Journal ofNeurochemistryLippincott—Raven Publishers, Philadelphia© 1998 International Society for Neurochemistry

Guai Cell Line-Derived Neurotrophic Growth Factor InhibitsApoptotic Death of Postnatal Substantia Nigra Dopamine

Neurons in Primary Culture

*Robe~E. Burke, *Megan Antonelli, and * ~tDavid Suizer

Departments of *Neurology, tPsychiatry, and ~Neuroscïence, New York State Psychiatric Institute, Columbia UniversityCollege of Physicians and Surgeons, New York, New York, U.S.A.

Abstract: GuaI cell line-derived neurotrophic factor(GDNF) was identified on the basis of its ability to en-hance the development of embryonic mesencephalic do-pamine neurons. lt remains unknown whether GDNF is aphysiologically relevant trophic factor for these neurons.We have shown that natural cell death among dopamineneurons of thesubstantia nigra occurs largely postnatally.To investigate whether GDNF may have the ability to sup-port these neurons during their period of natural celldeath, we have used a postnatal primary culture model.We find that GDNF is able to support the viability of post-natal nigral dopamine neurons by inhibiting apoptoticdeath. This ability of GDNF shows both regional specific-ity for the nigra and cellular specificity for the dopaminephenotype. Among eight other neurotrophic factors pre-viously reported to support embryonic dopamine neu-rons, GDNF was unique in this ability. Thus, GDNF meetsthis criterion for a physiologically relevant trophic factorfor dopamine neurons of the substantia nigra. KeyWords: Glial cell line-derived neurotrophic factor—Apoptosis—Substantia nigra— Dopamine—Parkinson‘sdisease—Trophic factors.J. Neurochem. 71, 51 7—525 (1998).

Guai cell line-derived neurotrophic factor (GDNF)was identified based on its ability to promote the differ-entiation of embryonic mesencephalic dopamine neu-rons (Lin et al., 1993). Although many other neuro-trophic factors have been shown to promote dopamineneurons in embryonic midbrain culture, includingbrain-derived neurotrophic factor (BDNF) (Hyman etal., 1991), transforming growth factors [TGF/32 andTGF/33 (Poulsen et al., 1994) and TGFa (Alexi andHefti, 1993)], epidermal growth factor (EGF) (Casperet al., 1991), and basic fibroblast growth factor(bFGF) (Knüsel et al., 1990), GDNF has shown theunique ability to protect or restore dopamine neuronsin a variety of in vivo injury models in adult rodentsand primates (Beck et al., 1995; Bowenkamp et al.,1995; Sauer et al., 1995; Tomac et al., 1995a; Gashet al., 1996). Thus, GDNF has been considered a can-

didate neurotrophic factor for the treatment of Parkin-son‘s disease, which is characterized pathologically bydegeneration of mesencephalic dopaminergic neurons.

It remains unknown whether GDNF plays a physio-logic role, either during development or in maturity,as a target-derived neurotrophic factor for the supportof nigrostriatal dopamine neurons, as envisioned byclassic neurotrophic theory (Barde, 1989). In supportof this possibility, GDNF mRNA is expressed in thetarget striatum during development (Schaar et al.,1993; Stromberg et al., 1993; Poulsen et al., 1994;Blum and Weickert, 1995; Choi-Lundberg and Bohn,1995), and mRNA for its receptor is expressed in sub-stantia nigra (SN) (Treanor et al., 1996). GDNF hasalso been shown to undergo specific retrograde axonaltransport to dopamine neurons of the mesencephalonfollowing intrastriatal injection (Tomac et al., 1995b).However, mice lacking GDNF show a normal comple-ment of mesencephalic dopamine neurons during em-bryogenesis and on postnatal day 1 (Moore et al., 1996;Pichel et al., 1996; Sanchez et al., 1996), casting doubton the possible physiologic role of GDNF as a factorfor these neurons.

Although there are several possible explanations forthe lack of an effect in GDNF null mice, one possibilityis that because the mice do not survive beyond the firstpostnatal day, they may have been examined prior tothe developmental period when dopamine neurons be-come dependent on their target for trophic support.

Received January 20, 1998; revised manuscript received March9, 1998; accepted March 13, 1998.

Address correspondence and reprint requests to Dr. R. E. Burkeat Department of Neurology, Box 67, 710 W. 168 St., New York,NY 10032, U.S.A.

Abbreviations used: BDNF, brain-derived neurotrophic factor;bFGF, basic fibroblast growth factor; DIV, days in vitro; EGF, cpi-dermal growth factor; GDNF, glial cell line-derived neurotrophicfactor; MEM, minimal essential medium; NT-3, neurotrophin-3;PBS, phosphate-buffered saline; PBS-T, 0.1% Triton in PBS; rHu,recombinant human; SN, substantia nigra; TBS-T, 0.2% Triton inTris-buffered saline; TGF, transforming growth factor; TH, tyrosinehydroxylase; VTA, ventral tegmental area.

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518 R. E. BURKE ET AL

We have shown that the natural cell death period fordopamine neurons of the SN takes place predominantlyin the postnatal period (Oo and Burke, 1997). Theseresults are compatible with a direct quantitative analy-sis that showed a decrement in the number of theseneurons postnatally (Tepper et al., 1994). In supportof the concept that SN dopamine neurons are depen-dent on their target, the striatum, during a critical post-natal period, we have shown that either axon-sparingtarget injury (Macaya et al., 1994; Kelly and Burke,1996) or destruction of intrastriatal dopamine termi-nals (Marti et al., 1997) results in an induced apoptoticevent in SN dopamine neurons.

