages. In

Ž .the present study this induction by BDNFrDA was found to be greatly amplified by adding forskolin fsk: 10 mM to the rat and humanŽ . Ž .cerebral cortex cultures together with DA 10 mM and BDNF 50 ngrml . This amplification was 14-fold for human tissue and 2-fold

for rat tissue treated over an 80% shorter period. Compared to treatment with BDNF alone, the additional fsk increased tyrosineŽ q.hydroxylase-positive TH cell numbers by 220-fold in the human and 26-fold in the rat tissue. Parallel reverse transcription–poly-Ž .merase chain reaction RT–PCR measurement of TH mRNA showed substantial increases above control levels when BDNFrDA or

Ž .BDNFrDArfsk treatments were applied. Since fsk boosts intracellular levels of cyclic AMP cAMP , its amplifying action when addedtogether with BDNFrDA is likely to be due to interactions via the cAMP response elementrcAMP response element binding proteinŽ .CRErCREB systems. This is discussed. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Dopaminergic phenotype; Tyrosine hydroxylase expression; Human and rat foetal tissue; BDNF; Forskolin

1. Introduction

Progressive loss of pigmented dopaminergic neuronesŽ .from the substantia nigra pars compacta SNC leads to theŽ .development of the Parkinson’s disease PD . One of the

current clinical and experimental approaches to treatingthis disease is to restore this impairment by implantingfresh foetal dopaminergic neurones into the striatum. How-ever, using cultured foetal human neurones for implanta-tion would provide a more practical approach than usingfresh tissue. Cultured tissue can be manipulated to improveits performance as graft tissue as well as cryostored in aviable state on a long term basis.

) Corresponding author. Fax: q44-171-225-0960; E-mail:h.bradford@ic.ac.uk

1 Present address: Universidad Autonoma del Estado de Mexico, Fac-ultad de Medicina, Paseo Tollocan, Toluca 50180, Mexico.

The foetal dopaminergic neurones used as a graft arecurrently those found in the small ventral mesencephalicŽ .VM region of the developing human foetal brain. Thesmall number of dopaminergic cells present requires thatthe VM from approximately 9–12 foetuses are used foreach graft. In contrast there is about 500 times morenon-dopaminergic tissue in the cerebral cortex of the hu-man foetus compared to the VM. Although cerebral cortexnormally contains only small numbers of dopaminergic

Žneurones, various studies have shown that the TH tyrosine.hydroxylase gene together with the full dopaminergic

w xphenotype 19 can be switched on by adding a variety ofŽ . w xtrophic factors and dopamine DA to rat 4,8,20–22 and

w xhuman 23 foetal cerebral cortex in culture.The present study investigated whether the synergistic

induction of TH gene expression in cerebral cortex couldbe amplified by increasing the cellular levels of cyclic

Ž .AMP cAMP , since the cAMP responsive element hasbeen shown to exert critical control of transcription of the

w xTH gene 7,17 .

0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-3806 99 00034-6

( )F.B. Pliego RiÕero et al.rDeÕelopmental Brain Research 114 1999 201–206202

2. Materials and methods

2.1. Tissue culture

2.1.1. Human tissueHuman foetal brain tissue aged between 10 and 14

weeks old was obtained from the Elizabeth Garrett-Ander-Ž . Ž .son EGA Hospital University College London after

routine curettage abortions, approved by the EGA Hospi-tal’s Ethical Committee and with the patients consent. Thebrain tissue was collected and then rinsed three times insterile Ca2q, Mg2q-free Hank’s balanced salt solutionŽ . Ž . ŽHBSS containing penicillin 5 mgrml , streptomycin 5

. Žmgrml and neomycin 10 mgrml: PSN; Life Technolo-.gies . After removing meningeal tissue, suspensions of

cells from cortex were prepared.Initially the tissue was cut into 1–2 mm pieces, then

Ž .incubated for 15 min at 378C in trypsin 0.5 grl , EDTAŽ . Ž .2 grl; Life Technologies and DNase 0.5 mgrml; Sigma .Subsequently, the trypsin supernatant was removed and 10volumes of Dulbecco’s minimum essential medium plus

Ž10% foetal calf serum and gentamycin 50 mgrml;.DMEMrFCS; Life Technologies per volume of tissue

was added to stop further trypsin proteolysis. With the aidŽ .firstly of 1 ml blue tip Scotlab , and then a fire-polished

glass Pasteur pipettes, the tissue was mechanically dissoci-ated with five gentle passages through each tip.

