Studies On The Function Of Nuclear Receptor-related Factor 1 And The Screening Of Its Regulated Genes

Background: Midbrain DA neurons, with their cell bodies localized in substantia nigra and the ventral tegmental area, play critical roles in the central regulation of motor and motivational behaviors. Dysfunction of the dopaminergic system of midbrain may lead to dopaminergic system diseases such as Parkinson disease (PD) and drug dependence (or drug addiction). DA is generated from the amino acid tyrosine in two enzymatic steps. The first rate-limiting step is mediated by tyrosine hydroxylase (TH), converting tyrosine to the DA precursor L-3, 4-dihydroxyphenylalanine (L-DOPA), which is converted to DA by L-amino acid decarboxylase (AADC). L-DOPA, which passes the blood brain barrier and is metabolized to DA by endogenous AADC, is the main treatment in PD. Nuclear receptor-related factor 1 (Nurr1/NR4A2/NOT/RNR-1/HZF-3) belongs to the family of nuclear receptors also including receptors for steroid hormones, retinoids, vitamin D, and thyroid hormone. Nurr1 is expressed predominantly in the central nervous system, especially in substantia nigra (SN), ventral tegmental area (VTA), midbrain and limbic areas. Several studies have indicated that Nurr1 is essential for development, migration and survival of dopaminergic neurons. Defects in Nurr1 or altered expression of the gene in SN have been found in association with PD and certain psychiatric disorders, such as schizophrenia, manic behavior, and predisposition to cocaine addiction. Hence, Nurr1 has been already considered as the candidate gene in the mechanism of PD and drug dependence. However, the role of Nurr1 in development and differentiation of dopaminergic neurons has not been elucidated. Especially, there are some disputes in the regulation of Nurr1 on the transcription of TH and maturation of dopaminergic neurons. In addition, our recently studies have indicated that GABA transporter-1 (GAT1) may play a critical role in morphine addiction and ethanol preference. However, the mechanism of these drugs addiction is still not elucidated. There are some evidences that Nurr1 is important for reward mechanisms in the reinforcement of ethanol or cocaine addiction. So, we propose that Nurr1 might play a same role in GAT1 related drug dependence.Materials and methods: Our study was divided into four parts. The first and second parts were all finished in vitro as following steps. (1) The first part was included in the followingthree steps. Firstly, we cloned the mouse Nurr1 gene by RT-PCR, and then it was been subcloned into multiple cloning site of pEGFP-C1 plasmid. Secondly, after being identified by digestion of restriction enzyme and sequencing, the right clones of Nurr1 expressing vector were transfected into MN9D cells with Lipofectamine^? 2000. Finally, the mRNA and total protein were extracted from the stably transfected MN9D cells, Nurr1 and TH mRNA or protein expression was detected using semi-quantitive RT-PCR, Real Time PCR and Western blot. Meanwhile, the neurite extension of MN9D cells was observed and photographed with inversion fluorescent microscope. (2) The second part included three steps too. Firstly, two target sequences in the middle of Nurr1 gene and one target sequence of unrelated gene as a negative control were selected, and then two complementary 72 mer oligonucleotides of each sequence were synthesized with the optimal 3' overhanging nucleotides. After annealed, the inserts were ligated into the linearized pSilenCircle plasmid. After transformation and amplification, the vectors were purified and identified by gel electrophoresis with restriction enzyme and sequencing. Secondly, the right clone of the constructed pSC-N1, pSC-N2 vectors and the vector of negative control were transfected into MN9D cells and selected with G418 for two weeks. Finally, Nurr1 and TH mRNA or protein expression in different groups were detected using semi-quantitive RT-PCR, Real Time PCR and Western blot. At the same time, the neurite extension of MN9D cells was observed and photographed. (3) The third part included in the following two parts: Firstly, to identify the correctness of the homologous recombination further, GAT1 protein expression in hippocampus were detected in nine GAT1 knockout mice (wide-type, heterozygous and homozygous are three respectively) using western blotting and immunohistochemistry. Secondly, using Real Time RT-PCR technique and immunohistochemistry method, we observed the changes of mRNA and protein expression of Nurr1 in the stratum and midbrain in GAT1 knockout mice (wide-type, heterozygous and homozygous are three respectively). (4) The fourth part was employed by using Affymetrix oligonucleotide microarrays to investigate changes in gene expression induced by Nurr1 in the MN9D cells. In this part, MN9D cells of the study group (Nurr1-overexpression MN9D cells) and the control group (Nurr1-downregulated MN9D cells by RNA interference) were cultured and the total RNA was extracted. After