The Crucial Roles of Pitx3 in Midbrain Dopaminergic Neuron Development and Parkinson's Disease-Associated Neurodegeneration.

The degeneration of midbrain dopaminergic (mDA) neurons, particularly in the substantia nigra pars compacta (SNc), is one of the most prominent pathological hallmarks of Parkinson's disease (PD). To uncover the pathogenic mechanisms of mDA neuronal death during PD may provide therapeutic targets to prevent mDA neuronal loss and slow down the disease's progression. Paired-like homeodomain transcription factor 3 (Pitx3) is selectively expressed in the mDA neurons as early as embryonic day 11.5 and plays a critical role in mDA neuron terminal differentiation and subset specification. Moreover, Pitx3-deficient mice exhibit some canonical PD-related features, including the profound loss of SNc mDA neurons, a dramatic decrease in striatal dopamine (DA) levels, and motor abnormalities. However, the precise role of Pitx3 in progressive PD and how this gene contributes to mDA neuronal specification during early stages remains unclear. In this review, we updated the latest findings on Pitx3 by summarizing the crosstalk between Pitx3 and its associated transcription factors in mDA neuron development. We further explored the potential benefits of Pitx3 as a therapeutic target for PD in the future. To better understand the transcriptional network of Pitx3 in mDA neuron development may provide insights into Pitx3-related clinical drug-targeting research and therapeutic approaches.

A Pitx3-deficient developmental mouse model for fine motor, olfactory, and gastrointestinal symptoms of Parkinson's disease.

Parkinson's disease (PD) is characterized by the selective death of substantia nigra pars compacta (SNpc) dopaminergic neurons and includes both motor and non-motor symptoms. While numerous models exist for the study of typical PD motor deficits, fewer exist for non-motor symptoms. Previous studies have shown that a Pitx3-/- mouse model (aphakia or ak mouse) has specific developmental failure of the dopaminergic neuron population in the SNpc and that it can be used for the study of PD-related gross motor dysfunction as well as cognitive functional deficits. It remains unclear whether the aphakia mouse, both male and female, might also be used to model fine motor deficits and for additional studies of non-motor deficits associated with PD. Here, using an extensive battery of behavioral tests, we demonstrate that the aphakia mouse shows both gross and fine motor functional deficits compared with control mice. Furthermore, aphakia mice show deficits of olfactory function in buried pellet, odor discrimination and odor habituation/dishabituation tests. We also found that aphakia mice suffer from gastrointestinal dysfunction (e.g., longer whole gut transit time and colon motility deficits), suggesting that the mutation also affects function of the gut-brain axis in this animal model. Moreover, our data demonstrate that in the aphakia mouse, L-DOPA, the gold standard PD medication, can rescue both gross and fine motor function deficits but neither olfactory nor gastrointestinal symptoms, a pattern much like that seen in PD patients. Altogether, this suggests that the aphakia mouse is a suitable model for fine motor, olfactory and gastrointestinal behavioral studies of PD as well as for the development of novel disease-modifying therapeutics. SIGNIFICANCE STATEMENT: While several animal models are available to study the major motor symptoms of PD, there are fewer that replicate non-motor symptoms, which constitute a major source of morbidity for patients. Moreover, available models often require manipulations resulting in sudden massive cell loss and inflammation, both of which may interfere with understanding of the direct effects of dopaminergic neuronal loss in the SNpc. We describe a model of congenital SNpc cell deficiency in a Pitx3-/- mouse and characterize it with a battery of behavioral tests suggesting that it closely mimics non-motor as well as motor symptoms of PD, providing a useful insight into the effects of the nigrostriatal dopamine deficit. Taken together, these data suggest that the ak mouse represents a useful model to study dopaminergic system function for both motor and non-motor symptoms of PD.

First evidence of SOX2 mutations in Peters' anomaly: Lessons from molecular screening of 95 patients.

