Danny Galleguillos, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorJosé A. Fuentealba, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Pharmacy, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorLuis M. Gómez, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorMathias Saver, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorAndrea Gómez, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorKevin Nash, Department of Molecular Pharmacology and Physiology University of South Florida, Tampa, Florida, USASearch for more papers by this authorCorinna Burger, Department of Neurology, University of Wisconsin-Madison, Madison, Wisconsin, USASearch for more papers by this authorKatia Gysling, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorMaría E. Andrés, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this author Danny Galleguillos, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorJosé A. Fuentealba, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Pharmacy, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorLuis M. Gómez, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorMathias Saver, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorAndrea Gómez, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorKevin Nash, Department of Molecular Pharmacology and Physiology University of South Florida, Tampa, Florida, USASearch for more papers by this authorCorinna Burger, Department of Neurology, University of Wisconsin-Madison, Madison, Wisconsin, USASearch for more papers by this authorKatia Gysling, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this authorMaría E. Andrés, Nucleus Millennium in Stress and Addiction, Santiago, Chile Department of Cell and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, ChileSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat AbstractJ. Neurochem. (2010) 114, 1158–1167.Abstract Genesis of midbrain dopamine (DA) neurons depends on Nurr1, a nuclear receptor expressed during development and adulthood in these neurons. Nurr1 is required for the expression of genes of dopaminergic phenotype such as tyrosine hydroxylase and DA transporter. The expression of the tyrosine kinase receptor RET also depends on Nurr1 during development. However, it is unknown whether RET expression is regulated by Nurr1 during adulthood, and the mechanism by which Nurr1 regulates RET expression. Using an adeno-associated vector-delivered anti-Nurr1 ribozyme, we knocked-down Nurr1 expression unilaterally in the substantia nigra (SN) of adult rats. Animals injected with the ribozyme displayed a 57.3% decrease in Nurr1 mRNA in the SN accompanied by decreased DA extracellular levels in the striatum. RET mRNA in the injected SN and RET protein in the ipsilateral striatum decreased 76.9% and 47%, respectively. Tyrosine hydroxylase and DA transporter mRNA did not change in Nurr1 knocked-down SN. Nurr1 induced the transcription of the human RET promoter in cell type and concentration-dependent manner. Nurr1 induction of RET promoter is independent of NBRE elements. These results show that the expression of RET in rat adult SN is regulated by Nurr1 and suggest that RET is a transcriptional target of this nuclear receptor. Abbreviations used: AAV5 Adeno-Associated Virus Serotype 5 CBA chicken β-actin DA dopamine DAT DA transporter FBS fetal bovine serum GDNF glial cell line-derived neurotrophic factor GFP Green Fluorescent Protein NBRE NGFI-B Response Element SN substantia nigra TH tyrosine hydroxylase Nurr1 (NR4A2) is an orphan member of the nuclear receptor superfamily of transcription factors critical for the generation of dopamine (DA) neurons of the substantia nigra (SN) and ventral tegmental area (Zetterstrom etal. 1997; Castillo etal. 1998; Saucedo-Cardenas etal. 1998). Precursor cells of DA neurons are born in Nurr1-null mice, however tyrosine hydroxylase (TH), the DA transporter (DAT), the tyrosine kinase receptor RET and the vesicular monoamine transporter-2 are not expressed in these precursor cells (Wallen etal. 2001; Smits etal. 2003; Perlmann and Wallen-Mackenzie 2004). Recently, it was demonstrated that Nurr1 is also essential to maintain midbrain DA neurons in the adult (Kadkhodaei etal. 2009). Indeed, specific ablation of Nurr1 in mature DA neurons induces TH, vesicular monoamine transporter-2 and DAT loss, and neuronal degeneration (Kadkhodaei etal. 2009). Heterozygous Nurr1 mice also show several impairments on dopaminergic markers, such as a reduction in TH and GTP cyclohydrolase expression (Eells etal. 2006). Newborn Nurr1+/− mice have