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Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands. However, the specific physiological roles of Nurr1 monomers and RXR-Nurr1 heterodimers remain to be elucidated. The aim of this study was to define structural requirements for RXR-Nurr1 heterodimerization. Several amino acid substitutions were introduced in both Nurr1 and RXR in the I-box, a region previously shown to be important for nuclear receptor dimerization. Single amino acid substitutions introduced in either Nurr1 or RXR abolished heterodimerization. Importantly, heterodimerization-deficient Nurr1 mutants exhibited normal activities as monomers. Thus, by introducing specific amino acid substitutions in Nurr1, monomeric and heterodimeric properties of Nurr1 can be distinguished. Interestingly, substitutions in the RXR I-box differentially affected heterodimerization with Nurr1, retinoic acid receptor, thyroid hormone receptor, and constitutive androstane receptor demonstrating that the dimerization interfaces in these different heterodimers are functionally unique. Furthermore, heterodimerization between RXR and Nurr1 had a profound influence on the constitutive activity of Nurr1, which was diminished as a result of RXR interaction. In conclusion, our data show unique structural and functional properties of RXR-Nurr1 heterodimers and also demonstrate that specific mutations in Nurr1 can abolish heterodimerization without affecting other essential functions.

Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.

The retinoid X receptor (RXR) is a nuclear receptor that functions as a ligand-activated transcription factor. Little is known about the ligands that activate RXR in vivo. Here, we identified a factor in brain tissue from adult mice that activates RXR in cell-based assays. Purification and analysis of the factor by mass spectrometry revealed that it is docosahexaenoic acid (DHA), a long-chain polyunsaturated fatty acid that is highly enriched in the adult mammalian brain. Previous work has shown that DHA is essential for brain maturation, and deficiency of DHA in both rodents and humans leads to impaired spatial learning and other abnormalities. These data suggest that DHA may influence neural function through activation of an RXR signaling pathway.

Nurr1, an orphan receptor of the nuclear receptor superfamily, is widely expressed in the central nervous system (CNS) including brain regions where dopaminergic neurons are abundant. Recent analyses of Nurr1 null mutant mice have shown that Nurr1 is essential for the development and survival of midbrain dopaminergic neurons. However, other dopaminergic neuronal populations do not seem to be affected by ablation of the Nurr1 gene. The purpose of the present study was to investigate the degree of co-existence of Nurr1 mRNA and tyrosine hydroxylase (TH) immunoreactivity in the brain of adult mice to better characterize the selective effects of Nurr1 on catecholaminergic neurons. Our results indicate that the majority of TH-immunoreactive neurons in the substantia nigra (SN; 96%), ventral tegmental area (VTA; 95%), retrorubral field (91%), olfactory bulb (85%), linear nucleus raphe (91%) and central grey (61%) express Nurr1. In contrast, dopaminergic cells of the paraventricular and periventricular hypothalamic nucleus showed only a few Nurr1/TH double labeled neurons, while TH-immunoreactive neurons in the arcuate nucleus and zona incerta did not express Nurr1 mRNA. Nurr1 expression was also excluded from (nor)adrenergic neurons of the brainstem. In conclusion, Nurr1 transcripts were not found in all CNS catecholaminergic neurons. Nurr1 expression was confined to periglomerular and midbrain dopaminergic neurons. These results suggest that within the adult mouse brain, Nurr1 may participate in dopaminergic functions of the olfactory bulb and midbrain.

Understanding nuclear receptor signaling in vivo would be facilitated by an efficient methodology to determine where a nuclear receptor is active. Herein, we present a feedback-inducible expression system in transgenic mice to detect activated nuclear receptor effector proteins by using an inducible reporter gene. With this approach, reporter gene induction is not limited to a particular tissue, and, thus, this approach provides the opportunity for whole-animal screens. Furthermore, the effector and reporter genes are combined to generate a single strain of transgenic mice, which enables direct and rapid analysis of the offspring. The system was applied to localize sites where the retinoic acid receptor ligand-binding domain is activated in vivo . The results identify previously discovered sources of retinoids in the embryo and indicate the existence of previously undiscovered regions of retinoic acid receptor signaling in vivo . Notably, the feedback-inducible nuclear-receptor-driven assay, combined with an independent in vitro assay, provides evidence for a site of retinoid synthesis in the isthmic mesenchyme. These data illustrate the potential of feedback-inducible nuclear-receptor-driven analyses for assessing in vivo activation patterns of nuclear receptors and for analyzing pharmacological properties of natural and synthetic ligands of potential therapeutic value.

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP−/−vim−/−) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP−/−, vimentin−/−, or GFAP−/−vim−/− mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP−/− or vimentin−/− mice, but was impaired in GFAP−/−vim−/− mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.

