Vera M. Nikodem

H-2RIIBP (RXR beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes.

H‐2RIIBP (RXR beta) is a member of the nuclear hormone receptor superfamily that activates transcription of MHC class I genes in response to retinoic acid (RA). Using chemical cross‐linking, co‐immunoprecipitation, gel mobility shift and streptavidin‐biotin DNA precipitation assays, we show that H‐2RIIBP formed heterodimers with thyroid hormone (T3) and RA receptors (T3R alpha and RAR alpha). H‐2RIIBP heterodimer formation required a conserved sub‐domain of its C‐terminal region, occurred independently of target DNA and was much more efficient than either T3R alpha/RAR alpha heterodimer or H‐2RIIBP homodimer formation. Heterodimers displayed enhanced binding to target DNA elements and contacted DNA in a manner distinct from that of homodimers. A functional role for heterodimers in vivo was demonstrated by synergistic enhancement of MHC class I transcription following co‐transfection of H‐2RIIBP with T3R alpha or RAR alpha. We provide biochemical evidence that H‐2RIIBP formed heterodimers with several naturally occurring nuclear proteins. The results suggest that H‐2RIIBP, by virtue of its ability to heterodimerize, enhances combinatorial diversity and versatility in gene regulation mediated by nuclear hormone receptors.

Dopamine Biosynthesis Is Selectively Abolished in Substantia Nigra/Ventral Tegmental Area but Not in Hypothalamic Neurons in Mice with Targeted Disruption of the Nurr1 Gene

To ascertain the function of an orphan nuclear receptor Nurr1, a transcription factor belonging to a large gene family that includes receptors for steroids, retinoids, and thyroid hormone, we generated Nurr1-null mice by homologous recombination. Mice, heterozygous for a single mutated Nurr1 allele, appear normal, whereas mice homozygous for the null allele die within 24 h after birth. Dopamine (DA) was absent in the substantia nigra (SN) and ventral tegmental area (VTA) of Nurr1-null mice, consistent with absent tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase, and other DA neuron markers. TH immunoreactivity and mRNA expression in hypothalamic, olfactory, and lower brain stem regions were unaffected. L-Dihydroxyphenylalanine treatments, whether given to the pregnant dams or to the newborns, failed to rescue the Nurr1-null mice. We were unable to discern differences between null and wild-type mice in the cellularity, presence of neurons, or axonal projections to the SN and VTA. These findings provide evidence for a new mechanism of DA depletion in vivo and suggest a unique role for Nurr1 in fetal development and/or postnatal survival.

Distribution of messenger RNAs for the orphan nuclear receptors Nurr1 and Nur77 (NGFI-B) in adult rat brain using in situ hybridization

Nurr1 and Nur77 (NGFI-B) are orphan nuclear receptors, belonging to the steroid/thyroid hormone receptor gene superfamily. They have conserved amino acid sequence in the zinc-finger DNA binding domains and similar COOH-terminal regions, but have no known ligands. However, different expression patterns during brain development and tissue distributions of these messenger RNAs imply that they might reflect a different transcriptional role in the brain. In this study, the regional and cellular expression of messenger RNAs encoding these two proteins in rat brain has been determined by in situ hybridization. Nurr1 messenger RNA is highly expressed in the piriform and entorhinal cortices, hippocampus, medial habenular and paraventricular thalamic nuclei. Moderate labeling was detected in layers II-V of most of the cerebral cortex, and in the dorsal lateral geniculate nucleus, substantia nigra (pars compacta and reticularis) and interpeduncular nucleus. No Nurr1 hybridization signal was seen in the rhombencephalon. In the cerebellum, Nurr1 messenger RNA is present in the internal granular cell layer and Purkinje cell layer. In contrast, Nur77 has a widespread distribution, with the highest level of expression in the cerebral cortex. Moderate hybridization signals were detected in the hippocampus, the lateral dorsal and posterior nuclei, reuniens thalamic nuclei, and paraventricular and supraoptic hypothalamic nuclei. In the rhombencephalon, higher signals were present in the medial and lateral vestibular, dorsal cochlear and facial, and raphe magnus nuclei. Nur77 signal was also detected in the nucleus of the spinal tract of the trigeminal nerve. In the cerebellum, Nur77 messenger RNA is highly expressed in the Purkinje cell layer and lateral deep nucleus of the cerebellum. Our results show that Nurr1 and Nur77 messenger RNAs have both overlapping and different distribution patterns within the brain, suggesting that they might regulate different sets of responsive genes.

Nurr1-null heterozygous mice have reduced mesolimbic and mesocortical dopamine levels and increased stress-induced locomotor activity.

Nurr1, an orphan nuclear receptor, is essential for the differentiation of the midbrain dopamine (DA) neurons; however, its function in adult midbrain DA neurons has not been determined. The present study compared regional brain levels of catecholamines and spontaneous and pharmacologically induced locomotor behaviors between mice heterozygous for the Nurr1-null allele (+/-) and wild type (+/+) littermates. The Nurr1 +/- mice had significantly lower levels of DA in whole brain, midbrain, prefrontal cortex and nucleus accumbens, although no significant differences were observed in the striatum, olfactory bulb or hippocampus. Nurr1 +/- mice displayed significantly greater locomotor activity in a novel open field and after saline injection with no significant difference in activity after treatment with amphetamine (2.5 or 5.0 mg/kg) or MK 801 (0.2 or 0.4 mg/kg). A similar elevation in locomotor activity was observed in Nurr1 +/- mice at 35 days old as was found in 70 days old adults. These data demonstrate that the loss of a single Nurr1 allele results in reduced DA levels in mesolimbic and mesocortical pathways and increased locomotor activity in response to mild stress. The involvement of Nurr1 in DA neurotransmission and the implications for schizophrenia are discussed.

