.Joiir,iaf ii/ /\Ieu,ochenijitrvLippincott—Raveii Puhlisher~. Philadelphia

199S International Society for Neurochemistry

Inhibition of Phosphatidylinositol 3-Kinase Activity BlocksCellular Differentiation Mediated by Glial Cell Line-Derived

Neurotrophic Factor in Dopaminergic Neurons

Kevin Pong, Ren Y. Xu, Will F. Baron, Jean-Claude Louis, and Klaus D. Beck

Department of Neuroscience, Amgen, Inc., Thousand Oaks, California, U.S.A

Abstract: Glial cell line-derived neurotrophic factor(GDNF) is a potent survival factor for midbrain dopamin-ergic neurons. To begin to understand the intracellularsignaling pathways used by GDNF, we investigated therole of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopa-minergic neurons. We found that treatment of dopaminer-gic neuron cultures with 10 ng/ml GDNF induced maximallevels of Ret phosphorylation and produced a profoundincrease in phosphatidylinositol 3-kinase activity, as mea-sured by western blot analysis and lipid kinase assays.Treatment with 1 pM 2-(4-morpholinyl)-8-phenylchro-mone (LY294002) or 100 nM wortmannin, two distinctand potent inhibitors of phosphatidylinositol 3-kinase ac-tivity, completely inhibited GDNF-induced phosphatidyl-inositol 3-kinase activation, but did not affect Ret phos-phorylation. Furthermore, we examined specific biologi-cal functions of dopaminergic neurons: dopamine uptakeactivity and morphological differentiation of tyrosine hy-droxylase-immunoreactive neurons. GDNF significantlyincreased dopamine uptake activity and promoted robustmorphological differentiation. Treatment with LY294002completely abolished the GDNF-induced increases of do-pamine uptake and morphological differentiation of tyro-sine hydroxylase-immunoreactive neurons. Our findingsshow that GDNF-induced differentiation of dopaminergicneurons requires phosphatidylinositol 3-kinase activa-tion. Key Words: Glial cell line-derived neurotrophic fac-tor—LY294002-—Neurotrophic factors—Phosphatidyl-inositol 3-kinase— Receptor tyrosine kinase—Wortman-nm.J. Neurochem. 71, 1912—1919 (1998).

potent trophic factor for motor neurons (Henderson etal., 1994; Oppenheim et al.. 1995; Yan et a!., 1995),as well as sensory and autonomic neurons of the PNS(Buj-Bello et a!., 1995; Trupp et al., 1995). Expressionof GDNF mRNA has been located in peripheral organsand tissues (Choi-Lundberg and Bohn, 1995; Nosratet al., 1996), suggesting a multifunctional and versatilerole for GDNF. Recently, the receptor for GDNF wasidentified as a multicomponent complex consisting ofa novel glycosylphosphatidylinositol-linked protein(Jing et a!., 1996; Treanor et al., 1996) and the func-tional tyrosine kinase receptor Ret (Durbec et al.,1996; Jing et al., 1996; Treanor et al., 1996; Trupp etal., 1996). As is the case with its ligand, Ret has beenfound outside the CNS and is required for kidney andgut morphogenesis (Schuchardt et a!., 1994).

Phosphatidylinositol 3-kinase (P1 3-K) is a hetero-dimer consisting of an 85-kDa regulatory subunit anda I l0-kDa catalytic subunit (Carpenter and Cantley,1990; Morgan et a!., 1990). The P1 3-K enzyme isbelieved to be involved in receptor tyrosine kinasesignal transduction pathways and intracellular traffick-ing. P1 3-K phosphorylates the D-3 position of phos-phatidylinositol, phosphatidylinositol 4-monophos-phate, and phosphatidyli nositol 4,5-bi sphosphate toproduce phosphatidylinositol 3-monophosphate, phos-phatidylinositol 3,4-bisphosphate, and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), respectively (Ka-peller and Cantley, 1994). It has been shown that thestimulation of various cell types by several differentgrowth factors activates P1 3-K (Carpenter and Cant-