Retinoic acid (RA), a retinoid metabolite, acts as a gene regulator via ligand-activated transcription factors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs), both existing in three different subtypes, alpha, beta and gamma. In the intracellular regulation of retinoids, four binding proteins have been implicated: cellular retinol binding protein (CRBP) types I and II and cellular retinoic acid binding protein (CRABP) types I and II. We have used in situ hybridization to localize mRNA species encoding CRBP- and CRABP I and II as well as all the different nuclear receptors in the developing and adult rat and mouse central nervous system (CNS), an assay to investigate the possible presence of RA, and immunohistochemistry to also analyse CRBP I- and CRABP immunoreactivity (IR). RXRbeta is found in most areas while RARalpha and -beta and RXRalpha and -gamma show much more restricted patterns of expression. RARalpha is found in cortex and hippocampus and RARbeta and RXRgamma are both highly expressed in the dopamine-innervated areas caudate/putamen, nucleus accumbens and olfactory tubercle. RARgamma could not be detected in any part of the CNS. Using an in vitro reporter assay, we found high levels of RA in the developing striatum. The caudate/putamen of the developing brain showed strong CRBP I-IR in a compartmentalized manner, while at the same time containing many evenly distributed CRABP I-IR neurons. The CRBP I- and CRABP I-IR patterns were closely paralleled by the presence of the corresponding transcripts. The specific expression pattern of retinoid-binding proteins and nuclear retinoid receptors as well as the presence of RA in striatum suggests that retinoids are important in many brain structures and emphasizes a role for retinoids in gene regulatory events in postnatal and adult striatum.

In the adult mammalian central nervous system lost nerve cells are not replaced and there is no regeneration of injured axons in white matter. Together, these two facts mean that there are no spontaneous reparative mechanisms in operation. Instead, the adult central nervous system copes with the risks of injuries and diseases by protective encapsulation in bone, by a multitude of neuroprotective mechanisms, and finally by the fact that many important functions are represented by a much larger number of neurons than minimally needed. The long life expectancy of a human being nevertheless means that the risk that the central nervous system is affected by disease, injury or other forms of insults for which it cannot fully compensate is relatively high. Experimentally, two strategies are being pursued in order to develop ways of minimizing various forms of CNS damage, namely neuroprotective and reparative strategies. Here we present a possible reparative intervention applicable to spinal cord injury based on multiple white-to-gray matter peripheral nerve bridge grafts and work based on the specific role of Nurr1 for dopamine neuron development, suggesting that development of ligands to transcription factor might be a new inroad to neuroprotective treatments in Parkinson's disease.

Nuclear receptors are ligand-modulated transcription factors that respond to steroids, retinoids, and thyroid hormones to control development and body physiology. Orphan nuclear receptors, which lack identified ligands, provide a unique, and largely untapped, resource to discover new principles of physiologic homeostasis. We describe the isolation and characterization of the vertebrate orphan receptor, BXR, which heterodimerizes with RXR and binds high-affinity DNA sites composed of a variant thyroid hormone response element. A bioactivity-guided screen of embryonic extracts revealed that BXR is activatable by low-molecular-weight molecules with spectral patterns distinct from known nuclear receptor ligands. Mass spectrometry and 1 H NMR analysis identified alkyl esters of amino and hydroxy benzoic acids as potent, stereoselective activators. In vitro cofactor association studies, along with competable binding of radiolabeled compounds, establish these molecules as bona fide ligands. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway.

The orphan nuclear receptor Nur77 has been implicated in thymic negative selection. We studied the effect of two T cell receptor (TCR) transgenes on positive selection and Nur77 mRNA expression in thymus. DO11.10 mice, expressing a transgenic TCR specific for an ovalbumin (OVA) 323-339 peptide presented by I-Ad, were found to have an enlarged thymus with a reduced apoptotic activity, measured by flow cytometry, reduced mitochondrial membrane potential and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) techniques. In contrast, in F5 mice expressing a transgenic TCR recognizing the influenza virus nucleoprotein (NP) 366-374 peptide restricted by Db, this positive selection effect was much less pronounced. Positive thymic selection in DO11.10 TCR+ mice correlated with a reduced level of Nur77 mRNA expression shown by Northern blot. F5 mice expressed levels close to those expressed by the wild type. Both transgenic mouse strains responded with extensive cortical apoptosis, and with up-regulation of Nur77 mRNA, to injection of cognate peptides. As 9-cis-Retinoic acid (9-cis-RA) inhibits Nur77-dependent apoptosis in T cell hybridomas in vitro, mice were pretreated with the drug to investigate a similar effect in vivo. However, the drug itself, at saturating concentrations, caused extensive apoptosis in immature CD4+/CD8+ thymocytes. The result demonstrates a correlation between Nur77 expression and thymic apoptotic activity, both during positive and negative selection events.

