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.

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 (

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.

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 longterm 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 transretinoic 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.V itamin A and its derivatives (the retinoids) activate signaling pathways necessary for development, differentiation, and homeostasis of several tissues, including the nervous system (1, 2). Known impairments caused by the lack of dietary vitamin A include blindness, infertility, embryonic malformations, and compromised immunity. Vitamin A deficiency (VAD) is currently a risk for over 100 million children in over 75 countries, and results in nearly 3.2 million associated childhood deaths annually (refs. 3 and 4, and http:͞͞www.unicef.org͞sowc98).The biological effects of retinoids are mediated by retinoid receptors, a subgroup of the nuclear receptor superfamily. The retinoid receptor family includes the retinoic acid receptors (RARs; ␣, ␤, and ␥), which bind all trans-retinoic acid and 9-cis retinoic acid, and the retinoid X receptors (RXRs; ␣, ␤, and ␥), which bind 9-cis retinoic acid only. RAR͞RXR heterodimers, and to some extent RXR homodimers, act as transcription factors by binding to retinoid response elements in the promoters of target genes and activating gene expression in the presence of ligand (1, 4, 5). Multiple combinations of RAR͞RXR heterodimers are possible, depending on the overlapping expression of receptor subtypes within tissues (1).Each RAR and RXR exhibits a specific expression pattern in the adult central nervous system (CNS), distinct from that found in the developing nervous system (2, 6-10), indicating that, in addition to the modulation of neuronal development during embryogenesis, retinoids are likely to regulate activities in the mature brain. Supporting this notion, evidence suggests that RAR␤, RXR␤, and RXR␥ modulate locomotor behavior by regulating the expression of dopamine receptors in the adult striatum (11). Additionally, retinoic acid production is required in distinct regions of the adult songbird brain for song maturation, a learned behavior (12). Moreover, we recently found that RAR␤ ϪϪ ...

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.

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 Dolle, 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.

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. O ur understanding of the biological functions of nuclear receptors has been hampered by difficulties in precisely localizing the cells and tissues in which a given nuclear receptor is active. To enable such analyses, we have recently used an assay in transgenic mice, which is based on the detection of a reporter gene induced by an effector protein, expressed under the control of an efficient transgenic promoter (1). The effector is a fusion protein consisting of the DNA-binding domain of the yeast transcription factor GAL4, linked in frame with the ligandbinding domain of the studied receptor. The reporter gene is a bacterial lacZ gene preceded by a promoter containing multiple binding sites for GAL4. Thus, activated effector protein will induce expression of the reporter gene in vivo. The assay has been used to study the activities of retinoic acid receptor (RAR) and the retinoid X receptor in vivo (1).Although useful for identifying sites of nuclear receptor activation, the strategy has limitations; in our previous experiments, the effector transgene was expressed under the control of a tissue-specific promoter, thus limiting the sites and stages where activated effector protein can be detected. A ubiquitous transgenic promoter driving the expression of the GAL4 effector protein would allow reporter gene detection in a broader range of tissues. The potential problem of phenotypic side effects, however, suggests that it would be beneficial to specify the expression to tissues in which the receptor is naturally signaling. Moreover, it is advantageous to combine the study of different effector and reporter genes in a single line of transgenic mice...

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