Role of c-Myc haploinsufficiency in the maintenance of HSCs in mice.

This study was conducted to determine the dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/ß-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence and by promoting apoptosis. We have identified Nr4a1, Nr4a2, and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2, and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediates the function of c-Myc in regulating HSC quiescence, whereas all 3 genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/ß-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induces a blockage of erythroid lineage differentiation through promoting secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc-heterozygous mice and to significantly prolong the survival of those mice. Furthermore, we showed that c-Myc-mediated Apc loss induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc-deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.

Role of the Orphan Nuclear Receptor NR4A Family in T-Cell Biology.

The nuclear orphan receptors NR4A1, NR4A2, and NR4A3 are immediate early genes that are induced by various signals. They act as transcription factors and their activity is not regulated by ligand binding and are thus regulated via their expression levels. Their expression is transiently induced in T cells by triggering of the T cell receptor following antigen recognition during both thymic differentiation and peripheral T cell responses. In this review, we will discuss how NR4A family members impact different aspects of the life of a T cell from thymic differentiation to peripheral response against infections and cancer.

Correlation between Nurr1 expression and drug resistance in the brain of rats with epilepsy.

OBJECTIVE: To investigate the correlation between nuclear receptor related 1 (Nurr1) expression and drug resistance in the brain of rats with epilepsy. MATERIALS AND METHODS: A total of 60 adult male Sprague-Dawley rats were selected, and the animal model of epilepsy was established by electrical stimulation. These rats were randomly divided into the control group and the drug-resistant group. The model of drug-resistant epilepsy was screened with phenytoin (PHT) and phenobarbital (PB); the hippocampus and temporal lobe cortex tissues were isolated from rats; the messenger ribonucleic acid (mRNA) and protein levels of Nurr1 in the hippocampus and cortex tissues of the two groups of rats were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, respectively. The budding tissue marker growth-associated protein-43 (GAP43) in hippocampus tissues were labeled by immunofluorescence staining; the mRNA and protein levels of indicators related to drug-resistant epilepsy, including leukemia-associated phenotype (LAP), multi-drug resistance protein (MRP), P-glycoprotein (P-gp) and monocyte chemoattractant protein-1 (MCP-1), were further detected. Pearson correlation analysis was used to analyze the correlations of the protein level of Nurr1 with drug-resistant indicators. RESULTS: The mRNA and protein levels of Nurr1 in hippocampus tissues of the drug-resistant group were significantly increased compared with those of the control group (p < 0.05). In temporal lobe cortex tissues, there was no significant difference in the mRNA level of Nurr1 between the control group and the drug-resistant group (p > 0.05). The immunohistochemistry results showed that the fluorescence intensity of GAP3 in hippocampus tissues of the drug-resistant group was significantly higher than that of the control group. The mRNA and protein levels of epilepsy-related indicators, LAP, MRP, P-gp and MCP-1 in hippocampus tissues of drug-resistant group were significantly up-regulated compared with those of the control group (p < 0.05). Pearson correlation analysis indicated that the protein level of Nurr1 in hippocampus tissues was positively correlated with those of LAP, MRP, P-gp and MCP-1 in the corresponding regions (p < 0.05). CONCLUSIONS: Nurr1 enhances the drug resistance of epilepsy in rats by up-regulating the expression of proteins related to drug resistance.

Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation.

Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.

Role of Nurr1 in the Generation and Differentiation of Dopaminergic Neurons from Stem Cells.

NURR1 is an essential transcription factor for the differentiation, maturation, and maintenance of midbrain dopaminergic neurons (DA neurons) as it has been demonstrated using knock-out mice. DA neurons of the substantia nigra pars compacta degenerate in Parkinson's disease (PD) and mutations in the Nurr1 gene have been associated with this human disease. Thus, the study of NURR1 actions in vivo is fundamental to understand the mechanisms of neuron generation and degeneration in the dopaminergic system. Here, we present and discuss findings indicating that NURR1 is a valuable molecular tool for the in vitro generation of DA neurons which could be used for modeling and studying PD in cell culture and in transplantation approaches. Transduction of Nurr1 alone or in combination with other transcription factors such as Foxa2, Ngn2, Ascl1, and Pitx3, induces the generation of DA neurons, which upon transplantation have the capacity to survive and restore motor behavior in animal models of PD. We show that the survival of transplanted neurons is increased when the Nurr1-transduced olfactory bulb stem cells are treated with GDNF. The use of these and other factors with the induced pluripotent stem cell (iPSC)-based technology or the direct reprogramming of astrocytes or fibroblasts into human DA neurons has produced encouraging results for the study of the cellular and molecular mechanisms of neurodegeneration in PD and for the search of new treatments for this disease.