If GDNF is a physiologically relevant trophic factorfor these neurons, then it should be possible to demon-strate its ability to support their viability during thiscritical postnatal period. All prior observations on theability of neurotrophic factors to support these neuronshave beenmade inembryonic cultures. To examinetheability of GDNF and other factors to support dopamineneurons during their period of maximal natural celldeath, we have made use of a well-characterized sys-tem for primary culture of postnatal mesencephalicdopamine neurons (Rayport et al., 1992; Sulzer et al.,1993; Cubells et al., 1994; Rayport and Sulzer, 1995;Pothos et al., 1996; Sulzer et al., 1996; Mena et al.,1997). We find that GDNF specifically supports theviability of these neurons during their period of naturalcell death, and it does so by diminishing apoptotic celldeath.

MATERIALS AND METHODS

Postnatal SN culturesCultures were established as previously described (Mena

et al., 1997). Timed pregnant rats were obtained fromCharles River Laboratories (Wilmington, MA, U.S.A.). Theday of delivery was defined as postnatal day 1. Postnatalday 2—3 animalswere used for the preparation of glial mono-layers. The rostral half of the cerebral cortex was dissectedin ice-cold calcium- andmagnesium-free phosphate-bufferedsaline (PBS) and cut into small pieces. The pieces of tissuewere dissociated with 20 U/ml papain in 1.0 mM cysteine,0.001% phenol red, 116 mM NaC1, 5.4 mM KC1, 26 mMNaHCO3, 2 mM NaH2PO4, 1 mM magnesium phosphate,500 ~jM EDTA, and 25 mM glucose at pH 7.3. Cells wereplated at 150,000/well (0.8 cm

2) andfed with medium con-taining 90% minimal essential medium (MEM), 10% calfserum, 0.33% glucose, 5 ~eg/m1bovine pancreatic insulin,500 j.tM glutamine, 12 U/mlpenicillin, and 12 ~ig/ml strepto-mycin. At 4—5 days after plating, cultures were treated with6.6 ng/ml 5-fluorodeoxyuridine/ 16.4 ng/ml uridine to sup-press growth of nonneuronal cells. Cells were plated ontoglass coverslips attached under 0.8-cm2 holes in the bottomof 50-mm snap-top polystyrene Petri dishes (Falcon) andpretreated with poly-D-lysine and laminin.

After 2 weeks, SN neurons were plated onto the glialmonolayers. Forty-eight hours prior to plating, glial growthmedium was removed, andserum-free neuronal medium wasadded to each dish following a single rinse with serum-freeneuronal medium. Serum-free neuronal medium contained

47% MEM, 40% Dulbecco‘s modified Eagle‘smedium, 10%Ham‘s F-12 nutrient medium, 3.4 mg/ml glucose, 0.25%albumin, 500 ‚uM glutamine, 100 ~.tg/mltransferrin, 15 ‚uMputrescine, 30 nM triiodothyronine, 25 ‚ug/ml insulin, 200nM progesterone, 115 nM corticosterone, 5 ‚ug/ml superox-ide dismutase, 432 U/ml catalase, and 500 ‚uM kynurenate.Twenty-four hours prior to plating, GDNF or other trophicfactors were added to each dish in 100 ~ilof fresh neuronalmedium. Control cultures received 100 ~.tlof fresh neuronalmedium alone. On the day of neuron plating, P2 rat pupswere anesthetized by hypothermia, the brain was removed,and a 2- to 3-mm coronal section of the mesencephalon wastaken. Meninges were removed. The coronal section waslaid on a Sylgardblock in ice-cold solution, and ahorizontalcut was made to remove dorsal mesencephalon. A parame-dian vertical cut was made left and right to separate left andright SN from the centrally located ventral tegmental area(VTA). SN neurons were then dissociated as described andplated at 80,000 cells/well. In experiments comparing ef-fects on SN and VTA, two pairs of paramedian vertical cutswere made to separate each SN from the centrally locatedVTA. Each pair of paramedian cuts was made ~ 1.0 mmapart, and the tissue between the cuts was discarded. Thisprocedure was done to avoid any cross-contamination be-tween separate SN and VTA cultures. The VTA neuronswere plated at the same concentration as the SN neurons.At 1 day in vitro (DIV 1), 5-fluorodeoxyuridine was addedto each dish. Cultures were maintained at 36.7°Cin 5% CO

2until use. Cultures were maintained in serum-free mediumat all times.

MaterialsRecombinant human (rHu) GDNF, rHu neurotrophin-3

(NT-3), and rHu BDNF were obtained from Intergen. rHuTGFß1, porcine TGF/32, rHu TGFß3, rHu EGF, and rHuTGFa were obtained from R&D Systems. rHu bFGF wasobtained from Boehringer—Mannheim.

HistologyIn preparation for immunostaining, cultures were washed

with normal saline andthen fixed with 4%paraformaldehydein 0.1 M phosphate buffer (pH 7.1), followed by washeswith PBS. For tyrosine hydroxylase (TH) peroxidase immu-nostaining, dishes were treated with 0.3% H202 in methanol,followed by 0.1% Triton in PBS (PBS-T) and then 10%horse serum in PBS-T. Cultures were incubated in a mousemonoclonal anti-TH antibody (Boehringer—Mannheim) at1:640 at 4°Cfor 24 h. Dishes were then rinsed with PB S-Tand incubated with biotinylated horse anti-mouse (Vector)at 1:200 in 10% horse serumlPBS-T at room temperaturefor 45 min. Following a PBS rinse, dishes were incubatedwith avidin—biotinylated horseradish peroxidase complexes(ABC; Vector) at 1:600 at roomtemperaturefor 1 h. Follow-ing arinse in PBS, dishes were incubated with diaminobenzi-dine (0.5 mg/ml Tris) in the presence of glucose oxidase,ammonium chloride, and D-glucose for the generation ofH202. The chromogen reaction was monitored under themicroscope and stopped with aTris wash when dark specificstaining had been achieved.

