Movies of dopamine transporter occupancy with ultra-high resolution focusing pinhole SPECT.

A pivotal question in neuropharmacology is how the function of neurotransmitter systems relates to psychiatric diseases. In experimental neuropharmacology, we have dreamt about a looking glass that would allow us to see neurotransmitter systems in action, and about animals that would faithfully serve us as models for human psychiatric disease. Analysis of animal models has been limited by the availability of methods to study in vivo neurotransmitter dynamics. Now, a single photon emission computed tomography system called U-SPECT can localize dopamine transporters in sub-compartments of the mouse brain during a range of points in time. Applied to the midbrain dopamine system of different models of disease, this will aid the understanding of dynamic processes of this neurotransmitter that underlie brain functions and human brain pathology.

The role of Pitx3 in survival of midbrain dopaminergic neurons.

Dopamine belongs to the most intensively studied neurotransmitters of the brain, because of its implications in psychiatric and neurological disorders. Although, clinical relevance of midbrain dopaminergic (mDA) neurons is well recognized and dopaminergic dysfunction may have a genetic component, the genetic cascades underlying developmental processes are still largely unknown. With the advances in molecular biology, mDA neurons and their involvement in psychiatric and neurological disorders are now subject of studies that aim to delineate the fundamental neurobiology of these neurons. These studies are concerned with developmental processes, cell-specific gene expression and regulation, molecular pharmacology, and genetic association of dopamine-related genes and mDA-associated disorders. Several transcription factors implicated in the post-mitotic mDA development, including Nurr1, Lmx1b, Pitx3, and En1/En2 have contributed to the understanding of how mDA neurons are generated in vivo. Furthermore, these studies provide insights into new strategies for future therapies of Parkinson's Disease (PD) using stem cells for engineering DA neurons in vitro. Here, we will discuss the role of Pitx3 in molecular mechanisms involved in the regional specification, neuronal specification and differentiation of mDA neurons.

Strategies to unravel molecular codes essential for the development of meso-diencephalic dopaminergic neurons.

Understanding the development of neuronal systems has become an important asset in the attempt to solve complex questions about neuropathology as found in Parkinson's disease, schizophrenia and other complex neuronal diseases. The development of anatomical and functional divergent structures in the brain is achieved by a combination of early anatomical patterning and highly coordinated neuronal migration and differentiation events. Fundamental to the existence of divergent structures in the brain is the early region-specific molecular programming. Neuronal progenitors located along the neural tube can still adapt many different identities. Their exact position in the developing brain, however, determines early molecular specification by region-specific signalling molecules. These signals determine time and region-specific expression of early regulatory genes, leading to neuronal differentiation. Here, we focus on a well-described neuronal group, the meso-diencephalic dopaminergic neurons, of which heterogeneity based on anatomical position could account for the difference in vulnerability of specific subgroups as observed in Parkinson's disease. The knowledge of their molecular coding helps us to understand how the meso-diencephalic dopaminergic system is built and could provide clues that unravel mechanisms associated with the neuropathology in complex diseases such as Parkinson's disease.

Neurohypophysial dysmorphogenesis in mice lacking the homeobox gene Uncx4.1.

A number of transcription factors have been implicated in the development of the hypothalamo-neurohypophysial system (HNS). Null mutations for these factors caused severe defects in proliferation, migration and survival during early embryogenesis. While they have informed about early events of HNS developments no insights in mechanisms of late development and maturation of this major peptidergic system have been obtained as yet. In a screen for adult-expressed homeobox genes we identified Uncx4.1 as a gene expressed in adult and embryonic magnocellular neurons of the (HNS). Null mutation of Uncx4.1 left these neurons viable and able to express neuropeptides. However, the connectivity of magnocellular neurons with posterior pituitary elements was compromised. As a consequence neuronal fibres traversed to the adenohypophysis. The penetrance of this phenotype was about 50%. The data show a selective role of Uncx4.1 in controlling the development of connections of hypothalamic neurons to pituitary elements, allowing central neurons to reach the peripheral blood circulation and to deliver hormones for control of peripheral functions.

Signalling through phospholipase C beta 4 is not essential for midbrain dopaminergic neuron survival.

The most prominent progressive neurodegenerative movement disorder, Parkinson's disease, is attributed to selective loss of dopamine neurons in the substantia nigra pars compacta, resulting in severe deficiency of dopamine. The homeo-domain gene, Pit x 3, is essential for proper development of midbrain dopaminergic neurons in the substantia nigra pars compacta and might be involved in midbrain dopaminergic survival pathways. The mGluR1-signaling downstream-effector phospholipase C beta 4 was identified in a suppression subtractive hybridization screen comparing wild-type and Pit x 3-deficient Aphakia midbrain dopaminergic neurons. Expression pattern analysis revealed that phospholipase C beta 4 was expressed in midbrain dopaminergic neurons of the substantia nigra pars compacta and part of the ventral tegmental area, whereas expression of mGluR1alpha was predominantly observed in the more vulnerable midbrain dopaminergic neurons in the lateral substantia nigra pars compacta. However, clear expression of phospholipase C beta 4 in spared midbrain dopaminergic neurons of Aphakia mice located in the ventral tegmental area, indicated that induction and maintenance of phospholipase C beta 4 expression is Pit x 3-independent in these neurons. Furthermore, we report here a normal distribution of midbrain dopaminergic cell bodies and axonal projection to the striatum in phospholipase C beta 4-/- mice, indicating that signaling of phospholipase C beta 4 is not essential for the survival of midbrain dopaminergic neurons.

Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in mu-opioid receptor knockout mice.

There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement.

Survey for paired-like homeodomain gene expression in the hypothalamus: restricted expression patterns of Rx, Alx4 and goosecoid.

Homeobox genes are important regulators of cellular identity. Several homeobox genes are known to be specifically expressed in subsets of neurons in the forebrain, exclusively, or in distinct combinations. In this study, we explored the expression of homeobox genes in the forebrain of the adult rat by a degenerate polymerase chain reaction cloning strategy. We identified the expression of 12 homeobox genes, several of which display a remarkable restricted expression pattern in the adult brain. We demonstrated the expression of goosecoid in a very small set of neurons in the hypothalamus. By using Otp as a marker, these goosecoid-positive cells were found to constitute a small area just beside the paraventricular nucleus. Furthermore, we found expression of Rx in the pineal gland, along with Alx4. Rx was additionally found in the posterior pituitary and in cells aligning the bottom of the third ventricle. These findings form a starting point to reveal functions of the described homeobox genes in the forebrain.

The homeobox genes Lhx7 and Gbx1 are expressed in the basal forebrain cholinergic system.

The specific combination of homeobox genes is proposed to be decisive in the terminal differentiation of neuronal systems. In order to identify combined expression of homeobox genes in the ventral forebrain, a reverse transcriptase-polymerase chain reaction strategy using degenerated primers was employed. We identified, amongst others, Lhx7 and Gbx1, displaying a marked overlapping expression in septal and pallidal areas. Gbx1 and Lhx7 were both expressed in those adult brain nuclei that collectively form the basal forebrain cholinergic system, a prime target of neurodegeneration in Alzheimer's disease. Indeed, we detected Lhx7 within cholinergic neurons, whereas the related Lhx6 gene was found in adjacent neurons. From these data we suggest that combined expression of Lhx7 and Gbx1 plays a role in the development of the cholinergic system of the basal forebrain. It is speculated that both genes remain participating in molecular processes in the adult cholinergic neurons, and can be employed to study regulation and survival of these neurons under normal and pathological conditions.

Analysis of three Ptx2 splice variants on transcriptional activity and differential expression pattern in the brain.

Three different transcripts of the homeodomain gene termed pituitary homeobox (Ptx) 2 (Pitx2/Brx/Rieg/Solurshin/Arp) were cloned from different species encoding proteins belonging to the paired-like family of homeodomain proteins. Ptx2a (324 amino acids), Ptx2b (271 amino acids), and Ptx2c (318 amino acids) share the C terminus, including the homeodomain, and have different N termini. Here we report the comparative analysis of all three different Ptx2 splice variants for their transcriptional activity and their expression pattern in the adult rat brain. Ptx2 is able to trans-activate via different model promoters in different cell lines. A mild difference in trans-activating potential is observed among the splice variants, but the underlying mechanism is at present unknown. It is surprising that all Ptx2 transcripts displayed an identical expression pattern in the brain. This markedly restricted pattern is limited to the following brain areas: the anterior and intermediate lobes of the pituitary gland, the subthalamic nucleus, the posterior hypothalamic nucleus, the mammillary bodies, the red nucleus, and the deep gray layer of the superior colliculus. The data presented suggest that all variants of Ptx2 are involved in the development and regulation of distinct neuronal cell groups and the pituitary gland.

A response element for the homeodomain transcription factor Ptx3 in the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area, which together form the mesencephalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position -50 to -45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3-containing nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight- to 12-fold enhancement of TH promoter activity in Neuro2A neuroblastoma cells, or a 60-80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3-binding site and the major cyclic AMP-response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP-modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type-dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons.

A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b.

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.

Molecular players in the development and maintenance of mesencephalic dopamine systems.

Several psychiatric diseases are considered to be neuro-developmental disorders. Amongst these are schizophrenia and autism, in which genetic and environmental components have been indicated. In these disorders intrinsic molecular mechanisms of brain development may be deranged due to genetic predispositions, or modified by external influences. Brain development is a delicate process of well-tuned cellular proliferation and differentiation of multipotent neural progenitor cells driven by spatiotemporal cues. One of the fundamental mechanisms is the interaction between external signals, e.g. growth factors, and internal regulators, e.g. transcription factors. An important transmitter system involved in behavioural and affective functions relevant for psychiatric disorders is the mesencephalic dopamine (DA) system. The mesencephalic DA system is organized in two anatomically and functionally different systems. DA neurons in the ventral tegmental area project to the mesolimbic system and are mostly related to control of behaviour. It has been implicated in drug addiction and affective disorders like dipolar disorder and schizophrenia. The dopamine system of the substantia nigra (nigro-striatal pathway) is implicated in movement control. Degeneration of this system, as in Parkinson's disease, or altered function in tardive dyskinesia have highlighted its importance in human disease. Recent findings in molecular neurobiology have provided the first clues to molecular mechanisms involved in developing and mature DA neurons. These may have clinical implications in novel therapeutic strategies.