Ž 5 .Cells 5=10 rwell were plated in 24-multiwell platesŽ . ŽNunc , pre-coated for 2–3 h at 378C with laminin 1

. Ž .mgrml and poly-L-lysine 5 mgrml; Sigma mixtureŽprepared in sterile phosphate buffered saline, pH 7.5 PBS;

.Life Technologies . For the first 12–13 h the brain cellsŽ .were cultured in 0.5 ml DMEMrFCS Life Technologies

Ž .to facilitate their attachment )90% to the lamininrpoly-L-lysine substrate. Then the DMEMrFCS was substituted

Žwith 0.5 mlrwell of defined DMEM DMEM supple-.mented with B27 and N2; Life Technologies diluted at

1:100 and containing recombinant brain-derived neu-Ž . Ž .rotrophic factor BDNF at 50 ngrml Regeneron . De-

fined DMEM was then replaced routinely every 3–5 dayswith fresh medium.

Human cortical neuronal cells were cultured for 5 weeksand treated every third day in triplicate with the following

Ž . Ž . Ž .additions: a control saline added vehicle ; b 10 mMŽ . Ž . Ž . Ž .DA Sigma ; c 10 mM forskolin fsk; Sigma ; and d 10

Ž .mM DA plus 10 mM fsk fskrDA .

2.1.2. Rat cortical culturesTo obtain rat cerebral cortical cultures pregnant female

Sprague–Dawley rats were used. Foetal cerebrocorticesŽ .were obtained on day 13 E13, E0sday of vaginal plug

of gestation, immediately after the rats were sacrificed bydecapitation. The embryos were dissected in HBSS and ifpossible the meninges were removed. Following three

Žwashes with HBSS the tissue was incubated in trypsin 0.5. Ž .grl and EDTA 2 grl for 10 min at 378C. Immediately

following trypsination the tissue was added to 1 ml ofDMEMrFCS and further dissociated by trituration using aglass pipette. Cell viability in the suspension of dissociatedcells was determined using the dye Trypan blue, whichviable cells exclude. The cells were plated out in 24-multi-well plates which had been pre-coated with poly-L-lysineŽ . w x5 mgrml as described previously 18 . The cells wereplated at 150 000 cellsrwell in 1 ml of DMEMrFCS andincubated at 378C in a 95%r5% CO humidified atmo-2

sphere. The medium was changed twice a week. The ratcortical cells were cultured for 7 days and triplicate wellswere treated every other day with the following additions:50 ngrml BDNF, 10 mM DA and 10 mM fsk.

2.2. Immunostaining of TH q neurones

Neuronal cultures were rinsed twice with PBS and fixedfor 30 min at room temperature with 4.5% paraformal-dehyde prepared in 100 mM PBS, pH 7.5. The fixativewas removed and the wells were rinsed three times withPBS. Endogenous peroxidase activity was inhibited byincubating the fixed cultures in 15% methanolrPBS plus1% H O for 20 min. Permeabilisation of the cell mem-2 2

brane was effected with 0.5% Triton X-100 in PBS for 30min. To block non-specific binding, the cells were incu-

Ž .bated for 1 h in a mixture of 3% non-fat dry milk MarvelŽ .and goat serum 1:1000 in PBS. Cells were incubated

overnight at 48C with the primary mouse monoclonalŽanti-TH antibody Sigma; 1:4000 in 0.5% milkrPBS and.1:1000 goat serum , and rinsed three times in PBS and

then washed for 5 and 10 min, followed by an hour ofincubation at room temperature with biotinylated goat

Ž .anti-mouse antibody Sigma; 1:400 in 0.5% milkrPBS .Following rinsing and washing as before, horseradish per-

Ž .oxidase–streptavidin Vector Labs; 1:2000 in PBS wasŽused to visualise the positive cells 60 minrroom tempera-

.ture followed by colour development with diaminobenzi-Ž .dine 1 mgrml in PBS plus 0.005% H O as the sub-2 2

Ž .strate. In the human cultures, only control BDNF andBDNFrDA-treated cultures were scanned and TH-labelledneurones were counted in the whole area of the well.However, because of the 200-fold larger numbers of THq

neurones present in the human cultures, the fskrBDNFand fskrDArBDNF-treated cultures were counted only ina sample area of 0.15 cm2rwell between two randomlychosen parallel diagonal lines which accounted for 8.2% ofthe total area of each well. In rat cultures all treatmentwells were scanned and THq cells were counted in thewhole area. Positive cells were counted if they werestained brown and had the morphology of a neurone,therefore, the great majority of the cells counted werelikely to be neurones.

