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Pitx3-Transfected Astrocytes Secrete Brain-Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor and Protect Dopamine Neurons in Mesencephalon Cultures Dehua Yang, 1 Changgeng Peng, 1 Xuping Li, 1 Xiaolan Fan, 1 Liang Li, 1 Ming Ming, 1 Sheng Chen, 2 and Weidong Le 1,2,3 * 1 Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China 2 Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China 3 Department of Neurology, Baylor College of Medicine, Houston, Texas The transcription factor Pitx3 is crucial for the develop- ment and differentiation of dopamine (DA) neurons. Our previous work has shown the Pitx3 can up-regulate the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in neuroblastoma cell line SH-SY5Y. Primary astrocytes are the major nonneuronal cells and can be easily modified genetically to deliver therapeutic molecules into the brain, so we investigated whether Pitx3 can increase the expres- sion and secretion of BDNF and GDNF in primary astro- cytes. We first transfected Pitx3 plasmid in purified rat astrocytes and collected the conditioned medium (CM) from the Pitx3-transfected cultures, and then we meas- ured the BDNF and GDNF levels from the CM and tested the protective effect of the CM against rotenone-induced DA neuron injury in ventral mesencephalon (VM) cultures. We found that the BDNF and GDNF levels were 1.4-fold and 1.5-fold higher in the CM from Pitx3-transfected astrocytes than empty vectors-transfected controls. Incu- bation with the CM from Pitx3-transfected astrocytes sig- nificantly attenuated the rotenone-induced DA neuron injury, and such protection can be significantly blocked by preincubation with antibodies against either BDNF or GDNF, whereas preincubation with purified BDNF or GDNF replicated the neuroprotection against rotenone- induced injury in VM cultures. These results demonstrate that Pitx3-transfection in astrocytes can up-regulate BDNF and GDNF expression and produce protective benefit to DA neurons, which might be a potential therapeutic alter- native for Parkinson’s disease. V V C 2008 Wiley-Liss, Inc. Key words: Pitx3; BDNF; GDNF; primary astrocytes; DA; neurodegeneration Pitx3, a homeodomain-containing transcription factor, is expressed from day E11 in mouse, which is highly restricted in mesencephalic dopamine (DA) neu- ron precursors of the substantia nigra (SNc) and ventral tegmental area (VTA; Smidt et al., 1997, 2004a). The close association of Pitx3 expression with an intact mes- encephalic DAergic system indicates that Pitx3 might be involved in the development and maintenance of mesen- cephalic DA neurons. Studies of Pitx3-deficient aphakia mice reveal that Pitx3 is required for the development of DA neurons in SNc (Hwang et al., 2003; Nunes et al., 2003; Smidt et al., 2004b) and for the postnatal survival of a subset of DA neurons in the VTA (van den Munckhof et al., 2003). Overexpression of Pitx3 in stem cells has been used to enhance DA neuron differentia- tion aiming at providing cell replacement therapy for Parkinson’s disease (Chung et al., 2005; Martinat et al., 2006). Our previous works demonstrated that overex- pression of Pitx3 up-regulated the expression of brain- derived neurotrophic factor (BDNF) and glial cell line- derived neurotrophic factor (GDNF) in SH-SY5Y cells and primary ventral mesencephalon (VM) cultures (Peng et al., 2007). Astrocytes are the major nonneuronal cells in the brain, and they have been implicated in the segregation, Contract grant sponsor: National Natural Science Foundation; Contract grant number: 30570560; Contract grant number: 30730096; Contract grant sponsor: Research Funds from Chinese Science and Technology Commission; Contract grant number: 863 project 2007AA02Z460; Con- tract grant number: National Basic Research Program 2007 CB947904. *Correspondence to: Dr. Weidong Le, Department of Neurology, Baylor College of Medicine, Houston, Texas 77030. E-mail: [email protected] Received 12 December 2007; Revised 8 March 2008; Accepted 24 March 2008 Published online 21 July 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21774 Journal of Neuroscience Research 86:3393–3400 (2008) ' 2008 Wiley-Liss, Inc.

