The transcription factor Nurr1 in human NT2 cells and hNT neurons

Human, neuronally committed hNT or NT2-N cells, originally derived from the Ntera2/D1 (NT2) clone after exposure to retinoic acid (RA), represent a potentially important source of cells to treat neurodegenerative diseases. Our previous in vitro experiments showed that hNT cells possess immunocytochemically detectable markers typical of dopaminergic (DA) ventral mesencephalic (VM) neurons, including tyrosine hydroxylase (TH), dopamine transporter (DAT), dopamine receptor (D2), and aldehyde dehydrogenase (AHD-2). In the current study, we sought to examine whether Nurr1, an orphan receptor of the nuclear receptor superfamily shown to be essential for the development, differentiation and survival of midbrain DA neurons, would be expressed in 3, 4, or 5 week RA-induced hNT neurons and their NT2 precursors. Our immunocytochemical analyses indicate that NT2 cells as well as hNT neurons independent of the length of RA-driven differentiation were Nurr1-immunoreactive. RT-PCR analysis confirmed the expression of Nurr1-specific mRNA in both NT2 precursors and the hNT neurons. Furthermore, immunocytochemical co-expression of Nurr1 and TH was detected in hNT neurons. The findings of this study suggest that Nurr1 may be important during the development of hNT neurons and involved in their differentiation into the dopaminergic phenotype.     

Fibroblast growth factor-20 promotes the differentiation of Nurr1-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons

Stem cells are currently considered as alternative cell resources for restorative transplantation strategies in Parkinson's disease. However, the mechanisms that induce differentiation of a stem cell toward the dopaminergic phenotype are still partly unknown thus hampering the production of dopaminergic neurons from stem cells. In the past, FGF-20 has been found to promote the survival of ventral mesencephalic (VM) dopaminergic (DA) neurons in culture. We hereby provide evidence that FGF-20, a growth factor of the FGF family, is expressed in the adult and 6-OHDA-lesioned striatum and substantia nigra, but is not expressed by VM glia or DA neurons, suggesting that FGF-20 may work on DA neurons in a paracrine- or target-derived manner. We also found that co-culture of Nurr1-NSCs with Schwann cells overexpressing FGF-20 induced the acquisition of a neuronal morphology by the NSCs and the expression of tyrosine hydroxylase (TH) as assessed by immunocytochemistry, cell ELISA, and Western blot analysis. RT-PCR showed, that both, Schwann cells and Nurr1-NSCs (differentiated or not), expressed the FGF-1 receptor suggesting that both direct and indirect actions of FGF-20 are possible. We show that differentiated Nurr1 cells retained both neuronal morphology and TH expression after transplantation into the striatum of 6-OHDA-lesioned postnatal or adult rats, but that neuritogenesis was only observed after postnatal grafts. Thus, our results suggest that FGF-20 promotes the differentiation of Nurr1-NSCs into TH-positive neurons and that additional factors are required for the efficient differentiation of DA neurons in the adult brain.     

Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon

In neural transplantation studies, there is an interest in identifying and isolating mesencephalic dopamine (mesDA) neuron precursors that have the capacity to differentiate into fully mature mesDA neurons after transplantation. We report here that in the developing ventral mesencephalon (VM) the proneural gene Neurogenin2 (Ngn2) is expressed exclusively in the part of the ventricular zone that gives rise to the migrating mesDA neuroblasts, but not in the differentiated mesDA neurons. From other studies, we know that Ngn2 is involved in the generation of mesDA neurons and that the development of mesDA neurons is severely compromised in Ngn2-null mutant mice. We show here that cells isolated by FACS from the developing VM of Ngn2-GFP knock-in mice are capable of generating mesDA neurons, both in vitro and after transplantation to the striatum of neonatal rats. All mesDA neuron precursors, but not the serotonergic or GABAergic neuron precursors, are contained in the Ngn2-GFP-expressing population. Moreover, all glial cells were generated from cells contained in the GFP-negative cell fraction. The results show that surviving mesDA neurons in VM grafts are derived from early postmitotic, probably Nurr1-expressing precursors before they have acquired their fully differentiated neuronal phenotype. The Ngn2-GFP reporter construct used here thus provides a tool for the identification of mesDA neuron precursors in the VM and selective isolation of transplantable mesDA neuron precursors for transplantation.     