For fluorescentTH and GABA double-labeling, cells werefixed as described and then treated with 0.2% Triton in Tris-buffered saline (TBS-T; pH 7.4) containing 2% goat serumand 2% horse serum at room temperature for 30 min. Cul-tures were then incubated with anti-TH (Boehringer) at1:625 and a rabbit anti-GABA antibody (Sigma) at 1:1,000

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GDNF INHIBITS APOPTOTIC DEATH 519

in 2% goat/2% horse sera in TBS-T at 4°Cfor 48 h. Follow-ing a rinse in TBS, cultures were incubated with Texas Redhorse anti-rabbit (Vector) at 1:75 and fluorescein goat anti-mouse (Vector) at 1:75 at room temperature for 1 h. Disheswere then washed with PBS and stained with 0.0004%Hoechst 33342 (Molecular Probes) prior to viewing to visu-alize apoptotic chromatin clumps.

Quantitative analysisDishes stained for TH by the immunoperoxidase technique

were counted by filling the culture well with Tris, placing aglass coverslipover the well, inverting thedish, andcountingpositive neurons at 400x on a Nikon Labophot microscope.An eyepiece reticle was used to guide successive verticalscans across the entire dish. Neurons were counted in eitherevery second or every fourth scan path and multiplied toprovide a count for the entire dish. TH-positive neuronswere identified by the presence of dark brown chromogendeposition (see Fig. 2). TH-negative neurons were identifiedby Nomarski optics as large polygonal cells with an abundantcytoplasm growing on top of the flat glial monolayer (seeFig. 2).

Dishes stained for TH and GABA using fluorescence im-munolabeling were viewed at 400x with a Zeiss Axiovert135 inverted microscope with xenon arc lamp illuminationand appropriate filters: excitation 540 ±25 nm and emission605 ±55 nm for Texas Red; 480 ± 30/555 ±40 nm forfluorescein; 365/390 nm (long pass) for Hoechst 33342.Each dish was scanned in its entirety, using the entire fieldto guide successive vertical scans and scanning every secondor fourth vertical path.

For determination of neuronal soma areas, the Nikon La-bophot was coupled to a Dage 81 series video camera, pro-files were digitized under a 60x objective, and area wasdetermined using a Loats Associates Inquiry image analysissystem.

Statistical analysis was performed in cases of multiple-groupcomparisons by ANOVA, followed by an all pairwisemultiple-comparison procedure, using the Student—New-man—Keuls method. Two-group comparisons were per-formed with the Student‘s t statistic.

RESULTS

GDNF increases viability and development ofpostnatal mesencephalic dopamine neurons inprimary culture

Effects of GDNF were initially assessed by adding10 ng/ml to glial monolayer cultures on the day beforeplating dissociated SN neurons, followed by additions(without medium change) on DIV 2 and 4. This regi-men resulted in no apparent effect on viability on DIV1 but a clear augmentation on DIV 4 and 6 (Fig. lA).The difference in TH-positive neuron numbers due toGDNF was established by DIV 4, with no further sig-nificant changes in number in either the GDNF or no-GDNF condition by DIV 6. Subsequent experimentsshowed that a single addition of 10 ng/ml GDNF on theday prior to plating led to a two- to fourfold increasein the number of TH-positive neurons on DIV 4. Aspreviously shown for embryonic mesencephalic cul-tures (Lin et al., 1993), the effect of GDNF appearedto be relatively specific for dopaminergic neurons be-

FIG. 1. GDNF increases the number and size of postnatal dopamin-ergic neurons of the SN in primary culture. A: GDNF (10 ng/ml) wasadded to the culture medium 24 h prior to plating dissociated SNneurons and at DIV 2 and 4. Cultures were fixed 2 h after plating (t= 0) and at DIV 1, 4, and 6. Cultures were immunostained forTH to identify SN dopaminergic neurons and counted. Counts areexpressed as total number of TH-positive neurons per dish. n = 3dishes were counted for each condition at each time point. GDNFtreatment resulted in an increased number of TH-positive neuronson DIV 4 and 6 (p = 0.01). This experiment was performed threetimes; a single representative experiment is shown. For the threeexperiments, the mean (±SEM) number of TH-positive neurons onDIV 4 was 410 ±20 with GDNF and 219 ± 11 without GDNF (a2.1-fold increase). On DIV 6 the mean number was 518 ±25 withGDNF and 175 ± 11 without GDNF (a threefold increase). Eachcolumn represents the mean + SEM. B: GDNF treatment resultedin an increase in the ratio of TH-positive to total neurons in the dish(p < 0.01). The dishes studied in A were analyzed. All three disheswere assessed for the ratio of TH-positive to total neurons for eachcondition on DIV 6. Each dish was examined in five randomly se-lected high-power fields, and the results are expressed as percentof TH-positive neurons per field. C: GDNF treatment induced anincrease in the mean somal area of TH-positive neurons (p <0.001).The dishes studied in A were analyzed. Fifteen randomly selectedTH-positive neurons in each of three dishes per condition (90 neu-rons total) on DIV 6 were chosen for area measurement. GDNFtreatment led to a 44% increase in mean somal area. D: Dose—response for GDNF-induced increases in TH-positive neuron num-ber. In two experiments, separate from those shown in A—C, dose—response was assessed in a total of 41 dishes. Cultures were treatedonce with the indicated concentrations of GDNF at 24 h prior toneuron plating and fixed on DIV 4. Each point represents the mean±SEM for n = 3—6 dishes. Data are here presented as ‘-foldincrease“ over the no-GDNF controls to facilitate presentation ofdose—response data from the two separate experiments, which hadslightly different numbers of TH-positive neurons in the controls. Anincrease in the number of TH-positive neurons was noted between0.1 and 1.0 ng/mI.

cause it led to a significant increase in the ratio of TH-positive neurons to total neurons by DIV 6 (Fig. lB).A dose—response analysis of the ability of GDNF to

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520 R. E. BURKE ET AL.