Glial cell line-derived neurotrophic factor (GDNF)was isolated from the rat B49 glial cell line and initiallycharacterized as a potent neurotrophic factor specificfor midbrain dopaminergic (DAergic) neurons (Lin etat., 1993). Animal studies showed GDNF to protectDAergic neurons of the substantia nigra against axot-omy- (Beck et a!., 1995), 6-hydroxydopamine- (Hof-fer et al., 1994), and l-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine- (Gash et al., 1996) induced degenera-tion. Later studies have also shown GDNF to be a

Received April 21. 1998: revised manuscript received June 2,1998: accepted June 3, 1998.

Address correspondence and reprint requests to Dr. K. Pong atDepartment of Ncuroscience, Amgen, line., One Amgen CenterDrive, M/S 5-I-C, Thousand Oaks, CA 91320, U.S.A.

Abbreviations used: DA. dopamine; DAergic, dopaminergic: Dlv,days in vitro: GDNF, glial cell line-derived neurotrophic factor:LY294002, 2- (4-morpholinyl ) -8-phenylchrornone: PIP3. phosphati-dylinositol 3,4,5-trisphosphate; P1 3-K, phosphatidylinosilol 3-Id-nase; SDS-PAGE, sodium dodecyl sulfate—polyacrylamide gel dee-trophoresis; TH, tyrostne hydroxylase.

1912

P1 3-KINASE ACTIVITY IS REQUIRED FOR GDNF SIGNALING 1913

ley, 1996). Activation of P1 3-K has also been shownto be involved in mitogenic signaling, chemotaxis, andmetabolism (Cantley et al., 1991).

The exact intracellular signaling pathway inducedby GDNF is still largely unknown. However, recentstudies have attempted to elucidate the signaling cas-cades of GDNF. van Weering and Bos (1997) reportedthat GDNF stimulation of SK-N-MC cells transfectedwith a full-length Ret construct resulted in P1 3-K acti-vation and P1 3-K-dependent formation of large lamel-lipodia, which is required for neuritogenesis. Upontreatment with wortmannin, a potent inhibitor of P1 3-K (Arcaro and Wymann, 1993; Yano et al., 1993; UiCt al., 1995), Ret-induced lamellipodia formation wasinhibited, suggesting the requirement of P1 3-K activa-tion for cellular differentiation. More recently, Cree-don et al. (1997) demonstrated that neurturin andGDNF activate PT 3-K in rat superior cervical ganglionneurons.

In the present study, we explored the role of PT 3-K activation in GDNF-induced intracellular signaling,cellular differentiation, and activity in cultured primaryDAergic neurons. We show that GDNF stimulation ofDAergic neurons caused a robust increase in P1 3-Kactivity, as measured by western blot analysis and lipidkinase assays. Treatment with either wortmannin or2-(4-morpholinyl ) -8-phenylchromone (LY294002),another potent inhibitor of PT 3-K activity (Sanchez-Margalet et a!., 1994; Vlahos et al., 1994), completelyabolished GDNF-induced P1 3-K activation. This inhi-bition of PT 3-K activity correlated with a completereduction of dopamine (DA) uptake activity and mor-phological differentiation in tyrosine hydroxylase(TH) -immunoreactive neurons. Thus, intracellularsignaling and cellular differentiation mediated byGDNF occur via P1 3-K-dependent pathways in DAer-gic neurons.

