Schizophrenia-relevant behaviors in a genetic mouse model of constitutive Nurr1 deficiency.

Nurr1 (NR4A2) is an orphan nuclear receptor highly essential for the dopaminergic development and survival. Altered expression of Nurr1 has been suggested as a potential genetic risk factor for dopamine-related brain disorders, including schizophrenia. In support of this, recent experimental work in genetically modified mice shows that mice with a heterozygous constitutive deletion of Nurr1 show a facilitation of the development of schizophrenia-related behavioral abnormalities. However, the behavioral characterization of this Nurr1-deficient mouse model remains incomplete. This study therefore used a comprehensive behavioral test battery to evaluate schizophrenia-relevant phenotypes in Nurr1-deficient mice. We found that these mice displayed increased spontaneous locomotor activity and potentiated locomotor reaction to systemic treatment with the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801). In addition, male but not female Nurr1-deficient mice showed significant deficits in the prepulse inhibition and prepulse-elicited reactivity. However, Nurr1 deletion did not induce overt abnormalities in other cardinal behavioral and cognitive functions known to be impaired in schizophrenia, including social interaction and recognition, spatial recognition memory or discrimination reversal learning. Our findings thus suggest that heterozygous constitutive deletion of Nurr1 results in a restricted phenotype characteristic of schizophrenia symptomatology, which primarily relates to motor activity, sensorimotor gating and responsiveness to the psychomimetic drug MK-801. This study further emphasizes a critical role of altered dopaminergic development in the precipitation of specific brain dysfunctions relevant to human psychotic disorder.

Neuronal cell replacement in Parkinson's disease.

Transplantation of foetal dopamine neurons into the striatum of Parkinson's disease patients can provide restoration of the dopamine system and alleviate motor deficits. However, cellular replacement is associated with several problems. As with pharmacological treatments, cell therapy can lead to disabling abnormal involuntary movements (dyskinesias). The exclusion of serotonin and GABA neurons, and enrichment of substantia nigra (A9) dopamine neurons, may circumvent this problem. Furthermore, although grafted foetal dopamine neurons can survive in Parkinson's patients for more than a decade, the occurrence of Lewy bodies within such transplanted cells and reduced dopamine transporter and tyrosine hydroxylase expression levels indicate that grafted cells are associated with pathology. It will be important to understand if such abnormalities are host- or graft induced and to develop methods to ensure survival of functional dopamine neurons. Careful preparation of cellular suspensions to minimize graft-induced inflammatory responses might influence the longevity of transplanted cells. Finally, a number of practical and ethical issues are associated with the use of foetal tissue sources. Thus, future cell therapy is aiming towards the use of embryonic stem cell or induced pluripotent stem cell derived dopamine neurons.

Adult mice with reduced Nurr1 expression: an animal model for schizophrenia.

The transcription factor Nurr1 (NR4A2) has been found to play a critical role in the development of midbrain dopaminergic neurons. Nurr1 heterozygous (+/-) male and female mice expressing 35-40% of normal levels of Nurr1 were generated and examined in animal models related to symptoms of schizophrenia. The Nurr1 (+/-) mice displayed hyperactivity in a novel environment, which persisted after administration of the dopamine-mimetic amphetamine and the N-methyl-D-aspartate receptor antagonist phencyclidine. The Nurr1 (+/-) mice were deficient in the retention of emotional memory and showed an enhanced response to swim stress. In addition, Nurr1 (+/-) male mice displayed a reduced dopamine turnover in the striatum and an enhanced dopamine turnover in the prefrontal cortex, while female mice showed an opposite pattern. These results show that Nurr1 (+/-) mice display a pattern of behaviors indicative of potential relevance for symptoms of schizophrenia combined with a gender-specific abnormal dopamine transmission in the striatum and prefrontal cortex, respectively. This suggests that the Nurr1 mutant mouse may be a potential animal model for studies on some of the behavioral and molecular mechanisms underlying schizophrenia.

Orphan nuclear receptor Nurr1 is essential for Ret expression in midbrain dopamine neurons and in the brain stem.