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Page 1: Pitx3-transfected astrocytes secrete brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor and protect dopamine neurons in mesencephalon cultures

Pitx3-Transfected Astrocytes SecreteBrain-Derived Neurotrophic Factor andGlial Cell Line-Derived NeurotrophicFactor and Protect Dopamine Neurons inMesencephalon Cultures

Dehua Yang,1 Changgeng Peng,1 Xuping Li,1 Xiaolan Fan,1 Liang Li,1

Ming Ming,1 Sheng Chen,2 and Weidong Le1,2,3*1Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,and Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China2Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai,People’s Republic of China3Department of Neurology, Baylor College of Medicine, Houston, Texas

The transcription factor Pitx3 is crucial for the develop-ment and differentiation of dopamine (DA) neurons. Ourprevious work has shown the Pitx3 can up-regulate theexpression of brain-derived neurotrophic factor (BDNF)and glial cell line-derived neurotrophic factor (GDNF) inneuroblastoma cell line SH-SY5Y. Primary astrocytes arethe major nonneuronal cells and can be easily modifiedgenetically to deliver therapeutic molecules into the brain,so we investigated whether Pitx3 can increase the expres-sion and secretion of BDNF and GDNF in primary astro-cytes. We first transfected Pitx3 plasmid in purified ratastrocytes and collected the conditioned medium (CM)from the Pitx3-transfected cultures, and then we meas-ured the BDNF and GDNF levels from the CM and testedthe protective effect of the CM against rotenone-inducedDA neuron injury in ventral mesencephalon (VM) cultures.We found that the BDNF and GDNF levels were 1.4-foldand 1.5-fold higher in the CM from Pitx3-transfectedastrocytes than empty vectors-transfected controls. Incu-bation with the CM from Pitx3-transfected astrocytes sig-nificantly attenuated the rotenone-induced DA neuroninjury, and such protection can be significantly blocked bypreincubation with antibodies against either BDNF orGDNF, whereas preincubation with purified BDNF orGDNF replicated the neuroprotection against rotenone-induced injury in VM cultures. These results demonstratethat Pitx3-transfection in astrocytes can up-regulate BDNFand GDNF expression and produce protective benefit toDA neurons, which might be a potential therapeutic alter-native for Parkinson’s disease. VVC 2008 Wiley-Liss, Inc.

Key words: Pitx3; BDNF; GDNF; primary astrocytes;DA; neurodegeneration

Pitx3, a homeodomain-containing transcriptionfactor, is expressed from day E11 in mouse, which is

highly restricted in mesencephalic dopamine (DA) neu-ron precursors of the substantia nigra (SNc) and ventraltegmental area (VTA; Smidt et al., 1997, 2004a). Theclose association of Pitx3 expression with an intact mes-encephalic DAergic system indicates that Pitx3 might beinvolved in the development and maintenance of mesen-cephalic DA neurons. Studies of Pitx3-deficient aphakiamice reveal that Pitx3 is required for the developmentof DA neurons in SNc (Hwang et al., 2003; Nuneset al., 2003; Smidt et al., 2004b) and for the postnatalsurvival of a subset of DA neurons in the VTA (van denMunckhof et al., 2003). Overexpression of Pitx3 in stemcells has been used to enhance DA neuron differentia-tion aiming at providing cell replacement therapy forParkinson’s disease (Chung et al., 2005; Martinat et al.,2006). Our previous works demonstrated that overex-pression of Pitx3 up-regulated the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cellsand primary ventral mesencephalon (VM) cultures (Penget al., 2007).

Astrocytes are the major nonneuronal cells in thebrain, and they have been implicated in the segregation,

Contract grant sponsor: National Natural Science Foundation; Contract

grant number: 30570560; Contract grant number: 30730096; Contract

grant sponsor: Research Funds from Chinese Science and Technology

Commission; Contract grant number: 863 project 2007AA02Z460; Con-

tract grant number: National Basic Research Program 2007 CB947904.

*Correspondence to: Dr. Weidong Le, Department of Neurology, Baylor

College of Medicine, Houston, Texas 77030.

E-mail: [email protected]

Received 12 December 2007; Revised 8 March 2008; Accepted 24

March 2008

Published online 21 July 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21774

Journal of Neuroscience Research 86:3393–3400 (2008)

' 2008 Wiley-Liss, Inc.