MATERIALS AND METHODS

DAergic neuron culturesExperimental protocols involving laboratory animals were

approved by NIH and Amgen, Inc. (Thousand Oaks, CA,U.S.A.) guidelines. Primary DAergic neuron cultures wereprepared as described previously (Pong et a!., 1997). Inbrief, embryonic day 15 rat fetuses (Sprague—Dawley;Charles River Laboratories) were collected, their brainswereremoved, and a small piece of tissue comprising the ventralmesencephalic DAergic region was dissectedout in ice-coldDulbecco’s phosphate-buffered saline without Ca2~ andMg2~(GibcoBRL, Grand Island, NY, U.S.A.). Dissectedpieces of tissue were pooled together, transferred to an enzy-matic dissociation medium containing 20 lU/rn! papain inEarle’s balanced salt solution (Worthington BiochemicalCorp., Freehold, NJ, U.S.A.), and incubated for 60 mm at3.7°C After enzymatic dissociation, the papain solution wasaspirated and the tissue mechanically triturated with a fire-polished glass Pasteur pipette in complete medium [equalvolumes of minimum essential medium and F-12 nutrientmixture(GibcoBRL) supplemented with 0.1 mg/ml apotrans-ferrinand 25 pg/ml insulin] containing 2,000 IU/ml DNase

and 10 mg/mI ovomucoid protease inhibitor. For DA uptakeexperiments and TH immunocytochemistry, single-cell sus-pensions in complete medium supplemented with 12% horseserum were seeded on poly-L-ornithine- (0.1 mg/ml) andlaminin- (1 pg/mI; GibcoBRL) coated 96-well plates at adensity of 0.3 >< l0~cells/well. For Ret phosphorylation,p85 phosphorylation, and lipid kinase assays, single-cell sus-pensions were seeded on similarly coated 100-mm dishes ata density of 2.0 )< l0~cells/dish. Recombinant humanGDNF wasproduced by Amgen. LY294002 and wortmanninwere purchased from Sigma (St. Louis, MO, U.S.A.).

High-affinity DA uptake assayDA uptake experiments were performed on 6 day in vitro

(DIV) cultures using a modified method described by Pro-chiantz et al. (1981). In brief, cultures were washed withmodified Krebs—Ringer’s—phosphate buffer (120 mMNaCI, 1.3 mM Na

4EDTA, 5.6 mM glucose, 4.7 mM KC1,1.8 mM CaCl2, 1.2mM MgSO4, 32mM sodium phosphate,1 mM ascorbic acid, and 50 pM pargyline) and incubatedfor 60 mm at 37°Cwith 50 nM [

3H]DA (31 Ci/mmol; DuPont/NEN, Wilmington, DE, U.S.A.). The cultures werewashed twice with buffer and incubated for 2 h at roomtemperature with Optiphase Supermix scintillation cocktail(Wallac Scintillation Products, Gaithersburg, MD, U.S.A.),and radioactivity was measured.

Determination of P1 3-K activity by lipid kinaseassay and western blot analysis

P1 3-Kactivity was determined by using a modified proto-col described by Soltoff et a!. (1994). Cultures were grownin 100-mm dishes at adensity of 2.0 X 1 o~cells/dish. Forty-eight hours after plating, medium was exchanged and cul-tures were returned to incubators. Replacement of culturemedium with serum-free medium was performed on 3 DIVcultures. Cultures from 4 DIV were exposed to 1 pMLY294002, 100 nM wortmannin, or control medium for Ih and then treated with 10 ng/ml GDNF or control mediumfor 10 mm. Cultures were washed with ice-cold buffer 1137mM NaCI, 20 mM Tris, 1 mM MgC1

2, 1 mM CaCl~,0.2mM vanadate (pH 7.5)] and subsequently lysed with lysisbuffer (rinse buffer plus 10% glycerol, 1% Nonidet P-40,and I mMphenylmethylsulfonyl fluoride). The lysates weremixed and centrifuged. Supernatants were transferred to mi-crocentrifuge tubes and incubated with the anti-phosphotyro-sine monoclonal antibody, 4G10 (6.6 pg/mI; UBI, LakePlacid, NY, U.S.A.) for 2 h and protein A—Sepharose (4pg/mI; Pharmacia Biotechnology, Piscataway, NJ, U.S.A.)for I h. The immunoprecipitates were pelleted and washedthree times in phosphate-buffered saline plus 1% Nonidet P-40, two times in 0.1 M Tris (pH 7.5)10.5 M LiCI, and twotimes in TNE [10 mM Tris, 100 mM NaC!, 1 mM EDTA(pH 7.5)]. All solutions contain 0.2mM vanadate. To assayP1 3-Kactivity, exogenous phosphatidylinositol 4,5-bisphos-phate (final concentration of 0.2 mg/mI) in 10mM HEPES/I mMEGTA (pH 7.5) and 20 pCi [y-