The orphan nuclear receptor Nurr1 is essential for development of midbrain dopamine (DA) cells. In Nurr1-deficient mice, DA precursor cells fail to migrate normally, are unable to innervate target areas, and only transiently express DA cell marker genes. In the search for Nurr1-regulated genes that might explain this developmental phenotype, we found that expression of the receptor tyrosine kinase Ret is deregulated in these cells of Nurr1-deficient embryos. In addition, our analyses establish Nurr1 as an early marker for the dorsal motor nucleus (DMN) of the vagus nerve. Interestingly, Ret expression is absent also in these cells in Nurr1-targeted mice. Neuronal innervation of vagus nerve target areas appeared normal apart from a subtle disorganization of the DMN-derived nerve fibers. In conclusion, regulation of Ret by Nurr1 in midbrain DA neurons and in the DMN has implications for both embryonal development and adult physiology in which signaling by neurotrophic factors plays important roles.

Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells.

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.

Vitamin A deprivation results in reversible loss of hippocampal long-term synaptic plasticity.

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 mutations in cases of schizophrenia and manic-depressive disorder.

Transgenic mice lacking the nuclear orphan transcription factor Nur-related receptor 1 (Nurr1) fail to develop mesencephalic dopamine neurons. There is a highly homologous NURR1 gene in humans (formerly known as NOT) which therefore constitutes a good candidate gene for neurologic and psychiatric disorders with an involvement of the dopamine neuron system, such as Parkinson's disease, schizophrenia, and manic-depression. By direct sequencing of genomic DNA, we found two different missense mutations in the third exon of NURR1 in two schizophrenic patients and another missense mutation in the same exon in an individual with manic-depressive disorder. All three mutations caused a similar reduction of in vitro transcriptional activity of NURR1 dimers of about 30-40%. Neither of these amino acid changes, nor any sequence changes whatsoever, were found in patients with Parkinson's disease or control DNA material of normal populations. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:808-813, 2000.

Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain.

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.

Activity of the Nurr1 carboxyl-terminal domain depends on cell type and integrity of the activation function 2.

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.

Fate of mesencephalic AHD2-expressing dopamine progenitor cells in NURR1 mutant mice.

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.

Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice.

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.

Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes.

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.

Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin.

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.

Role of retinoids in the CNS: differential expression of retinoid binding proteins and receptors and evidence for presence of retinoic acid.

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.

Retinoids are produced by glia in the lateral ganglionic eminence and regulate striatal neuron differentiation.

In order to identify molecular mechanisms involved in striatal development, we employed a subtraction cloning strategy to enrich for genes expressed in the lateral versus the medial ganglionic eminence. Using this approach, the homeobox gene Meis2 was found highly expressed in the lateral ganglionic eminence and developing striatum. Since Meis2 has recently been shown to be upregulated by retinoic acid in P19 EC cells (Oulad-Abdelghani, M., Chazaud, C., Bouillet, P., Sapin, V., Chambon, P. and Dollé, P. (1997) Dev. Dyn. 210, 173-183), we examined a potential role for retinoids in striatal development. Our results demonstrate that the lateral ganglionic eminence, unlike its medial counterpart or the adjacent cerebral cortex, is a localized source of retinoids. Interestingly, glia (likely radial glia) in the lateral ganglionic eminence appear to be a major source of retinoids. Thus, as lateral ganglionic eminence cells migrate along radial glial fibers into the developing striatum, retinoids from these glial cells could exert an effect on striatal neuron differentiation. Indeed, the treatment of lateral ganglionic eminence cells with retinoic acid or agonists for the retinoic acid receptors or retinoid X receptors, specifically enhances their striatal neuron characteristics. These findings, therefore, strongly support the notion that local retinoid signalling within the lateral ganglionic eminence regulates striatal neuron differentiation.

A selective group of dopaminergic neurons express Nurr1 in the adult mouse brain.

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.

Retinoid-X receptor signalling in the developing spinal cord.

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 receptors. Retinoids are of fundamental importance during development, 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 RAR, 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.

On CNS repair and protection strategies: novel approaches with implications for spinal cord injury and Parkinson's disease.

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.

BXR, an embryonic orphan nuclear receptor activated by a novel class of endogenous benzoate metabolites.

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 1H 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.

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.

Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.