In Parkinson disease (PD), affected midbrain dopamine (DA) neurons lose specific dopaminergic properties before the neurons die. How the phenotype of DA neurons is normally established and the ways in which pathology affects the maintenance of cell identity are, therefore, important considerations. Orphan nuclear receptor NURR1 (NURR1, also known as NR4A2) is involved in the differentiation of midbrain DA neurons, but also has an important role in the adult brain. Emerging evidence indicates that impaired NURR1 function might contribute to the pathogenesis of PD: NURR1 and its transcriptional targets are downregulated in midbrain DA neurons that express high levels of the disease-causing protein α-synuclein. Clinical and experimental data indicate that disrupted NURR1 function contributes to induction of DA neuron dysfunction, which is seen in early stages of PD. The likely involvement of NURR1 in the development and progression of PD makes this protein a potentially interesting target for therapeutic intervention.

Studies in model organisms constitute the basis of our understanding of the principal molecular mechanisms of cell fate determination in the developing central nervous system. Considering the emergent applications in stem cell-based regenerative medicine, it is important to demonstrate conservation of subtype specific gene expression programs in human as compared to model vertebrates. We have examined the expression patterns of key regulatory genes in neural progenitor cells and their neuronal and glial descendants in the developing human spinal cord, hindbrain, and midbrain, and compared these with developing mouse and chicken embryos. As anticipated, gene expression patterns are highly conserved between these vertebrate species, but there are also features that appear unique to human development. In particular, we find that neither tyrosine hydroxylase nor Nurr1 are specific markers for mesencephalic dopamine neurons, as these genes also are expressed in other neuronal subtypes in the human ventral midbrain and in human embryonic stem cell cultures directed to differentiate towards a ventral mesencephalic identity. Moreover, somatic motor neurons in the ventral spinal cord appear to be produced by two molecularly distinct ventral progenitor populations in the human, raising the possibility that the acquisition of unique ventral progenitor identities may have contributed to the emergence of neural subtypes in higher vertebrates.

Several nuclear receptors including the all-trans retinoic acid receptor RAR, form heterodimers with the 9-cis retinoic acid receptor, RXR. RXR-RAR heterodimers show an impressive flexibility in DNA binding and can recognize palindromic, inverted palindromes and direct repeats of the core half-site sequence AGGTCA. Dimerization interfaces in the DNA-binding domains of RXR, RAR, and thyroid hormone receptor (TR) that promote selective binding to strictly spaced direct repeats have previously been identified. However, an additional dimerization domain is present within the ligand-binding domains (LBDs) of these receptors. Here we localize a transferable 40-amino acid region within the LBDs of RXR, RAR, TR, and chicken ovalbumin upstream promoter transcription factor that is critical for determining identity in the heterodimeric interaction and for high-affinity DNA binding. This region overlaps almost perfectly with a helical segment in the RXR LBD crystal structure that was recently demonstrated to be part of the dimer interface. Our data suggest a sequential pathway for nuclear receptor dimerization whereby the LBD dimerization interface initiates the formation of solution heterodimers that, in turn, acquire the capacity to bind to a number of differently organized repeats. Formation of a second dimer interface within the DNA-binding domain (DBD) restricts receptors to direct repeat targets. Accordingly, the combination of an obligatory (LBD) and an optional (DBD) dimerization domain imparts a dynamic DNA-binding potential to the heterodimerizing receptors that both increases the diversity of the hormonal response as well as providing a restricted set of target sequences in direct repeat elements that ensures physiological specificity.

Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones. A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands. Known as orphan receptors, these proteins represents targets for novel signaling molecules. We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor. A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex. Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins. Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast. Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.

Glial cell line-derived neurotrophic factor (GDNF) and its close relative neurturin are currently in clinical trials for neuroprotection in patients with Parkinson disease (PD). However, in animal models of PD, GDNF fails to protect nigral dopamine (DA) neurons against α-synuclein-induced neurodegeneration. Using viral vector delivery of human wild-type α-synuclein to nigral DA neurons in rats, we show that the intracellular response to GDNF is blocked in DA neurons that overexpress α-synuclein. This block is accompanied by reduced expression of the transcription factor Nurr1 and its downstream target, the GDNF receptor Ret. We found that Ret expression was also reduced in nigral DA neurons in PD patients. Conditional knockout of Nurr1 in mice resulted in reduced Ret expression and blockade of the response to GDNF, whereas overexpression of Nurr1 restored signaling, providing protection of nigral DA neurons against α-synuclein toxicity. These results suggest that Nurr1 is a regulator of neurotrophic factor signaling and a key player in the cellular defense against α-synuclein toxicity.

The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b. Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons (RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the differentiation of the DN lineage.

Many previous studies have focused on understanding how midbrain dopamine neurons, which are implicated in many neurological conditions, are generated during embryogenesis. One of the remaining questions concerns how different dopamine neuron subtypes are specified. A recent paper in Neural Development has revealed features of a spatial and temporal lineage map that, together with other studies, begins to elucidate the developmental origin of distinct neuronal subtypes within the developing midbrain. See research article http://www.neuraldevelopment.com/content/6/1/29

DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.