reduced DA levels in the striatum (Zetterstrom etal. 1997; Saucedo-Cardenas etal. 1998), but this abnormality is corrected when animals reach adulthood (Le etal. 1999). Thus, Nurr1 is required for DA neuron survival in the adult and for the expression of critical genes of the DA neurochemical phenotype. Unlike other genes lost in DA precursors of Nurr1 null mice, RET has been shown to be important in the survival of DA neurons, but not directly involved in the synthesis and storage of DA. RET, a member of the receptor tyrosine kinase superfamily, is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (GDNF). It has been shown that GDNF protects and repair dopaminergic neurons from insults such as MPTP and 6-hydroxydopamine toxicity, and axotomy (Beck etal. 1995; Bowenkamp etal. 1995; Kearns and Gash 1995; Tomac etal. 1995). Moreover, it has been demonstrated that GDNF is essential for the survival of midbrain DA neurons during post-natal development (Pascual etal. 2008). It has also been reported that knock-in mice expressing a constitutive active mutant RET gene have increased number of midbrain TH neurons (Mijatovic etal. 2007). Moreover, Kramer etal. (2007) showed that mice lacking RET suffer progressive and late degeneration of dopaminergic nigro-striatal system. These mice also show impaired capacity to regenerate dopaminergic axon terminals (Kowsky etal. 2007). Nurr1 heterozygous mice share several of these characteristics, such as reduced capacity to compensate for damage and loss of DA and DAT in aged animals (Le etal. 1999; Jiang etal. 2005). These data suggest that RET could be a key target of Nurr1 in the maintenance of midbrain DA neurons during adulthood. Our objectives were to study if RET is a Nurr1 gene target in the adult midbrain DA neurons and the mechanism by which Nurr1 regulates RET expression. Male Sprague Dawley rats weighing 180 g were used. Rats were maintained in the Animal Care Facility of the Faculty of Biological Sciences, Catholic University of Chile, under the advice of a veterinarian, on a 14/10 h light/dark schedule (lights on between 7:00 am and 9:00 pm) Rats were housed in a colony room in groups of four per cage, where they were maintained under constant temperature with food and water available ad libitum. All experiments were conducted in accordance with institutional and international guidelines [National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and Society for Neuroscience Guidelines for the Use of Animals in Neuroscience Research]. The ribozyme Rz1844 was cloned in an Adeno-Associated Virus Serotype 5 (AAV5) backbone containing the chicken β-actin (CBA) promoter (Niwa etal. 1991). A second promoter follows this cassette and controls expression of the humanized renilla reniformis Green Fluorescent Protein (GFP) gene (Stratagene, La Jolla, CA, USA). Virus preparation is described in Appendix S1. Vector titers were determined by dot-blot assay as described (Wu and Ataai 2000) and were 2.08 × 1013 genome copies/mL (gc/mL) (rAAV5-GFP-Rz1844) and 4.5 × 1013 gc/mL (rAAV5-GFP). Rats were anesthetized with xylacine/ketamine ip (xylacine: 10 mg/kg; ketamine: 50 mg/kg) and placed in a stereotaxic frame (Stoelting, Wood Dale, IL, USA). Virus injections were performed with a 10-μL Hamilton syringe at a rate of 1 μL/min. Each vector-treated rat received the injection in the right SN of either the rAAV5-GFP (2 μL) or rAAV5-GFP-Rz1844 (4.3 μL). The coordinates for injections were: 5.3 mm posterior to bregma, 1.5 mm lateral and 6.5 mm under dura. Following the injection, the needle was left in place an additional 2 min before being slowly removed from the brain. Eight weeks following the injection rats were decapitated, brains rapidly removed and placed in an ice-cooled block, the brains were then sectioned coronally at the level of the cerebral peduncles. A 2 mm thick coronal section posterior to the cut, containing the midbrain, was isolated and each SN dissected and placed in TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The left and right striata were dissected from the region anterior to the cut: the left and right hemispheres were separated, and each striatum isolated from the surrounding tissue. The striatum from each hemisphere was homogenized separately and quickly frozen in liquid nitrogen. The tissue was thereafter stored in −80°C until further use. Experiments were performed as previously described (Ojeda etal. 2003, see Appendix S1). Thirty micrometer brain slides were incubated overnight at 4°C with a specific polyclonal TH antibody (H-196, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 5000. After extensive washing, slides were incubated during 2 h with ant-rabbit IgG Alexa-596 (1 : 1000). Finally, slides immunofluorescence was visualized in a microscope Olympus FV-1000 with a QImaging Micropublisher 5.0 camera coupled to QCapture Pro software (QImaging, Surrey, BC, Canada). Tissue samples from striatum were prepared in radioimmunoprecipitation buffer. 