FIG. 2. Morphologic features of GDNF-induced augmentation in the growth of postnatal dopamine neurons in primary culture, visualizedby Nomarski optics. A: A typical TH-positive neuron following treatment with 10 ng/mI GDNF. GDNF was added to the dish 24 h priorto neuron plating, and the culture was fixed at DIV 4. A TH-positive neuron is indicated by the filled arrow and a TH-negative neuronby the open arrow. Numerous TH-positive processes and varicosities surrounding the single neuron have been marked by arrowheads.B: A non-GDNF-treated TH-positive neuron, cultured and stained in parallel. A TH-positive neuron is indicated by the filled arrow anda TH-negative neuron by the open arrow. Bar = 25 ~tm.

increase the number of TH-positive neurons showedthat the effect required between 0.1 and 1.0 ng/ml(Fig. 1D). Effects were near maximal at 1.0 ng/ml.

GDNF also augmented the differentiation of postnatalSN dopamine neurons. It led to a 44% increase in the areaof the cell body (Fig. 1C), which was visually apparentin the cultures (Fig. 2). Also apparent was an increase inthe elaboration of m-positive processes (Fig. 2).

GDNF has regional, cellular, and molecularspecificity

Prior studies of the effects of GDNF on embryonicmesencephalic dopaminergic neurons have not beenable to differentiate between dopamine neurons of thenigrostriatal (A9) and mesolimbic (Al 0) systems. ThemRNA for GDNF is abundant in dorsal striatum, atarget of the A9 system. Lower levels of expressionare observed in frontal cortex, entorhinal cortex, amyg-dala, and hippocampus, some of the major targets ofthe AlO system, during the early postnatal period(Poulsen et al., 1994). This observation suggests that

ifGDNF is a physiologically relevant factor, its effectsmay be regionally specific. To assess this possibility,we examined the relative ability of GDNF to supportthe viability of dopamine neurons in cultures derivedfrom SN (A9) and VTA (Ab), obtained by microdis-section from the same animals and cultured in parallelunder identical conditions. GDNF selectively sup-ported the viability of SN neurons (Fig. 3).

Whereas our previous finding that GDNF increasedthe ratio of TH-positive to total neurons in these cul-tures suggested that its effects were specific for dopa-mine neurons, we also examined its cellular specificityfor this neuronal phenotype in comparison with GA-BAergic neurons, the other major neuronal phenotypein these cultures (Przedborski et al., 1996). We foundthat GDNF did not augment the viability of GABAer-gic neurons; on the contrary, it led to a reduction intheir number after 4 days in culture (Fig. 4). Thus, inpostnatal SN cultures, GDNF effects are specific atthe cellular level for dopamine neurons, as previouslyshown for embryonic cultures (Lin et al., 1993).

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GDNF INHIBITS APOPTOTIC DEATH 521

FIG. 3. Regional specificity of the effect of GDNF on mesence-phalic dopaminergic neurons. GDNF induced a significant in-crease in the number of 1H-positive neurons derived from theSN but not from the VTA. SN and VTA were dissected simultane-ously from individual animals and plated into medium in eitherthe presence or the absence of a 24-h pretreatment with GDNF.n = 4 cultures for each condition were fixed and processed for1H immunostaining at DIV 4. Columns represent the means +SEM for the total number of TH-positive neurons per dish. Forthree experiments performed, the mean -fold increase in TH-positive neurons in the SN cultures was 3.2 ±0.3. In the VTAcultures, the mean -fold increase was 1 .5 ±0.2, which was nota significant effect.

To examine the molecular specificity of GDNF, wecompared it with three other members of the TGFfamily litwo of which, TGF/32 and TGFß3, have beenshown to enhance the viability of dopamine neuronsin embryonic mesencephalic culture (Poulsen et al.,1994)1 and five other growth factors previously shownto support the viability of embryonic dopamine neu-rons. Among these factors, GDNF was uniquely ableto increase the viability of dopamine neurons (Fig. 5).Following treatment with EGF 1 day prior to neuronplating, there was a clear increase in the density of theglial monolayer by DIV 1. However, this increase inglial number was not associated with a change in thenumber of TH-positive neurons.

FIG. 4. Cellular specificity of the effect of GDNF on SN dopa-mine neurons. A single dose of GDNF (10 ng/mI) was added toneuronal medium 24 h prior to neuron plating; cultures were thenfixed on DIV 4 and processed for double-immunofluorescencestaining of TH and GABA. n = 5 dishes were examined for eachcondition, and a single representative experiment is shown.GDNF led to an increase in the number of TH-positive neuronsby DIV 4, as previously observed, and a significant decrease inthe number of GABA-positive neurons (p = 0.02). A secondexperiment revealed an identical effect on GABA-positive neu-rons, a reduction in their number to 0.7-fold of that irr the no-GDNF condition.

FIG. 5. Pharmacologic specificity of the effect of GDNF on SNdopaminergic neurons. A: Each of the indicated factors wasadded to the neuronal medium at a concentration of 10 ng/mI24 h prior to plating. On DIV 4, the cultures were fixed andprocessed for 1H immunostaining. n = 4 dishes were examinedfor each factor, and two experiments were performed. Data fromboth experiments (n = 8 dishes) are shown. Only GDNF led to anincrease in the number of TH-positive neurons. TGFß1, TGF/32,TGFß3, and BDNF were without significant effect. Data are ex-pressed as “-fold change“ in comparison with the no-GDNFcondition to facilitate directcomparisons of effects across exper-iments. There were 125 ±14TH-positive neurons in the controldishes. B: In a second set of experiments, similar in design tothat described in A, EGF, TGFa, bFGF, and NT-3 were alsowithout effect. n = 4—5 dishes for each factor were analyzed intwo separate experiments (a total of 8—9 dishes). There were204 ±29 TH-positive neurons in the control dishes.