Recently, we have shown that receptors for vitamin D3 (VDR), thyroid hormone (TR), and retinoic acid (RAR) activate preferentially through direct repeats (DRs) spaced by 3, 4, and 5 nucleotides, respectively. In addition, the RAR can activate weakly through DRs spaced by 2 nucleotides. A common feature of RAR, TR, and VDR is their ability to heterodimerize with the retinoid X receptor (RXR) through their ligand-binding domains (LBDs) to form high-affinity DNA-binding complexes that are specific for appropriately spaced repeats. In this paper we demonstrate that selective binding of RAR-RXR and TR-RXR heterodimers to their cognate DRs is a consequence of a novel cooperative dimer interaction within the DNA-binding domains (DBDs). Accordingly, a region in the first zinc finger of the TR and RAR DBDs interacts with the second zinc finger in the RXR DBD to promote selective DNA-binding to DRs spaced by 4 and 5 nucleotides, respectively. The resulting polarity established by this interaction places RXR in the 5' position of the direct repeats. These data provide a mechanism for selective receptor recognition of a restricted set of target sequences in DR DNA and explains the structural basis for physiological specificity.

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARbeta/delta signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARbeta/delta and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

The preservation of a pool of neural precursors is a prerequisite for proper establishment and maintenance of a functional central nervous system(CNS). Both Notch signaling and SoxB1 transcription factors have been ascribed key roles during this process, but whether these factors use common or distinct mechanisms to control progenitor maintenance is unsettled. Here, we report that the capacity of Notch to maintain neural cells in an undifferentiated state requires the activity of SoxB1 proteins, whereas the mechanism by which SoxB1 block neurogenesis is independent of Notch signaling. A common feature of Notch signaling and SoxB1 proteins is their ability to inhibit the activity of proneural bHLH proteins. Notch represses the transcription of proneural bHLH genes, while SoxB1 proteins block their neurogenic capacity. Moreover, E-proteins act as functional partners of proneural proteins and the suppression of E-protein expression is an important mechanism by which Notch counteracts neurogenesis. Interestingly, in contrast to the Hes-dependent repression of proneural genes, suppression of E-protein occurs in a Hes-independent fashion. Together, these data reveal that Notch signaling and SoxB1 transcription factors use distinct regulatory mechanisms to control proneural protein function and to preserve neural cells as undifferentiated precursors.

Originally published in: Cellular Proteins and Their Fatty Acids in Health and Disease. Edited by Asim K. Duttaroy and Friedrich Spener. Copyright © 2003 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30437-0 The sections in this article are Retinoids in Development Retinoid Receptors Transduce Retinoic Acid Signals Retinoid Receptors Belong to the Nuclear Hormone Receptor Family Nuclear Receptors Share a Common Structure The LBD and Ligand-dependent Transactivation Cross-talk Co-activators Co-repressors Nuclear Receptors from an Evolutionary Perspective Fatty acids as Endogenous Ligands for RXR Conclusions Acknowledgements Keywords: retinoid receptors; RAR; RXR; nuclear hormone receptor family; ligand-dependent transactivation; activators; repressors; fatty acids

Purified glucocorticoid receptor (GR) from rat liver was used for a quantitative analysis of the protein-DNA interaction at specific GR-binding segments within the 5'-long terminal repeat of the mouse mammary tumor virus. A truncated receptor was generated and used to demonstrate formation of heterodimeric GR, which furthermore was shown to be in rapid equilibrium with receptor-monomer. The relative affinity for GR binding to specific GR sites versus random calf thymus DNA was approximately 2 x 10(3). At equilibrium a free GR concentration of 3 x 10(-10) M was required for half-maximal saturation of the two functionally important DNA sites within the mouse mammary tumor virus 5'-long terminal repeat. Although these two DNA segments act synergistically in mediating hormonal response, we did not detect cooperative GR binding to these regions in vitro. However, GR bound cooperatively within the downstream binding region. Similarly, GR was unable to facilitate factor binding to a neighboring nuclear factor 1 site, another essential element in the promoter. In contrast, nuclear factor 1 binding was inhibited slightly by GR.

Nurr1 is an orphan nuclear receptor regulating transcription both as a monomer and as a heterodimer with retinoid X receptor (RXR). RXR-Nurr1 heterodimers are permissive RXR heterodimers as they activate transcription in response to RXR ligands. In contrast, heterodimers formed by RXR and retinoic acid receptor (RAR) are non-permissive as they activate transcription only upon RAR ligand binding. We studied the mechanism mediating permissiveness and non-permissiveness by creating receptor chimeras between Nurr1 and RAR. We show that the amino-terminal part of the Nurr1 ligand binding domain conveys permissiveness to RXR-Nurr1 heterodimers. This region is involved in interactions with the corepressors SMRT and NcoR. The corepressors were released from RXR-Nurr1 heterodimers by RXR ligand binding. In contrast, RXR ligand increased the interaction between RXR-RAR heterodimers and the corepressors. The corepressors were released only upon binding of RAR ligand. In conclusion, corepressor interaction differentiates the permissive RXR-Nurr1 heterodimers from the non-permissive RXR-RAR heterodimers.