50 μg of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gels. Membranes were thereafter blocked for 1 h at 22°C and incubated with the primary antibody (rabbit anti-TH 1 : 2500; mouse anti-RET 1 : 5000, Neuromics GT15002) in blocking buffer overnight at 4°C. Secondary antibodies were incubated for 1 h in blocking buffer. Immunoreactive bands were detected using an enhanced chemoluminiscence kit (Santa Cruz Biotechnology). Immediately after dissecting SN, the tissue from each hemisphere was placed in 0.5 mL of ice-cold TRIzol reagent. Total RNA was extracted following manufacturer’s instructions. About 500 ng of RNA were subjected to reverse transcription using MMLV-RT (Fermentas International Inc., Burlington, ON, Canada). Quantitative Real-Time PCR analysis was performed using a LightCycler (Roche Applied Science, Mannheim, Germany) in 10 μL of FastStart DNA Master SYBR Green I. Briefly, 1 μL of each sample was subjected to PCR using the following primers to amplify targets cDNAs: Nurr1-F: 5′-CCTGACTATCAGATGAGTGG-3′, Nurr1-R: 5′-ATTGCAACCTGTGCAAGACC-3′; TH-F: 5′-CCTGGAGTATTTTGTGCGCT-3′, TH-R: 5′-CACATGGGGAATTGGTTCAC-3′; DAT-F: 5′-AATGCTCCGTGGGACCAATG-3′, DAT-R: 5′-CAATAACCATGAAGAGCAGG-3′; RET-F: 5′-ACAAGCACACTACTCTCAGG-3′, RET-R: 5′-CATTGACCAGGACTACTAGC-3′; CYC-F: 5′-TGCTCTGAGCACTGGGGAGAAA-3′, CYC-R: 5′-CATGCCTTCTTTCACCTTCCCAAAGAC-3′. SYBR Fluorescence was acquired 2°C over the Tm of each amplicon. The amount of cDNA in each sample was calculated using the method described by Pffafl (2001), briefly, by performing PCR using serial dilutions of SN cDNA we calculate the efficiency (E) of each target and reference gene amplification. To quantify the relative abundance of each gene we calculate the crossing point of each PCR reaction using the Fit Point Method without baseline adjustment (LightCycler Software 3.0, Roche Applied Science, Mannheim, Germany). For each rat we compare the abundance of each target gene in the ipsilateral SN versus its abundance in the contralateral side. Cyclophilin A was used as the internal reference gene. Statistical comparison was performed between ipsilateral sides of AAV-GFP injected rats versus ipsilateral sides of AAV-GFP-Rz1844 injected rats. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus (Stoelting). A concentric microdialysis probe (MD-2200 BAS America, West Lafayette, IN, USA) was lowered into the striatum using the following coordinates: 0.4 mm anterior to bregma, 3 mm lateral and 6.5 mm under dura. Body temperature was maintained between 37.3°C and 37.6°C by a thermostatically controlled electric heating pad. Supplemental chloral hydrate was given as needed to maintain deep anesthesia during all the experiment. Microdialysis sampling and DA quantification was performed as previously described (Fuentealba etal. 2006, see also Appendix S1). SH-SY5Y cell line was cultured in Minimal Essential Medium : Ham’s F12 (1 : 1) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine. HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS; both cell lines were maintained at 37°C and 5% CO2. PC12 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum and 5% FBS and maintained at 37°C and 10% CO2. All cell lines were supplemented with 1% penicillin/streptomycin. pCMX-Nurr1 encoding full length Nurr1 was kindly donated by Kazuto Kobayashi (Fukushima Medical University, Japan). Expression vectors pACT-Nurr1 encoding full-length rat Nurr1 fused to the activation domain of VP16 was subcloned by recombinant technology. Reporter plasmid pGL3 −453/+222 RET was kindly donated by Scott Andrew (Queen’s University, Kingston, ON, Canada). HEK293, PC12 and SH-SY5Y cells (1 × 105 cells/well, 24-well plates) were transfected with 0.4 μg of total DNA using LipofectAMINE 2000 reagent (Invitrogen). Control experiments were carried out using equivalent molar amounts of empty vectors. DNA was kept constant by adding pBluescript SK (Stratagene) and, in every experiment, 0.025 μg of pCMX-βgal reporter vector was co-transfected as control of transfection efficiency. Luciferase and β-galactosidase activity were quantified 48 h after transfection. One-way analysis of variance (anova) was