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Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD.

Cellular systems for DNA repair ensure prompt removal of DNA lesions that threaten the genomic stability of the cell. Transcription factors (TFs) have long been known to facilitate DNA repair via transcriptional regulation of specific target genes encoding key DNA repair proteins. However, recent findings identified TFs as DNA repair components acting directly at the DNA lesions in a transcription-independent fashion. Together this recent progress is consistent with the hypothesis that TFs have acquired the ability to localize DNA lesions and function by facilitating chromatin remodeling at sites of damaged DNA. Here we review these recent findings and discuss how TFs may function in DNA repair.

Nuclear receptors comprise a large family of proteins that shares a common structure and mechanism of action. Members of this family, first cloned 20 years ago, are regulated by small lipophilic signaling molecules such as steroid hormones, retinoids and thyroid hormone. More recently, the characterization of proteins that resemble nuclear receptors (referred to as orphan receptors) has resulted in the determination of novel signaling pathways. However, many orphan-receptor ligands remain unidentified, and recent structural studies of the binding domains for orphan-receptor ligands suggest that not all of these receptors use ligand binding in a classical way. Notably, it is now evident that some orphan receptors lack the capacity for ligand binding, which suggests that they are regulated by alternative, ligand-independent mechanisms.

Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. Activation of Nurr1 depends on two so-called activation functions (AFs) situated in the N- and C-terminal regions, respectively. The region important for activation within the C-terminal domain has been shown to promote activation in a highly cell-type specific fashion in the absence of added exogenous ligands. In contrast, the region in the N-terminal domain (AF1) has been much less characterized. Here we mutagenized the N-terminal domain of Nurr1 to define essential activation regions. The results identified a short core activation region localized close to the N-terminus of Nurr1. In addition, cell-type specific influences by other signaling pathways were analyzed by mutagenesis of specific conserved phosphorylation sites. The results indicate that mitogen-activated protein kinase activity (MAPK) positively influences Nurr1 AF1-dependent transcriptional activation via a conserved phosphorylation site outside the core activation region.

The identity of motor neurons diverges markedly at different rostrocaudal levels of the spinal cord, but the signals that specify their fate remain poorly defined. We show that retinoid receptor activation in newly generated spinal motor neurons has a crucial role in specifying motor neuron columnar subtypes. Blockade of retinoid receptor signaling in brachial motor neurons inhibits lateral motor column differentiation and converts many of these neurons to thoracic columnar subtypes. Conversely, expression of a constitutively active retinoid receptor derivative impairs the differentiation of thoracic motor neuron columnar subtypes. These findings provide evidence for a regionally restricted role for retinoid signaling in the postmitotic specification of motor neuron columnar identity.

Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands. However, the specific physiological roles of Nurr1 monomers and RXR-Nurr1 heterodimers remain to be elucidated. The aim of this study was to define structural requirements for RXR-Nurr1 heterodimerization. Several amino acid substitutions were introduced in both Nurr1 and RXR in the I-box, a region previously shown to be important for nuclear receptor dimerization. Single amino acid substitutions introduced in either Nurr1 or RXR abolished heterodimerization. Importantly, heterodimerization-deficient Nurr1 mutants exhibited normal activities as monomers. Thus, by introducing specific amino acid substitutions in Nurr1, monomeric and heterodimeric properties of Nurr1 can be distinguished. Interestingly, substitutions in the RXR I-box differentially affected heterodimerization with Nurr1, retinoic acid receptor, thyroid hormone receptor, and constitutive androstane receptor demonstrating that the dimerization interfaces in these different heterodimers are functionally unique. Furthermore, heterodimerization between RXR and Nurr1 had a profound influence on the constitutive activity of Nurr1, which was diminished as a result of RXR interaction. In conclusion, our data show unique structural and functional properties of RXR-Nurr1 heterodimers and also demonstrate that specific mutations in Nurr1 can abolish heterodimerization without affecting other essential functions.

Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.