Page 2: Pitx3-transfected astrocytes secrete brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor and protect dopamine neurons in mesencephalon cultures

maintenance, and support of neurons. There is emergingevidence that astrocytes are an important source of neu-roactive substances such as growth factors, eicosanoids,and neurosteroids, which may subsequently influenceneuronal development, survival, and neurosecretion(Kabbadj et al., 1993; Ojeda et al., 2000; Araque et al.,2001; Azcoitia et al., 2001). Many studies have demon-strated the therapeutic potential of astrocyte transplanta-tion in neurodegenerative disease (Smith et al., 1987;Lundberg et al., 1996; Ridet et al., 2003; Ericson et al.,2005). The aim of this study is to evaluate further therole of Pitx3 in regulating BDNF and GDNF expressionand the therapeutic potential of genetically engineeredastrocytes for the treatment of Parkinson’s disease. In thepresent study, we investigate whether overexpression ofPitx3 in primary astrocytes can regulate BDNF andGDNF levels and protect rotenone-induced injury ofDA neurons in primary VM cultures. We demonstratethat Pitx3 transfection in astrocytes can up-regulateBDNF and GDNF expression and produce protectivebenefit to DA neurons, which might be a potential ther-apeutic alternative for Parkinson’s disease.

MATERIALS AND METHODS

Plasmid Construction

Total RNA of adult mouse midbrain tissue was isolatedusing Trizol Reagent (Invitrogen, Carlsbad, CA). Pitx3cDNA was synthesized from the total RNA using a RT-PCRsystem (Promaga, Madison, WI) according to the protocolprovided by the manufacturer. The primers for Pitx3 cDNAamplification were: forward 50ACAGCCACCACCCGGAGT30, reverse 50GCACCCCTTTCAGACCCT30. ThePCR product was cloned into pUCm T-vector (Sangon,China). The expressed Pitx3 gene was then excised frompUCm T-vector by EcoRI/BamHI and cloned into the cor-responding sites of pEGFP-C2. The identity of the cDNAwas confirmed by the sequencing analysis according to thePitx3 mRNA sequence published in the NCBI database(NM_008852).

Astrocytes Cultures, Transfection, and Preparationof CM

The rat primary astrocytes were prepared according topreviously described procedures (McCarthy and de Vellis,1980; Du et al., 2005; Liu et al., 2005). Briefly, primary astro-cyte cultures were grown from dissociated cells derived fromthe cerebral cortex of 24-hr-old Sprague-Dawley (SD) rats(Experimental Animal Center of Shanghai). After the cerebralcortex was removed and meninges and microvessels werecleaned, the cortex tissues were cut into 1–2-mm3 pieces andthen digested with 0.025% trypsin-EDTA (Sigma, St. Louis,MO) and 0.01% DNase I (Sigma) at 378C for 15 min, thenterminated with culture media (DMEM; Gibco BRL, GrandIsland, NY) containing 10% fetal bovine serum (PAA, Vienna,Austria). After mechanical dissociation, centrifugation, andresuspension, the cells were seeded in 75-cm2 tissue cultureflasks (Coning) at a density of 2 3 107 cells per flask. Afterculture for 2 weeks, the astrocytes in flasks were shaken at

190 rpm for 18 hr at 378C to remove microglia and oligoden-drocytes, and the adherent cells were allowed to grow foranother 3 days. Then, flasks were shaken again under thesame conditions to remove the remaining microglia and oligo-dendrocytes. Next, the attached cells were digested with0.25% trypsin-EDTA and transferred to six-well plates at adensity of 1.5 3 105/cm2. Animal care and procedures wereperformed in accordance with the Laboratory Animal CareGuidelines approved by Shanghai Institutes for Biological Sci-ences of Chinese Academy of Sciences.

For transient transfection, Pitx3-pEGFP-C2 (CP) orcontrol plasmid pEGFP-C2 (C2) at two doses (150 ng or300 ng) was transfected into astrocytes in six-well dishes usinglipofectamine 2000 (Invitrogen), with the guidance of themanufacturers’ instruction. To prepare CM, the mediumderived from astrocytes that had been cultured for 5 days aftertransfection was collected and centrifuged at 1,000g for 10min at 48C to remove cells and debris. The supernatant wasconcentrated fivefold by centrifuging at 4,500g for 15 min at48C in an Amicon Ultra tube (10 kDa; Millipore, Beedford,MA). Concentrated medium was diluted to the desired con-centration using serum-free medium. The final reconstitutedsupernatant, namely, CM, was filtered through Millex-GVFilter Units (0.22-lm pore size, Millipore) and stored at –808C until use.

