Differentiation Of Midbrain Dopaminergic Neurons Research Articles

Expression and role of nicotinic acetylcholine receptors during midbrain dopaminergic neuron differentiation from human induced pluripotent stem cells.

Nicotinic acetylcholine receptors (nAChRs) expressed in midbrain dopaminergic (mDA) neurons modulate mDA neuronal activity. However, their expression patterns and functional roles during mDA neuronal development remain unknown. Here, we profiled the expression and function of nAChR subtypes during mDA neuron differentiation from human induced pluripotent stem cells (hiPSCs). Midbrain dopaminergic neurons were differentiated from hiPSCs using a recently developed proprietary method that replicates midbrain development. The expression patterns of developmental marker proteins were monitored during mDA neuronal differentiation using immunohistochemical analysis. Gene expression of nAChR subtypes was analyzed by reverse transcription polymerase chain reaction. Pharmacological nAChR agonists and antagonists were used to reveal the role of the α6 nAChR subunit in the differentiation of mDA neurons from hiPSCs. CHRNA4 expression was detected at the mDA neural progenitor stage, whereas CHRNA6 expression began during the mDA neuronal stage. CHRNA7 was expressed throughout the differentiation process, including in the undifferentiated hiPSCs. We also found that LMO3, a gene expressed in a subset of substantia nigra pars compacta (SNC) DA neurons in the midbrain, showed increased expression following nicotine treatment in a concentration-dependent manner. Additionally, 5-iodo A85380, a selective α6 nAChR agonist, also increased LMO3 expression in hiPSC-derived mDA neurons, and this increase was suppressed by simultaneous treatment with bPiDi, a selective α6 nAChR antagonist. Our findings suggest that stimulating the α6 nAChR subunit on hiPSC-derived mDA neurons may induce neuronal maturation that is biased toward SNC DA neurons.

Ferulic Acid Induces NURR1 Expression and Promotes Dopaminergic Differentiation in Neural Precursor Cells.

Degeneration of dopaminergic (DA) neurons in the substantia nigra is known as the main cause of Parkinson's disease (PD). Preventing the loss of DA neurons alongside the cell-replacement therapy have brought tremendous hope for the treatment of PD. For this purpose, various studies have been done to find the specific DA neuro-protective compounds or progressing DA-differentiation methods. Ferulic acid (FA) has strong neuro-protective effects, but at this point its role on protection and differentiation of DA neurons is not well-defined. Mouse neural stem cells (mNSCs) were treated with FA and expressions of TH (tyrosine hydroxylase) and NURR1 as the DA neuron specific markers were determined using real time qRT-PCR and immunostaining assays . Finally, efficacy of FA on DA differentiation was evaluated in comparison with other methods using fibroblast growth factor 8b (FGF8b) and sonic hedgehog (SHH). Treatment with FA could increase the Th and Nurr1 gene expressions in mNSCs. Also, it enhanced β - tubullin - III expression and increased the neurite length in treated groups. Real time qRT-PCR and immunostaining assays showed that FA could increase DA differentiation in mNSCs effectively. Also, gene expression profile in some groups showed that FA can raise the differentiation rate of other neuronal subtypes such as cholinergic neurons. FA effectively induces the DA differentiation in neural precursor cells by its ability to increase the expression of the NURR1 transcription factor, which is a known transcription factor for differentiation of midbrain DA neurons.

Dose-Dependent and Subset-Specific Regulation of Midbrain Dopaminergic Neuron Differentiation by LEF1-Mediated WNT1/b-Catenin Signaling.

The mesodiencephalic dopaminergic (mdDA) neurons, including the nigrostriatal subset that preferentially degenerates in Parkinson’s Disease (PD), strongly depend on an accurately balanced Wingless-type MMTV integration site family member 1 (WNT1)/beta-catenin signaling pathway during their development. Loss of this pathway abolishes the generation of these neurons, whereas excessive WNT1/b-catenin signaling prevents their correct differentiation. The identity of the cells responding to this pathway in the developing mammalian ventral midbrain (VM) as well as the precise progression of WNT/b-catenin action in these cells are still unknown. We show that strong WNT/b-catenin signaling inhibits the differentiation of WNT/b-catenin-responding mdDA progenitors into PITX3+ and TH+ mdDA neurons by repressing the Pitx3 gene in mice. This effect is mediated by RSPO2, a WNT/b-catenin agonist, and lymphoid enhancer binding factor 1 (LEF1), an essential nuclear effector of the WNT/b-catenin pathway, via conserved LEF1/T-cell factor binding sites in the Pitx3 promoter. LEF1 expression is restricted to a caudolateral mdDA progenitor subset that preferentially responds to WNT/b-catenin signaling and gives rise to a fraction of all mdDA neurons. Our data indicate that an attenuation of WNT/b-catenin signaling in mdDA progenitors is essential for their correct differentiation into specific mdDA neuron subsets. This is an important consideration for stem cell-based regenerative therapies and in vitro models of neuropsychiatric diseases.