2.3. RNA extraction

Total RNA was extracted from the cultures using TRI-Ž . w xzol Life Technologies 3 according to the manufacturers

( )F.B. Pliego RiÕero et al.rDeÕelopmental Brain Research 114 1999 201–206 203

Table 1Sequences of primers used in RT–PCR

Ž . Ž .Gene Primers Product size bp Number of cycles Temperature 8CX Ž .b-actin 5 GCT CAT TGC CGA TAG TGA TGA CCT 2531–2554 632 21 63X Ž .3 GAT GGT GGG TAT GGG TCA GAA GGA 1457–1480X Ž .TH 5 AGT GCA CCC AGT ATA TCC GC 945–964 414 30 63X Ž .3 AGC TCC TGA GCT TGT CCT TG 1339–1358

Indicated are the unique sequences, chosen to ensure only the target genes were amplified, and the predicted size of the PCR product. Also shown is thenumber of cycles of PCR that was carried out and the annealing temperature used.

instructions. Briefly, 250 ml TRIzol was added to the cellsharvested from each well. A total of 50 ml chloroform wasthen added to extract the RNA into the aqueous phase

Ž .following centrifugation 12 000=g at 48C for 15 min .The aqueous phase was removed and the RNA was col-lected by the addition of isopropanol and incubated aty308C for 30 min followed by centrifugation at 12 000=gat 48C for 30 min. The RNA pellet was then washed threetimes in 70% ethanol and resuspended in 35 ml diethyl

Ž .pyrocarbonate-treated H O DEPC H O .2 2

2.4. ReÕerse transcription–polymerase chain reaction( )RT–PCR

cDNA was synthesised by RT. A 20-ml RT reactionwas set up as follows: 1–5 mg of total RNA was incubated

Ž . Ž .with 0.2 mg of pd N primers Pharmacia and heated at6

708C for 10 min and then immediately stored on ice. Toeach tube a master mix of 7 ml was added to give finalconcentrations of: 50 mM Tris–HCl pH 8.3, 75 mM KCl,3 mM MgCl , 10 mM DTT and 0.5 mM of each 2Xde-2

X Ž .oxynucleoside 5 triphosphates dNTPs; Life Technologies .The tubes were then heated at 428C for 2 min before 200

Ž .units of Superscript II RT Life Technologies were added.Synthesis occurred for 50 min at 428C, followed by treat-ment at 708C for 15 min to inactivate the RT enzyme.

The RT product was amplified using PCR. The PCRw xprimers used were for TH and b-actin 14 and the se-

quences can be seen in Table 1. The TH primers were

designed to span the intron 10rexon 11 boundary. Thiswould allow the detection of contaminating DNA whichwould produce a larger band. Initially a 10% solution ofRT product and 125 ngrml of each primer in DEPC H O2

was made. The cDNA was denatured by heating to 948Cfor 2 min and then held at 808C. The reaction mixture wasthen added, also at 808C to give final concentrations of: 20mM Tris–HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl , 0.22

ŽmM dNTPs and 1 unit Taq polymerase Life Technolo-.gies in a final volume of 25 ml. The cDNA was amplified

under the following reaction conditions: 948C for 1 min,638C for 45 s, 728C for 1 min. This cyclic process wasperformed 21 times for b-actin and 30 times for TH. A5-ml aliquot of each PCR product was visualised byluminescence following electrophoresis through a 1.5%agarose gel containing 0.5 mgrml ethidium bromide. Foreach sample, both the b-actin and TH products were addedto the same lane. To semi-quantify the level of TH mRNAin rat cortical cells following the various treatments, the

Ž .gel was photographed Kodak Digital camera and in-Ž .verted. The relative optical density ROD of the bands for

both TH and b-actin were measured using NIH imageŽ .Scioncorp and corrected for background levels. A densityratio was then calculated, for each sample, for the compar-ison of levels of TH PCR product with the levels ofb-actin PCR product. It was assumed that the level ofb-actin would remain constant during the treatments, andso any relative changes in TH mRNA would be detectedw x1 .