Real-Time RT-PCR Analysis

Total RNA from cultures (2 days after transfection) wasprepared using Trizol reagent (Invitrogen) and digested withRNase-free DNase for 30 min to exclude genomic DNAcontamination. For RT-PCR analysis, 2 lg of RNA wastranscribed into cDNA with the Reverse Transcription System(Promega) and oligo(dT) primers in a 20-ll volume. Theexpression levels of Pitx3, BDNF, and GDNF against internalcontrol GAPDH were quantitatively measured by real-timePCR assay using the primers: Pitx3 (forward: 50TGAGGATGGCTCCCTGAAGA30, reverse: 50AAGGCGAACGGGAAGGTC30), BDNF (forward: 50GCCTCCTCTGCTCTTTCTGC30, reverse: 50ATGGGATTACACTTGGTCTCGTAG 30), GDNF (forward: 50ATGAAGTTATGGGATGTCGTGG30, reverse: 50GCCGCTTGTTTATCTGGTGA30), and GAPDH (forward: 50CCATGTTCGTCATGGGTGTGAACCA30, reverse: 50GCCAGTAGAGGCAGGGATGATGTTC30). PCR amplification was carried out ina mixture of 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 3 mMMgCl2, 0.2 mM deoxynucleotides, 0.8 lM of each primers,13 Sybr green (Molecular Probes, Eugene, OR), and 1 UTaq DNA Polymerase (Fermentas, Lithuania) in a final vol-ume of 50 ll. Thermocycling was conducted using an Opti-con DNA Engine (MJ Research Inc., Toronto, Ontario, Can-ada). Ct values were determined by the Opticon Monitor 2software using fluorescence manual threshold for all runs andexported into an Excel workbook for analysis.

Western Blot Analysis

Astrocytes (3 days after transfection) were lysed in ice-cold 13 sample buffer. Lysates were then centrifuged for10 min at 13,000 rpm at 48C. After the measurement of pro-

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tein concentration by the Bradford method, 40 lg of proteinfrom each sample was loaded onto 12% SDS-PAGE and trans-ferred to polyvinylidene difluoride, blocked for 1 hr in 5%nonfat milk, and incubated with primary antibodies [anti-Pitx3 (Zymed, South San Francisco, CA), anti-b-actin(Sigma), anti-BDNF (Promega), and anti-GDNF (Promega)]overnight at 48C. After washing with TBST, samples wereincubated with peroxidase-conjugated secondary antibody.The immunoreaction was developed using Super Signal WestDura Extended Duration Substrate (Pierce Biotechnology,Rockford, IL), and the signal was quantified by measuring op-tical density of the bands.

Measurement of BDNF and GDNF by ELISA

The medium from astrocytes that were cultured 5 daysafter the transfection was concentrated up to fivefold in anAmicon Ultra tube (10 kDa; Millipore). The protein concen-tration of the test samples was adjusted to equality, and 100 llof each sample was applied into each well (Maxisorpa 96-wellplate; Nunc, Roskilde, Denmark) for the immunoassay. Thelevels of BDNF and GDNF were measured by using theBDNF or GDNF Emax ImmunoAssay System (Promega), andthe procedures were performed according to the manufac-turers’ instruction.

