Nurr1 regulates dopamine synthesis and storage in MN9D dopamine cells

doi:10.1016/S0014-4827(03)00216-7

Experimental Cell Research

Volume 288, Issue 2, 15 August 2003, Pages 324-334

https://doi.org/10.1016/S0014-4827(03)00216-7 Get rights and content

Abstract

Nurr1, a transcription factor belonging to the nuclear receptor family, is essential for the generation of midbrain dopamine (DA) cells during embryonic development. Nurr1 continues to be expressed in adult DA neurons but the role for Nurr1 in inducing and regulating basic dopaminergic functions such as dopamine synthesis and storage has remained unknown. We have previously used MN9D dopamine cells to analyze the role of Nurr1 and retinoids in DA cell maturation. These studies demonstrated that both Nurr1 and retinoids induce cell cycle arrest and a mature morphology. Here we used MN9D cells to investigate how Nurr1 regulates dopaminergic functions. Our results demonstrate that Nurr1, but not retinoids, increases DA content and the expression of aromatic L-amino acid decarboxylase (AADC) and vesicular monoamine transporter-2 (VMAT2) in MN9D cells. In a Nurr1-inducible cell line upregulation of VMAT2 is dependent on continuous Nurr1 expression. Moreover, AADC and VMAT2 are deregulated in midbrain DA cells of Nurr1 knockout embryos as revealed by in situ hybridization. Together, the results provide evidence indicating an instructive role for Nurr1 in controlling DA synthesis and storage.

Introduction

Midbrain DA neurons, with their cell bodies localized in substantia nigra and the ventral tegmental area, play critical roles in the central regulation of motor and motivational behaviors [1]. DA neurons project axons to striatum and other forebrain targets, and progressive degeneration of DA cells leads to the cardinal symptoms of Parkinson’s disease [2]. DA is generated from the amino acid tyrosine in two enzymatic steps. The first rate-limiting step is mediated by tyrosine hydroxylase (TH), converting tyrosine to the DA precursor l-3,4-dihydroxyphenylalanine (l-DOPA), which is converted to DA by l-amino acid decarboxylase (AADC). l-DOPA, which passes the blood brain barrier and is metabolized to DA by endogenous AADC, is the main treatment in Parkinson’s disease. However, l-DOPA supplementation gradually becomes less effective, partly due to the loss of DA terminals that can store and synthesize DA. Vesicular monoamine transporter 2 (VMAT2) also plays a critical role in regulating DA content by packaging DA into synaptic vesicles.

Nurr1 (NR4A2) belongs to the family of nuclear receptors also including receptors for steroid hormones, retinoids, vitamin D, and thyroid hormone [3]. Nuclear receptors are ligand-inducible transcription factors that bind to DNA and modulate expression of target genes [4]. However, ligands for Nurr1 and several additional orphan receptors remain unknown. Nurr1, and its close relatives NGFI-B and Nor1, can bind as monomers to specific DNA target sites in promoters of regulated genes [5], [6]. Nurr1 and NGFI-B can form heterodimers with the retinoid X receptor (RXR) and bind to certain types of retinoic acid-response elements [7], [8]. Moreover, all three receptors recognize DNA as homodimers and bind to a specific DNA-binding site within the pro-opiomelanocortin gene promoter [9], [10].

Nurr1 is highly expressed in the developing and adult ventral midbrain, and is essential for the generation of midbrain DA cells as revealed by analyses of Nurr1 gene-targeted mice [11], [12], [13], [14], [15]. In the absence of Nurr1, developing DA cells fail to express TH and the receptor tyrosine kinase subunit Ret [13], [16]. In contrast, many other dopaminergic markers of DA cells such as Ptx3, Lmx1b, En-1, and En-2 are initially expressed in the absence of Nurr1 but are lost at later stages of development [17]. Moreover, Nurr1-deficient embryonic ventral midbrain cells fail to migrate normally and are unable to innervate their striatal target areas [17]. Thus, although not required for initial specification of DA neurons, Nurr1 is critical from an early stage of DA cell maturation.

The dopamine cell line MN9D was generated by a somatic cell fusion of primary neurons from mouse Embryonic Day 14 rostral mesencephalic tegmentum and the neuroblastoma cell line N18TG2 [18]. MN9D cells synthesize catecholamines, have embryonic properties, express neuron-specific markers, and are sensitive to the DA cell toxin N-methyl-4-phenylpyridinium (MPP⁺) [19], [20]. Furthermore, these cells can be induced to extend neurites by various stimuli [21], [22], [23], [24]. Thus, these cells provide a useful in vitro model for studying developing and mature DA neurons.

Recently, we have shown that both Nurr1 and retinoids can induce cell cycle arrest and morphological differentiation of MN9D cells [25]. Here we used MN9D cells to study how Nurr1 influences basic dopaminergic functions such as DA content and production. Our results distinguish Nurr1 from retinoid-induced maturation events and provide evidence linking Nurr1 to the control of DA synthesis and storage.