Table 2The effect of BDNF, DA and fsk alone and in combination on the number of THq cells in human cerebral cortical cells

q qTreatment TH cellsrwell Percent of TH cellscompared to control

Ž .Control 50 ngrml BDNF 0.278"0.180 10050 ngrml BDNFq10 mM DA 16"10 1600

UUU50 ngrml BDNFq10 mM fsk 78.505"4.896 7851UUU50 ngrmlq10 mM DAq10 mM fsk 219.506"15.849 21 950

Human cortical neurones were grown in the presence of 50 ngrml BDNF for 5 weeks. The cultures were treated three times a week from DIV 0 by 10 mMfsk and 10 mM DA either alone or together and then stained for THq cells. THq cells were counted in each well and expressed as a percentage of thecontrol. Numbers represent mean"S.E.M. from four to six separate experiments carried out in triplicate. Significant difference, compared with controlgroup, is indicated: UUU P-0.001.

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3. Results

3.1. Human cell cultures

The cortical cells obtained from human foetal braintissue were kept in culture for 5 weeks. During this timethey were treated with DA, fsk or a combination of both,always in the presence of BDNF. Immunocytochemistrywas then used to identify cells expressing TH proteinŽ . qTable 2 . Relatively few TH cells were seen in controlŽ .BDNF and in BDNFrDA-treated cultures of human

Žcortical neurones averages of 0.278"0.180 S.E.M.: con-. Ž .trol and 16"10 S.E.M.: BDNFrDA being recorded. In

contrast, in BDNF and fsk-treated cultures the number ofTHq cortical neurones increased to an average of 78.5

Ž . Žneuronesrwell "4.89 S.E.M. Fs180.8, P-0.0001.ANOVA; P-0.001 Tukey . The combined treatment with

BDNF, fsk and DA induced a large further increase in theq Žnumbers of TH cortical neurones present up to 219.5"

15.84 S.E.M. neuronesrwell; F s 180.8, P - 0.0001.ANOVA; P-0.001 Tukey equivalent to a 220-fold in-

Ž .crease, above the mean level in controls Table 2 , assum-ing a minimum of 1 THq cellrwell. The amplification byfsk above the THq cell numbers in cultures treated withBDNFrDA was 14-fold. Percentage changes based onmean values are also shown in Table 2.

3.2. Rat cell culture

Following plating, the rat cell cultures were treatedevery other day with BDNF, DA and fsk, either alone or incombination. After 7 days in culture the cells were stained

q Ž . Žto detect TH cells Table 3 . In control untreated, minus. ŽBDNF , or treated with DA or fsk alone i.e., minus. qBDNF there were relatively few TH cells. Adding BDNF

Ž .alone, BDNFrfsk or DArfsk without BDNF causedŽ .large increases 10–11-fold over control in the mean

number of THq cells. This increase in mean cell numberswas not a significant increase. Treatment with a combina-

Table 4The effect of BDNF and DA in the absence and presence of fsk on thelevel of TH mRNA in rat cerebral cortical cells from E13 rat embryos

Conditions in culture ROD THrROD b-actin

Control 0.233"0.03450 ngrml BDNFq10 mM DA 0.395"0.021

UU50 ngrml BDNFq10 mM DAq10 mM fsk 0.583"0.112

Cultures were grown for 7 days and treated every other day from DIV 0.Total RNA was extracted, RT–PCR was performed and the opticaldensities of the PCR product bands on an agarose gel were quantifiedusing NIH Image. A comparison of the ROD of TH to b-actin were madefor each condition. Numbers represent mean"S.E.M. from five separateexperiments. Significant difference compared to control is indicated:UU P -0.01.

tion of BDNFrDA or BDNFrDArfsk produced furtherŽ .increases ;20-fold and ;26-fold, respectively in the

q Ž .number of TH cells compared to control Table 3 . Thusfsk amplified the effect of BDNFrDA by 2-fold.

To accommodate the variation in cell numbers betweenexperiments, the number of THq cells in each culture wasexpressed as a percentage of the number of THq cells in

Ž .the control untreated and these percentage changes for ratcultures are shown in Table 3. It can be seen that the threetreatments caused a significant increase in the percentageof THq cells compared to control, whereas increases inmean THq cell numbers were not always significant.Thus, the addition of BDNFrfsk caused an 11-fold in-

Ž .crease Fs23.62, P-0.0001 ANOVA; P-0.01 Tukeyin the percentage of THq cells compared to control,

Žwhilst BDNFrDA caused a 17-fold increase Fs23.62,.P-0.0001 ANOVA; P-0.001 Tukey in the percentage

of THq cells compared to control, which is similar tow xprevious findings 20–22 . However, the greatest increase

in the percentage of THq cells compared to control wasinduced by treatment with BDNFrDArfsk all added to-

Žgether, which resulted in a 28-fold increase Fs23.62,.P-0.0001 ANOVA; P-0.001 Tukey in the percentage

of THq cells compared to control.