used to determine statistically significant differences among groups when appropriate, followed by a Dunn’s post hoc test. When only two groups were compared, Mann–Whitney U-test was applied. Transduction of rAAV5-GFP-Rz1844 ribozyme efficiently reduces Nurr1 mRNA levels in the SN of adult rats To investigate the role of Nurr1 in adult DA neurons of the SN, we designed and tested ribozymes that specifically recognize and cleave Nurr1 rat mRNA. We identified ribozyme, Rz1844 (FigureS1a), displaying fast kinetic digestion of rat Nurr1 mRNA in vitro (TableS1) (Fritz etal. 2002). Rz1844 was cloned into a rAAV5 vector under the control of the synthetic CBA promoter (Burger etal. 2005). This vector also contains a GFP reporter gene under the control of the herpes simplex virus thymidine kinase promoter down-stream from the ribozyme cassette (FigureS1b). In a first set of experiments, rats injected in the right SN (FigureS1c) with AAV5-GFP or AAV5-GFP-Rz1844 were killed after 4 weeks of injection and brains processed for histology and fluorescence microscopy. GFP fluorescence signal is observed in cells of the SN pars compacta and adjacent ventral tegmental area, of rats injected with rAAV5-GFP (FigureS2). Similar results were observed in rats injected with rAAV5-GFP-Rz1844 (not shown). No GFP signal was detected in the uninjected contralateral SN (not shown). To confirm that vectors were transducing dopaminergic neurons, dual GFP and TH immunofluorescence labeling was performed in the SN and in the striatum. Double-labeled cells (FigureS2c) were seen in the injected SN of brain sections processed for double labeling with GFP (FigureS2a) and TH (FigureS2b). Extensive GFP and TH co-expression was observed throughout the striatum ipsilateral to the injected SN, while no GFP signal was detected in the contralateral striatum (FigureS2d–i). Thus, dopaminergic neurons of the SN were efficiently transduced with rAAV5. To analyze the efficiency of the rAAV5-GFP-Rz1844 in knocking-down Nurr1 mRNA expression in the injected SN, rats were killed 6–8 weeks after injection. Quantitative PCR analysis indicated that Nurr1 expression was not modified in the SN injected with rAAV5-GFP (94.04 ± 6.11% of total) when compared with the contralateral SN. In contrast, a significant reduction of Nurr1 mRNA was found in the SN infected with rAAV5-GFP-Rz1844 (42.71 ± 7.51% of total) compared with SN injected with rAAV-GFP (Fig.1a). To ensure that dissected tissue corresponded to SN, the specific DA midbrain gene Pitx3 was amplified from each sample. Pitx3 was equivalently amplified from ipsi and contralateral SN of animals injected with either rAAV5-GFP or rAAV5-GFP-Rz1844 (Fig.1b). In the same samples, GFP expression was exclusively detected in SN injected with rAAV5-GFP or rAAV5-GFP-Rz1844, while no signal for GFP was observed in the contralateral SN (Fig.1c). Effect of the ribozyme RZ1844 in the expression of Nurr1. Eight weeks after infection, each substantia nigra (SN) of every rat was dissected out and cDNA prepared from total RNA. (a) Relative abundance of the Nurr1 mRNA in the SN in control-injected (rAAV5-GFP; n = 5) and ribozyme-injected (rAAV5-Rz1844; n = 5) rats. Nurr1 mRNA levels were calculated comparing the abundance of its cDNA in the injected SN (ipsi) versus the contralateral SN (contra). For each sample, cyclophilin mRNA was used as reference gene. *p = 0.0079. (b) PCR demonstrating the expression of the mesodiencephalic specific gene Pitx3 in the dissected tissue. (c) PCR showing the specific expression of GFP in the injected side in rats. Ip: ipsilateral, Co: contralateral. Unilateral Nurr1 knock-down results in decreased extracellular levels of DA in the ipsilateral striatum As Nurr1 regulates genes essential for the DA neurochemical phenotype, we studied DA extracellular levels by microdialysis in the ipsilateral striatum of rats injected in the right SN, 6–8 weeks after the injection. Figure2a and b show that basal DA extracellular levels were significantly lower in the striatum of rats injected with rAAV5-GFP-Rz1844 than in control rats injected with rAAV5-GFP. The amount of extracellular levels of DA induced by high K+ (40 mM) was similar between rats injected with rAAV5-GFP-Rz1844 and rAAV5-GFP (Fig.2a and c). At the end of the experiments, the striatum from each brain hemisphere were dissected out and total DA tissue content quantified. Figure2d shows that there were no changes in the total DA tissue content in the ipsilateral striatum of rats injected with rAAV5-GFP-Rz1844 relative to control striatum, or