Half of the members of the nuclear receptors superfamily are so-called "orphan" receptors because the identity of their ligand, if any, is unknown. Because of their important biological roles, the study of orphan receptors has attracted much attention recently and has resulted in rapid advances that have helped in the discovery of novel signaling pathways. In this review we present the main features of orphan receptors, discuss the structure of their ligand-binding domains and their biological functions. The paradoxical existence of a pharmacology of orphan receptors, a rapidly growing and innovative field, is highlighted.

Preliminary tissue content data obtained by High Performance Liquid Chromatography showed that DAT −/− KO rats have markedly decreased total tissue DA (down to 20% of wild-type levels) and 3−4 fold increased levels of DA metabolites − 3,4-dihydroxyphenylacetic acid (DOPAC) and Homovanillic acid (HVA) without major differences observed in serotonin and its main metabolite 5-hydroxyindolacetic acid (5-HIAA) levels. A preliminary Fast Scan Cyclic Voltammetry analysis showed that DA clearance in DAT −/− is much longer (over 60 seconds) when compared with DAT +/+, suggesting that the increase in the spontaneous locomotor activity is a direct consequence of the extended length of time that DA spends in the extracellular space following release. The DAT knockout rats should be an excellent and improved tool for the study and development of drugs used in the management of drugs used in dopaminergic dysfunctions, providing many similarities with DAT knockout mice but at the same time a better model for the study of complex behaviors. DAT −/− rats could provide a complex and translational model for several human diseases involving aberrant DA function or mutations affecting DAT or altered DAT regulatory mechanisms in vivo such as schizophrenia, ADHD and newly discovered Dopamine Transporter Deficiency Syndrome (DTDS) [3].

Dopamine neurons of the substantia nigra and ventral tegmental area regulate movement and affective behavior and degenerate in Parkinson’s disease. The orphan nuclear receptor Nurr1 was shown to be expressed in developing dopamine neurons before the appearance of known phenotypic markers for these cells. Mice lacking Nurr1 failed to generate midbrain dopaminergic neurons, were hypoactive, and died soon after birth. Nurr1 expression continued into adulthood, and brains of heterozygous animals, otherwise apparently healthy, contained reduced dopamine levels. These results suggest that putative Nurr1 ligands may be useful for treatment of Parkinson’s disease and other disorders of midbrain dopamine circuitry.

Electrospray (ES) mass spectrometry data is presented showing that agonist binding to the nuclear receptor (NR), retinoid X receptor α (RXRα), is competitive. The competitive nature of agonist binding can be used to discriminate between the specific and non-specific binding of small lipophilic molecules to NRs. Further, data is presented which show that high-affinity ligand binding to the RXRα ligand-binding domain (LBD) stabilises the domain homodimer. The results indicate that homodimerisation, a functional property of the receptor associated with the binding of agonist ligands, could be used to discriminate between specific and non-specific binding events. Additionally, we report on the remarkable stability of the gas-phase complex between the RXRα LBD protein and endogenous bile acids. Protein–bile acid interactions in the gas phase were found to be surprisingly strong, withstanding ‘in-source’ fragmentation in the ES interface, and, in the case of taurocholic acid (TCA) and lithocholic acid-3-sulphate (LCA-3-sulphate), collision-induced dissociation within the collision cell of a tandem mass spectrometer. Bile acids were found to be inactive towards RXRα in transfection assays, and have not been reported to be ligands for the RXRα, although lithocholic acid (LCA) has been found to be a competitor in the photoaffinity labelling of RXRβ with 9-cis-retinoic acid (9-cis-RA). The observation of strong RXRα-bile acid non-covalent complexes in ES mass spectrometry highlight the danger of extrapolating gas-phase binding data to the solution phase and further to a possible biological activity, particularly when surface-active compounds such as bile acids are involved. The introduction of a competitive ligand-binding experiment can alleviate this problem and allow the differentiation between specific and non-specific binding. Copyright © 2005 John Wiley & Sons, Ltd.

The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the "canonical" non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors.

Nurr1 is a transcription factor that is expressed in the embryonic ventral midbrain and is critical for the development of dopamine (DA) neurons. It belongs to the conserved family of nuclear receptors but lacks an identified ligand and is therefore referred to as an orphan receptor. Recent structural studies have indicated that Nurr1 belongs to a class of ligand-independent nuclear receptors that are unable to bind cognate ligands. However, Nurr1 can promote signaling via its heterodimerization partner, the retinoid X receptor (RXR). RXR ligands can promote the survival of DA neurons via a process that depends on Nurr1–RXR heterodimers. In developing DA cells, Nurr1 is required for the expression of several genes important for DA synthesis and function. However, Nurr1 is probably also important for the maintenance of adult DA neurons and plays additional less-well-elucidated roles in other regions of the central nervous system and in peripheral tissues.