From protocol to product: ventral midbrain dopaminergic neuron differentiation for the treatment of Parkinson's disease.

Current cell therapy product limitations include the need for in-depth product understanding to ensure product potency, safety and purity. New technologies require development and validation to address issues of production scale-up to meet clinical need; assays are required for process control, validation and release. Prior to clinical realization, an understanding of production processes is required to implement process changes that are essential for process control. Identification of key parameters forms the basis of process tolerances, allowing for validated, adaptive manufacturing processes. This enables greater process control and yield while withstanding regulatory scrutiny. This report summaries key milestones in specifically for ventral midbrain dopaminergic neuroprogenitor differentiation and key translational considerations and recommendations to enable successful, robust and reproducible current cell therapy product-manufacturing.

Dopaminergic and behavioural changes in a loss-of-imprinting model of Cdkn1c.

The imprinted gene Cdkn1c is expressed exclusively from the maternally inherited allele as a consequences of epigenetic regulation. Cdkn1c exemplifies many of the functional characteristics of imprinted genes, playing a role in foetal growth and placental development. However, Cdkn1c also plays an important role in the brain, being key to the appropriate proliferation and differentiation of midbrain dopaminergic neurons. Using a transgenic model (Cdkn1c BACx1) with a twofold elevation in Cdkn1c expression that mimics loss‐of‐imprinting, we show that increased expression of Cdkn1c in the brain gives rise to neurobiological and behavioural changes indicative of a functionally altered dopaminergic system. Cdkn1c BACX1 mice displayed altered expression of dopamine system‐related genes, increased tyrosine hydroxylase (Th) staining and increased tissue content of dopamine in the striatum. In addition, Cdkn1c BACx1 animals were hypersensitive to amphetamine as showed by c‐fos expression in the nucleus accumbens. Cdkn1c BACX1 mice had significant changes in behaviours that are dependent on the mesolimbic dopaminergic system. Specifically, increased motivation for palatable food stuffs, as indexed on a progressive ratio task. In addition, Cdkn1c BACX1 mice displayed enhanced social dominance. These data show, for the first time, the consequence of elevated Cdkn1c expression on dopamine‐related behaviours highlighting the importance of correct dosage of this imprinted gene in the brain. This work has significant relevance for deepening our understanding of the epigenetic factors that can shape neurobiology and behaviour.

The Shh coreceptor Cdo is required for differentiation of midbrain dopaminergic neurons

Sonic hedgehog (Shh) signaling is required for numerous developmental processes including specification of ventral cell types in the central nervous system such as midbrain dopaminergic (DA) neurons. The multifunctional coreceptor Cdo increases the signaling activity of Shh which is crucial for development of forebrain and neural tube. In this study, we investigated the role of Cdo in midbrain DA neurogenesis. Cdo and Shh signaling components are induced during neurogenesis of embryonic stem (ES) cells. Cdo−/− ES cells show reduced neuronal differentiation accompanied by increased cell death upon neuronal induction. In addition, Cdo−/− ES cells form fewer tyrosine hydroxylase (TH) and microtubule associated protein 2 (MAP2)-positive DA neurons correlating with the decreased expression of key regulators of DA neurogenesis, such as Shh, Neurogenin2, Mash1, Foxa2, Lmx1a, Nurr1 and Pitx3, relative to the Cdo+/+ ES cells. Consistently, the Cdo−/− embryonic midbrain displays a reduction in expression of TH and Nurr1. Furthermore, activation of Shh signaling by treatment with Purmorphamine (Pur) restores the DA neurogenesis of Cdo−/− ES cells, suggesting that Cdo is required for the full Shh signaling activation to induce efficient DA neurogenesis.