32P]ATP was addedto the immunoprecipitates for 10 mm at room temperature.The reaction was stopped by the addition of 80 p,1 of I MHCI and 160 plot methanol/chloroform (1:1, vol/vol). Thelipid-containing organic phase was loaded on TLC plates(Silica gel 60, EM Science) precoated with 1% sodium oxa-late, and resolved in chloroformlmethanol/water/ammo-nium hydroxide (60:40:11 .3:2, by volume). Radiolabeledspots were quantified by using a Molecular Dynamics Phos-phorimager System (Image Quant, Version 3.3).

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1914 K. PONG ET AL.

Immunoprecipitates were run on sodium dodecy! sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) and trans-ferred to nitrocellulose membranes. Nitrocellulose mem-branes were blocked with 5% bovine serum albumin. Theamount of p85 immunoprecipitated by 4GlO was determinedby probing with an anti-p85 polyclonal antibody (UBI).An anti-rabbit secondary antibody (Boehringer Mannheim,Indianapolis, IN, U.S.A.) directly conjugated to horseradishperoxidase and ECL (Amersham, Arlington Heights, IL,U.S.A.) were used to visualize p85.

Ret phosphorylation and western blotRet autophosphorylation experiments were performed us-

ing a modified protocol described by Jing eta!. (1996). Inbrief, cultures were seeded on 100-mm dishes at a densityof 2.0 x I0~cells/dish. Forty-eight hours after plating, me-dium was exchanged and cultures were returned to incuba-tors. On DIV 3, medium was exchanged with serum-freemedium. Cultures from 4 DIV were pretreated with I pMLY294002, 100 nM wortmannin, or control medium for Ih and then exposed to 10 ng/ml GDNF or control mediumfor 10 mm. Cells were lysed with Triton X-l00 lysis bufferand immunoprecipitated with anti-Ret antibodiesand proteinA—Sepharose. Immunoprecipitates were run on SDS-PAGEand transferred to nitrocellulose membranes. Nitroce!!ulosemembranes were blocked with 5% bovine serum albumin,and tyrosine phosphorylation levels of Ret were determinedby probing with the anti-phosphotyrosine monoc!onal anti-body, 4G10 (UBI) at roomtemperature for 2 h. A rabbitanti-mouse secondary antibody (Boehringer Mannheim) directlyconjugated to horseradish peroxidase and ECL (Amersham)were used to visualize corresponding levels of phosphoryla-tion. The amount of Ret protein in each lane was then deter-mined by stripping the membrane and reprobing with ananti-Ret antibody (Amgen). A sheep anti-mouse secondaryantibody (Boehringer Mannheim) directly conjugated tohorseradish peroxidase and ECL (Amersham) were used tovisualize Ret.

TH immunocytochemistrySister cultures were fixed for 45 mm at 37°Cwith 4%

paraformaldehyde. Nonspecific binding was blocked by in-cubating with Superblock (Pierce, Rockford, IL, U.S.A.)containing 1% Nonidet P-40 for 90 mm at 37°C.Cultureswere then incubated overnight at 4°Cwith an anti-TH rabbitantibody (Eugene Tech, Ridgefleld Park, NJ, U.S.A.) diluted1:1,500 in 10 mg/ml bovine serum albumin in Tris-bufferedsaline. Following three washes, cultures were processed anddeveloped to visualize TH immunoreactivity with a Vecta-stain anti-rabbit Ig peroxidase ABC kit (Vector Laboratories,Burlingame, CA, U.S.A.) and diaminobenzidine substrate.Measurement of morphological parameters was done by us-ing MetaMorph (version 2.5; Dallas, TX, U.S.A.).