Table 3The effect of BDNF, DA and fsk alone and in combination on the number of THq cells in cerebral cortical cells from E13 rat embryos

q q qConditions in culture TH cellsrwell Percent of TH cells Percent of TH cellscompared to control compared to BDNF

treatment alone

Control 18.0"9.547 100"0 11.150 ngrml BDNF 200.14"122.344 901.3"141.6 10010 mM DA 48.29"32.433 194.7"45.2 21.610 mM fsk 39.71"23.886 169.4"35.6 18.8

UUU50 ngrml BDNFq10 mM DA 360.29"233.076 1709.8"299.9 189.6UU50 ngrml BDNFq10 mM fsk 173.14"88.768 1140.8"194.4 126.5

10 mM DAq10 mM fsk 202.57"151.54 512.9"200.9 56.9UUU UUU50 ngrml BDNFq10 mM DAq10 mM fsk 468.71"247.744 2806.7"326.2 311.4

Cultures were grown for 7 days and treated every other day from DIV 0 and then stained for THq cells. THq cells were counted in each well andexpressed as a percentage of the control. Numbers represent mean"S.E.M. from seven separate experiments carried out in duplicate. Significantdifference, compared with control group, is indicated: UUU P-0.001 and UU P-0.01.

( )F.B. Pliego RiÕero et al.rDeÕelopmental Brain Research 114 1999 201–206 205

To determine the levels of TH mRNA in rat foetaltissue, RT–PCR was performed on 7-day old cultures.RT–PCR can give semi-quantitative data by comparing thelevels of mRNA for the gene of interest to a constitutivelyexpressed gene. In this case TH mRNA was compared to

Ž .the level of the constitutive gene, b-actin Table 4 . Itappears that although the level of TH mRNA increased50% following the treatment with BDNFrDA, it was not asignificant increase. However, following treatment with

ŽBDNFrDArfsk a significant 2.5-fold Fs6.46, P-0.05.ANOVA; P-0.01 Tukey increase in the level of TH

mRNA compared to control was seen.

4. Discussion

In a series of papers this laboratory has reported that thefull dopaminergic phenotype can be induced in culturedfoetal rat cerebral cortex by exogenously applied DA andBDNF working synergistically and in a narrow time-

Ž . w xwindow E12–14 20–22 . The effect was a 17-fold in-crease in neurones which expressed TH compared to con-trols. This phenotype was also induced in foetal human

w x Ž .cerebral cortex 23 . We now report that in both rat E13Ž .and human 10–14 weeks old cortical foetal cells in

culture the appearance of THq neurones in much largernumbers can be induced by treatment with fsk and DA inthe presence of BDNF. Control treatment with vehiclealone produced no such changes. In the present study theeffect was a 3-fold increase in the number of rat THq cellsand a 220-fold increase in human THq cells which repre-sent much larger degrees of expression of the dopaminer-gic phenotype than were induced by BDNFrDA mixturealone. It is not clear why the THq cells produced duringthe treatment of human foetal tissue with BDNFrDArfskshould be substantially greater than for rat tissue, but this

Žmay be linked to the 5 times longer treatment i.e., 7 days.as 5 weeks . This longer treatment was obligatory to

produce the THq response in the human tissue as no THq

cells were seen after 2 or 4 weeks of treatment, and almostŽ .certainly reflects the much longer 9-fold gestation period

compared to rat. Equally important may be the differencein embryonic ages of the human and rat tissues when takenfor tissue culture and this needs to be a focus for furtherexperimentation.