hnRNP U Inhibits Carboxy-Terminal Domain Phosphorylation by TFIIH and Represses RNA Polymerase II Elongation

ABSTRACT This study describes a potential new function of hnRNP U as an RNA polymerase (Pol II) elongation inhibitor. We demonstrated that a subfraction of human hnRNP U is associated with the Pol II holoenzyme in vivo and as such recruited to the promoter as part of the preinitiation complex. hnRNP U, however, appears to dissociate from the Pol II complex at the early stage of transcription and is therefore absent from the elongating Pol II complex. When tested in the human immunodeficiency virus type 1 transcription system, hnRNP U inhibits elongation rather than initiation of transcription by Pol II. This inhibition requires the carboxy-terminal domain (CTD) of Pol II. We showed that hnRNP U can bind TFIIH in vivo under certain conditions and inhibit TFIIH-mediated CTD phosphorylation in vitro. We find that the middle domain of hnRNP U is sufficient to mediate its Pol II association and its inhibition of TFIIH-mediated phosphorylation and Pol II elongation. The abilities of hnRNP U to inhibit TFIIH-mediated CTD phosphorylation and its Pol II association are necessary for hnRNP U to mediate the repression of Pol II elongation. Based on these observations, we suggest that a subfraction of hnRNP U, as a component of the Pol II holoenzyme, may downregulate TFIIH-mediated CTD phosphorylation in the basal transcription machinery and repress Pol II elongation. With such functions, hnRNP U might provide one of the mechanisms by which the CTD is maintained in an unphosphorylated state in the Pol II holoenzyme.

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 Parkinsons disease.

Early postnatal isolation reduces dopamine levels, elevates dopamine turnover and specifically disrupts prepulse inhibition in Nurr1-null heterozygous mice

Sensorimotor gating is a phenomenon that is linked with dopamine neurotransmission in limbic and cortical areas, and disruption of sensorimotor gating has been consistently demonstrated in schizophrenia patients. The nuclear receptor Nurr1 is essential for development of dopamine neurons and, using Nurr1-null heterozygous mice, has been found to be important for normal dopamine neurotransmission as null heterozygous mice have reduced limbic and cortical dopamine levels and elevated open-field locomotor activity. The current investigation compared sensorimotor gating, as measured by prepulse inhibition of the acoustic startle response, in Nurr1 wild-type and null heterozygous mice. When mice were weaned between 19 and 21 days of age either into isolation or groups of three to five and tested 12 weeks later, prepulse inhibition was elevated in group-raised null heterozygous mice and significantly disrupted in isolated null heterozygous mice as compared with isolation-raised wild-type mice and group-raised null heterozygous mice. Isolation had no effect on prepulse inhibition in wild-type mice. Isolation reduced tissue dopamine levels and elevated dopamine turnover in the nucleus accumbens and striatum in both wild-type and null heterozygous mice. In the prefrontal cortex, isolation reduced dopamine and 3,4-dihydroxyphenylacetic acid levels in null heterozygous as compared with isolation-raised wild-type mice, whereas no differences were observed between group-raised wild-type and null heterozygous mice. Neither the null heterozygous genotype nor isolation had any effect on basal or stress-induced corticosterone levels. These data suggest that the Nurr1 null heterozygous genotype predisposes these mice to isolation-induced disruption of prepulse inhibition that may be related to the interactions between intrinsic deficiencies in dopamine neurotransmission as a result of the null heterozygous genotype and isolation-induced changes in dopamine neurotransmission. Post-weaning isolation of Nurr1 null heterozygous mice provides a model to explore the interactions of genetic predisposition and environment/neurodevelopment on dopamine function that has important relevance to neuropsychiatric disorders.

Differential expression of tyrosine hydroxylase in catecholaminergic neurons of neonatal wild-type and nurr1-deficient mice

The orphan nuclear receptor Nurr1 is a transcription factor that belongs to the steroid/thyroid hormone receptor superfamily and is expressed in many regions of the brain. To determine 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 h after birth. Subsequent analysis revealed the absence of neurotransmitter dopamine and tyrosine hydroxylase immunoreactivity in the central dopaminergic area of newborn pups. Herein, using in situ hybridization histochemistry, we show that Nurr1 is expressed only in subset of catecholamine producing neurons (A2 partly, A8-A10 and A11 catecholaminergic cell groups), and is excluded from the norepinephrine producing neurons (A1, A2, A5-A6 catecholaminergic cell groups). Nurr1 was not expressed in the dopamine synthesizing cell groups (A12-A16 catecholaminergic cell groups) of the diencephalon and the olfactory bulb. As previously shown and confirmed in this study, tyrosine hydroxylase immunoreactivity was absent in the substantia nigra and ventral tegmental area of Nurr1-deficient mice. However, the loss of Nurr1 expression in A2 and A11 dopaminergic neurons did not affect their tyrosine hydroxylase immunoreactivity. This study begins to dissect cues necessary for understanding the complex regulation of the catecholaminergic biosynthetic pathway with regard to local, chemical and developmental changes in the brain.

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.

Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3‐E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3‐E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real‐time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA‐treated MC3T3‐E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation. J. Cell. Biochem. 99: 986–994, 2006.