RESULTS

GDNF induction of Ret phosphorylation is notaffected by LY294002 or wortmannin

Embryonic mesencephalic DAergic neuron cultureswere prepared and maintained in 96-well tissue cultureplates as previously described (Pong et al., 1997). Adose—response analysis was initially done withLY294002 and wortmannin to determine relative lev-els of cytotoxicity in our cultures. LY294002 did not

FIG. 1. Dose—response analysis of LY294002 and wortmanninin DAergic neuron cultures. DAergic neuron cultures were pre-pared from embryonic day 15 rat embryos. A dose—responseanalysis was done with LY294002 and wortmannin ranging from10 pM to 1 mMon DIV 0. Cultures were processed for DA uptakeactivity or TH-positive immunoreactivity on DIV 6. A: DA uptakeactivity in cultures treated with LY294002 or wortmannin, or un-treated control cultures. B: Number of TH-positive immunoreac-tive neurons in treated or untreated cultures. Data are means±SEM of triplicate samples.

have any toxic or trophic effects at concentrationsranging from 10 pM to 1 ~M. Moderate toxicity wasapparent at concentrations above 5 ~M. At concentra-tions above 100 ,uM, LY294002 was highly toxic tocultures, as measured by DA uptake activity (Fig. 1A)and the number of TH-immunoreactive neurons (Fig.IB). Concentrations of wortmannin ranging from 10pM to 100 nM were not toxic or trophic when DAuptake activity was measured and TH-positive neuronswere counted; however, concentrations above 500 nMwere toxic in a dose—dependent manner (Fig. 1).

Cultures were maintained for 3 DIV in serum-con-

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P1 3-KINASE ACTIVITY IS REQUIRED FOR GDNF SIGNALING 1915

FIG. 2. GDNF induction of Ret phosphorylation is not affectedby LY294002 or wortmannin. DAergic neuron cultures weregrown and maintained for 2 DIV. Cultures were treated withLY294002, wortmannin, or control medium for 1 h and then with10 ng/ml GDNF or control medium for 10 mm. Cells were lysedand immunoprecipitated with anti-Ret antibodies, subjected toSDS-PAGE, transferred to nitrocellulose membranes, andprobed with an anti-phosphotyrosine antibody. Arrows indicatethe 1 70-kDa band that corresponds to Ret. Experiments wererepeated in triplicate, with the third experiment shown here. Lane1, 10 ng/ml GDNF; lane 2, control; lane 3, 10 ng/ml GDNF + 1pM LY294002; lane 4, 1 pM LY294002; laneS, 10 ng/ml GDNF+ 100 nM wortmannin; lane 6, 100 nM wortmannin.

taming medium. Medium was changed to the serum-free formulation on DIV 4. Cultures were pretreatedwith 1 p.M LY294002, 100 nM wortmannin, or controlmedium for I h followed by a 10-mm exposure to 10ng/ml GDNF or control medium. Cultures were thenprocessed and visualized for phosphotyrosine and Retprotein levels. Exposure to 10 ng/ml GDNF producedmaximal phosphorylation levels (Fig. 2A, lane 1)when compared with basal phosphorylation levels(Fig. 2A, lane 2). As expected, LY294002 and wort-mannin did not have any effects on GDNF-inducedRet phosphorylation (Fig. 2A, lanes 3 and 5) or Retprotein levels (Fig. 2B, lanes 3 and 5). Treatment witheither LY294002 or wortmannin alone had no effecton both Ret phosphorylation levels (Fig. 2A, lanes 4and 6) and protein levels (Fig. 2B, lanes 4 and 6).