Peters' anomaly (PA) is a rare anterior segment dysgenesis characterized by central corneal opacity and irido-lenticulo-corneal adhesions. Several genes are involved in syndromic or isolated PA (B3GLCT, PAX6, PITX3, FOXE3, CYP1B1). Some copy number variations (CNVs) have also been occasionally reported. Despite this genetic heterogeneity, most of patients remain without genetic diagnosis. We retrieved a cohort of 95 individuals with PA and performed genotyping using a combination of comparative genomic hybridization, whole genome, exome and targeted sequencing of 119 genes associated with ocular development anomalies. Causative genetic defects involving 12 genes and CNVs were identified for 1/3 of patients. Unsurprisingly, B3GLCT and PAX6 were the most frequently implicated genes, respectively in syndromic and isolated PA. Unexpectedly, the third gene involved in our cohort was SOX2, the major gene of micro-anophthalmia. Four unrelated patients with PA (isolated or with microphthalmia) were carrying pathogenic variants in this gene that was never associated with PA before. Here we described the largest cohort of PA patients ever reported. The genetic bases of PA are still to be explored as genetic diagnosis was unavailable for 2/3 of patients. Nevertheless, we showed here for the first time the involvement of SOX2 in PA, offering new evidence for its role in corneal transparency and anterior segment development.

Unilateral buphthalmos, corneal staphyloma and corneal fistula caused by pathogenic variant in the PITX3 gene: a case report.

INTRODUCTION: PITX3 has been reported to be associated with congenital cataracts, anterior segment mesenchymal dysgenesis, Peters' anomaly, and microphthalmia. In this case, an infant with unilateral buphthalmos, corneal staphyloma and corneal fistula carrying a variant in PITX3 was reported. CASE DESCRIPTION: We describe a 4-month-old female infant who was referred to our Eye Clinic because of gradual enlargement of the eyeball in the right eye and whitish opacity in both eyes. Buphthalmos with long axial length (22.04 mm), macrocornea with diffuse corneal oedema and opacity (14.50 mm*14.50 mm) and high intraocular pressure (23.78 mmHg) were detected in the right eye. Microphthalmia with short axial length (16.23 mm), microcornea with diffuse corneal oedema and opacity (7.50 mm*6.50 mm) were detected in the left eye. A 360° trabeculotomy was performed for the right eye. However, corneal staphyloma and corneal fistula in the right eye were detected 6 months after the surgery. A variant in exon 4 of PITX3 (c.640_656dup (p. Gly220Profs*95)) was identified in the proband but was not detected in her healthy parents. CONCLUSION: A novel phenotype characterized by unilateral buphthalmos, corneal staphyloma and corneal fistula in an infant were reported to be associated with PITX3 in our study. Our study expands the scope of the clinical heterogeneity of PITX3 variants. It also improves our understanding and increases the attention given to patients with PITX3 variants.

Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease.

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.

Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Bioluminescence-driven optogenetic activation of transplanted neural precursor cells improves motor deficits in a Parkinson's disease mouse model.

The need to develop efficient therapies for neurodegenerative diseases is urgent, especially given the increasing percentages of the population living longer, with increasing chances of being afflicted with conditions like Parkinson's disease (PD). A promising curative approach toward PD and other neurodegenerative diseases is the transplantation of stem cells to halt and potentially reverse neuronal degeneration. However, stem cell therapy does not consistently lead to improvement for patients. Using remote stimulation to optogenetically activate transplanted cells, we attempted to improve behavioral outcomes of stem cell transplantation. We generated a neuronal precursor cell line expressing luminopsin 3 (LMO3), a luciferase-channelrhodopsin fusion protein, which responds to the luciferase substrate coelenterazine (CTZ) with emission of blue light that in turn activates the opsin. Neuronal precursor cells were injected bilaterally into the striatum of homozygous aphakia mice, which carry a spontaneous mutation leading to lack of dopaminergic neurons and symptoms of PD. Following transplantation, the cells were stimulated over a period of 10 days by intraventricular injections of CTZ. Mice receiving CTZ demonstrated significantly improved motor skills in a rotarod test compared to mice receiving vehicle. Thus, bioluminescent optogenetic stimulation of transplanted neuronal precursor cells shows promising effects in improving locomotor behavior in the aphakia PD mouse model and encourages further studies to elucidate the mechanisms and long-term outcomes of these beneficial effects.

Human Mesenchymal Stromal Cells Unveil an Unexpected Differentiation Potential toward the Dopaminergic Neuronal Lineage.