reverse transcription, doubled stranded cDNA was synthesized and then was further done to produce biotin-labeled cRNA. The cRNA was fragmented and hybridized. Immediately following hybridization, the probe array undergoes an automated washing and staining protocol on the fluidics station. After that, it was scaned and analyzed with GCOS software.Results: The following are the results of the experiments in three parts. (1) Enzyme digestion analysis and DNA sequencing confirmed that the Nurr1 expressing vectors were constructed successfully. Nurr1 mRNA and protein expression in MN9D cells was significantly increased in Nurr1-overexpression MN9D cells. Meanwhile, the expression of TH mRNA and protein is also increased significantly. Moreover, TH expression was upregulated more significantly. The average numbers, average process length and total process length of the neurite extension increased more significantly in Nurr1 overexpression MN9D cells and it is suggested that Nurr1 could induce cell maturation and morphological differentiation, characterized by neurite extension. (2) Enzyme digestion analysis and DNA sequencing confirmed that the Nurr1 or unrelated gene shRNA expression vectors were constructed successfully. Nurr1 mRNA expression in MN9D cells was specifically suppressed after the transfection of pSC-N1 and pSC-N2, the silencing effect is 62.3% and 45.6% respectively using Real Time RT-PCR. The downstream gene TH mRNA is also suppressed significantly and the silencing effect is 76.3% and 62.6% respectively. Meanwhile, the protein expression of Nurr1 and TH were suppressed too, and also the TH protein expression was downregulated more significantly. The silencing effect of Nurr1 and TH protein is 57.4%, 72.0% and 79.1%, 70.1% respectively. While the negative control and liposome has no effect on the mRNA or protein expression of the two genes. Neurite extension of Nurr1 RNA interference group was shorter but has no statistic significantly. (3) The expression of GAT1 protein is deficient in hippocampus in both heterozygous and homozygous mice, while its expression is normally in wide-type mice; it indicates the correctness of GAT1 homologous recombination. Compared to wide-type mice, Nurr1 mRNA expression in stratum is increased significantly in both heterozygous and homozygous mice (p<0.05), while Nurr1 mRNA in the midbrain of the homozygous mice is increased more significantly (p<0.01). Meanwhile, Nurr1 immune positive cells in stratum increased more significantly in both heterozygous and homozygous mice than those in wide-type mice by using immunohistochemistry method (p<0.01). Although Nurr1 immune positive cells in midbrain increased slightly in both heterozygous and homozygous mice, it has no statistic significantly (p>0.05). Moreover, the staining of Nurr1 immune positive cells in midbrain of homozygous mice and in stratum of heterozygous mice is extremely deep than other mice. (4) Eighty genes were found to be upregulated and eight genes downregulated in the study. They mainly encode proteins for signal transduction, development and differentiation, biosynthesis, metabolite, inflammation and immune response, etc. In addition, there are 22 genes encode proteins whose function have not yetbeen known.Conclusions: (1) Nurr1 overexpression in MN9D cells could promote the expression of TH mRNA and protein and increase neurite extension of the cells. It suggested that Nurr1 was essential for development of dopaminergic neurons and played a major role in the maintenance of the maturation of dopaminergic neurons. The fact that Nurr1 is important for the development of DA neurons has important implications in the gene therapy of PD, as these cells regulate motor control and their degeneration in PD. (2) Nurr1 shRNA expressing vectors had been successfully constructed and could specifically suppress mRNA and protein expression of Nurr1. Meanwhile it could downregulate the expression of TH more significantly. Therefore, it could be concluded that Nurr1 has a critical role in the transcription of TH in dopaminergic neurons. So, Nurr1 specific shRNA expression vector may provide a novel applicable strategy for the function study of genes associated with PD and the development of dopaminergic neuron. (3) The increasing expression of Nurr1 mRNA and protein in stratum and midbrain maybe a protective responses to disequilibrium of GABA system due to the deficient of GABA-transporter 1. Nurr1 could enhance the reward mechanism in the reinforcement of ethanol and cocaine dependence and it might play a positive role in the GAT1 related drug dependence. Moreover, Nurr1 might be a link between DA system and GABA system in the mechanism of drug dependence. (4) The results indicate that Nurr1 might be involved in the regulation of those signal transduction, development and differentiation, biosynthesis and metabolite, etc. Although it needs to be further studied, the data provides clues for further scrutiny into the role of Nurr1 and its regulated genes in the dopaminergic cells.