Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the f loor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.

The cerebral cortex of Alzheimer's and Down syndrome patients is characterized by the presence of protein deposits in neurofibrillary tangles, neuritic plaques, and neuropil threads. These structures were shown to contain forms of beta amyloid precursor protein and ubiquitin-B that are aberrant (+1 proteins) in the carboxyl terminus. The +1 proteins were not found in young control patients, whereas the presence of ubiquitin-B+1 in elderly control patients may indicate early stages of neurodegeneration. The two species of +1 proteins displayed cellular colocalization, suggesting a common origin, operating at the transcriptional level or by posttranscriptional editing of RNA. This type of transcript mutation is likely an important factor in the widely occurring nonfamilial early- and late-onset forms of Alzheimer's disease.

We identified the LIM homeodomain transcription factor Lmx1b in the mesencephalic dopamine (mesDA) systems of embryos and adults. Analysis of spatiotemporal expression in Lmx1b null mutants and wild-type mice implicated a cascade involving Lmx1b in the early development of mesDA neurons. Although disruption of this cascade did not block induction of tyrosine hydroxylase (TH), a key enzyme in DA synthesis, or Nurr1, a nuclear hormone receptor, Lmx1b knockout mice failed to induce the mesDA-specific homeodomain gene Ptx3 in TH-positive neurons. Eventually, this small set of TH-positive neurons was lost during embryonic maturation. The data suggest that at least two molecular cascades operate during the specification of the mesDA system, one specifying neurotransmitter phenotype and another essential for other aspects of mesDA neuron differentiation.

The mesencephalic dopamine (mesDA) system is involved in the control of movement and behavior. The expression of Pitx3 in the brain is restricted to the mesDA system and the gene is induced relatively late, at E11.5, a time when tyrosine hydroxylase (Th) gene expression is initiated. We show here that, in the Pitx3-deficient aphakia(ak) mouse mutant, the mesDA system is malformed. Owing to the developmental failure of mesDA neurons in the lateral field of the midbrain,mesDA neurons are not found in the SNc and the projections to the caudate putamen are selectively lost. However, Pitx3 is expressed in all mesDA neurons in control animals. Therefore, mesDA neurons react specifically to the loss of Pitx3. Defects of motor control where not seen in the ak mice,suggesting that other neuronal systems compensate for the absence of the nigrostriatal pathway. However, an overall lower activity was observed. The results suggest that Pitx3 is specifically required for the formation of the SNc subfield at the onset of dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral mesodiencephalon are essential for the control of voluntary movement and the regulation of emotion, and are severely affected in neurodegenerative diseases such as Parkinson's disease. Recent advances in molecular biology and mouse genetics have helped to unravel the mechanisms involved in the development of mesodiencephalic dopaminergic (mdDA) neurons, including their specification, migration and differentiation, as well as the processes that govern axonal pathfinding and their specific patterns of connectivity and maintenance. Here, we follow the developmental path of these neurons with the goal of generating a molecular code that could be exploited in cell-replacement strategies to treat diseases such as Parkinson's disease.

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxydopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.The patterning of the developing mammalian brain is thought to involve cascades of signaling molecules and transcription factors, but the mechanisms for generation of distinct neuronal cell types during terminal differentiation are still largely speculative (1, 2). Yet, the specification of individual neuronal phenotypes underlies the assembly of neural circuits essential for brain function. The mesencephalic dopaminergic (mesDA) system consists of a limited set of neurons that are well defined anatomically and functionally (3-5). Their specific degeneration in Parkinson disease reveals their functional properties in control of behavior and movement as well as a unique vulnerability (6-10). In a search for homeobox genes associated with a unique neuronal lineage, we isolated a cDNA encoding a bicoid-related homeobox gene Ptx3, a member of the Ptx subfamily (11)(12)(13)(14). This gene is strictly expressed in mesDA neurons. METHODS AND MATERIALSCloning of Ptx3 Gene Transcripts. Poly(A) ϩ RNA from hypothalamic fragments of the adult rat brain were subjected to reverse transcriptase-PCR with primers based on brainexpressed homeobox genes: upstream, 5Ј-GMRSCGM-SAVMGSACMMBCTTYAC-3Ј; downstream, 5Ј-TGGT-TYMRVAAYCGYHGMGCMARRTG-3Ј. The annealing temperature was 40°C. The PCR product was used to screen an adult rat hypothalamus library in gt11. Isolated phage DNA was cut with EcoRI, the insert of Ϸ1.2 kb was subcloned into pGEM7Zf(ϩ), and both strands of the insert were sequenced.Northern Analysis. Total RNA extracted from tissues of the adult rat by Rnazol (Biotecx Laboratories, Houston) was fractionated on formaldehyde-agarose gels and transferred onto a nylon membrane (Hybond-N, Amersham) by downward capillary blotting. Blots were hybridized with a 32 P-randomprimed-labeled complete Ptx3 cDNA at 65°C overnight (15). Autoradiography was performed with a Fujix BAS1000 phosphor-imager (Fuji).Cell Culture, Transfection, and Gel Retardation Assays. Murine fibroblast L cells were grown in DMEM supplemented with 10% FCS. L cells were transfected by the calcium phosphate method (16). Precipitate containing 3 g of reporter plasmid, 1 g of effector plasmid, 1 g of RSV-human growth hormone (hGH) internal control plasmid, and 5 g of carrier DNA (pSP64, Promega) was app...