GDNF decreases apoptotic cell death in dopamineneurons

There are several possible mechanisms by whichGDNF may induce an increase in the number of TH-positive neurons. Although induction of cell divisionamong dopamine neurons is formally possible, it seemsunlikely in these cultures, which were established withpostmitotic dopamine neurons. Moreover, the cultureswere treated with 5-fluorodeoxyuridine to block mito-sis. The two major possibilities that apply to this cul-ture system are enhanced differentiation (resulting inan increased abundance of TH-positive profiles) anda reduction in death rate among dopaminergic neurons.To directly examine the latter possibility, we first dem-onstrated that apoptotic cell death, such as occurs dur-ing the natural cell death period of postnatal dopamineneurons (Janec and Burke, 1993; Oo and Burke,1997), occurs in these cultures on both DIV 1 and 4.Apoptotic profiles were identified by Hoechst 33342

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522 R. E. BURKE ET AL.

FIG. 6. Apoptotic cell death in postnataldopamine neurons. Cultures were fluo-rescence immunostained for TH and thendouble-labeled for chromatin staining withHoechst 33342. A: Hoechst stain. Thelarge filled arrow to the right indicates aprofile with two rounded, distinct, and in-tensely fluorescent apoptotic chromatinclumps. The open arrow to the left indi-cates the faintly fluorescent, mottled ap-pearance of a normal neuronal nucleus.The small filled arrow indicates the ab-sence of chromatin staining at the positionof a cytoplasmic apoptotic body demon-strated by THstaining in B. B: TH immuno-stain. The large filled arrow indicates a TH-positive profile that corresponds to theapoptotic profile shown in A. lt has arounded appearance typical of apoptoticprofiles. The open arrow indicates a TH-positive neuronal profile with normal mor-phology. The small filled arrow indicates aTH-positive apoptotic body that does notcontain nuclear chromatin; it is thereforeprobably a cytoplasmic fragment. Bar =

10 /.tm.

staining, which revealed bright fluorescent, rounded,distinct intranuclear chromatin clumps in both TH-neg-ative and TH-positive cells (Fig. 6). Addition of 10ng/ml GDNF to the cultures 1 day prior to neuronplating resulted in a highly significant reduction inthe total number of apoptotic profiles per dish (29%decrease; p = 0.003) and in the number of apoptoticdopaminergic neurons on DIV 1 (Fig. 7). With timein culture, the total number of apoptotic profiles de-creased in the no-GDNF condition from 125 ±10.8/dish on DIV 1 to 71.2 ±10.8 on DIV 4. By DIV 4,there was no longer a difference in the number ofapoptotic profiles between the GDNF and no-GDNFconditions, and the number of apoptotic dopaminergicneurons was negligible. This observation is compatiblewith our data on the timecourse of the effect of GDNF,as shown in Fig. lA. The difference in number ofTH-positive neurons between conditions is establishedbetween days 1 and 4; after day 4, there is little changein the relative numbers. Thus, a significant differencein death rates, reflected in the prevalence of apoptoticprofiles, would not be expected 4 days and after. Al-though it remains possible that the effect of GDNF

may be partially mediated by up-regulation of, or con-version to, the TH-positive phenotype, these resultsshow that the effect is certainly mediated, at least inpart, by a reduction in apoptotic cell death.

FIG. 7. Effect of GDNF on the prevalence of 1H-positive apo-ptotic profiles. On DIV 1, cultures were fixed, immunostained forTH, and fluorescence stained for apoptotic chromatin clumpswith Hoechst 33342. The total numbers of TH-positive apoptoticprofiles were determined for n = 5 dishes for each condition ineach experiment. The experiment was repeated three times, andthe composite result shown is expressed as a -fold change.GDNF led to a 58% reduction in the prevalence of TH-positiveapoptotic profiles (p < 0.001).

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FIG. 8. The effect of GDNF on SN dopaminergic neurons inthe absence of a glial monolayer. Dissociated SN neurons wereplated onto glass coverslips treated with poly-D-Iysine and lami-nm and maintained for 4 days. n = 5 dishes were examined foreach condition. On DIV 4, the cultures were fixed and immuno-stained for 1H with the peroxidase technique. In spite of theabsence of a glial monolayer, GDNF treatment resulted in anincreased number of 1H-positive neurons (p = 0.003). Notethat for both the GDNF and the non-GDNF conditions, there areconsiderably fewer surviving 1H-positive neurons in comparisonwith the glial monolayer growth conditions (Fig. lA). A secondexperiment with n = 5 dishes in each condition yielded a similarresult, a 2.4-fold increase in TH-positive neurons in the GDNFcondition in spite of the absence of a glial monolayer.

GDNF may act directly on dopamine neurons, or itseffects may be mediated indirectly via an action onglia. To assess the possibility that GDNF is acting byan effect on the glial monolayer, its effect was assessedin postnatal cultures grown in the absence of the mono-layer. Whereas survival of dopamine neurons wasmuch reduced in the absence of a monolayer, therewas still a significant enhancement in viability affordedby GDNF treatment (Fig. 8). Although an effect ofGDNF on dopamine neurons mediated indirectly byan effect on the monolayer is excluded by this experi-ment, we cannot exclude the possibility that it is actingthrough a small number of glia carried into the culturewith the dissociated SN neurons. The proliferation ofsuch glia was inhibited by the addition of 5-fluoro-deoxyuridine, but they nevertheless populate the cul-tures in small numbers.

DISCUSSION

These results demonstrate that GDNF promotes theviability of postnatal dopamine neurons of the SN invitro during a time period when they would normallyundergo their maximal level of natural cell death (00and Burke, 1997). The ability of GDNF to promotethe viability of these neurons is due, at least in part,to suppression of apoptotic cell death, which is mor-phologically identical to that observed during naturalcell death in vivo. Classic concepts of neurotrophictheory would propose that GDNF is a target-derivedfactor, present in limiting quantities, which is taken upby the terminals of those neurons that successfullycompete for target contact and maintains their viabilityby suppressing apoptotic death. Our results indicatethat GDNF may meet this latter important criterionfor a physiologically relevant neurotrophic factor for

dopamine neurons of the SN. However, we must alsopoint out that more dopamine neurons die in postnatalculture (‘—.~75%between DIV 1 and DIV 4) than arelikely to die in vivo (Tepper et al., 1994), so much ofthe in vitro death is an induced event and possiblytherefore a different process from natural cell death. Itis conceivable that GDNF acts to suppress this inducedevent rather than natural cell death. Since the lightmicroscopic appearance of apoptosis is identical forinduced death in vitro and natural cell death in vivo,it is not possible to distinguish between the two pro-cesses. Ultimately, in vivo studies will be requiredto assess the effect of GDNF on natural cell death.Nevertheless, the present results are consistent withthe possibility that an in vivoeffect may be anticipated.