in animals injected with rAAV5-GFP. Effect of decreased Nurr1 expression in dopamine (DA) of the striatum. (a) Temporal course of extracellular levels of DA in the striatum ipsilateral to control-injected (rAAV5-GFP; n = 5) and ribozyme-injected (rAAV5-GFP-Rz1844; n = 3) substantia nigra. The bar indicates time of the K+ infusion through the microdyalisis probe. (b) Basal extracellular levels of DA in the striatum of control-injected (rAAV5-GFP) and ribozyme-injected (rAAV5-GFP-Rz1844) rats. *p = 0.0005. (c) Net DA release induced by depolarizing stimulus (K+ 40 mM). (d) Tissue DA content in each striatum of control-injected (rAAV5-GFP) and ribozyme-injected (rAAV5-GFP-Rz1844) rats. Knock-down of Nurr1 expression in the SN of adult rats induces a specific decrease of RET expression As demonstrated by other groups, Nurr1 is essential for the expression of TH, DAT, and RET, in DA neurons of the ventral mesodiencephalon during development (Wallen etal. 2001; Smits etal. 2003). Thus, we hypothesized that these genes could be also under the regulation of Nurr1 in the nigro-striatal system in adult animals. Thus, we measured the mRNA expression of these genes by real-time RT-PCR in the SN of injected animals, 8 weeks after injection with rAAV5-GFP-Rz1844 or rAAV5-GFP. Nurr1 mRNA expression was also measured in the same samples. Only samples with 50% or more reduction of Nurr1 mRNA expression (as compared with contralateral levels) were included in the analysis. We did not find significant differences in the expression levels of TH or DAT mRNAs in the SN of rats with decreased levels of Nurr1, when compared with controls (Fig.3a and b). However, a significant decrease of RET mRNA levels was found in the SN of rats injected with rAAV5-GFP-Rz1844 relative to control rats injected in SN with rAAV5-GFP (Fig.3c). To determine whether decreased RET mRNA levels results in a decrease in RET protein levels; we performed western blot analysis from total tissue extracts from striatum. As it is shown in Fig.3d, RET protein significantly decreased in the striatum ipsilateral to injected SN with rAAV5-GFP-Rz1844. There were no changes in TH (Fig.3d) or in β-actin (data not shown). Effect of the decreased Nurr1 expression in the abundance of its putative target genes. Relative abundance of (a) tyrosine hydroxylase (TH) mRNA, (b) DA transporter (DAT) mRNA and (c) RET mRNA in control-injected (rAAV5-GFP; n = 5) and ribozyme-injected (rAAV5-GFP-Rz1844; n = 5) substantia nigra, 8 weeks after the infection. Target gene mRNA levels were calculated by Q-PCR with cyclophilin mRNA as the reference gene, using the Pfaff method. *p = 0.048 (d) Relative abundance of RET protein in the striatum of control-injected (rAAV5-GFP) and ribozyme-injected (rAAV5-GFP-Rz1844) rats. Relative abundance of RET was calculated using the immunoreactivity of actin as a loading control, *p = 0.0095. Bottom panel shows a representative western blot analysis depicting the expression of RET and TH in the striatum ipsilateral (R) and contralateral (L) of control-injected (rAAV5-GFP; n = 3) and ribozyme-injected (AAV5-GFP-Rz1844; n = 3) rats. RET is expressed in several tissues (Sariola and Saarma 2003). Accordingly, the human RET gene promoter shows significant basal activity in different cell types. As it is shown in Fig.4a, a luciferase reporter driven by the human RET gene promoter (−453/+222) is active in PC12, HEK293 and SH-SY5Y cell lines. To test the capacity of Nurr1 to induce RET expression, we over-expressed Nurr1 in each cell line along with the luciferase human RET reporter gene. Nurr1 induced a 6-fold increase of RET promoter activity in SH-SY5Y cells, while this effect was absent in PC12 and HEK293 cells (Fig.4b). These results suggest that while human RET promoter is active in each cell line, the mechanism by which Nurr1 induces RET expression is cell-type dependent. To better characterize the effect of Nurr1 over RET promoter activity; a dose-response curve was carried out. Increasing Nurr1 over-expression in SH-SY5Y cells dose-dependently increased human RET reporter transcription (Fig.4c). The transactivating effect of the hybrid Nurr1-VP16 was specific for RET reporter in SH-SY5Y (Fig.4d). Taken together this data demonstrate that Nurr1 specifically transactivates human RET promoter activity in a cell-type and dose-dependent manner. Nurr1 activates the human RET promoter in cell-type and dose-dependent manner. (a) Transcriptional activity of the human RET promoter in PC12, HEK293 and SH-SY5Y cell lines. Cells were transfected with equimolar