Preparation of Primary VM Cultures and ExperimentalTreatments

Primary embryonic rat VM cells were isolated and cul-tured according to a method described previously (Li et al.,2004), with minor modifications. Briefly, pregnant SD rats(embryonic day 14, E14; Experimental Animal Center ofShanghai) were anesthetized with chloral hydrate (400 mg/kg). VM tissues were dissected and incubated in Ca21- andMg21-free Hank’s balanced salt solution (HBSS; Gibco) con-taining 0.025% trypsin-EDTA for 8 min at room temperature.After washing in DMEM containing 10% fetal bovine serum,the tissues were mechanically dissociated into a single-cell sus-pension using sterilized micropipette tips. The cell suspensionwas resuspended in serum-containing medium (DMEM andHam’s F12 at 1:1, supplemented with 10% fetal bovine serum)and plated at a final density of 5 3 105 viable cells/cm2 in96-well plates (Nunc) precoated with poly-l-lysine (Sigma).The cells were incubated at 378C in a 5% CO2-humidifiedatmosphere for 12 hr and then switched to the serum-freemedium, consisting of DMEM/Ham’s F12 (1:1) with theaddition of 2% B27 supplement (Gibco), and incubated for 6days with medium change every 3 days. Animal care and pro-cedures were performed in accordance with the LaboratoryAnimal Care Guidelines approved by Shanghai Institutes forBiological Sciences of Chinese Academy of Sciences.

To injure the DA neurons, 500 lM rotenone dissolvedin DMSO and diluted with culture medium was added in theVM cultures (final concentration 25 nM) for 8 hr. Some ofthe VM cultures were preincubated with CM or with purifiedBDNF (PeproTech) and GDNF (R&D Systems, Minneapolis,MN) with a final concentration of 10 ng/ml for 4 hr beforerotenone treatment to prevent the injury. To block the neu-

rotrophic effects of CM, 1 lg/ml antibodies against BDNF(Promega) or GDNF (Promega) was added in CM of Pitx3-transfected astrocytes for 2 hr at room temperature before CMpreincubation.

Immunocytochemistry and Cell Counts

Cells were fixed in 4% formaldehyde for 30 min, rinsedwith PBS, and then incubated with blocking buffer (PBS; 5%normal horse serum; Vector, Burlingame, CA) for 30 min.Astrocytes for immunofluorescent staining were then incu-bated overnight at 48C with rabbit anti-Pitx3 antibody (1:200;Zymed). After additional rinsing in PBS, cells were incubatedwith Cy3-conjugated anti-rabbit antibody for 2 hr at roomtemperature. After rinsing for 3 3 10 min in PBS, cells wereincubated with Hoechst for 20 min and examined underthe fluorescent microscope; VM cultures for diaminobenzidine(DAB) staining were incubated overnight at 48C with mouseantityrosine hydroxylase (TH) antibody (1:3,000; Sigma). Thesecond antibody was biotinylated anti-mouse IgG (Elite ABCkit; Vector). After rinsing for 3 3 10 min in PBS, the cellswere incubated with avidin-biotinylated-peroxidase complexfor 1 hr (Elite ABC kit; Vector). Immunoreactivity wasvisualized by DAB substrate (Vector).

TH-positive cells in each well were counted in a blindway by an unrelated investigator to count 10 random opticalfields per well (32 mm2 surface area). The size of each exami-nation field was 0.4 mm2, and 10 fields that consisted of about12% of the whole surface of the cultured well were examined.Experiments were performed on three or four independentcultures.

Statistical Analysis

All values were shown as mean 6 SEM. Data analysiswas performed by using one-way ANOVA, followed by post-hoc LSD multiple comparisons with the SPSS 13.0 program(SPSS). P < 0.05 was considered significant.

RESULTS

Transfected-Pitx3 Is Expressed in the Nucleus ofPrimary Astrocytes

By using the astrocyte purification methoddescribed above, we found that the purity of astrocyteswas at least 95% based on immunocytochemical stainingof a specific astrocyte marker, GFAP (Liu et al., 2005).Pitx3-expressing vector was transfected into primaryastrocytes. Immunofluorescent staining with anti-Pitx3antibody was performed 24 hr after transient transfectionusing lipofectamine 2000, which showed that about 10–15% astrocytes were positively transfected (Fig. 1C).Double staining with anti-Pitx3 antibody and Hoechstillustrated that Pitx3 was located only in the nuclei ofastrocytes (Fig. 1A,B).