Tiam1 Regulates the Wnt/Dvl/Rac1 Signaling Pathway and the Differentiation of Midbrain Dopaminergic Neurons

Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.

The transcription factor Uncx4.1 acts in a short window of midbrain dopaminergic neuron differentiation

BackgroundThe homeobox containing transcription factor Uncx4.1 is, amongst others, expressed in the mouse midbrain. The early expression of this transcription factor in the mouse, as well as in the chick midbrain, points to a conserved function of Uncx4.1, but so far a functional analysis in this brain territory is missing. The goal of the current study was to analyze in which midbrain neuronal subgroups Uncx4.1 is expressed and to examine whether this factor plays a role in the early development of these neuronal subgroups.ResultsWe have shown that Uncx4.1 is expressed in GABAergic, glutamatergic and dopaminergic neurons in the mouse midbrain. In midbrain dopaminergic (mDA) neurons Uncx4.1 expression is particularly high around E11.5 and strongly diminished already at E17.5. The analysis of knockout mice revealed that the loss of Uncx4.1 is accompanied with a 25% decrease in the population of mDA neurons, as marked by tyrosine hydroxylase (TH), dopamine transporter (DAT), Pitx3 and Ngn2. In contrast, the number of glutamatergic Pax6-positive cells was augmented, while the GABAergic neuron population appears not affected in Uncx4.1-deficient embryos.ConclusionWe conclude that Uncx4.1 is implicated in the development of mDA neurons where it displays a unique temporal expression profile in the early postmitotic stage. Our data indicate that the mechanism underlying the role of Uncx4.1 in mDA development is likely related to differentiation processes in postmitotic stages, and where Ngn2 is engaged. Moreover, Uncx4.1 might play an important role during glutamatergic neuronal differentiation in the mouse midbrain.

<em>In ovo</em> Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders. Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick β-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

A Small Synthetic Cripto Blocking Peptide Improves Neural Induction, Dopaminergic Differentiation, and Functional Integration of Mouse Embryonic Stem Cells in a Rat Model of Parkinson's Disease

Cripto is a glycosylphosphatidylinositol-anchored coreceptor that binds Nodal and the activin type I (ALK)-4 receptor, and is involved in cardiac differentiation of mouse embryonic stem cells (mESCs). Interestingly, genetic ablation of cripto results in increased neuralization and midbrain dopaminergic (DA) differentiation of mESCs, as well as improved DA cell replacement therapy (CRT) in a model of Parkinson's disease (PD). In this study, we developed a Cripto specific blocking tool that would mimic the deletion of cripto, but could be easily applied to embryonic stem cell (ESC) lines without the need of genetic manipulation. We thus screened a combinatorial peptide library and identified a tetrameric tripeptide, Cripto blocking peptide (BP), which prevents Cripto/ALK-4 receptor interaction and interferes with Cripto signaling. Cripto BP treatment favored neuroectoderm formation and promoted midbrain DA neuron differentiation of mESCs in vitro and in vivo. Remarkably, Cripto BP-treated ESCs, when transplanted into the striatum of PD rats, enhanced functional recovery and reduced tumor formation, mimicking the effect of genetic ablation of cripto. We therefore suggest that specific blockers such as Cripto BP may be used to improve the differentiation of ESC-derived DA neurons in vitro and their engraftment in vivo, bringing us closer towards an application of ESCs in CRT.