As observed for embryonic mesencephalic cultures(Lin et al., 1993), GDNF is able to enhance the mor-phologic differentiation of these postnatal dopamineneurons, evidenced by an increase in soma size andthe number and extent of TH-positive processes. Thesemorphologic changes may have functional conse-quences, including elevated dopamine release due toincreased quantal size (Pothos et al., 1997).

The ability of GDNF to increase the number of TH-positive neurons in these postnatal cultures requireddoses ranging from 0.1 to 1.0 ng/ml, somewhat higherthan the doses used in the studies of Lin and colleaguesin embryonic cultures in which an ED5() of 36 pg/mlwas required. There are a number of possible explana-tions for this disparity. We utilized a response measureof increase in TH-positive neurons, whereas Lin et al.used a biochemical measure of dopamine uptake. Inaddition, the physical and developmental characteris-tics of our postnatal cultures differ from those of theembryonic cultures. Furthermore, our GDNF was ob-tained through a commercial supplier, and its potencymay differ from that used by Lin and associates. Otherinvestigators have found that doses similar to thosethat we used were required: Hou et al. (1996) foundthat a maximum effect on TH-positive neuron numberin their embryonic cultures required 1.0 ng/ml.

In these postnatal cultures, we found that the GDNFeffect was regionally specific for dopamine neurons ofthe SN in comparison with those of the VTA. Thisfinding is consistent with the greater abundance ofGDNF mRNA in the postnatal striatum (Schaar et al.,1993; Stromberg et al., 1993; Poulsen et al., 1994;Blum and Weickert, 1995; Choi-Lundberg and Bohn,1995).

In postnatal culture, the effect of GDNF on neuronalviability appeared to be specific for the dopamine cel-lular phenotype. This specificity was reflected in a sig-nificant increase in the ratio of TH-positive to totalneurons in the GDNF-treated cultures. In addition,GDNF actually decreased the number of GABAergicneurons. We do not know whether this effect is dueto negative regulation of the GABAergic phenotype oran induction of death among GABAergic neurons. Weare not aware of a precedent for either possibility for

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GDNF. However, there are precedents for other neuro-trophic factors to induce apoptotic neuron death: Bothleukemia inhibitory factor and ciliary neurotrophic fac-tor induce death in cultured sympathetic neurons (Kess-ler et al., 1993). Thus, it is conceivable that GDNFmay play a role in the induction of deathof GAB Aergicneurons of the SN if, during development, they wereto send aberrant afferents to inappropriate targets suchas the striatum.

GDNF is quite specific in its ability to support post-natal dopamine neurons, as it was the only factoramong nine tested that showed this ability. This speci-ficity is especially remarkable in view of the fact thatmost of the other factors examinedhad previously beenshown to have effects on embryonic mesencephaliccultures. We cannot exclude the possibility that otherfactors may show synergistic effects when combinedwith GDNF or one another. In view of our finding thatother factors do not support dopamine neurons duringtheir period of natural cell death, the physiologic sig-nificance of their ability to support embryonic dopa-mine neurons becomes unclear. It is unknown whetherembryonic dopamine neurons at embryonic days 14—18 undergo programmed cell death. Our studies haveshown that at embryonic day 19 they do not, but earlierages have not been studied. In addition, at embryonicday 14, SN dopamine neuron contact with the targetstriatum is minimal (Specht et al., 1981; Kalsbeek etal., 1992). Thus, the ability of other factors to supportembryonic dopamine neurons may not be relevant toany possible role as target-derived factors that act tosuppress a natural cell death event, as envisioned byclassic neurotrophic theory (Clarke, 1985; Barde,1989). Alternatively, it is possible that these factorsmay serve a role locally in the mesencephalon to sup-port dopamine neurons. The observations that GDNFis quite specific in its ability to support dopamine neu-rons in postnatal culture and that it has emerged as afactor uniquely potent in its ability to protect or restoredopamine neurons in a number of adult injury models(Beck et al., 1995; Bowenkamp et al., 1995; Sauer etal., 1995; Tomac et al., 1995a; Gash et al., 1996) maysuggest that this culture system is especially useful forits ability to predict such restorative effects.

Our results show that, as predicted for a physiologi-cally relevant target-derived factor, GDNF supportsviability by suppressing apoptotic cell death. Our stud-ies do not rule out the additional possibility that GDNFmay also act to increase TH-positive neuron numbereither by augmenting expression of TH or by con-verting nondopaminergic neurons in the dish to theTH-positive phenotype.

Although we cannot strictly exclude the possibilitythat GDNF is acting indirectly through glia rather thandirectly on neurons, we believe that it is unlikely. Linand colleagues (1993) noted no effect on the densityor glial fibrillary acidic protein expression of astrocytesin embryonic cultures. In our studies, GDNF exertedeffects in the presence of an antimitotic agent, added

on DIV 1, and was effective in the absence of a glialmonolayer. On the other hand, whereas EGF had avisually apparent effect on the density of the glialmonolayer, it had no effect on dopamine neuron num-ber. Furthermore, expression of mRNA for the GDNFreceptor is localized regionally to the SN pars com-pacta, suggesting a cellular localization to dopamineneurons (Treanor et al., 1996). In addition, it is knownthat dopamine neurons are capable of taking up GDNFat their terminals and retrogradely transporting it (To-mac et al., 1995b). Thus, there is evidence that GDNFis capable of acting directly on dopamine neurons. Inour studies, we have not examined where GDNF actsat a cellular level. Although it may be bound at termi-nals and retrogradely transported, it may also act di-rectly at the level of the cell soma.