Section snippets

MN9D cells

MN9D cells [18] were maintained at 37°C, with 5% CO₂ in DMEM/F12 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells were grown in poly-d-lysine-(Sigma) coated flasks.

MN9D-Nurr1^Tet-On cells

MN9D cells were transfected with the pTet-On vector using lipofectamine PLUS technique. Individual G418-resistant colonies were isolated, expanded, and screened for expression of the Tet-inducible transcription factor. One of the clones (MN9D

Nurr1 increases DA content and expression of VMAT2 and AADC in MN9D-Nurr1^Tet-On cells

To study how Nurr1 influences developing and mature DA cells we have generated stable subclones of MN9D cells(MN9D-Nurr1^Tet-On) in which Nurr1 expression is under the control of a tetracycline-inducible promoter (see Materials and methods). We used semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analyses to investigate gene expression levels in these cells (see materials and methods for details). Nurr1 mRNA is rapidly upregulated already 1 h after addition of the

Discussion

Although it is well established that Nurr1 is essential in DA neuron development, its exact regulatory role has remained unclear. We have shown previously that both Nurr1 and retinoids can induce cell cycle arrest and a mature neuronal morphology in MN9D cells [25]. These findings indicated that Nurr1 and retinoids might trigger a similar genetic program for cell maturation. Here we show that Nurr1 and retinoids have different effects in MN9D cells. Whereas both retinoids and Nurr1 induce cell

Acknowledgements

We thank Dr. Alfred Heller for providing MN9D cells and Drs. Ludmila Solomin and Mariette Arvidsson for valuable comments on the manuscript. E.H. was supported by a fellowship from the Swedish National Network in Neuroscience (NNN). L.O. was supported by the Swedish Research Council and USPH grants.

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Citation Excerpt :
The nuclear receptor related-1 protein, Nurr1 (NR4A2), is a transcription factor that regulates the expression of genes critical for the development, maintenance, and survival of dopaminergic neurons (Alavian et al., 2014; Decressac et al., 2013; Dong et al., 2016; Jankovic et al., 2005; Johnson et al., 2011; Kadkhodaei et al., 2009; Luo, 2012; Zetterstrom et al., 1997). In particular, Nurr1 plays a fundamental role in maintaining dopamine homeostasis by regulating transcription of the genes governing dopamine synthesis (TH, tyrosine hydroxylase; DDC, dopa decarboxylase), packaging (SLC18A2, vesicular monoamine transporter 2, VMAT2), and reuptake (DAT, dopamine transporter, also known as SLC6A3) (Hermanson et al., 2003; Iwawaki et al., 2000; Johnson et al., 2011; Sacchetti et al., 2001) (Figure 1A). Nurr1 also regulates the survival of dopaminergic neurons by stimulating the transcription of genes coding for neurotrophic factors (brain-derived neurotrophic factor, nerve growth factor), anti-inflammatory responses (glial cell-derived neurotrophic factor receptor c-Ret), and oxidative stress management (SOD1), as well as repressing the transcription of pro-inflammatory genes (tumor necrosis factor α, inducible nitric oxide synthase, interleukin-1β) (Galleguillos et al., 2010; Johnson et al., 2011; Kadkhodaei et al., 2013; Kim et al., 2003; Saijo et al., 2009; Sakurada et al., 1999; Volpicelli et al., 2007).
Nurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson's disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and X-ray crystallography, we show that DHI binds to the ligand-binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson's disease.

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¹: Present address: Institute of Environmental Medicine, Karolinska Institute, P.O. Box 210, Stockholm, Sweden.

²: Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Ilkirch Cedex, France.

³: Present address: Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Finland.

⁴: Present address: AstraZeneca (ATCG), AstraZeneca R&D Mölndal, Mölndal, Sweden.

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regular articleNurr1 regulates dopamine synthesis and storage in MN9D dopamine cells

Abstract

Introduction

Section snippets

MN9D cells

MN9D-Nurr1Tet-On cells

Nurr1 increases DA content and expression of VMAT2 and AADC in MN9D-Nurr1Tet-On cells

Discussion

Acknowledgements

Exp. Neurol.

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Cell

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Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Brain Res.

Neurosci. Lett.

J. Biol. Chem.

Biochim. Biophys. Acta

Neuron

Biochem. Biophys. Res. Commun.

Brain Res. Mol. Brain Res.

Identification of a new brain-specific transcription factor, NURR1

Mol. Endocrinol.

regular article
Nurr1 regulates dopamine synthesis and storage in MN9D dopamine cells

MN9D-Nurr1^Tet-On cells

Nurr1 increases DA content and expression of VMAT2 and AADC in MN9D-Nurr1^Tet-On cells