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Overexpression of Pitx3 Increases the mRNAExpression of Both BDNF and GDNF in PrimaryAstrocytes

With real-time RT-PCR assays, we analyzed theeffects of Pitx3 transfection on the gene expression ofBDNF and GDNF in primary astrocytes. It was clearlyshown that transfection with Pitx3 gene at 150 ng or300 ng plasmid remarkably up-regulated the mRNAexpression of Pitx3, whereas transfection with emptyvector had no such effect (Fig. 2A). It was found thatmRNA levels of BDNF and GDNF were elevated by4.2-fold and 3.7-fold in 150 ng plasmid of Pitx3 gene-transfected astrocytes compared with empty vector-trans-fected controls (Fig. 2A,B), and such elevation of BDNFand GDNF was further increased to 7.7-fold and 6.7-

fold by a higher dose (300 ng plasmid) of Pitx3 genetransfection (Fig. 2A,B).

Up-Regulation of BDNF and GDNF Proteins byOverexpression of Pitx3

Consistent with the results of mRNA measure-ment, Western blot revealed that the protein levels ofBDNF and GDNF were increased by 2.8-fold and 2.7-fold, respectively, in the Pitx3-transfected (300 ng plas-mid) astrocytes compared with empty vector-transfected

Fig. 2. Effects of Pitx3 transfection on mRNA levels of BDNF andGDNF. Primary astrocytes were transiently transfected with an emptyvector (astro-C2) or Pitx3 gene (astro-CP, 150 ng or 300 ng plasmid)for 48 hr. Total RNA was extracted with Trizol reagent, and cDNAwas synthesized by reverse transcription. mRNA levels of Pitx3, BDNF,and GDNF in each group were measured by real-time PCR. Overex-pression of Pitx3 in astrocytes up-regulated gene expression of BDNFand GDNF in a dose-dependent manner (A,B). Data shown aremean 6 SEM values from three independent experiments (*P < 0.01,**P < 0.001, compared to Astro-C2).

Fig. 1. Immunofluorecent staining with anti-Pitx3 antibody and Hochest to detect the Pitx3 expressionand transfection efficiency. Pitx3-pEGFP-C2 was transiently transfected into primary astrocytes withlipofectamine 2000. A: Pitx3-expressing astrocytes. B: Hoechst staining of nucleus. C: Overlay. Scalebar 5 20 lm.

Fig. 3. Up-regulation of protein levels of BDNF and GDNF inPitx3-transfected (300 ng plasmid) astrocytes. Upper panel: Westernblot analysis showed that protein levels of BDNF and GDNF wereincreased 2.8-fold and 2.7-fold in Pitx3-transfected astrocytes (astro-CP; A,B), respectively, compared with empty vector-transfectedcontrols. b-Actin served as an internal control. Lower panel: Increaseof BDNF and GDNF proteins secreted by Pitx3-transfected (300 ngplasmid) astrocytes. The content of BDNF (C: astro-CP, 26.1 6 1.2pg/ml; astro-C2, 18.4 6 0.59 pg/ml) and GDNF (D: astro-CP,186.3 6 5.0 pg/ml; astro-C2, 128.0 6 3.3 pg/ml) released into theculture medium (pg/ml) was measured by ELISA. Data shown aremean 6 SEM values from three independent experiments performedin duplicate (*P < 0.05, **P < 0.01, compared to Astro-C2).

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controls (Fig. 3A,B). In addition, ELISA showed thatthe protein levels of BDNF and GDNF in the culturemedium secreted by Pitx3-transfected (300 ng plasmid)astrocytes were increased 1.4-fold and 1.5-fold overtransfection with empty vector (Fig. 3C,D), whereas thetotal cell number did not show any significant differencebetween Pitx3-transfected astrocytes and control cells(data not shown).

CM From the Pitx3-Transfected AstrocyteCultures Protects Against the Rotenone-InducedDA Neuron Loss in VM Cultures

After being exposed to 25 nM rotenone for 8 hr,the VM cultures were fixed and immunostainied for DAneurons using TH antibody. In contrast with vehicletreatment (Con, Fig. 4A,E), the number of TH-positivecells in the rotenone-treated VM cultures was decreasedby 67.4% (Fig. 4B,E). Pretreatment with CM from thePitx3-transfected astrocytes for 4 hr significantly attenu-ated the rotenone-induced cell loss by 48.6% comparedwith the rotenone-treated VM cultures (Fig. 4D,E),whereas the CM from the cultures of empty vector-transfected astrocytes (astro-C2) showed only a moderateprotection against rotenone-induced TH-positive cellloss by 18.7% (Fig. 4C,E). In addition, these protectiveeffects of CM against rotenone-induced cytotoxicitywere concentration dependent, showing a greater pro-

tection when a higher concentration of CM was incu-bated in the VM cultures (0.53 astro-CP, 13 astro-CP,23 astro-CP, Fig. 4F–J).