Degeneration of dopaminergic neurons represents the cause of many neurodegenerative diseases, with increasing incidence worldwide. The replacement of dead cells with new healthy ones may represent an appealing therapeutic approach to these pathologies, but currently, only pluripotent stem cells can generate dopaminergic neurons with high efficiency. However, with the use of these cells arises safety and/or ethical issues. Human mesenchymal stromal cells (hFM-MSCs) are perinatal stem cells that can be easily isolated from the amniochorionic membrane after delivery. Generally considered multipotent, their real differentiative potential is not completely elucidated. The aim of this study was to analyze their stemness characteristics and to evaluate whether they may overcome their mesenchymal fate, generating dopaminergic neurons. We demonstrated that hFM-MSCs expressed embryonal genes OCT4, NANOG, SOX2, KLF4, OVOL1, and ESG1, suggesting they have some features of pluripotency. Moreover, hFM-MSCs that underwent a dopaminergic differentiation protocol gradually increased the transcription of dopaminergic markers LMX1b, NURR1, PITX3, and DAT. We finally obtained a homogeneous population of cells resembling the morphology of primary midbrain dopaminergic neurons that expressed the functional dopaminergic markers TH, DAT, and Nurr1. In conclusion, our results suggested that hFM-MSCs retain the expression of pluripotency genes and are able to differentiate not only into mesodermal cells, but also into neuroectodermal dopaminergic neuron-like cells.

Carbon Fibers as a New Type of Scaffold for Midbrain Organoid Development.

The combination of induced pluripotent stem cell (iPSC) technology and 3D cell culture creates a unique possibility for the generation of organoids that mimic human organs in in vitro cultures. The use of iPS cells in organoid cultures enables the differentiation of cells into dopaminergic neurons, also found in the human midbrain. However, long-lasting organoid cultures often cause necrosis within organoids. In this work, we present carbon fibers (CFs) for medical use as a new type of scaffold for organoid culture, comparing them to a previously tested copolymer poly-(lactic-co-glycolic acid) (PLGA) scaffold. We verified the physicochemical properties of CF scaffolds compared to PLGA in improving the efficiency of iPSC differentiation within organoids. The physicochemical properties of carbon scaffolds such as porosity, microstructure, or stability in the cellular environment make them a convenient material for creating in vitro organoid models. Through screening several genes expressed during the differentiation of organoids at crucial brain stages of development, we found that there is a correlation between PITX3, one of the key regulators of terminal differentiation, and the survival of midbrain dopaminergic (mDA) neurons and tyrosine hydroxylase (TH) gene expression. This makes organoids formed on carbon scaffolds an improved model containing mDA neurons convenient for studying midbrain-associated neurodegenerative diseases such as Parkinson's disease.

Differences in the spatiotemporal expression and epistatic gene regulation of the mesodiencephalic dopaminergic precursor marker PITX3 during chicken and mouse development.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral mesencephalon and caudal diencephalon of all tetrapod species studied so far. They are the most prominent DA neuronal population and are implicated in control and modulation of motor, cognitive and rewarding/affective behaviors. Their degeneration or dysfunction is intimately linked to several neurological and neuropsychiatric human diseases. To gain further insights into their generation, we studied spatiotemporal expression patterns and epistatic interactions in chick embryos of selected marker genes and signaling pathways associated with mdDA neuron development in mouse. We detected striking differences in the expression patterns of the chick orthologs of the mouse mdDA marker genes Pitx3 and Aldh1a1, which suggests important differences between the species in the generation/generating of these cells. We also discovered that the sonic hedgehog signaling pathway is both necessary and sufficient for the induction of ectopic PITX3 expression in chick mesencephalon downstream of WNT9A-induced LMX1a transcription. These aspects of early chicken development resemble the ontogeny of zebrafish diencephalic DA neuronal populations, and suggest a divergence between birds and mammals during evolution.

A novel mutation in the OAR domain of PITX3 associated with congenital posterior subcapsular cataract.

BACKGROUND: Congenital cataract is the most common cause of blindness among children worldwide. The aim of this study was to identify causative mutations in a Chinese family with isolated autosomal dominant posterior subcapsular cataract. METHODS: The proband and her parents underwent full ophthalmological examinations. DNA was extracted from the participants' peripheral venous blood. The mutation was identified via panel-based next-generation sequencing (NGS) and was validated via Sanger sequencing. RESULTS: Posterior subcapsular lenticular opacity was observed in both of the proband's eyes. The novel deletion mutation c.797_814del, p.Ser266_Ala271del in the PITX3 gene was identified in the proband and her father. This mutation is located within the otp/aristaless/rax (OAR) domain at the COOH-terminus of the protein, which functions in DNA binding and transactivation. This mutation would result in a deletion of 6 amino acid residues at the C terminal of the protein. CONCLUSIONS: The mutation c.797_814del, p.Ser266_Ala271del is a novel mutation in the conserved DNA-binding OAR domain of PITX3 that causes congenital cataract.