amounts of luciferase reporter plasmid driven by the human RET promoter (pGL3 −453/+222 RET). White bars correspond to pGL3 transcriptional activity. Black bars correspond to pGL3 −453/+222 RET activity. *p   0.0286 (b) Effect of over-expressed Nurr1 over the activity of the human RET promoter in different cell lines. pGL3 −453/+222 RET plasmid or empty reporter vector pGL3 were cotransfected in HEK293, PC12 and SH-SY5Y cells along with an expression plasmid encoding full-length Nurr1 (pCMX-Nurr1, black bars) or the empty vector (pCMX, white bars). Nurr1 activity over RET promoter was normalized against its activity over the empty reporter plasmid pGL3, *p = 0.0159. (c) Dose-dependent response of the human RET promoter in SH-SY5Y cells. pCMX-Nurr1 (0–1.7 μg) was co-transfected with pGL3 reporter vector (white bars) or with pGL3 −453/+222 RET (black bars). Decreasing amounts of pCMX vector were co-transfected to keep constant the amount of expression plasmid. (d) Analysis of the activity of Nurr1-VP16 over the human RET gene promoter activity in SH-SY5Y and HEK293 cells. VP16-Nurr1 corresponds to the viral transcriptional activator VP16 fused to the full length Nurr1 protein. SH-SY5Y (black bars) and HEK293 (white bars) cells were co-transfected with the indicated expression vectors and pGL3 −453/+222 RET. For every experiment, luciferase activity was measured 48 h post-transfection. Data correspond to the mean ± SE of at least three independent experiments performed in triplicate. We evaluated the basal activity of a family of reporters driven by different segments of human RET promoter (Fig.5a). As shown in Fig.5b, the family of RET reporters shows different basal activity in SH-SY5Y cells. The highest basal activity was shown by −243/+222 segment of human RET promoter, which is significantly higher than +99/+222 (Fig.5b), suggesting that besides core promoter, transcriptional activation elements relevant for inducing activity in these cells are located between −243/+99 of human RET promoter. The activity of RET promoter is significantly reduced expanding towards 5′ (−453/+222, Fig.5b), indicating that a strong repressive element is located between −453/−243. Albeit the basal activity of the different promoter fragments is different, they respond similarly to over-expressed Nurr1. As shown in Fig.5c, over-expressed Nurr1 induced an 80% increment in the activity of the reporters driven by the different segments of the human RET promoter. As deleting the region containing an NGFI-B Response Element (NBRE)-like sequence found in RET promoter does not modify Nurr1-dependent transactivation, our data suggest that Nurr1 transactivate human RET promoter in an NBRE independent way. Nurr1 activates the human RET promoter independently of NBRE element. Serial deletions of the human RET promoter was generated to evaluate the contribution of a NBRE-like element in the Nurr1-induced activity. These reporter plasmids were transfected in the absence or presence of Nurr1 and the reporter gene activity evaluated 48 h later. (a) Schematic representation of serial deletions of the human RET promoter, black box represent the detected putative NBRE element. (b) Activity of the different RET promoter fragments in SH-SY5Y cells. Cells were transfected with equimolar quantities of the indicated reporter vectors derived from pGL3 −453/+222 RET. Values represent luciferase activity relative to the activity of pGL3 empty vector. (c) Effect of Nurr1 on the activity of the different fragments of the human RET promoter in SH-SY5Y cells. The different reporter vectors were co-transfected with equimolar amounts of pCMX or pCMX-Nurr1. White bars depict the effect of pCMX, and black bars the effect of pCMX-Nurr1 over the indicated reporter vector. Data correspond to the mean ± SEM of at least three independent experiments performed in triplicate. *p   0.05. In this report, we show that Nurr1 regulates RET expression in the SN of adult rats. In addition, we provide evidence that Nurr1 regulates the transcription of RET gene. During development, Nurr1 is essential for RET expression in midbrain DA neurons (Wallen etal. 2001), but the mechanisms involved in this regulation and whether Nurr1 is required for RET expression in adult DA neurons has remained elusive. By using a ribozyme against Nurr1, a 57% reduction of Nurr1 mRNA levels were induced in the SN of adult rats. We found that this reduction of Nurr1 levels resulted in a significant decrease of RET mRNA in the SN and a significant decrease of RET protein in the striatum ipsilateral to