Blockade of BDNF and GDNF Inhibits theProtective Effects of CM From Pitx3-TransfectedAstrocytes

To determine whether the neurotrophic effects ofCM were due to the BDNF and GDNF secreted fromthe astrocytes, we incubated the CM from Pitx3-trans-fected astrocytes with specific antibodies against BDNFand GDNF for 2 hr at room temperature. Primary VMcultures were incubated in the medium for 8 hr in thepresence or absence of rotenone. Blockade of eitherBDNF or GDNF with specific antibodies (1 lg/ml) dra-matically abolished the CM-induced neuroprotectioneffects (Fig. 5A–E), whereas antibodies themselves at thisconcentration did not affect the rotenone-induced cellloss (data not shown).

Protective Effects of the Purified NeurotrophicFactors on Rotenone-Induced Neuronal Injury inVM Cultures

To confirm further that the neurotrophic effectswere due to BDNF and GDNF, we added purifiedBDNF or GDNF in VM cultures and determined theireffect on the rotenone-induced dopamine neuron injury.

Fig. 4. CM of Pitx3-tranfected astrocytes protected against the rote-none-induced DA neuron loss in primary VM cultures. Primary VMcultures were treated with vehicle (Con, A), rotenone (Rot, B), rote-none 1 astro-C2 (astro-C2, C), or rotenone 1 astro-CP (astro-CP,D). The statistical results for A–D are shown in E. Data are mean 6SEM (n 5 3); P < 0.05 was considered significant (*P < 0.01, com-pared to Con; #P < 0.05, compared to Rot). F–I: Representativephotomicrographs of TH-positive cells in VM cultures treated with 25

nM rotenone (Rot; F), rotenone 1 0.53 CM from Pitx-3-transfectedastrocytes (0.53 astro-CP, G), rotenone 1 13 CM (13 astro-CP,H), and rotenone 1 23 CM from Pitx-3-transfected astrocytes (23astro-CP, I). J: Statistical results for F–I. Data are mean 6 SEM valuesfrom three independent experiments performed in duplicate (*P <0.05, **P < 0.01, compared to Rot). The number of TH-positivecells was counted in 10 random optical fields, which consisted of 12%of the area of a well in a 96-well dish. Scale bar 5 40 lm.

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Both neurotrophic factors (10 ng/ml each) had signifi-cant neuroprotection against the rotenone-induced do-pamine neuronal injury (Fig. 6B,C) compared with rote-none-treated VM cultures (Fig. 6A). Statistical analysisshowed that the number of TH-positive cells in rote-none-treated VM cultures was decreased to 46.1% com-pared with vehicle control, whereas the cotreatmentwith BDNF or GDNF increased the TH-positive cellsto 71.4% and 75.1%, respectively (Fig. 6E).

DISCUSSION

Homeodomain proteins affect a number of targetgenes functioning as either activators or repressors of eu-

karyotic transcription. In the brain, Pitx3 is strictlyexpressed in the DA neurons in SNc (Smidt et al.,2004b). Many studies have reported the close associationof Pitx3 with TH expression during development andadulthood (Smidt et al., 2004b; Zhao et al., 2004; Max-well et al., 2005), indicating that DA neurons in SNcare specifically regulated by Pitx3. A recent paperdescribed the developmental cascade in mice in whichAldh1a1 is under the transcriptional control of Pitx3(Jacobs et al., 2007). These studies indicated a prominentrole for Pitx3 in the late differentiation and maintenanceof DA neurons. Our previous work has demonstrated

Fig. 5. Blockade of BDNF and GDNF inhibited the protection ofthe CM from the Pitx3-transfected astrocytes against rotenone-induced DA neuron injury. Antibodies against either BDNF orGDNF (1 lg/ml) were added in the CM of the Pitx3-transfectedastrocytes (astro-CP) for 2 hr at room temperature. A: Rot (rotenonetreatment). B: BDNF-Ab (BDNF antibodies incubation). C: GDNF-Ab (GDNF antibodies incubation) markedly reduced the neuropro-tective effects of CM (D) from Pitx3-transfected astrocytes. Thenumber of TH-positive cells was counted in 10 random optical fields,which consisted of 12% of the area of a well in a 96-well dish. Thestatistical results are shown in E (*P < 0.01, compared to Astro-CP).Scale bar 5 40 lm.