Dysregulation of Dopaminergic Regulatory Factors TH, Nurr1, and Pitx3 in the Ventral Tegmental Area Associated with Neuronal Injury Induced by Chronic Morphine Dependence.

The ventral tegmental area (VTA), a critical portion of the mesencephalic dopamine system, is thought to be involved in the development and maintenance of addiction. It has been proposed that the dopaminergic regulatory factors TH, Nurr1, and Pitx3 are crucial for determining the survival and maintenance of dopaminergic neurons. Thus, the present study investigated whether abnormalities in these dopaminergic regulatory factors in the VTA were associated with neuronal injury induced by chronic morphine dependence. Rat models with different durations of morphine dependence were established. Thionine staining was used to observe morphological changes in the VTA neurons. Immunohistochemistry and western blot were used to observe changes in the expression of the dopaminergic regulatory proteins TH, Nurr1, and Pitx3. Thionine staining revealed that prolonged morphine dependence resulted in dopaminergic neurons with edema, a lack of Nissl bodies, and pyknosis. Immunohistochemistry showed that the number of TH⁺, Nurr1⁺, and Pitx3⁺ cells, and the number of TH⁺ cells expressing Nurr1 or Pitx3, significantly decreased in the VTA after a long period of morphine dependence. Western blot results were consistent with the immunohistochemistry findings. Chronic morphine exposure resulted in abnormalities in dopaminergic regulatory factors and pathological changes in dopaminergic neurons in the VTA. These results suggest that dysregulation of dopaminergic regulatory factors in the VTA are associated with neuronal injury induced by chronic morphine dependence.

Nurr1, Pitx3, and α7 nAChRs mRNA Expression in Nigral Tissue of Rats with Pedunculopontine Neurotoxic Lesion.

Background and Objectives: The knowledge that the cholinergic neurons from pedunculopontine nucleus (PPN) are vulnerable to the degeneration in early stages of the Parkinson disease progression has opened new perspectives to the development of experimental model focused in pontine lesions that could increase the risk of nigral degeneration. In this context it is known that PPN lesioned rats exhibit early changes in the gene expression of proteins responsible for dopaminergic homeostasis. At the same time, it is known that nicotinic cholinergic receptors (nAChRs) mediate the excitatory influence of pontine-nigral projection. However, the effect of PPN injury on the expression of transcription factors that modulate dopaminergic neurotransmission in the adult brain as well as the α7 nAChRs gene expression has not been studied. The main objective of the present work was the study of the effects of the unilateral neurotoxic lesion of PPN in nuclear receptor-related factor 1 (Nurr1), paired-like homeodomain transcription factor 3 (Pitx3), and α7 nAChRs mRNA expression in nigral tissue. Materials and Methods: The molecular biology studies were performed by means of RT-PCR. The following experimental groups were organized: Non-treated rats, N-methyl-D-aspartate (NMDA)-lesioned rats, and Sham operated rats. Experimental subjects were sacrificed 24 h, 48 h and seven days after PPN lesion. Results: Nurr1 mRNA expression, showed a significant increase both 24 h (p < 0.001) and 48 h (p < 0.01) after PPN injury. Pitx3 mRNA expression evidenced a significant increase 24 h (p < 0.001) followed by a significant decrease 48 h and seven days after PPN lesion (p < 0.01). Finally, the α7 nAChRs nigral mRNA expression remained significantly diminished 24 h, 48 h (p < 0.001), and 7 days (p < 0.01) after PPN neurotoxic injury. Conclusion: Taking together these modifications could represent early warning signals and could be the preamble to nigral neurodegeneration events.

Xenopus pitx3 target genes lhx1 and xnr5 are identified using a novel three-fluor flow cytometry-based analysis of promoter activation and repression.

BACKGROUND: Pitx3 plays a well understood role in directing development of lens, muscle fiber, and dopaminergic neurons; however, in Xenopus laevis, it may also play a role in early gastrulation and somitogenesis. Potential downstream targets of pitx3 possess multiple binding motifs that would not be readily accessible by conventional promoter analysis. RESULTS: We isolated and characterized pitx3 target genes lhx1 and xnr5 using a novel three-fluor flow cytometry tool that was designed to dissect promoters with multiple binding sites for the same transcription factor. This approach was calibrated using a known pitx3 target gene, Tyrosine hydroxylase. CONCLUSIONS: We demonstrate how flow cytometry can be used to detect gene regulatory changes with exquisite precision on a cell-by-cell basis, and establish that in HEK293 cells, pitx3 directly activates lhx1 and represses xnr5. Developmental Dynamics 246:657-669, 2017. © 2017 Wiley Periodicals, Inc.