Nuclear receptors (NRs) constitute a large and highly conserved family of ligand-activated transcription factors that regulate diverse biological processes such as development, metabolism, and reproduction. As such, NRs have become important drug targets, and the identification of novel NR ligands is a subject of much interest. The retinoid X receptor (RXR) belongs to a subfamily of NRs that bind vitamin A metabolites (i.e. retinoids), including 9-cis-retinoic acid (9-cis-RA). However, although 9-cis-RA has been described as the natural ligand for RXR, its endogenous occurrence has been difficult to confirm. Recently, evidence was provided for the existence of a different natural RXR ligand in mouse brain, the highly enriched polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) (Mata de Urquiza et al. (2000) Science 290, 2140-2144). However, the results suggested that supra-physiological levels of DHA were required for efficient RXR activation. Using a refined method for ligand addition to transfected cells, the current study shows that DHA is a more potent RXR ligand than previously observed, inducing robust RXR activation already at low micromolar concentrations. Furthermore, it is shown that other naturally occurring PUFAs can activate RXR with similar efficiency as DHA. In additional experiments, the binding of fatty acid ligands to RXRalpha is directly demonstrated by electrospray mass spectrometry of the noncovalent complex between the RXR ligand-binding domain (LBD) and its ligands. Data is presented that shows the noncovalent interaction between the RXR LBD and a number of PUFAs including DHA and arachidonic acid, corroborating the results in transfected cells. Taken together, these results show that RXR binds PUFAs in solution and that these compounds induce receptor activation, suggesting that RXR could function as a fatty acid receptor in vivo.

The retinoid X receptor (RXR) is essential as a common heterodimerization partner of several nuclear receptors (NRs). However, its function as a bona fide receptor for endogenous ligands has remained poorly understood. Such a role would depend on the existence of RXR activating ligands in vivo and on the ability of such ligands to influence relevant biological functions. Here we demonstrate the presence of endogenous RXR ligands in the embryonic central nervous system (CNS) and show that they can activate heterodimers formed between RXR and the orphan NR Nurr1 in vivo. Moreover, RXR ligands increase the number of surviving dopaminergic cells and other neurons in a process mediated by Nurr1-RXR heterodimers. These results provide evidence for a role of Nurr1 as a ligand-independent partner of RXR in its function as a bona fide ligand-activated NR. Finally, our findings identify RXR-Nurr1 heterodimers as a potential target in the treatment of neurodegenerative disease.

Cyclin-dependent kinase inhibitors of the Cip/Kip family play critical roles in regulating cell proliferation during embryogenesis. However, these proteins also influence cell differentiation by mechanisms that have remained unknown. Here we show that p57 Kip2 is expressed in postmitotic differentiating midbrain dopamine cells. Induction of p57 Kip2 expression depends on Nurr1, an orphan nuclear receptor that is essential for dopamine neuron development. Moreover, analyses of p57 Kip2 gene-targeted mice revealed that p57 Kip2 is required for the maturation of midbrain dopamine neuronal cells. Additional experiments in a dopaminergic cell line demonstrated that p57 Kip2 can promote maturation by a mechanism that does not require p57 Kip2 -mediated inhibition of cyclin-dependent kinases. Instead, evidence indicates that p57 Kip2 functions by a direct protein–protein interaction with Nurr1. Thus, in addition to its established function in control of proliferation, these results reveal a mechanism whereby p57 Kip2 influences postmitotic differentiation of dopamine neurons.

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.

Recent studies have identified several factors that influence the development of midbrain dopamine (DA) neurons. The identity of early proliferating DA progenitor cells are specified by the secreted factors sonic hedgehog and fibroblast growth factor 8, derived from the floor plate of the ventral midline and the mid/hindbrain border, respectively. While transcription factors specifically expressed in the proliferating DA progenitor cells remain to be identified, several transcription factors important for postmitotic DA cell development have been characterized. These include Nurr1, Lmx1b, Pitx3, and En1/En2. The studies of these transcription factors have not only increased the understanding of how DA neurons are generated in vivo, but also allowed the development of new strategies using stem cells for engineering DA neurons in vitro, results that may have significance in future therapies of patients with Parkinson's disease.