Age-related decreases in Nurr1 immunoreactivity in the human substantia nigra

Nuclear receptor-related factor 1 (Nurr1), a member of the nuclear receptor superfamily, is associated with the induction of dopaminergic (DA) phenotypes in developing and mature midbrain neurons. It is well established that dopaminergic nigrostriatal function decreases with age. Whether age-related deficits in DA phenotypic markers are associated with alterations in Nurr1 expression is unknown. The present study found that virtually all of tyrosine hydroxylase-immunoreactive (TH-ir) neurons within the young adult human substantia nigra were Nurr1-immunoreactive (Nurr1-ir) positive. Stereologic counts revealed a significant reduction in the number of Nurr1-ir nigral neurons in middle-aged (23.13%) and aged (46.33%) individuals relative to young subjects. The loss of Nurr1-ir neurons was associated with a similar decline in TH-ir neuron number. In this regard, TH-ir neuronal number was decreased in middle-aged (11.10%) and in aged (45.97%) subjects, and this loss of TH-ir neurons was highly correlated (r = 0.92) with the loss of Nurr1-ir neurons. In contrast, the number of melanin-containing nigral neuron number was generally stable across age groups, indicating that changes in Nurr1 and TH reflect phenotypic age-related changes and not frank neuronal degeneration. In support of this concept, confocal microscopic analyses of Nurr1-ir and TH-ir fluorescence intensity revealed parallel decreases in Nurr1- and TH-immunofluorescence as a function of age. These data demonstrate that age-related decline of DA phenotypic markers is associated with down-regulation of Nurr1 expression in the SN.     

NR4A2 controls the differentiation of selective dopaminergic nuclei in the zebrafish brain

The orphan nuclear receptor NR4A2/Nurr1 is mandatory for the terminal differentiation of mesencephalic dopamine neurons in mammals, but a similar role has remained elusive in the homologous area of the fish brain, the posterior tuberculum. Using loss- and gain-of-function experiments in zebrafish, we show that NR4A2 is indeed responsible for the expression of tyrosine hydroxylase (TH) in selective subpopulations of dopamine cells in the posterior tuberculum, as well as in the pretectum, preoptic area and telencephalon. Cross sections of the neural tube reveal that cells expressing the proliferation marker PCNA, NR4A2 and TH are aligned along a mediolateral progression rather than overlapping populations, suggesting that NR4A2 does not simply regulate TH expression but also controls more general steps of progenitor commitment towards the fully differentiated DA neuronal state. Finally, in line with NR4A2+/− heterozygote mice, NR4A2 morphant fish are hyperactive. This behavioural phenotype is maintained throughout life, pointing to a developmental control of locomotor activity by NR4A2. Our results shed new light on NR4A2 function in the DA differentiation pathway, and stress the effect of DA dysregulation on the control of locomotor activity.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.     

Neurogenesis in the circumventricular organs of adult mouse brains

The circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median eminence (ME), and area postrema (AP), allow parenchyma cells to sense a variety of blood‐derived substances and/or secreted peptides into blood circulation. In the present study, we examined continuous neurogenesis in the CVOs of adult mice. The immunohistochemistry of neural progenitor cell (NPC) marker proteins revealed that Math1‐ and Mash1‐positive cells were observed in the discrete regions of CVOs, including the capillary plexus in the OVLT, the internal zone of the ME, and the lateral zone in the AP. A few Mash1‐ and Math1‐positive cells were seen throughout the SFO, and many Math1‐ but not Mash1‐positive cells were observed at the arcuate nucleus. Math‐positive cells were often seen to localize in close proximity to the vasculature. Bromodeoxyuridine (BrdU) immunohistochemistry revealed the incorporation of BrdU in a subpopulation of Mash1‐, Math1‐, HuC/D‐, and microtubule‐associated protein 2 (MAP2)‐positive cells. Mash1‐ and Math1‐positive cells expressed exclusively high level of plasminogen, whereas a subpopulation of HuC/D‐ and MAP2‐positive neurons expressed low or undetectable level of plasminogen. Thus, the present study demonstrates that newborn cells express NPC marker proteins and plasminogen to localize closely at vascular matrix and moreover differentiate into neurons expressing mature neuron marker proteins, indicating that new neurons are possibly generated to integrate into new neural circuits. © 2013 Wiley Periodicals, Inc.     

Transplantation of neural stem cells co-transfected with Nurr1 and Brn4 for treatment of Parkinsonian rats

Neural stem cells (NSCs) tranplantation has great potential for the treatment of neurodegenerative disease such as Parkinson's disease (PD). However, the usage of NSCs is limited because the differentiation of NSCs into specific dopaminergic neurons has proven difficult. We have recently demonstrated that transgenic expression of Nurr1 could induce the differentiation of NSCs into tyrosine hydroxylase (TH) immunoreactive dopaminergic neurons, and forced co-expression of Nurr1 with Brn4 caused a dramatic increase in morphological and phenotypical maturity of these neurons. In this study, we investigated the effect of transplanted NSCs in PD model rats. The results showed that overexpression of Nurr1 promoted NSCs to differentiate into dopaminergic neurons in vivo, increased the level of dopamine (DA) neurotransmitter in the striatum, resulting in behavioral improvement of PD rats. Importantly, co-expression of Nurr1 and Brn4 in NSCs significantly increased the maturity and viability of dopaminergic neurons, further raised the DA amount in the striatum and reversed the behavioral deficit of the PD rats. Our findings provide a new potential and strategy for the use of NSCs in cell replacement therapy for PD.     