Correlation between orphan nuclear receptor Nurr1 expression and amyloid deposition in 5XFAD mice, an animal model of Alzheimer's disease.

The functional roles of the orphan nuclear receptor, Nurr1, have been extensively studied and well established in the development and survival of midbrain dopamine neurons. As Nurr1 and other NR4A members are widely expressed in the brain in overlapping and distinct manners, it has been an open question whether Nurr1 has important function(s) in other brain areas. Recent studies suggest that up-regulation of Nurr1 expression is critical for cognitive functions and/or long-term memory in forebrain areas including hippocampal formation. Questions remain about the association between Nurr1 expression and Alzheimer's disease (AD) brain pathology. Here, using our newly developed Nurr1-selective antibody, we report that Nurr1 protein is prominently expressed in brain areas with Aß accumulation, that is, the subiculum and the frontal cortex, in the 5XFAD mouse and that Nurr1 is highly co-expressed with Aß at early stages. Furthermore, the number of Nurr1-expressing cells significantly declines in the 5XFAD mouse in an age-dependent manner, accompanied by increased plaque deposition. Thus, our findings suggest that altered expression of Nurr1 is associated with AD progression. Using our newly developed Nurr1-selective antibody, we show that Nurr1 protein is prominently expressed in brain areas accumulating amyloid-beta (Aß) in the transgenic mouse model of Alzheimer's disease (AD) and that Nurr1 is highly co-expressed with Aß at early stages (upper panel). Furthermore, in the AD brain the number of Nurr1-expressing cells significantly declines in an age-dependent manner concomitant with increased Aß accumulation (lower diagram) highlighting a possible Nurr1 involvement in AD pathology.

Low dose bexarotene treatment rescues dopamine neurons and restores behavioral function in models of Parkinson's disease.

Nurr1 is a nuclear hormone receptor (NucHR) strongly implicated in the growth, maintenance, and survival of dopaminergic neurons. Nurr1 may be unable to bind ligands directly, but it forms heterodimers with other NucHRs that do. Using bioluminescence resonance energy transfer (BRET) assays to directly monitor interactions of Nurr1 with other NucHRs, we found the cancer drug bexarotene (Targretin, also LGD1069) displayed biased interactions with Nurr1-RXR heterodimers compared with RXR-RXR homodimers. Remarkably, at doses up to 100-fold lower than those effective in rodent cancer models, bexarotene rescued dopamine neurons and reversed behavioral deficits in 6-hydroxydopamine (6-OHDA) lesioned rats. Compared to the high doses used in cancer therapy, low doses of bexarotene have significantly milder side effects including a reduced increase in plasma triglycerides and less suppression of thyroid function. On the basis of extrapolations from rat to human doses, we hypothesize that low oral doses of bexarotene may provide an effective and tolerated therapy for Parkinson's disease (PD).

Vitamin D and Parkinson's disease.

Parkinson's disease (PD) is the second most common form of neurodegeneration among the elderly population. PD is clinically characterized by tremors, rigidity, slowness of movement, and postural imbalance. Interestingly, a significant association has been demonstrated between PD and low levels of vitamin D in the serum, and vitamin D supplement appears to have a beneficial clinical effect on PD. Genetic studies have provided the opportunity to determine which proteins link vitamin D to PD pathology, e.g., Nurr1 gene, toll-like receptor, gene related to lipid disorders, vascular endothelial factor, tyrosine hydroxylase, and angiogenin. Vitamin D also exerts its effects on cancer through nongenomic factors, e.g., bacillus Calmette-Guerin vaccination, interleukin-10, Wntß-catenin signaling pathways, mitogen-activated protein kinase pathways, and the reduced form of the nicotinamide adenine dinucleotide phosphate. In conclusion, vitamin D might have a beneficial role in PD. Calcitriol is best used for PD because it is the active form of the vitamin D(3) metabolite and modulates inflammatory cytokine expression. Further investigation with calcitriol in PD is needed.

The function and mechanisms of Nurr1 action in midbrain dopaminergic neurons, from development and maintenance to survival.