Expression pattern of NuIP gene in adult mouse brain

We previously reported the identification of the Nurr1 interacting protein (NuIP) that was demonstrated to modulate the transcriptional activity of Nurr1, the orphan nuclear receptor required for midbrain dopaminergic neuron differentiation. NuIP was also cloned by others and referred to as a small G protein signaling modulators. The open reading frame of NuIP predicts a protein with an N-terminal RUN domain (RPIP8, UNC-14, and NESCA) and a C-terminal TBC domain (Tre-2, Bub2, and Cdc16) both of which are found in proteins of the GTPase activating protein (GAP) family, involved in the GTPase signaling pathway. To characterize the NuIP gene product, we developed a polyclonal antibody. Since NuIP gene is expressed most abundantly in adult and the level of expression during development is below the detection limit of immunohistochemistry, we now report the expression pattern of NuIP in adult mouse brain compared with the expression pattern of Nurr1 protein. Many regions co-expressed Nurr1 and NuIP including cortex, hippocampus, substantia nigra, and the cerebellum. However, there are also regions that exclusively express NuIP such as striatum, septum, globus pallidus, and the reticular thalamic nucleus. We also find that NuIP protein expresses mainly in NeuN-positive (neuronal nuclei) neurons but can be detected in GFAP-positive (glial fibrillary acidic protein) glial cells in hippocampus. Interestingly, NuIP is expressed in high levels in midbrain dopaminergic neurons including ventral tegmental area (VTA) and substantia nigra (SN) dopaminergic neurons but is not expressed or expressed in low levels in other dopaminergic neurons such as olfactory bulb and hypothalamus. Overall, the expression pattern of NuIP in adult mouse brain suggests that it may be involved in motor activity control in basal ganglia as well as higher central nervous system (CNS) functions such as cognition and memory in cortex and hippocampus.

Foxa1andFoxa2regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner

The role of transcription factors in regulating the development of midbrain dopaminergic (mDA) neurons is intensively studied owing to the involvement of these neurons in diverse neurological disorders. Here we demonstrate novel roles for the forkhead/winged helix transcription factors Foxa1 and Foxa2 in the specification and differentiation of mDA neurons by analysing the phenotype of Foxa1 and Foxa2 single- and double-mutant mouse embryos. During specification, Foxa1 and Foxa2 regulate the extent of neurogenesis in mDA progenitors by positively regulating Ngn2 (Neurog2) expression. Subsequently, Foxa1 and Foxa2 regulate the expression of Nurr1 (Nr4a2) and engrailed 1 in immature neurons and the expression of aromatic l-amino acid decarboxylase and tyrosine hydroxylase in mature neurons during early and late differentiation of midbrain dopaminergic neurons. Interestingly, genetic evidence indicates that these functions require different gene dosages of Foxa1 and Foxa2. Altogether, our results demonstrate that Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner.

Desire, Disease, and the Origins of the Dopaminergic System

The dopaminergic neurons in the midbrain region of the central nervous system project an extensive network of connections throughout the forebrain, including the neocortex. The midbrain-forebrain dopaminergic circuits are thought to regulate a diverse set of behaviors, from the control of movement to modulation of cognition and desire--because they relate to mood, attention, reward, and addiction. Defects in these pathways, including neurodegeneration, are implicated in a variety of psychiatric and neurological diseases, such as schizophrenia, attention-deficit/hyperactivity disorder, drug addiction, and Parkinson disease. Based on the importance of the midbrain dopaminergic neurons to normal and pathological brain function, there is considerable interest in the molecular mechanisms that regulate their development. The goal of this short review is to outline new methods and recent advances in identifying the molecular networks that regulate midbrain dopaminergic neuron differentiation and fate. Midbrain dopaminergic neurons are descended from progenitor cells located near the ventral midline of the neural tube floor plate around the cephalic flexure. It is now clear that their initial formation is dependent on interactions between the signaling molecules Sonic hedgehog, WINGLESS 1, and FIBROBLAST growth factor 8, but there is still an extensive wider network of molecular interactions that must be resolved before the complete picture of dopaminergic neuron development can be described.

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.

Symposium on ‘Neural stem and progenitor cells: biology and clinical potential’, part one