GDNF induction of P1 3-K activity is inhibited byLY294002 and wortmannin

DAergic cultures were maintained for 3 DIV beforeswitching to a defined medium without serum supple-mentation. On DIV 4, cultures were treated with 10ng/ml GDNF for 10 mm and processed for western blotanalysis. Treatment with 10 ng/ml GDNF effectivelyproduced an increased amount of p85 immunoprecipi-tated by the anti-phosphotyrosine antibody 4GlO (Fig.3). Sister cultures were treated with 1 ,uM LY294002,100 nM wortmannin, or control medium for I h andthen exposed to 10 ng/ml GDNF for 10 mill. Lipidkinase assays were performed to measure the inductionof P1 3-K activity. Quantification of radiolabeled spotsrepresenting PIP3, the main product of the lipid kinaseassay, revealed that GDNF treatment increased PT 3-K activity fourfold above baseline levels (Fig. 4). Pre-

FIG. 3. Immunoprecipitation of p85, theregulatory subunit of P1 3-K. DAergic cul-tures were prepared and maintained for 3DIV. On DIV 3, medium was exchanged withfresh serum-free medium. On DIV 4, cul-tures were treated with 10 ng/mI GDNF orcontrol medium for 10 mm. Cells werelysed, and lysates were immunoprecipi-tated with an anti-phosphotyrosine anti-body, subjected to SDS-PAGE, transferred to nitrocellulosemembranes, and probed with an anti-p85 antibody. The arrowindicates the location of p85. These experiments were repeatedin triplicate with the third experiment shown here. Lane 1, 10ng/ml GDNF; lane 2, control. IP, immunoprecipitated; IB, immu-noblotted.

treatment with LY294002 or wortmannin completelyinhibited the GDNF induced production of PIP3 via P13-K activity (Fig. 4).

P1 3-K activity is required for GDNF-induced DAuptake activity

Cultures were treated on DIV 3 with various concen-trations of LY294002. Twenty-four hours later, GDNFat I ng/ml, a concentration known to promote maximalactivity in these cultures (Pong et al., 1997), was added.A second treatment of LY294002 was administered onDIV 5. High-affinity DA uptake activity was measuredon DIV 6. LY294002 at concentrations ranging from10 pM to 10 nM had no effect on DA uptake activity.However, DA uptake activity began to decrease at 50nM LY294002. Treatment with I p.M LY294002 com-pletely abolished GDNF-induced DA uptake activity

FIG. 4. GDNF induction of P1 3-K activity is inhibited byLY294002 and wortmannin. DAergic cultures were switched toserum-free medium on DIV 3. On DIV 4, cultures were treatedwith 1 pM LY294002, 100 nM wortmannin, or control mediumfor 1 h and then exposed to 10 ng/ml GDNF or control mediumfor 10 mm. Cells were lysed, and lysates were immunoprecipi-tated with an anti-phosphotyrosine antibody. P1 3-K activity wasmeasured using phosphatidylinositol 4,5-bisphosphate as thesubstrate. PIP3, the main product of the lipid kinase assay, wasidentified using TLC. Radiolabeled PIP3 dots were quantified byusing a Molecular Dynamics Phosphorlmager. Results areshown as fold increase above untreated control. Data are means±SEM oftriplicate samples. *p <0.0001, vs. control (two-tailedStudent’s t test).

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1916 K. PONG ET AL.

DA uptake assay. On DIV 6, cultures were fixed andprocessed for TH immunoreactivity (Fig. 6). Morpho-metric analysis of TH-immunopositive neuronsshowed that GDNF at 1 ng/ml increased cell bodysize and the number of primary neurites, i.e., neuritesprojecting directly from the cell body (Fig. 6B), witha mean neuron soma area of 331.0 ± 33 p.m2 and amean number of primary neurites per neuron of 3.7±0.8, when compared with untreated control cultures(Fig. 6A, Table 1). As described in our previous stud-ies (Pong et a!., 1997), GDNF-treated TH-positiveneurons exhibit shorter neurites than untreated neuronswhile promoting more primary neurites. Untreatedcontrol cultures, exhibiting slender cell bodies and longprimary neurites, yielded a mean soma area of 109.4±25 p.m2 and 1.7 ±0.5 primary neurites per neuron.Treatment of cultures with GDNF and 1 p.M LY294002inhibited morphological differentiation (Fig. 6C) anddecreased mean neuron soma size and number of pri-mary neurites to baseline levels (153.6 ±43 p.m2 and1.7 ±0.5, respectively). Addition of LY294002 alonehad no effects on morphological differentiation (Fig.6D, Table I).