Nurr1, an orphan receptor of the nuclear receptor superfamily, is widely expressed in the central nervous system (CNS) including brain regions where dopaminergic neurons are abundant. Recent analyses of Nurr1 null mutant mice have shown that Nurr1 is essential for the development and survival of midbrain dopaminergic neurons. However, other dopaminergic neuronal populations do not seem to be affected by ablation of the Nurr1 gene. The purpose of the present study was to investigate the degree of co-existence of Nurr1 mRNA and tyrosine hydroxylase (TH) immunoreactivity in the brain of adult mice to better characterize the selective effects of Nurr1 on catecholaminergic neurons. Our results indicate that the majority of TH-immunoreactive neurons in the substantia nigra (SN; 96%), ventral tegmental area (VTA; 95%), retrorubral field (91%), olfactory bulb (85%), linear nucleus raphe (91%) and central grey (61%) express Nurr1. In contrast, dopaminergic cells of the paraventricular and periventricular hypothalamic nucleus showed only a few Nurr1/TH double labeled neurons, while TH-immunoreactive neurons in the arcuate nucleus and zona incerta did not express Nurr1 mRNA. Nurr1 expression was also excluded from (nor)adrenergic neurons of the brainstem. In conclusion, Nurr1 transcripts were not found in all CNS catecholaminergic neurons. Nurr1 expression was confined to periglomerular and midbrain dopaminergic neurons. These results suggest that within the adult mouse brain, Nurr1 may participate in dopaminergic functions of the olfactory bulb and midbrain.

In the adult mammalian central nervous system lost nerve cells are not replaced and there is no regeneration of injured axons in white matter. Together, these two facts mean that there are no spontaneous reparative mechanisms in operation. Instead, the adult central nervous system copes with the risks of injuries and diseases by protective encapsulation in bone, by a multitude of neuroprotective mechanisms, and finally by the fact that many important functions are represented by a much larger number of neurons than minimally needed. The long life expectancy of a human being nevertheless means that the risk that the central nervous system is affected by disease, injury or other forms of insults for which it cannot fully compensate is relatively high. Experimentally, two strategies are being pursued in order to develop ways of minimizing various forms of CNS damage, namely neuroprotective and reparative strategies. Here we present a possible reparative intervention applicable to spinal cord injury based on multiple white-to-gray matter peripheral nerve bridge grafts and work based on the specific role of Nurr1 for dopamine neuron development, suggesting that development of ligands to transcription factor might be a new inroad to neuroprotective treatments in Parkinson's disease.

Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones. A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands. Known as orphan receptors, these proteins represents targets for novel signaling molecules. We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor. A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex. Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins. Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast. Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.

Preliminary tissue content data obtained by High Performance Liquid Chromatography showed that DAT −/− KO rats have markedly decreased total tissue DA (down to 20% of wild-type levels) and 3−4 fold increased levels of DA metabolites − 3,4-dihydroxyphenylacetic acid (DOPAC) and Homovanillic acid (HVA) without major differences observed in serotonin and its main metabolite 5-hydroxyindolacetic acid (5-HIAA) levels. A preliminary Fast Scan Cyclic Voltammetry analysis showed that DA clearance in DAT −/− is much longer (over 60 seconds) when compared with DAT +/+, suggesting that the increase in the spontaneous locomotor activity is a direct consequence of the extended length of time that DA spends in the extracellular space following release. The DAT knockout rats should be an excellent and improved tool for the study and development of drugs used in the management of drugs used in dopaminergic dysfunctions, providing many similarities with DAT knockout mice but at the same time a better model for the study of complex behaviors. DAT −/− rats could provide a complex and translational model for several human diseases involving aberrant DA function or mutations affecting DAT or altered DAT regulatory mechanisms in vivo such as schizophrenia, ADHD and newly discovered Dopamine Transporter Deficiency Syndrome (DTDS) [3].

Purified glucocorticoid receptor (GR) from rat liver was used for a quantitative analysis of the protein-DNA interaction at specific GR-binding segments within the 5'-long terminal repeat of the mouse mammary tumor virus. A truncated receptor was generated and used to demonstrate formation of heterodimeric GR, which furthermore was shown to be in rapid equilibrium with receptor-monomer. The relative affinity for GR binding to specific GR sites versus random calf thymus DNA was approximately 2 x 10(3). At equilibrium a free GR concentration of 3 x 10(-10) M was required for half-maximal saturation of the two functionally important DNA sites within the mouse mammary tumor virus 5'-long terminal repeat. Although these two DNA segments act synergistically in mediating hormonal response, we did not detect cooperative GR binding to these regions in vitro. However, GR bound cooperatively within the downstream binding region. Similarly, GR was unable to facilitate factor binding to a neighboring nuclear factor 1 site, another essential element in the promoter. In contrast, nuclear factor 1 binding was inhibited slightly by GR.