Nurr1 is critical for the development and maintenance of midbrain dopaminergic (DA) neurons in mouse. Loss of Nurr1 function early during development in mice leads to the absence of midbrain DA neurons. Reduction of Nurr1 function in adulthood leads to a slowly progressive loss of striatal DA and markers for DAergic neurons, supporting its selective roles in the maintenance of DAergic neuronal survival and function. To understand the molecular mechanisms of Nurr1 action, our group has identified VIP as a potential target gene of Nurr1. Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. Nurr1 loss of function leads to the decrease of VIP mRNA level in developing midbrain, suggesting that Nurr1 is involved in the in vivo regulation of VIP expression in midbrain. Our group has also cloned a novel protein interactor for Nurr1. We identified a family of gene products that interact and regulate the activity of Nurr1 by screening yeast two-hybrid library and termed the longest splicing form, NuIP. In vivo NuIP protein is largely colocalized with Nurr1 in adult midbrain dopaminergic neurons. NuIP interacts and positively regulates the activity of Nurr1 protein and could also possibly mediate cross talk between Nurr1 and GTPase mediated signaling pathways. Other recently identified potential target genes and interacting proteins of Nurr1 are also summarized and discussed in this review.

Nurr1 regulates RET expression in dopamine neurons of adult rat midbrain.

Genesis of midbrain dopamine (DA) neurons depends on Nurr1, a nuclear receptor expressed during development and adulthood in these neurons. Nurr1 is required for the expression of genes of dopaminergic phenotype such as tyrosine hydroxylase and DA transporter. The expression of the tyrosine kinase receptor RET also depends on Nurr1 during development. However, it is unknown whether RET expression is regulated by Nurr1 during adulthood, and the mechanism by which Nurr1 regulates RET expression. Using an adeno-associated vector-delivered anti-Nurr1 ribozyme, we knocked-down Nurr1 expression unilaterally in the substantia nigra (SN) of adult rats. Animals injected with the ribozyme displayed a 57.3% decrease in Nurr1 mRNA in the SN accompanied by decreased DA extracellular levels in the striatum. RET mRNA in the injected SN and RET protein in the ipsilateral striatum decreased 76.9% and 47%, respectively. Tyrosine hydroxylase and DA transporter mRNA did not change in Nurr1 knocked-down SN. Nurr1 induced the transcription of the human RET promoter in cell type and concentration-dependent manner. Nurr1 induction of RET promoter is independent of NBRE elements. These results show that the expression of RET in rat adult SN is regulated by Nurr1 and suggest that RET is a transcriptional target of this nuclear receptor.

Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons.

Transcription factors involved in the specification and differentiation of neurons often continue to be expressed in the adult brain, but remarkably little is known about their late functions. Nurr1, one such transcription factor, is essential for early differentiation of midbrain dopamine (mDA) neurons but continues to be expressed into adulthood. In Parkinson's disease, Nurr1 expression is diminished and mutations in the Nurr1 gene have been identified in rare cases of disease; however, the significance of these observations remains unclear. Here, a mouse strain for conditional targeting of the Nurr1 gene was generated, and Nurr1 was ablated either at late stages of mDA neuron development by crossing with mice carrying Cre under control of the dopamine transporter locus or in the adult brain by transduction of adeno-associated virus Cre-encoding vectors. Nurr1 deficiency in maturing mDA neurons resulted in rapid loss of striatal DA, loss of mDA neuron markers, and neuron degeneration. In contrast, a more slowly progressing loss of striatal DA and mDA neuron markers was observed after ablation in the adult brain. As in Parkinson's disease, neurons of the substantia nigra compacta were more vulnerable than cells in the ventral tegmental area when Nurr1 was ablated at late embryogenesis. The results show that developmental pathways play key roles for the maintenance of terminally differentiated neurons and suggest that disrupted function of Nurr1 and other developmental transcription factors may contribute to neurodegenerative disease.

Generation of dopamine neurons with improved cell survival and phenotype maintenance using a degradation-resistant nurr1 mutant.

Nurr1 is a transcription factor specific for the development and maintenance of the midbrain dopamine (DA) neurons. Exogenous Nurr1 in neural precursor (NP) cells induces the differentiation of DA neurons in vitro that are capable of reversing motor dysfunctions in a rodent model for Parkinson disease. The promise of this therapeutic approach, however, is unclear due to poor cell survival and phenotype loss of DA cells after transplantation. We herein demonstrate that Nurr1 proteins undergo ubiquitin-proteasome-system-mediated degradation in differentiating NP cells. The degradation process is activated by a direct Akt-mediated phosphorylation of Nurr1 proteins and can be prevented by abolishing the Akt-target sequence in Nurr1 (Nurr1(Akt)). Overexpression of Nurr1(Akt) in NP cells yielded DA neurons in which Nurr1 protein levels were maintained for prolonged periods. The sustained Nurr1 expression endowed the Nurr1(Akt)-induced DA neurons with resistance to toxic stimuli, enhanced survival, and sustained DA phenotypes in vitro and in vivo after transplantation.