the injected SN. Because DA neurons projecting from SN to striatum express RET in their terminals (Trupp etal. 1997), decreased RET protein in the striatum is most plausibly a direct effect of decreased RET mRNA levels in SN. This effect on RET expression in adult animals with reduced levels of Nurr1 has not been reported before. Our results identify RET as a Nurr1-regulated gene in mature DA neurons. Our analysis of gene expression in nigro-striatal system after specific SN Nurr1 knock-down did not show changes in either TH or DAT expression. These data are similar to those reported in adult Nurr1 heterozygous mice. Although, recently born Nurr1 heterozygous mice have decreased TH expression levels and DA content in the striatum (Zetterstrom etal. 1997; Saucedo-Cardenas etal. 1998; Eells etal. 2006), these effects are corrected in adulthood. Le etal. (1999) described a 45% reduction of Nurr1 in adult Nurr1 heterozygous mice, which is accompanied by normal number of TH positive cells in SN, and normal DA content in striatum. On the other hand, recent evidence from adult Nurr1 knock-out mice (Kadkhodaei etal. 2009) demonstrated that Nurr1 is necessary for the maintenance of TH and DAT in midbrain DA neurons. This divergence could be as a result of a dosage dependent effect of Nurr1: while a 50% reduction in its expression alters the expression of RET, the remnant Nurr1 can support the expression of TH and DAT. Several authors have shown an increase in damage susceptibility of dopaminergic neurons associated to a reduction of Nurr1 (Le etal. 1999; Jiang etal. 2005). These animals share several characteristics with RET deficient animals (Kowsky etal. 2007; Kramer etal. 2007) such as a reduced capacity to compensate for damage and loss of DA and DAT in aged animals. Our findings suggest that a decreased expression of RET because of a Nurr1 decrease could be part of the mechanisms responsible of the higher susceptibility in Nurr1 heterozygous animals. In addition to the decreased expression of RET induced by knocking-down Nurr1 in SN, we observed a significant decrease in the basal extracellular levels of DA in the striatum ipsilateral to the SN injected with the ribozyme. In Nurr1 heterozygous mice, Moore etal. (2008) showed a significant reduction in 3,4-dihydroxyphenylacetic acid (DOPAC) levels in striatum and lower DA levels, although not significantly different from wild type mice. Compensatory mechanisms (Calne and Zigmond 1991; Zigmond and Hastings 1998) during early developmental periods could account for this difference. Species-specific or methodological effect can not be discarded. Is the decreased RET expression related to decreased DA basal extracellular levels? It has been shown that RET regulates DA neurotransmission. In vivo voltammetry experiments performed in MENB2 mice, which have a permanently active RET, showed increased DA release (Mijatovic etal. 2008). RET ablation in midbrain DA neurons decreased striatal evoked DA release (Kramer etal. 2007). These reports support the possibility that reduced RET expression in the striatum results in decreased basal DA extracellular levels. We cannot discard that other genes regulated by Nurr1 could be responsible for the decrease in DA extracellular levels observed in the striatum, however our data support a role of RET regulating DA neurotransmission. We did not detect any difference on tissue DA levels in striatum between control and ribozyme treated animals. This is similar to the unchanged levels of tissue DA in the striatum between wild type and heterozygous Nurr1 mice reported by Eells etal. (2006). Mice lacking RET expression in the dopaminergic system have normal DA tissue content in the striatum, even though there is a modest decrease in the number of TH positive cells in the SN (Kramer etal. 2007). Similarly, our Nurr1 knock-down experiments in the SN show that the decrease of RET protein levels in the striatum do not result in changes in DA content or in TH expression in the striatum. We provide evidence indicating that RET is a transcriptional target of Nurr1. This is further supported by the evidence showing that forced expression of Nurr1 in cultured cells induces (Li etal. 2009) and increases RET mRNA expression (Sonntag etal. 2004). We show that Nurr1 induces the expression of a reporter gene driven by the human RET promoter in a cell type and dose-dependent manner. Cell type specificity of Nurr1-dependent transactivation of genes important in dopaminergic neurons has been shown for TH (Iwawaki etal. 2000; Kim etal. 2003), DAT (Sacchetti