Our developmental studies have shown that naturalcell death within the dopamine neuron population oc-curs largely postnatally (Oo and Burke, 1997).Whereas recent observations in GDNF null animalshave brought into question the possible physiologicrole of GDNF in supporting dopamine neurons (Mooreet al., 1996; Pichel et al., 1996; Sanchez et al., 1996),these observations may be incomplete because of theearly lethality caused by the mutation. The currentstudies show that in vitro GDNF is capable of sup-pressing apoptotic cell death, which is morphologicallyidentical to that observed in vivo, at a time when theseneurons would undergo their natural cell death eventin brain. It remains unknown whether GDNF regulatesthis death event in vivo. Future studies of its possiblerole in vivo will require more spatially and temporallyselective knock-out models or anti-sense “knock-down“ approaches.

Acknowledgment: We are grateful to Ms. Pat White forexcellent secretarial assistance. We are also grateful for dili-gent preparation and analysis of cultures provided by Ms.Irma Ryjak and Mr. William J. Kelly. Additionally, we ac-knowledge the assistance of Ms. Uzma Khan and Dr. SergePrzedborski in establishing the double-immunofluorescencestaining of TH and GABA. This work was supported byNS 26836, Shannon Award NIDA 07418, NIDA 10154, theParkinson‘ s Disease Foundation, the Smart Family Founda-tion, and the Lowenstein Foundation.

REFERENCES

Alexi T. and Hefti F. (1993) Trophic actions of transforming growthfactor a on mesencephalic dopaminergic neurons developing inculture. Neuroscience 55, 903—918.

Barde Y. A. (1989) Trophic factors and neuronal survival. Neuron2, 1525—1534.

Beck K. D., Valverde J., Alexi T., Poulsen K., Moffat B., VandienR. A., Rosenthal A., and Hefti F. (1995) Mesencephalic dopa-minergic neurons protected by GDNF from axotomy-induceddegeneration in the adult brain. Nature 373, 339—341.

Blum M. and Weickert C. S. (1995) GDNF mRNA expression innormal postnatal development, aging, and in weaver mutantmice. Neurobiol. Aging 16, 925—929.

Bowenkamp K. E., Hoffman A. F., Gerhardt G. A., Henry M. A.,Biddle P. T., Hoffer B. J., and Granholm A-C. E. (1995) Glial

J. Neurochem., Vol. 71, No. 2, 1998

GDNF INHIBITS APOPTOTIC DEATH 525

cell line-derived neurotrophic factor supports survival ofinjuredmidbrain dopaminergic neurons. J. Camp. Neural. 355, 479—489.

Casper D., Mytilineou C., and Blum M. (1991) EGF enhances thesurvival of dopamine neurons in rat embryonic mesencephalonprimary cell culture. J. Neurosci. Res. 30, 372—381.

Choi-Lundberg D. L. and Bohn M. C. (1995) Ontogeny and distribu-tion of glial cell line-derived neurotrophic factor (GDNF)mRNA in rat. Dey. Brain Res. 85, 80—88.

Clarke P. G. H. (1985) Neuronal death in the development of thevertebrate nervous system. Trends Neurosci. 8, 345—349.

Cubells J. F., Rayport S., Rajendran G., and Sulzer D. (1994) Meth-amphetamine neurotoxicity involves vacuolation of endocyticorganelles and dopamine-dependent intracellular oxidativestress. J. Neurosci. 14, 2260—2271.

Gash D. M., Zhang Z. M., Ovadia A., Cass W. A., Yi A., Sim-merman L., Russell D., Martin D., Lapchak P. A., Collins F.,Hoffer B. J., and Gerhardt G. A. (1996) Functional recovery inparkinsonian monkeys treated with GDNF. Nature 380, 252—255.

Hou J-G. G., Lin L-F. H., and Mytilineou C. (1996) Glial cellline-derived neurotrophic factor exerts neurotrophic effects ondopaminergic neurons in vitro and promotes their survival andregrowth after damage by 1-methyl-4-phenylpyridinium. J.Neurochem. 66, 74—82.

Hyman C., Hofer M., Barde Y. A., Juhasz M., Yancopoulos G. D.,Squinto S. P., and Lindsay R. M. (1991) BDNF is a neuro-trophic factor for dopaminergic neurons of the substantia nigra.Nature 350, 230—232.

Janec E. and Burke R. E. (1993) Naturally occurring cell deathduring postnatal development of the substantia nigra of the rat.Mol. Cell. Neurosci. 4, 30—35.

Kalsbeek A., Voorn P., and Buijs R. M. (1992) Development ofdopamine-containing systems in the CNS, in Handbook ofChemical Neuroanatomy, Vol. 10: Ontogeny of Transmittersand Peptides in the CNS (Bjorklund A., Hokfelt T., and Toh-yama M., eds), pp. 63—112. Elsevier Science, Amsterdam.

Kelly W. J. and Burke R. E. (1996) Apoptotic neuron death in ratsubstantia nigra induced by striatal excitotoxic injury is devel-opmentally dependent. Neurosci. Lett. 220, 85—88.

Kessler J. A., Ludlam W. H., Freidin M. M., Hall D. H., MichaelsonM. D., Spray D. C., Dougherty M., and Batter D. K. (1993)Cytokine induced programmed death of cultured sympatheticneurons. Neuron 11, 1123—1132.

Knüsel B., Michel P. P., SchwaberJ. S., and Hefti F. (1990) Selec-tive and nonselective stimulation of central cholinergic and do-paminergic development in vitro by nerve growth factor, basicfibroblast growth factor, epidermal growth factor, insulin andthe insulin-like growth factors I and II. J. Neurosci. 10, 558—570.

Lin L-F. H., Doherty D. H., Lile J. D., Bektesh S., and Collins F.(1993) GDNF: a guaI cell line-derived neurotrophic factor formidbrain dopaminergic neurons. Science 260, 1130—1132.