Fig. 6. Purified BDNF and GDNF protected against the rotenone-induced neuronal injury. BDNF at 10 ng/ml or GDNF at 10 ng/mlwas added to the medium of VM cultures and incubated for 4 hrbefore rotenone treatment (25 nM rotenone for 8 hr). Both neuro-trophic factors significantly attenuated the rotenone-induced loss ofTH-positive cells in VM cultures compared with the rotenone-treated group. The number of TH-positive cells was counted in 10random optical fields, which consisted of 12% of the area of a well ina 96-well dish. A: Rot (rotenone treatment). B: BDNF 1 Rot(BDNF treatment). C: GDNF 1 Rot (GDNF treatment). D: Con(vehicle control). E: Statistical analysis of data from A–D. Data aremean 6 SEM values from four independent experiments (*P < 0.01,compared to Rot). Scale bar 5 40 lm.

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that overexpression of Pitx3 up-regulated the expressionof BDNF and GDNF in SH-SY5Y cells and in primaryVM cultures (Peng et al., 2007), which broadens ourinsight by demonstrating that Pitx3 is not only regulatingDA neurons phenotype but also is involved in the pro-duction of growth factors to promote the developmentand survival of DA neurons.

We have demonstrated that overexpression ofPitx3 in primary astrocytes up-regulated BDNF andGDNF expression at both transcriptional and transla-tional levels, which is consistent with our previous workin SH-SY5Y cells and primary VM cultures. BDNF andGDNF are the two most important neurotrophic factorsfor the differentiation and survival of midbrain DA neu-rons (Knusel et al., 1991; Lin et al., 1993; Seroogyet al., 1994). These two neurotrophic factors can protectDA neurons against neurotoxin-induced injury in vivoand in vitro (Skaper et al., 1993; Gash et al., 1996; Faw-cett et al., 1998). Our results showed that CM fromPitx3-transfected astrocytes contained high levels ofBDNF and GDNF and significantly protected the rote-none-induced injury in DA neurons. Furthermore, wereported that the protection of CM was significantlydiminished when BDNF or GDNF antibodies wereadded to neutralize the biological effects of these twoneurotrophic factors. We also demonstrated that purifiedneurotrophic factors BDNF and GDNF protected theDA neurons in VM cultures against rotenone-inducedneuronal injury. These studies suggest that the neurotro-phic factors secreted from the Pitx3-transfected astrocytesto protect against oxidative stress-induced DA neuroninjury are mainly BDNF and GDNF.

Astrocytes have several unique properties that makethem an ideal resource for cell or gene therapy. 1)Astrocytes can provide growth factors or other moleculesto support the neurons growth and maintain their func-tions (Ridet et al., 1997; Araque et al., 2001); 2) theyare excellent carriers to help deliver therapeutic mole-cules into the brain; and 3) they have the ability to sur-vive for prolonged periods when the cells are trans-planted into their native environment (Andersson et al.,1993). The transplantation of GDNF-delivered astrocytesinto the striatum has demonstrated the curative effect inParkinson’s disease (Ericson et al., 2005). We reporthere that CM from Pitx3-transfected astrocytes signifi-cantly protected against the rotenone-induced injury ofDA neurons in primary VM cultures. Interestingly, thetransfection of Pitx3 did not significantly alter the cellproliferation. This work suggests that Pitx3-expressedastrocytes might be a good resource for cell replacementtherapy for Parkinson’s disease.

In conclusion, we report that astrocytes transfectedwith Pitx3 have a neuroprotective effect against the ro-tenone-induced DA neuron injury in VM cultures viaenhancing the synthesis and secretion of BDNF andGDNF from the cells. This finding suggests that Pitx3-transfected astrocytes might be an active derivation ofgrafted cells for ex vivo gene therapy of Parkinson’s dis-ease.

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