In July 2004 the Department of Anatomy at University College Cork, Ireland, hosted the summer meeting of the Anatomical Society of Great Britain and Ireland in conjunction with a meeting of the Irish National Neuroscience Network. In addition to the normal ASGBI summer meeting proceedings, a special international symposium entitled ‘Neural stem and progenitor cells: biology and clinical potential’ was held. The meeting also included update talks and posters from members of the National Neuroscience Network (NNN), an ongoing nationwide interdisciplinary research programme funded by the Irish Government's National Development Plan. The meeting and symposium were organized by Professor John Fraher, Dr Aideen Sullivan, Dr Kieran McDermott and Professor Arthur Butt. The symposium brought together international researchers who have contributed to recent developments in neural stem cell biology. Throughout the two days of the symposium, the invited experts, from a diverse range of backgrounds from basic biology to clinical applications, addressed outstanding issues relating to neural stem and progenitor cell characterization and nomenclature in development and in adulthood, as well as novel approaches to cell replacement therapies for neurological disorders. Review articles resulting from this symposium comprise this special issue of the Journal of Anatomy, and part of one issue that will follow later in the year. The symposium articles presented in this issue focus on the characterization and potential clinical application of neural stem and progenitor cells, as well as papers presented by members of the NNN. The October issue will contain review articles from the symposium that addressed the related issue of defining NG2-expressing cells in the CNS and their roles in regeneration and remyelination. The first six papers presented in this issue relate to neural stem cell biology and the potential of stem cells to be used in therapeutic approaches to neurological disorders. The article by Watts et al. discusses the biology of neural stem cells in the adult brain, their relevance to brain injury and disease, and their potential to be used for the development of novel treatments for neurological diseases. The papers by Maxwell & Li and Sullivan & O’Keeffe focus on midbrain dopaminergic neurons, which are of much interest in the development of new therapies for Parkinson's disease. Maxwell & Li have identified a number of regulatory factors, including Pitx3, that influence the development of these neurons in vivo. They also describe the generation of dopaminergic neurons in vitro from embryonic stem cells, which may have the potential to be used in transplantation approaches to Parkinson's disease. Sullivan & O’Keeffe describe a neurotrophic protein, GDF5, which has actions on the induction, survival and differentiation of midbrain dopaminergic neurons. These properties could be used to induce neuroprotection and/or to enhance transplantation in Parkinson's disease. Zietlow et al. describe some of their recent work involving the transplantation of embryonic neural precursor cells into the adult rat striatum. Their finding that the long-term expansion of these cells in vitro impairs their ability to survive long term after transplantation has implications for the potential use of these cells for neural transplantation strategies. The paper by McDermott et al. is concerned with radial glial cells – their roles as neuronal progenitor cells, as well as their involvement in cell migration and axonal growth in the developing brain and spinal cord. Zhao et al. discuss the biology of cells that respond to demyelinating disorders of the central nervous system, such as multiple sclerosis. They describe the types of adult stem and/or progenitor cells that may be activated following demyelination and may contribute to remyelination and repair. The four papers presented by invited members of the NNN demonstrate a few of the research themes under investigation by the neuroscience research community in Ireland. Dinan & Scott describe the involvement of the hypothalamic–pituitary–adrenal axis in melancholic depression. In particular, the roles of the hypothalamic hormones, corticotrophin-releasing hormone and vasopressin, are discussed. The paper by Moynagh reviews the contribution of interleukin-1 signalling in astrocytes in neuropathological conditions such as multiple sclerosis and Alzheimer's disease, and the potential of this signalling pathway to be exploited as a therapeutic target in the treatment of many neurological diseases. O'Mara's paper is concerned with synaptic plasticity in the subiculum, as well as the roles of this brain area in spatial navigation, mnemonic processing and the control of responses to stress. Finally, the article by Hennessy et al. demonstrates how facial dysmorphogenesis can be measured using three-dimensional laser scanning and geometric morphometrics. These measurements can serve as an accessible and informative index of brain dysmorphogenesis in neurological and psychiatric disorders of early developmental origin.

Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain

Estrogen plays an important role during differentiation of midbrain dopaminergic neurons. This is indicated by the presence of estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase within the dopaminergic cell groups. We have previously shown that estrogen regulates the plasticity of dopamine cells through the stimulation of neurite growth/arborization. In this study, we have analyzed the capability of estrogen to influence the activity of developing mouse dopamine neurons. The expression of tyrosine hydroxylase (TH) was assessed by competitive RT-PCR and Western blotting. The developmental expression of TH in the ventral midbrain was studied from embryonic day 15 until postnatal day 15 and revealed highest TH levels early postnatally. This profile coincides with the transient aromatase expression in this brain area. Using cultured midbrain cells, we found that estrogen increased TH mRNA/protein levels. The application of the estrogen receptor antagonist ICI 182,780 resulted in a complete inhibition of estrogen effects. To verify these data in vivo, fetuses were exposed in utero from E15 until birth to the aromatase inhibitor CGS 16949A or to CGS supplemented with estrogen. CGS caused a robust reduction in TH mRNA/protein levels in the midbrain, which could be restored by estrogen substitution. Taken together, our data strongly suggest that estrogen controls dopamine synthesis in the developing nigrostriatal dopaminergic system and support the concept that estrogen is implicated in the regulation of ontogenetic steps but also in the function of midbrain dopamine neurons.