Gene targeting experiments, in which both alleles of the Nurr1 gene were deleted, have shown that this molecule plays an essential role in the development of midbrain dopaminergic neurons, as shown by the loss of dopaminergic markers and the neurotransmitter dopamine (DA) in the ventral mesencephalon of Nurr1 null mutant mice. Nurr1-deficient mice die within a few hours of birth. Herein, we investigated whether adult mice (12–15-month-old), heterozygous for the Nurr1 mutation ( Nurr1 +/− ), show alterations in locomotor function and in the nigrostriatal dopaminergic system after acute exposure to methamphetamine. We first evaluated spontaneous and amphetamine-induced (5 mg/kg) locomotor response of >12-month-old wildtype ( Nurr1 +/+ ) and Nurr1 +/− mice. Both, spontaneous and methamphetamine-induced locomotor behavior was significantly increased in the Nurr1 +/− animals as compared to Nurr1 +/+ mice. Striatal DA and DA metabolite levels were measured in untreated animals and methamphetamine-treated animals. No significant differences in striatal dopamine levels or its metabolites DOPAC and HVA were found in the Nurr1 +/− as compared to Nurr1 +/+ mice in untreated or methamphetamine-treated animals. These data show that deletion of a single allele of the Nurr1 gene alters the locomotor activity of 12–15-month-old Nurr1 +/− animals. While total dopamine levels were not altered in the striatum of Nurr1 +/− mice, future studies will be necessary to determine if processes involved with the dynamics of DA release/clearance within the nigrostriatal system may be altered in Nurr1 +/− mutant mice.

Nurr1 (Nr4a2) is a transcription factor expressed in dopamine cells from early development and throughout life. Null mutants for Nurr1 lack the ventral midbrain dopamine neurons and die soon after birth. Animals with a heterozygous deletion are viable and display no apparent abnormality. We have investigated the impact of heterozygous deletion of Nurr1 on ethanol consumption in adult mice as a model for drug-induced reward and on wheel running as a model for natural reward. Interestingly, Nurr1 heterozygous mice never developed high ethanol consumption nor did they develop as much running behaviour as did the wild-type animals. Thus, Nurr1 appears to have a key role for the reinforcing properties of ethanol and running that underlies the development of excessive reward-seeking behaviours characteristic for addiction. Quantitative trait loci mapping using C57Bl/6 and DBA/2 mice describe a locus for ethanol preference on chromosome 2, wherein Nurr1 is located. We found two dinucleotide repeats in the Nurr1 promoter that were longer in mice with low preference for ethanol (DBA/2 and 129/Sv) than in mice with high preference for ethanol (C57Bl/6J and C57Bl/6NIH). These sequential data are compatible with Nurr1 as a candidate gene responsible for the quantitative trait loci for ethanol preference on mouse chromosome 2. Together, our data thus imply involvement of Nurr1 in the transition to a state of high ethanol consumption as well as in the development of a high amount of wheel running in mice.

Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 A resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a 'classical' binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stable conformation. Our findings highlight a unique structural class of NRs and define a model for ligand-independent NR function.

Publisher Summary This chapter focuses on the in vivo and in vitro reporter systems for studying nuclear receptor and ligand activities. Nuclear receptor (NR) ligands are generally small and lipophilic molecules making NRs highly attractive drug targets. These same properties render ligands challenging to the researchers interested in NR biology. Novel ligands for orphan NRs are not easily identified by biochemical methods and often cannot be identified by expression cloning or related strategies. The chapter describes alternative strategies that are based on transgenic mouse technology allowing NR activities to be assayed in vivo . It provides examples of complementary in vitro assays that are useful in verifying and extending results obtained in transgenic mouse experiments. It is well established that several nuclear receptors are activated by ligand independent mechanisms, which in transgenic experiments could lead to reporter induction in the absence of ligand. NR ligands may be unstable and/or sensitive to light. Therefore, all preparations should be kept on ice, and care should be taken to avoid unnecessary exposure to light. Further improvements in established methods, as well as the development of techniques to search for natural NR ligands in vivo , will be an essential complement to the screening efforts currently in progress, in particular within pharmaceutical and biotech companies. Although synthetic ligands are invaluable as tools, the identification of natural ligands will undoubtedly prove vital for achieving a complete understanding of the roles played by NRs in vivo .

Nurr1 is an orphan nuclear receptor belonging to the family of evolutionary conserved steroid/thyroid hormone receptors. It has been shown that Nurr1 is required for development of ventral mesencephalic dopaminergic cells in vivo and that Nurr1 regulates the tyrosine hydroxylase (TH) gene. The aim of this study was to investigate the possibility of finding ventral mesencephalic TH-positive neurons in Nurr1 deficient tissue when developed in the presence of wild type (WT) striatum. Therefore, fetal ventral mesencephalic tissue from embryonic day (E) 9.5–10.5 fetuses from Nurr1 mutant mice was co-cultured with lateral ganglionic eminence (LGE) from WT fetuses using the ‘roller-drum’ culture technique. TH-immunohistochemistry revealed similar number of positive neurons in WT, heterozygous, and Nurr1 deficient tissue, respectively. When ventral mesencephalon, dissected from E10.5 fetuses, was cultured alone without the presence of LGE, significantly more TH-immunoreactive neurons were found in WT and Nurr1 +/− than that seen in Nurr1 −/− cultures. In single ventral mesencephalic cultures dissected from E15.5, TH-positive neurons were found in all tissue cultures derived from knockout animals. Interestingly, the formation of TH-positive nerve fiber bundles was obvious in WT cultures while not observed in cultures of knockout tissue. When ventral mesencephalon was cultured alone in serum-free medium, almost no TH-positive neurons were found in cultures of knockout tissue. The addition of the growth factors epidermal growth factor and fibroblast growth factor-8 did not induce TH-immunoreactivity in serum-free Nurr1 −/− tissue cultures. In conclusion, TH-positive neurons may be generated in ventral mesencephalic tissue of Nurr1 deficient mice, suggesting that Nurr1 is not required for TH gene expression in ventral midbrain in vitro.