The TH gene contains several cis-acting elements up-stream of the start site and these elements are similar in

w x w xboth the rat 2,6 and human 13 . Several studies haveŽ .shown that the cAMP response element CRE is the most

important in controlling both basal and cAMP-inducedw xtranscription of the TH gene 7,10–12,15–17 . cAMP lev-

els can be modulated by altering the activity of adenylatecyclase or phosphodiesterase. The fsk activates adenylatecyclase causing increased cAMP levels. Increases in levels

Ž .of cAMP activate cAMP-dependent protein kinase PKA ,phosphorylating several proteins including trans-acting el-

ements. Trans-acting elements are factors which bindspecifically to DNA sequences—cis-acting elements. One

Ž .such element is the CRE binding protein CREB , a tran-scription factor, which when activated by phosporylation,binds to CRE and activates transcription.

Thus, by manipulating the intracellular levels of cAMPŽ . qlarge 3- or 220-fold amplifications of TH neurone

induction can be effected in rat or human foetal cerebralcortex, respectively. This clearly shows the critical controlexerted by the cAMP-responsive element in TH geneexpression in developing rat and brain tissue. Moreoverthis artificially-induced dopaminergic tissue can produce

w xDA 21 . The rat tissue has been shown to maintain itsdopaminergic phenotype for at least 8 weeks in grafts

w ximplanted into rat striatum 22 .The relatively large quantities of human foetal cerebral

Ž .cortex available compared to VM 500-fold suggests thatit would provide a useful tissue source when artificiallyinduced to become dopaminergic and secrete DA. Thus theequivalent tissue from foetal rat brain has been to shown to

w xcorrect asymmetric locomotion in rat models of PD 22 .Although DA synthesis from rat and human foetal cerebralcortex was not demonstrated in the present study, DAsynthesis was clearly demonstrated when cerebral cortextissue was treated with BDNFrDA in a previous studyw x21 .

By measuring the levels of mRNA, involvement ofcAMP in the mechanism of induction was measured. Whentreating with BDNF and DA together the increase in thelevels of mRNA for TH compared to control was notsignificant but may be due to the lack of sensitivity of theassay—as an increase of 50% in the mean values formRNA levels after the treatment with DA and BDNF wasseen. However, when this dual treatment was combinedwith fsk the large and significant 2.5-fold increase in thelevel of TH mRNA demonstrated that boosting intra-cellular cAMP levels correlated closely with the novel

Ž .appearance of TH protein in 3-fold rat and 220-foldŽ .human more cells. These increases are likely to be due tomore transcripts being produced through the modulation oftranscription factors andror cis-acting elements on the THgene. The increases in mRNA may also be due to adecrease in the decay of transcripts in the cell through the

w xelongation of the poly-A tail 9 . The rises were mostprobably due to an increase in mRNA synthesis becausefsk is increasing the cAMP levels and so upregulating THmRNA synthesis through the CRE on the TH gene. It maybe a combination of both degradation and synthesis pro-cesses, but these cannot be distinguished from the experi-mental results reported here.

Previous studies have shown that BDNF-induced genew xexpression involves CREB 5 . It appears that BDNF phos-

phorylates CREB which then binds to CRE on the THw xgene and thus activates transcription 5 . It can be seen that

in rat foetal cortical cultures BDNF alone does produce anincrease in THq cells following 7 days treatment com-

( )F.B. Pliego RiÕero et al.rDeÕelopmental Brain Research 114 1999 201–206206

pared to control suggesting the involvement of CREB andCRE in the regulation of TH gene expression. The additionof DA with BDNF causes a significant increase in thenumber of THq cells suggesting that DA causes an inter-action with CREB andror CRE and thus increases theexpression of TH. The addition of fsk to this treatmentfurther increases the number of THq cells. This suggeststhat CREB is a major component in the regulation of THexpression because by increasing the level of phosphory-lated CREB results in increased THq cells. This workshows that CREB and CRE are important in the regulationof TH expression and that the expression may be increasedsignificantly in tissue that does not normally express thegene. This obviously has huge implications for the treat-ment of PD with cortical foetal tissue and thus reducingthe amount of tissue required per procedure.

There is clearly a quantitative difference between THmRNA and protein levels following dopaminergic pheno-type induction. This large disparity is not surprising sinceit is well known that the volume of mRNA resulting fromgene expression commonly does not lead to equivalentquantities of the target protein.

Acknowledgements

We would like to thank Paul Hamlyn and his founda-tion for his generous financial support of Dr. BernadoPliego-Rivero and the costs all the research materials usedto carry out this project. Wendy McCormack was sup-ported by a BBSRC-CASE award with Merck, Sharp &Dohme. We would also like to thank Brenda Miles and theCapel Singers from Watford for financial support of thisresearch.

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