Finding the ligand : retinoid receptor activation in the CNS

Nuclear hormone receptors (NRs) comprise a large family of ligand activated transcription factors, found in vertebrates, arthropods and nematodes. Among the members of the NR family are the receptors for small, lipophilic ligands such as the steroid hormones, vitamins A and D, thyroid hormone oxysterols and bile acids. These lipophilic molecules easily pass through the plasma membrane and enter cells where they encounter and bind their cognate receptor, thereby affecting transcription of target genes.

Retinoids, the biologically active vitamin A metabolites, have been shown to be essential in processes such as organogenesis, neurogenesis, vision and reproduction. The two most important retinoids m vivo, namely all-trou retinoic acid (atRA) and 9-cis retinoic acid (9cRA), exert their functions by binding and activating two classes of NRs, the retinoic acid receptors (RARs) and the retionid X receptors. Knockout studies of die genes encoding RARs and RXRs fully recapitulate the phenotypes seen in mice lacking vitamin A, confirming the receptors' roles in transducing retinoid signals. In addition, RXR also functions as a heterodimer partner for several other NRs. For example, RARs, thyroid hormone receptors (TRs), vitamin D receptor (VDR) and several orphan receptors, are known to mediate their function as heterodimers with RXRs.

An important issue to better ~stand the roles played by nuclear receptors, is to establish ~c the receptor is active in vivo. We have previously developed a transgenic mouse model, where RAR and RXR activity in mouse embryos was directly reflected by the transcription of a 1acZ reporter gene. Here, we present a refined version of the so called effector-reporter system (referred to as die FIND expression system), where ligand-activated, receptor not only will induce the expression of the reporter, but also of the receptor itself in an auto-inductive loop. Transgenic mice expressing GAL4-RAR were analyzed, and the resulting reporter expression pattern is consistent with our previous data. Strong expression was detected m the developing spinal cord, forebrain and pro~ for~ buds. In addition, reporter activation was detected at the midbrain/hindbrain boundary, a region not previously reported to contain retinoids at this stage. The FIND expression system can potentially be used with any given NR of interest and should provide a powerful tool when searching for novel NR ligands.

Expression of the homeobox gene Meis2 is enriched in a ventral structure of the developing forebrain (the lateral ganglionic eminence, LGE) and is induced by retinoic acid in PC12 cells. Thus, we became interested in investigating a potential role for retinoids during the development of the forebrain. By making use of the effector-reporter assay described above we show that radial glial cells within the LGE are responsible for producing retinoids. Hence, we suggest a model where radial glia not only play a vital role as guiding cells in neuronal migration, but also as direct contributors to differentiation of LGE neurons by local retinoid synthesis.

The importance of retinoids during development is well established, whereas their role in postnatal and adult tissues is less clear. By studying the expression of all known retinoid binding proteins and retinoid receptors, we show that most of these proteins are still expressed after birth. Co- culture experiments show that postnatal striatum but not hippocampus and cortex, produces retinoids, implicating retinoids as important factors in this structure also after birth.

A surprising result when analyzing different adult brain tissues for RAR and RXR activation, was that most tissues activated RXR stronger than RAR. The fact that 9cRA, the natural ligand for RXR, has been difficult to detect in vivo prompted us to go ahead and purify the RXR ligand(s) present in the adult brain. Biochemical isolation, purification and mass spectrometry thus identified the activity as docosahexaenoic acid (DHA), a polyunsaturated fatty acid highly enriched in postnatal central nervous system. Deficiency studies have shown that DHA is essential for normal growth and development, as deprivation of this fatty acid leads to neurological problems, learning disabilities and growth retardation. Certain RXR mutant mice display similar defects and both DHA and RXR ligands have beneficial effects on blood cholesterol levels and insulin sensitivity. We therefore suggest that RXR activation by DHA is a novel signaling mechanism, which may m part explain how DHA influences neural processes and maturation of the mammalian brain.

List of scientific papers

I. Mata De Urquiza A, Solomin L, Perlmann T (1999). "Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice. " Proc Natl Acad Sci U S A 96(23): 13270-5
https://pubmed.ncbi.nlm.nih.gov/10557310

II. Toresson H, Mata de Urquiza A, Fagerstrom C, Perlmann T, Campbell K (1999). "Retinoids are produced by glia in the lateral ganglionic eminence and regulate striatal neuron differentiation." Development 126(6): 1317-26
https://pubmed.ncbi.nlm.nih.gov/10021349

III. Zetterstrom RH, Lindqvist E, Mata de Urquiza A, Tomac A, Eriksson U, Perlmann T, Olson L (1999). "Role of retinoids in the CNS: differential expression of retinoid binding proteins and receptors and evidence for presence of retinoic acid. " Eur J Neurosci 11(2): 407-16.
https://pubmed.ncbi.nlm.nih.gov/10051741

IV. Mata de Urquiza A, Liu S, Sjoberg M, Zetterstrom RH, Griffiths W, Sjovall J, Perlmann T (2000). "Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain." Science 290: 2140-44