The bZip transcription factor vitellogenin-binding protein is post transcriptional down regulated in chicken liver.

The vitellogenin-binding protein (VBP) is a member of the proline and acidic-region rich (PAR) family of bZip transcription factors. PAR is located N-terminally to the DNA-binding domain. VBP binds to specific sites within the 300-bp 5'-flanking region of the chicken-liver-specific estrogen-dependent very-low-density apolipoprotein gene (apoVLDL II). One of these binding sites (site D) resembles the albumin site D and is positioned in close proximity of the major estrogen-responsive element. Previous studies showed that VBP can bind simultaneously with the estrogen receptor to the putative complex regulatory element E1D. To investigate whether VBP is involved in apoVLDL II gene expression, we examined its capacity to enhance apoVLDL II transcription and its presence in liver. We show that VBP is capable of enhancing transcription in transfection experiments. However, VBP could not be detected in liver by Western-blots or immuno-electro mobility shift assays (EMSAs) using antibodies against different moieties of the protein. We examined the possible reduction in translation efficiencies due to a small upstream open reading frame in the VBP leader sequence, but did not find any. Although VBP binds to the proximal apoVLDL II promoter region and enhances transcription in co-transfection experiments, the protein is unlikely to be involved in apoVLDL II gene transcription because of its undetectable low level in liver nuclei.

Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons.

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 floor 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.

Hypothalamic transcription factors and the regulation of the hypothalamo-neurohypophysial system.

The transcription factors that confer high level expression and regulate the genes encoding neurohypophysial hormones are largely unknown. A number of different approaches have been taken to identify these factors and to elucidate molecular mechanisms of physiological gene regulation. In this chapter two transcription factor families are considered: homeodomain proteins and nuclear receptors. Their identification in the hypothalamus and actions on the OT gene are addressed here.

Homeobox gene Prx3 expression in rodent brain and extraneural tissues.

Different cDNA clones encoding a rat homeobox gene and the mouse homologue OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains). Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the "aristaless domain." Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum revealed marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also expressed the Prx3 gene. In the adult rat brain, Prx3 gene expression was restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation.

A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons.

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.

Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed.

Binding of a bZip protein to the estrogen-inducible apoVLDL II promoter.

Activation of the very low density apolipoprotein II (apoVLDL II) gene in chicken liver by estrogen results in the binding of a variety of nuclear proteins including members of the steroid receptor superfamily and the bZip superfamily to the immediate 5' flanking region. In the present study, we have identified a bZip protein from chicken liver as one of the potential binding activities. Its cognate cDNA was cloned from an expression library using a recognition site DNA probe corresponding to part of the apoVLDL II promoter region. By footprinting and gel shift analysis with the recombinant protein from a prokaryotic expression system we have established that the protein binds to at least three different sites in the apoVLDLII promoter region. One of these sites partially overlaps with the major estrogen response element of the gene. Despite the proximity of their binding sites, the estrogen receptor and the bZip protein can bind simultaneously to the very region. Possible implications of this intimate arrangement of binding sites for the activation of the apoVLDL II promoter are discussed.

Identification of chloroacetaldehyde dehydrogenase involved in 1,2-dichloroethane degradation.

The degradation of 1,2-dichloroethane and 2-chloroethanol by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde, a reactive and potentially toxic intermediate. The organism produced at least three different aldehyde dehydrogenases, of which one is plasmid encoded. Two mutants of strain GJ10, designated GJ10M30 and GJ10M41, could no longer grow on 2-chloroethanol and were found to lack the NAD-dependent aldehyde dehydrogenase that is the predominant protein in wild-type cells growing on 2-chloroethanol. Mutant GJ10M30, selected on the basis of its resistance to 1,2-dibromoethane, also had lost haloalkane dehalogenase activity and Hg resistance, indicating plasmid loss. From a gene bank of strain GJ10, different clones that complemented one of these mutants were isolated. In both transconjugants, the aldehyde dehydrogenase that was absent in the mutants was overexpressed. The enzyme was purified and was a tetrameric protein of 55-kDa subunits. The substrate range was rather broad, with the highest activity measured for acetaldehyde. The K(m) value for chloroacetaldehyde was 160 muM, higher than those for other aldehydes tested. It is concluded that the ability of GJ10 to grow with 2-chloroethanol is due to the high expression level of an aldehyde dehydrogenase with a rather low activity for chloroacetaldehyde.