Studies in nuclear receptor Nurr1 : identification of Nurr1-regulated genes

Abstract

The nuclear receptor family comprises more than sixty members, including receptors for steroids, thyroid hormone and retinoids. Many nuclear receptors function as ligand- activated transcription factors that regulate the expression of specific target genes. The family also includes nuclear receptors that lack identified ligands, and these receptors are therefore referred to as orphan receptors. It has recently been shown that some of these orphan receptors are ligand- independent.

Nurr1 (NR4A2) is a constitutively active nuclear receptor that belongs to this category of nuclear receptors. Nurr1 is expressed in the central nervous system (CNS) from early embryogenesis into adulthood. Interestingly, Nurr1 is an early expression marker of midbrain dopamine (DA) cells, and gene targeting of Nurr1 in mice leads to agenesis of midbrain DA neurons. The fact that Nurr1 is important for the development of DA neurons has important implications, as these cells regulate motor control and their degeneration is the cause of Parkinson's disease. The main focus of this thesis was to identify genes that are regulated by Nurr1 and in this way increase our understanding of the role of this transcription factor in DA cells and in other cells that express Nurr1. We used the DA cell line MN9D, which store and produce DA. Expression of Nurr1 in this cell line resulted in an increased DA content of the cells. Furthermore, Nurr1 increased the expression of the DA producing enzyme aromatic L-amino acid decarboxylase (AADC) and the DA transporter vesicular monoamine transporter 2 (VMAT2). Expression analyses showed that the levels of expression of AADC and VMAT2 were deregulated in developing midbrain DA cells of mice in which the Nurr1 gene had been selectively disabled, which suggests that Nurr1 plays a role in DA production and storage.

Using cDNA microarrays, we investigated changes in gene expression induced by Nurr1 in the MN9D cells. This analysis revealed that neuropilin-1 (Nrp1), a receptor for secreted neuronal guidance polypeptides, is regulated by Nurr1 in the cells. Furthermore, Nurr1 regulated the Nrp1 promoter in reporter gene assays. In situ hybridization experiments revealed that Nrp1 expression was diminished in Nurr1 knockout mice in the dorsal motor nucleus of the vagus nerve, which suggests that Nurr1 plays a role in the regulation of Nrp1 in vivo. We used MN9D cells to study the effects of Nurr1 on DA cell differentiation. Expression of Nurr1 in the MN9D cells induced cell cycle arrest and morphological differentiation, characterized by neurite extension. We determined the functional requirements for Nurr1-induced differentiation using different Nurr1 derivatives, and the results provide valuable information about the functional role of Nurr1 in vivo. We elucidated the function of Nurr1 in developing DA cells by experiments on embryonic ventral midbrains cultured in vitro." Expression of the DA cell marker tyrosine hydroxylase, which is never expressed in the ventral midbrains of Nurr1 knockouts in vivo, could be induced in the knockout cultures. Moreover, well-defined nerve fiber bundles were formed in wild-type cultures, but did not form in Nurr1-deficient cultures, giving further evidence that Nurr1 is important for target innervation. To understand the role of Nurr1 in adult physiology we investigated the impact of a heterozygous deletion of Nurr1 on rewarding behavior in mice. Mice heterozygous for the Nurr1 gene had a lower tendency than wild-type mice to become dependent on ethanol drinking and wheel running, which suggests that Nurr1 is important for reward mechanisms.