Despite its long history, the central effects of progressive depletion of vitamin A in adult mice has not been previously described. An examination of vitamin-deprived animals revealed a progressive and ultimately profound impairment of hippocampal CA1 long-term potentiation and a virtual abolishment of long-term depression. Importantly, these losses are fully reversible by dietary vitamin A replenishment in vivo or direct application of all trans -retinoic acid to acute hippocampal slices. We find retinoid responsive transgenes to be highly active in the hippocampus, and by using dissected explants, we show the hippocampus to be a site of robust synthesis of bioactive retinoids. In aggregate, these results demonstrate that vitamin A and its active derivatives function as essential competence factors for long-term synaptic plasticity within the adult brain, and suggest that key genes required for long-term potentiation and long-term depression are retinoid dependent. These data suggest a major mental consequence for the hundreds of millions of adults and children who are vitamin A deficient.

Nurr1, a member of the nuclear hormone receptor superfamily, was recently demonstrated to be of critical importance in the developing central nervous system, where it is required for the generation of midbrain dopamine cells. Nuclear receptors encompass a transcriptional activation function (activation function 2; AF2) within their carboxyl-terminal domains important for ligand-induced transcriptional activation. Since a Nurr1 ligand remains to be identified, the role of the Nurr1 AF2 region in transcriptional activation is unclear. However, here we show that the Nurr1 AF2 contributes to constitutive activation independent of exogenously added ligands in human embryo kidney 293 cells and in neural cell lines. Extensive mutagenesis indicated a crucial role of the AF2 core region for transactivation but also identified unique features differing from previously characterized receptors. In addition, Nurr1 did not appear to interact with, and was not stimulated by, several previously identified coactivators such as the steroid receptor coactivator 1. In contrast, adenovirus protein E1A, stably expressed in 293 cells, was shown to contribute to AF2-dependent activation. Finally, while the AF2 core of RXR is required for ligand-induced transcriptional activation by Nurr1-RXR heterodimers, the functional integrity of Nurr1 AF2 core is not critical. These results establish that the ligand binding domain of Nurr1 has intrinsic capacity for transcriptional activation depending on cell type and mode of DNA binding. Furthermore, these results are consistent with the possibility that gene expression in the central nervous system can be modulated by an as yet unidentified ligand interacting with the ligand binding domain of Nurr1.

The orphan nuclear receptor NURR1 was previously demonstrated to be required for the generation of mesencephalic dopamine (DA) cells. However, even in the absence of NURR1, which is normally expressed as cells become postmitotic, neuronal differentiation is induced and expression of several genes detected in developing dopamine cells appears normal during early stages of development. These include the homeobox transcription factors engrailed and Ptx-3 as well as aldehyde dehydrogenase 2, here defined as the earliest marker identified in developing DA cells, expressed already in mitotic DA progenitors. We have used the expression of these dopaminergic markers, retrograde axonal tracing, and apoptosis analyses to study the fate of the DA progenitor cells in the absence of NURR1. We conclude that NURR1 plays a critical role in the maturation, migration, striatal target area innervation, and survival of differentiating mesencephalic DA cells.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.

Retinoids regulate gene expression through the action of retinoic acid receptors (RARs) and retinoid-X receptors (RXRs), which both belong to the family of nuclear hormone receptors1,2. Retinoids are of fundamental importance during development2, but it has been difficult to assess the distribution of ligand-activated receptors in vivo. This is particularly the case for RXR, which is a critical unliganded auxiliary protein for several nuclear receptors, including RAR1, but its ligand-activated role in vivo remains uncertain. Here we describe an assay in transgenic mice, based on the expression of an effector fusion protein linking the ligand-binding domain of either RXR or RAR to the yeast Gal4 DNA-binding domain, and the in situ detection of ligand-activated effector proteins by using an inducible transgenic lacZ reporter gene. We detect receptor activation in the spinal cord in a pattern that indicates that the receptor functions in the maturation of limb-innervating motor neurons. Our results reveal a specific activation pattern of Gal4–RXR which indicates that RXR is a critical bona fide receptor in the developing spinal cord.