Selective neuronal loss in the substantia nigra (SNc), as described for Parkinson's disease (PD) in humans and for Pitx3 deficiency in mice,highlights the existence of neuronal subpopulations. As yet unknown subset-specific gene cascades might underlie the observed differences in neuronal vulnerability. We identified a developmental cascade in mice in which Ahd2 (Aldh1a1) is under the transcriptional control of Pitx3. Interestingly, Ahd2 distribution is restricted to a subpopulation of the meso-diencephalic dopaminergic (mdDA) neurons that is affected by Pitx3 deficiency. Ahd2 is involved in the synthesis of retinoic acid(RA), which has a crucial role in neuronal patterning, differentiation and survival in the brain. Most intriguingly, restoring RA signaling in the embryonic mdDA area counteracts the developmental defects caused by Pitx3 deficiency. The number of tyrosine hydroxylase-positive (TH+)neurons was significantly increased after RA treatment in the rostral mdDA region of Pitx3-/- embryos. This effect was specific for the rostral part of the developing mdDA area, and was observed exclusively in Pitx3-/- embryos. The effect of RA treatment during the critical phase was preserved until later in development, and our data suggest that RA is required for the establishment of proper mdDA neuronal identity. This positions Pitx3 centrally in a mdDA developmental cascade linked to RA signaling. Here, we propose a novel mechanism in which RA is involved in mdDA neuronal development and maintenance, providing new insights into subset-specific vulnerability in PD.

In recent years, the meso-diencephalic dopaminergic (mdDA) neurons have been extensively studied for their association with Parkinson's disease. Thus far, specification of the dopaminergic phenotype of mdDA neurons is largely attributed to the orphan nuclear receptor Nurr1. In this study, we provide evidence for extensive interplay between Nurr1 and the homeobox transcription factor Pitx3 in vivo. Both Nurr1 and Pitx3 interact with the co-repressor PSF and occupy the promoters of Nurr1 target genes in concert. Moreover, in vivo expression analysis reveals that Nurr1 alone is not sufficient to drive the dopaminergic phenotype in mdDA neurons but requires Pitx3 for full activation of target gene expression. In the absence of Pitx3, Nurr1 is kept in a repressed state through interaction with the co-repressor SMRT. Highly resembling the effect of ligand activation of nuclear receptors, recruitment of Pitx3 modulates the Nurr1 transcriptional complex by decreasing the interaction with SMRT, which acts through HDACs to keep promoters in a repressed deacetylated state. Indeed, interference with HDAC-mediated repression in Pitx3 -/-embryos efficiently reactivates the expression of Nurr1 target genes, bypassing the necessity for Pitx3. These data position Pitx3 as an essential potentiator of Nurr1 in specifying the dopaminergic phenotype, providing novel insights into mechanisms underlying development of mdDA neurons in vivo, and the programming of stem cells as a future cell replacement therapy for Parkinson's disease.KEY WORDS: DA phenotype, Dat, Dopamine, Parkinson's disease, VMAT2, mdDA Development 136, 531-540 (2009) DEVELOPMENT MATERIALS AND METHODS Cell culture MN9D-Nurr1Tet On 13N (MN9D) cells were cultured and transfected as described previously (Jacobs et al., 2007). Identification of Pitx3 interacting proteinsMN9D cells were transfected with 0.5 μg Pitx3-pcDNA3.1(-)myc-His or an equivalent molar amount of pcDNA3.1(-)myc-His expression vector and His-tagged proteins were purified using Ni-NTA magnetic agarose beads (Qiagen) according to manufacturer's protocol. Purified proteins were separated by SDS PAGE and visualized by protein gel silver-staining. Subsequently, the gel was fixed in 50% methanol (2ϫ15 minutes) and 5% methanol (10 minutes), rinsed with water (3ϫ10 seconds), soaked in 10 μM DTT (20 minutes), incubated in 0.1% AgNO 3 (20 minutes), rinsed with water and incubated in developer solution (3% NaCO 3 , 0.05% formaldehyde) until protein bands were visible. The reaction was stopped by adding citric acid and the gel was washed in water. Protein bands of interest were excised from the gel and subjected to nanoLC-ESI-MS Mass Spectrometry analysis (Proteome Factory). Immunoprecipitation (IP) and western blottingMN9D cells or tissue was homogenized in lysis buffer [50 mM Tris HCl (pH 8), 150 mM NaCl, 5 mM MgCl 2 , 0.5 mM EDTA, 0.2% NP-40, 5% glycerol and 0.5 mM DTT + Complete Protease Inhibitor Cocktail (Roche)], and subjected to IP. Two dishes (10 cm in diameter) of MN9D cells or five ventral midbrains ...