DISCUSSION

FIG. 5. P1 3-K activity is required for GDNF-induced DA uptakeactivity. DAergic cultures were prepared and maintained for 3DIV. On DIV 3, cultures were treated with LY294002. On DIV4, cultures were treated with GDNF. LY294002 treatment wasrepeated on DIV 5. DA uptake activity was measured on DIV 6.A: Titration of LY294002 treated with 1 ng/mI GDNF. B: Titrationof GDNF treated with 1 pM LY294002. Control cultures wereuntreated sister cultures. Data are means ±SEM of triplicatesamples.

(Fig. 5A). A further decrease in DA uptake at concen-trations above 1 p.M was due to dose-dependent toxicityas indicated (Fig. I). A similar treatment paradigm wasused to observe the inhibitory effects of 1 p.MLY294002 on a dose—response treatment of GDNF.LY294002 at! p.M effectively inhibited GDNF-inducedactivity at all concentrations. Resulting activity was sim-ilar to that of basal levels (Fig. SB).

GDNF induction of morphological differentiationof TH-immunoreactive neurons requiresP1 3-K activity

Cultures used for morphological analysis weregrown and treated identically to those used for the

Our study provides evidence of a central role of P13-K in the signal transduction pathway used by GDNFin cultured DAergic neurons. We demonstrated thattreatment with LY294002 and wortmannin did not af-fect GDNF-induced phosphorylation of Ret. These re-sults were in line with the fact that P1 3-K is down-stream of Ret, but ensured us that LY294002 and wort-mannin did not have any nonspecific activities thatwould interfere with GDNF-induced Ret signaling. Inwestern blots, we showed that treatment with GDNFstimulated an increase in the phosphorylation of p85.The increase in p85 phosphorylation correlated wellwith data from the lipid kinase assays. Furthermore,LY294002 inhibited the GDNF-induced increase ofDA uptake activity and abolished the promotion ofmorphological differentiation of DAergic neurons.These findings suggest a critical role for PT 3-K activityin GDNF-induced promotion of cellular differentiationin DAergic neurons.

The intracellular cascade of signal transductionmechanisms involved in the mediation of the biologicalactivities of neurotrophic factors is still largely un-known. However, there is some evidence suggestingthat PT 3-K is involved in the cellular trafficking andin the downstream signal transduction pathways in-duced by neurotrophic factor activation of tyrosine ki-nase receptors. It has been demonstrated that the neuro-trophic activities of nerve growth factor (Yao and Coo-per, 1995; Spear et al., 1997), insulin-like growthfactor-I (D’Mello et al., 1997; Kulik et al., 1997; Parri-zas et al., 1997), platelet-derived growth factor (Ran-kin et al., 1996; Thomas et al., 1997), the trk B ligandsbrain-derived neurotrophic factor and neurotrophin-4!

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P1 3-KINASE ACTiVITY 15 REQUIRED FOR GDNF SIGNALING 1917

FIG. 6. Morphological differentiation induced by GDNF requires P1 3-K activity. DAergic cultures were prepared and maintained for 3DIV. On DIV 3, cultures were treated with LY294002. On DIV 4, cultures were treated with GDNF. LY294002 treatment was repeatedon DIV 5. Cultures were fixed and prepared for immunohistochemistry. DAergic neurons were identified with an anti-TH antibody. A:Untreated control cultures. B: GDNF (1 ng/mI). C: GDNF (1 ng/ml) + LY294002 (1 pM). D: LY294002 (1 pM). Scale bar = 50 pm.