The differentiation potential of human foetal neuronal progenitor cells in vitro

Previously, this laboratory has shown that human foetal progenitor cells derived from ventral mesencephalon (VM) can be developmentally directed towards a dopaminergic lineage. In the present study, the effects are reported of several as yet untested differentiation/survival factors on the controlled conversion of neural progenitor cells to dopaminergic neurons. Positive immunoreactivity to tyrosine hydroxylase (TH) and raised levels of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), secreted into culture medium, were used to indicate the presence of the dopaminergic neuronal phenotype, i.e., active TH. Incubation with retinoic acid (RA) (0.5 microM) lead to an increase in the number of cultured cells showing positive immunoreactivity for the neuronal marker, microtubule-associated protein (MAP)-2ab. A concomitant increase in TH-positive immunoreactivity was also demonstrated. The brain-derived neurotrophic factor (BDNF) (50 ng/ml), glial-derived neurotrophic factor (GDNF) (10 ng/ml) and interleukin-1 beta (IL-1 beta) (10 ng/ml) also had positive effects in promoting neural progenitor cell differentiation towards the dopaminergic phenotype in the presence of dopamine (10 microM) and forskolin (Fsk) (10 microM). There was no synergy in this effect when progenitor cells were incubated with all of these agents simultaneously. The trans-differentiation potential of the progenitor cells to be directed towards other neurotransmitter phenotypic lineages was also investigated. It was found that, with the right cocktails of agents, serotonin (Ser) (75 microM), acidic fibroblast growth factor (aFGF) (10 ng/ml), BDNF (50 ng/ml) and forskolin (10 microM), these same cells could be directed down the serotonergic cell lineage pathway (as judged by the appearance of tryptophan hydroxylase (TPH) positive immunoreactivity, and synthesis of 5-HT and its metabolites, secreted into the culture medium). However, no cocktail containing noradrenaline (10 nM-500 microM), BDNF (50 ng/ml) and forskolin (10 microM) was found which promoted differentiation towards the noradrenergic cell phenotype as judged by the absence of any TH or D beta H positive immunoreactivity, and no formation of 3,4-dihydroxyphenylethyleneglycol (DOPEG), the principal metabolite of noradrenaline. The controlled trans-differentiation potential of these cell could pave the way for development and harvesting of large numbers of neurons of the appropriate neurotransmitter phenotype for future transplantation therapies for the treatment of neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease.     

Nurr1在MN9D细胞中的过表达及其对酪氨酸羟化酶的影响

目的 :观察MN9D细胞中Nurr1的表达和分布及Nurr1过表达对酪氨酸羟化酶的影响。 方法 :应用高压液相色谱、免疫细胞化学、转基因、Westernblot和Northernblot等方法检测了MN9D细胞中多巴胺及其代谢物的水平和Nurr1蛋白在细胞中的分布 ,建立了过表达Nurr1的细胞株并观察了Nurr1过表达对TH基因的影响。结果 :MN9D细胞裂解液中含有大量多巴胺及其代谢物 ;Nurr1蛋白主要分布在MN9D细胞的胞质中 ,尤以核周最多 ;MN9D细胞中有Nurr1的mRNA存在和蛋白质的表达 ;过表达Nurr1的MN9D细胞A1株酪氨酸羟化酶表达增高。结论 :Nurr1与多巴胺能神经元发育有关 ,有可能用于帕金森病的基因治疗。     

Generation of tyrosine hydroxylase-immunoreactive neurons in ventral mesencephalic tissue of Nurr1 deficient mice

Nurr1 is an orphan nuclear receptor belonging to the family of evolutionary conserved steroid/thyroid hormone receptors. It has been shown that Nurr1 is required for development of ventral mesencephalic dopaminergic cells in vivo and that Nurr1 regulates the tyrosine hydroxylase (TH) gene. The aim of this study was to investigate the possibility of finding ventral mesencephalic TH-positive neurons in Nurr1 deficient tissue when developed in the presence of wild type (WT) striatum. Therefore, fetal ventral mesencephalic tissue from embryonic day (E) 9.5-10.5 fetuses from Nurr1 mutant mice was co-cultured with lateral ganglionic eminence (LGE) from WT fetuses using the 'roller-drum' culture technique. TH-immunohistochemistry revealed similar number of positive neurons in WT, heterozygous, and Nurr1 deficient tissue, respectively. When ventral mesencephalon, dissected from E10.5 fetuses, was cultured alone without the presence of LGE, significantly more TH-immunoreactive neurons were found in WT and Nurr1 +/- than that seen in Nurr1 -/- cultures. In single ventral mesencephalic cultures dissected from E15.5, TH-positive neurons were found in all tissue cultures derived from knockout animals. Interestingly, the formation of TH-positive nerve fiber bundles was obvious in WT cultures while not observed in cultures of knockout tissue. When ventral mesencephalon was cultured alone in serum-free medium, almost no TH-positive neurons were found in cultures of knockout tissue. The addition of the growth factors epidermal growth factor and fibroblast growth factor-8 did not induce TH-immunoreactivity in serum-free Nurr1 -/- tissue cultures. In conclusion, TH-positive neurons may be generated in ventral mesencephalic tissue of Nurr1 deficient mice, suggesting that Nurr1 is not required for TH gene expression in ventral midbrain in vitro.