Macaya A., Munell F., Gubits R. M., and Burke R. E. (1994)Apoptosis in substantia nigra following developmental striatalexcitotoxic injury. Proc. Natl. Acad. Sd. USA 91, 8117—8121.

Marri M. J., James C. J., Oo T. F., Kelly W. J., and Burke R. E.(1997)Early developmental destruction of terminals in the stri-atal target induces apoptosis in dopamine neurons of the sub-stantia nigra. J. Neurosci. 17, 2030—2039.

Mena M. A., Davila V., and Sulzer D. (1997) Neurotrophic effectsof L-DOPA in postnatal midbrain dopamine neuron/corticalastrocyte cocultures. J. Neurochem. 69, 1398—1408.

Moore M. W., Klein R. D., Farinas I., Sauer H., Armanini M., Phil-lips H., Reichardt L. F., Ryan A. M., Carver-Moore K., andRosenthal A. (1996) Renal and neuronal abnormalities in micelacking GDNF. Nature 382, 76—79.

Oo T. F. and Burke R. E. (1997) The time course of developmentalcell death in phenotypically defined dopaminergic neurons ofthe substantia nigra. Dey. Brain Res. 98, 191—196.

Pichel J. G., Shen L., Sheng H. Z., Granholm A-C., Drago J., Grin-berg A., LeeE. J., Huang S. P., Saarma M., HofferB. J., SariolaH., and Westphal H. (1996) Defects in enteric innervation andkidney development in mice lacking GDNF. Nature 382, 73—76.

Pothos E., Desmond M., and Sulzer D. (1996) L-3,4-Dihydroxyphe-nylalanine increases the quantal size of exocytic dopamine re-lease in vitro. J. Neurochem. 66, 629—636.

Pothos E. N., Davila V., and Suizer D. (1997) Glial-derived neuro-trophic factor increases quantal size in midbrain dopamine neu-rons. Soc. Neurosci. Abstr. 23, 1173.

Poulsen K. T., Armanini M. P., Klein R. D., Hynes M. A., PhillipsH. S., and Rosenthal A. (1994) TGF/32 and TGFß3 are potentsurvival factors for midbrain dopaminergic neurons. Neuron 13,1245—1252.

Przedborski S., Kihan U., Kostic V., Carlson E., Epstein C. J., andSuizer D. (1996) Increased superoxide dismutase activity im-proves survival of cultured postnatal midbrain neurons. J. Neu-rochem. 67, 1383—1392.

Rayport S. and Sulzer D. (1995) Visualization of antipsychotic bind-ing to living mesolimbic neurons reveals D2 receptor mediated,acidotropic and lipophilic components. J. Neurochem. 65, 691—703.

Rayport S., Sulzer D., Shi W. X., Sawasdikosol S., Monaco J., Bat-son D., and Rajendran G. (1992) Identified postnatal mesolim-bic dopamine neurons in culture morphology and electrophysi-ology. J. Neurosci. 12, 4264—4280.

Sanchez M. P., Silos-Santiago 1., Frisen J., He B., Lira S. A., andBarbacid M. (1996) Renal agenesis and the absence of entericneurons in mice lacking GDNF. Nature 382, 70—73.

Sauer H., Rosenblad C., and Bjorklund A. (1995) Glial cell line-derived neurotrophic factor but not transforming growth factor/13 prevents delayed degeneration of nigral dopaminergic neu-rons following striatal 6-hydroxydopamine lesion. Proc. NatI.Acad. Sd. USA 92, 8935—8939.

Schaar D. G., Sieber B. A., Dreyfus C. F., and Black I. B. (1993)Regional and cell specific expression of GDNF in rat brain.Exp. Neural. 124, 368—371.

Specht L. A., Pickel V. M., Joh T. H., and Reis D. J. (1981) Light-microscopic immunocytochemical localization of tyrosine hy-droxylase in prenatal brain. I. Early ontogeny. J. Camp. Neural.199, 233 —253.

Stromberg 1., Bjorklund L., Johansson M., Tomac A., Collins F.,Olson L., Hoffer B., and Humpel C. (1993) GuaI cell linederived neurotrophic factor is expressed in the developing butnot adult striatum and stimulates developing dopamine neuronsin vivo. Exp. Neurol. 124, 401—412.

Sulzer D., Maidment N. T., and Rayport S. (1993) Amphetamineand other weak bases act to promote reverse transport of dopa-mine in ventral midbrain neurons. J. Neurochem. 60, 527—535.

Suizer D., St. Remy C., and Rayport S. (1996) Reserpine inhibitsamphetamine action in ventral midbrain culture. Mol. Pharma-col. 49, 338—342.

Tepper J. M., Damlama M., and Trent F. (1994) Postnatal changesin the distribution and morphology of rat substantia nigra dopa-minergic neurons. Neuroscience 60, 469—477.

Tomac A., Lindqvist E., Lin L.-F. H., Ogren S. O., Young D., HofferB. J., and Olson L. (I995a) Protection and repair of the nigro-striatal dopaminergic system by GDNF in vivo. Nature 373,335 —339.

Tomac A., Widenfalk J., Lin L. H., Kohno T., Ebendal T., HofferB. J., and Olson L. (1995b) Retrograde axonal transport of glialcell line-derived neurotrophic factor in the adult nigrostriatalsystem suggests a trophic role in the adult. Proc. Natl. Acad.Sd. USA 92, 8274—8278.

Treanor J. J. S., Goodman L., deSauvage F., Stone D. M., PoulsenK. T., Beck C. D., Gray C., Armanini M. P., Pollock R. A.,Hefti F., Phillips H. S., Goddard A., Moore M. W., Buj-BelloA., Davies A. M., Asai N., Takahashi M., Vandlen R., Hender-son C. E., and Rosenthal A. (1996)Characterization of amulti-component receptor for GDNF. Nature 382, 80—83.

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