Nurr1 is a transcription factor that structurally resembles members of the superfamily of nuclear hormone receptors. Nurr1 expression appears to be predominantly brain specific detected in embryonic stages with the peak expression at birth or soon thereafter. To investigate the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Nurr1-null mice appear to develop normally but die within 12 hours of birth. Subsequent analysis revealed the absence of neurotransmitter dopamine, dopamine biosynthetizing enzymes, transporters and receptors for dopamine utilization in the central dopaminergic area of newborn pups. We have also shown that in the absence of Nurr1, 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. Recently, we have also detected the expression of another dopaminergic marker, homeobox gene Ptx3, a member of the Ptx subfamily. Furthermore, using double in situ hybridization we detected the message for cholecystokinin. This brain peptide coexists in 80% tyrosine hydroxylase positive dopaminergic neurons in the wild type brain. In addition, using the NIH image program we quantitatively analyzed the TUNEL positive nuclei in the ventral midbrain of Nurr1 null mice. No difference was detected in the number of apoptotic cells between Nurr1-null mice 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 in other areas of the brain, a procedure for primary neuronal cultures was established. The system includes midbrain cultures with dopaminergic neurons, olfactory bulb and hippocampal neuronal cultures. Other techniques such as detection of dopamine using high pressure liquid chromatography, immunohistochemistry, in situ hybridization, and in situ cell death detection using the TUNEL reaction in primary brain cultures were setup as well. Currently, these techniques are being utilized to examine the differential expression of various genes and cell survival in primary brain cultures prepared from Nurr1 knockout pups compared to wild-type controls. Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. Having primary brain cultures on hand, the aim is to introduce the Nurr1 gene into midbrain cultures and subsequently into the brain of Nurr1 knockout mice to further study the Nurr1 function. Nurr1 sequences, wild type and mutated, were used to generate recombinant adenoviruses. Viral production was followed with green fluorescent protein. In parallel, we are further investigating the physiological role of Nurr1, using PC12 cells. Preliminary results have shown that in PC12 cells, endogenous Nurr1 expression can be induced by several factors. For our in vitro studies we have constructed and will purify hemagluttinin tagged Nurr1 (HA-Nurr1) from HA-Nurr1 overexpressing bacteria. Purified HA-Nurr1 will be used to define and characterize the DNA target of Nurr1 using footprint and mobility shift experiments. The identity of any Nurr1 accessory proteins, such as RXR, will be investigated by immunoprecipitation and supershift studies. It has been proposed that a Nurr1 ligand is secreted by midbrain specific astrocytes. We will add fractionated conditioned glial cell media to mobility shift experiments attempting to identify the ligand. - nuclear receptors; Nurr1; dopamine biosynthesis: midbrain neurons