Ontogenetic expression and splicing of estrogen receptor- α and β mRNA in the rat midbrain

Several studies have shown that estrogen is important for the differentiation of midbrain dopaminergic neurons. This is supported by the previous demonstration of estrogen synthesis in the perinatal ventral midbrain. The present study attempts to characterize the expression pattern of nuclear estrogen receptors (ER-α/β) mRNAs in the ventral rat midbrain during development. By applying primers specific for the hormone-binding domain, ER-α mRNA was detected from embryonic day (E) 14 until postnatal day (P) 20, whereas considerable levels of ER-β mRNA were found from P3 to P20. In contrast, primers spanning the DNA-binding domain demonstrated the presence of transcripts for ER-α as well as ER-β after birth. These findings indicate that both ERs are expressed in the developing midbrain. The presence of ER-α transcripts devoid of the DNA-binding region is discussed in the context of ‘non-genomic’ estrogen signaling possibly by membrane receptors.

Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid-induced seizure activity

Nurr1 is an immediate early gene encoding a member of the steroid–thyroid hormone receptor family. In PC12 cells, Nurr1 is readily induced by membrane depolarization, but not by growth factors. Nurr1 is predominantly expressed in the brain, and is essential to the differentiation of midbrain dopaminergic neurons. However, Nurr1 is also expressed in brain regions unrelated to dopaminergic neurons, e.g., hippocampus and cerebral cortex, and its immediate induction following seizure activity suggests a potential involvement of this transcription factor in modulating gene expression in the nervous system. To investigate the response of Nurr1 to neuronal activation, we analyzed Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid (KA)-induced seizure. In P7 animals, systemic injection of KA increased Nurr1 mRNA levels in a few hilar cells of the dentate gyrus and some pyramidal cells of the CA3 region of the hippocampus. In older animals, Nurr1 induction progressively expanded to all hippocampal regions (P14, P21) and eventually to cortical regions (adult). The increase was rapid and transient in the dentate gyrus, a structure resistant to the neurotoxic effect of KA, and was more prolonged in other regions more susceptible to KA toxicity. Induction of Nurr1 at early postnatal stages and rapid increase in the dentate gyrus following KA-induced seizure, suggest that Nurr1 expression is modulated by neuronal activity. On the other hand, prolonged Nurr1 induction in regions sensitive to KA toxicity indicates a possible involvement of Nurr1 in selective neuronal vulnerability.

GDNF mRNA levels are induced by FGF-2 in rat C6 glioblastoma cells

Glia cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-β (TGF-β) superfamily, has been implicated in the survival, morphological and functional differentiation of midbrain dopaminergic neurons and motoneurons in vitro and in vivo. The factor may thus have utility in the treatment of various human neurodegenerative disorders. Mechanisms regulating expression of GDNF in normal and diseased brain as a possible means to increase the local availability of GDNF are only beginning to be explored. We have established and employed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to study and compare levels of expression of GDNF mRNA in several cell types and to investigate its regulation. GDNF expression was clearly evident in primary cultured astrocytes, the glioma B49 and c6 cell, but less pronounced in the Schwannoma RN22 cell lines. Little or no signal could be observed in neuroblastoma cell lines (IMR32, LAN-1) or the pheochromocytoma cell line PC12, emphasizing the glial character of this factor. Using the C6 cell line we found that fibroblast growth factor-2 (FGF-2; bFGF) can increase GDNF mRNA levels, whereas FGF-1, platelet-derived growth factor (PDGF), and vasoactive intestinal polypeptide (VIP) are apparently ineffective. Several other factors (forskolin, kainic acid, triiodothyronine, dexamethasone, GDNF, TGF-β1, and interleukin-6) appear to have slightly negative effects on GDNF mRNA levels at the concentrations tested. To further explore the relationship between FGF-2 and GDNF, we also addressed the question whether GDNF, like FGF-2, may have an effect on C6 cell proliferation. We conclude that (1) glial and glial tumor cells, rather than neuronal cell lines, express GDNF, (2) that FGF-2 has a prominent inductive effect on GDNF expression and (3) that GDNF stimulates C6 cell proliferation. Finally, these data suggest that neurotrophic actions of FGF-2 in mixed glial-neuronal cell cultures might be mediated in part by GDNF.