5 (Skaper et al., 1998), and epiderma! growth factor(Soltoff et al., 1994) are mediated through P1 3-Kactivation. The activation of PT 3-K results in the con-

TABLE 1. LY294002 inhibits GDNF-inducedmorphological differentiation of DAergic neuron.s

through the blockade of P1 3-K

Treatment

Neuronsoma area

(pm2)

Primaryneurites/neuron

I ng/ml GDNF 331.0 ±33° 3.7 ± 0.8°Untreated control 109.4 ± 25 1.7 ±0.5I ng/ml GDNF + 1 pM LY294002 153.6 ±43 1.7 ±0.51 pM LY294002 122.8 ±20 1.8 ±0.4

P1 3-K activity is required for GDNF-induced morphological pa-rameters. Sister cultures were grown under the indicated conditions.Cultures were prepared immunohistochemically, and TH-positiveneurons were identified. Image analysis was performed by Meta-Morph to measure the neuron soma area (pm2) and number of pri-mary neurites per neuron. Data are means ±SEM of 30 neuronsper independent experiment, repeated in triplicate.

“p < 0.001, vs. control (Student’s t test).

version of phosphatidylinositol 4,5-bisphosphate toPIP

3, which has been demonstrated to be a key secondmessenger generated by receptor tyrosine kinases(Carpenter and Cantley, 1990; Parker and Waterfield,1992; Hawkins et a!., 1992). It has been further dem-onstrated that PIP3 is required for cellular maintenance,differentiation, proliferation, and prevention of apo-ptotic activity in various cell types (Cantley et al.,1991; Kapeller and Cantley, 1994). The current beliefis that PIP3 initiates its activity through the protoonco-gene serine/threonine protein kinase B, also called Akt(for review, see Hemmings, 1997).

The intracellular signaling cascade induced byGDNF is largely unknown. It was only recently thatthe multicomponent receptor for GDNF was identified.This receptor is composed of a glycosy!phosphatidyl-inositol-linked accessory component and the protoon-cogene tyrosine kinase receptor Ret (Durbec et al.,1996; Jing et a!., 1996; Treanor et al., 1996; Trupp etal., 1996). Recently, van Weering and Bos (1997)demonstrated that GDNF stimulation of SKF5 andSKP2 cell lines, transfected with the GDNF putativereceptor Ret, induced activation of PT 3-K. Pretreat-

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1918 K PONG ET AL.

ment with wortmannin inhibited GDNF-induced la-mellipodia formation, a crucial event in neurite out-growth. In another study, Creedon et al. (1997) dem-onstrated the activation of PT 3-K upon treatment witheither neurturin or GDNF in superior cervical ganglioncultures. Our study expands these initial findings intransfected cell lines and PNS neurons into the CNSby focusing on mesencephalic DAergic neurons. Inparticular, we demonstrate that P1 3-K plays a centralrole in the GDNF-induced biochemical and morpho-logical differentiation of these neurons.

We conclude that GDNF, a potent neurotrophic fac-tor for DAergic neurons of the substantia nigra, exertsits trophic effects through the activation of PT 3-Kand that these effects can be inhibited entirely by theaddition of LY294002 or wortmannin. Our results fur-ther elucidate the signaling cascade and mechanismsmediating the trophic actions of GDNF on CNS neu-rons and demonstrate the central role of P1 3-K in thiscascade. In addition, these findings strongly suggestthe conservation of signaling components, in this casePT 3-K, among various neurotrophic agents and its cen-tral role in mechanisms promoting maintenance of cellviability and function. These findings further suggestthat the activation of tyrosine kinase receptors initiatesintracellular signaling cascades that include PT 3-K ac-tivity (Otsu et al., 1991; Hiles et al., 1992).

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