etal. 2001), GTP cyclohydroxylase I (Gil etal. 2007) and RET (this work), as well as for genes in other systems such as osteopontin (Lammi etal. 2004). Even though it has been demonstrated that Nurr1 naturally adopts a transcriptional active conformation (Wang etal. 2003), the data presented here along with other reports allow suggesting that Nurr1-dependent transcriptional activity and its binding to NBRE elements are highly regulated. For instance, our results show that Nurr1 is inactive over human RET proximal promoter in HEK293 cells. However, in this cell line, an artificial promoter driven by three NBRE elements in tandem is highly induced by forced expression of Nurr1 (Castro etal. 1999; Galleguillos etal. 2004). The data indicates that in HEK293 cells there are co-activators that interact and mediate Nurr1-dependent transactivation through strong NBRE elements, but not in an indirect manner (RET). In addition, the fact that the hybrid VP16-Nurr1 does not activate human RET promoter in HEK293 cells, supports the idea that Nurr1 binding to RET promoter is also tissue specific, suggesting that Nurr1 binding and activation of RET promoter require additional factors complementing the activity of Nurr1. Our results show that besides cell specificity, the transactivating effect of Nurr1 over human RET promoter is independent of NBRE elements. The fragment of human RET promoter that mediates Nurr1 activity is located in the most proximal promoter (−163/+99) which lacks NBRE elements. This indirect form of Nurr1-dependent activation is common to DAT (Sacchetti etal. 2001), GTP cyclohydroxylase (Gil etal. 2007), 1 alpha hydroxylase (Zierold etal. 2007) and RET (this work). DAT was the first gene identified as up regulated in an indirect way by Nurr1 (Sacchetti etal. 2001). In this case, it was shown that a mutant form of Nurr1 lacking the DNA binding domain was still able to induce DAT transcription (Sacchetti etal. 2001). The authors proposed a model in which the effect of Nurr1 was mediated by another factor that would bind DAT promoter. More recently, it was also shown that Nurr1 regulates GTP cyclohydroxylase expression in an indirect way (Gil etal. 2007). Similarly to DAT gene, the NBRE-like elements on GTP cyclohydroxylase promoter do not contribute to the activity induced by Nurr1. Indeed, the proximal promoter of GTP cyclohydroxylase gene, which lacks Nurr1-binding sites, was activated by Nurr1 over-expression (Gil etal. 2007). Taken together, we conclude that the RET gene is a target of Nurr1, but its induction may require an as-yet-unidentified cellular factor for bringing Nurr1 to RET promoter. We gratefully acknowledge Dr. Kazuto Kobayashi (Fukushima Medical University, Japan) for the pCMX-Nurr1 plasmid. We thank Dr. Scott Andrew (Queen’s University, Canada) for the human RET reporter plasmid. This work was supported by FONDECYT Project 1070349 and Millennium Nucleus in Stress and Addiction MSI N° P06/008-F. DG and AG were supported by Conicyt and VRAID Ph.D. fellowships. Appendix S1. Supplementary Materials and methods. Table S1. Kinetic parameters for Nurr1 ribozyme Rz 1844. Figure S1. (a) Hammerhead ribozyme conformationemerging from hybridization between hammerhead ribozyme Rz1844 andNurr1 (Target) mRNA as predicted by the mfold algorithm. (b)Schematic representation of rAAV5-GFP and rAAV5-GFP-Rz1844 vectors.Expression of the ribozyme is driven by the synthetic CBA promotergenomic backbones. CMVe: Cytomegalovirus enhancer. Bap, chicken® actin promoter. Exon 1-intron, from rabbit ® globin. pA polyadenylation signal. WPRE, Woodchuck hepatitis virus post-transcriptional regulatory Element. PYF441e: Polioma Virus Enhancer F441, HSV-tk: Herpes Simplex Virus Thymidine Kinase Promoter, hrGFP: humanized renilla reniformis Green Fluorescent Protein (Stratagene). TR5: Terminal Repeat 5. (c) Diagram showing a coronal section of the rat midbrain. The arrow indicates the location of injection site. Figure S2. rAAV5 transduction in the SN(a–c). Coronal sections illustrate the distribution of theexpression of GFP in the SN of rats infected with rAAV5-GFP,4weeks after the injection. Microphotographs correspond to:(a) GFP fluorescence and (b) TH-immunoreactivity. (c) Merged imagesshow that most GFP expressing cells in the SN correspond to TH+neurons. (d–i) Coronal sections of the striatum (CE) depictthe presence of GFP in the terminals of the DA neurons only in theinfected side (ipsi) of the brain (d–f) as compared with thecontralateral (con) side (g–i). 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