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.     

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.     

Nigrostriatal innervation is preserved in Nurr1-null mice, although dopaminergic neuron precursors are arrested from terminal differentiation

Various factors, including the orphan nuclear receptor Nurr1, have been implicated in dopamine biosynthesis, but many of the specific events involved in this process have to be determined. Using genetic manipulations in mice, the obligatory role for Nurr1 in dopamine (DA) biosynthesis has been documented; however, the mechanism remains unclear. DA biosynthetic enzymes, transporters and receptors are absent in the substantia nigra (SN) and the ventral tegmental area (VTA) of Nurr1-null neonates. The current study establishes that the loss of Nurr1 function does not affect the normal ventralization of neuroepithelial cells to the ventral midbrain, their differentiation into neurons, and their topographical pattern in the SN and VTA. Futhermore, the absence of Nurr1 does not affect the survival of these DA precursor cells in the ventral midbrain, as determined by quantitative analysis of cells, expressing the general neuronal nuclear marker (NeuN) and the TUNEL assay for apoptosis. These neurons express cholecystokinin (CCK), a co-transmitter of dopaminergic neurons in this area. The untranslated exon 1-2 of the Nurr1 gene, which remains intact after homologous recombination, revealed the presence of dopaminergic precursors in the ventral midbrain of the Nurr1-null mice. In addition, these neurons establish their nigrostriatal projections, as shown by axonal transport of a fluorescent tracer, DiI. These results provide evidence that Nurr1 is essential for terminal differentiation of the dopaminergic neurons in the ventral midbrain but does not affect the early steps of their neurogenesis, migration, survival and striatal projections. Our findings suggest that activation of Nurr1 might be therapeutically useful in Parkinson's disease.     

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.