Nurr1 is a transcription factor, a member of the superfamily of nuclear hormone receptors. To investigate the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Subsequent analysis revealed the absence of neurotransmitter dopamine, dopamine biosynthesizing enzymes, transporters and receptors for dopamine utilization in the central dopaminergic area of newborn pups. We have also shown that the neuroepithelial cells undergo normal ventralization and migration. These dopaminergic neuron precursors express general neuronal markers such as a neuronal nuclear marker and project to the striatum. The TUNEL assay showed no difference in the number of apoptotic cells between Nurr1-null and wild type mice. Hence, our results show that the transcription factor Nurr1 is required for terminal maturation of mesencephalic dopaminergic neurons, while genesis of dopaminergic precursors, their survival, and selection of specific target innervation are not affected at the time of birth in mice lacking Nurr1 gene function. In order to investigate the role of Nurr1, a procedure for primary neuronal cultures was established. Recently, using primary midbrain neuronal cultures from newborn Nurr1 knockouts, we have shown that a population of neurons could be induced to express tyrosine hydroxylase, a key enzyme in dopamine biosynthesis, in the presence of forskolin with a synergistic increase in the number of tyrosine hydroxylase expression neurons when combined with brain-derived neurotrophic factor and dopamine. These data indicate that midbrain neurons from Nurr1 knockout pups retain the capacity for the induced expression of tyrosine hydroxylase even though in vivo tyrosine hydroylase expression is absent. Thus, the factors, such as forskolin, brain-derived neurotrophic factor, and dopamine induce tyrosine hydroxylase expression via a pathway independent of Nurr1.Recombinant adenoviruses provide a versatile system for gene expression studies. The Nurr1 sequence, wild type and mutated, were used to generate recombinant adenoviruses. Recombinant adenovirus-Nurr1 was employed to identify the Nurr1 target genes in PC12 cells. These cells are of neuronal origin and synthesize dopamine. In addition in these cells endogenous expression of Nurr1 can be induced by several factors suggesting a physiological role of Nurr1 in these cells. The PC12 cells were infected with recombinant virus, followed by flow-activated cell sorting analysis, resulting in a homogenous population of infected cells. Two dimensional gel analyses revealed several protein spots affected by the presence of recombinant wild Nurr1 virus. An attempt will be made to identify the protein composition of these spots using mass spectrophotometry. The results should provide information on Nurr1 target genes. Continuing to investigate the mechanism(s) of action of the steroid/thyroid hormone receptor superfamily, we isolated a novel cofactor from a rat embryonic brain library using a yeast two hybrid system and a thyroid hormone receptor as bait. Sequence analysis revealed the presence of two leucine-rich motifs, a signature of cofactors of the nuclear receptors. This cofactor is highly expressed in rat testis in the spermatogonia and Leydig cells and in the hippocampus. In vitro binding assays revealed that a point mutation in the leucine motif interferes with estrogen receptor interaction. Consequently, this cofactor represses estrogen inducibility of the reporter gene. Interaction of this cofactor with other members of the superfamily is under investigation. """"""""