From eyeless to neurological diseases.

Age-related cataracts are frequently associated with degenerative changes in the ocular lens including the aggregation of proteins - mainly crystallins, but also other proteins including amyloids (Aß) leading to the hypothesis that cataracts could be used as "biomarkers" for Alzheimer disease. Even if this hypothesis was rejected by David Beebe's last paper (Bei et al., Exp. Eye Res., 2015), it is a fascinating aspect to look for commonalities between eye diseases and neurological disorders. In this review, I discuss such commonalities between eye and brain mainly from a developmental point of view. The finding of the functional homology of the Drosophila eyeless gene with the mammalian Pax6 gene marks a first highlight in the developmental genetics of the eye - this result destroyed the "dogma" of the different evolutionary routes of eye development in flies and mammals. The second highlight was the finding that Pax6 is also involved in the development of the forebrain supporting the pleiotropic role of many genes. These findings opened a new avenue for research showing that a broad variety of transcription factors, but also structural proteins are involved both, in eye and brain development as well as into the maintenance of the functional integrity of the corresponding tissue(s). In this review recent findings are summarized demonstrating that genes whose mutations have been identified first to be causative for congenital or juvenile eye disorders are also involved in regenerative processes and neurogenesis (Pax6), but also in neurodegenerative diseases like Parkinson (e.g. Pitx3) or in neurological disorders like Schizophrenia (e.g. Crybb1, Crybb2).

Genetic analysis of PITX3 variants in patients with essential tremor.

BACKGROUND: A clinical overlap between essential tremor (ET) and Parkinson's disease (PD) has prompted investigation whether these disorders share common genetic factors. The paired-like homeodomain transcription factor 3 gene (PITX3) has been shown to play an important role for the differentiation and survival of midbrain dopaminergic neurons in the substantia nigra pars compacta. The preferential degeneration of those dopaminergic neurons is the pathological hallmark in PD. AIMS OF THE STUDY: The purpose of this study was to evaluate whether PITX3 variants are related to susceptibility of ET in Chinese Han population. METHODS: Genetic analysis of two variants rs3758549 and rs4919621 of the PITX3 gene was conducted in 200 Chinese Han patients with ET and 426 controls. RESULTS: We did not identify any statistically significant difference in either genotypic or allelic frequencies of variants between the ET patients and control cohort (all P > 0.05). Haplotype analysis of two variants in the PITX3 gene showed no potential association between the haplotypes and risk of ET (all P > 0.05). CONCLUSIONS: Our data suggest that PITX3 variants rs3758549 and rs4919621 are not associated with ET in Chinese Han population.

A WNT1-regulated developmental gene cascade prevents dopaminergic neurodegeneration in adult En1(+/-) mice.

The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) in the mammalian midbrain is a hallmark of human Parkinson's Disease (PD) and of certain genetic mouse models of PD, such as mice heterozygous for the homeodomain transcription factor Engrailed 1 (En1(+/-) mice). Neurotoxin-based animal models of PD, in contrast, are characterized by the fast and partly reversible degeneration of the SNc and VTA DA neurons. The secreted protein WNT1 was previously shown to be strongly induced in the neurotoxin-injured adult ventral midbrain (VM), and to protect the SNc and VTA DA neurons from cell death in this context. We demonstrate here that the sustained and ectopic expression of Wnt1 in the SNc and VTA DA neurons of En1(+/Wnt1) mice also protected these genetically affected En1 heterozygote (En1(+/-)) neurons from their premature degeneration in the adult mouse VM. We identified a developmental gene cascade that is up-regulated in the adult En1(+/Wnt1) VM, including the direct WNT1/ß-catenin signaling targets Lef1, Lmx1a, Fgf20 and Dkk3, as well as the indirect targets Pitx3 (activated by LMX1A) and Bdnf (activated by PITX3). We also show that the secreted neurotrophin BDNF and the secreted WNT modulator DKK3, but not the secreted growth factor FGF20, increased the survival of En1 mutant dopaminergic neurons in vitro. The WNT1-mediated signaling pathway and its downstream targets BDNF and DKK3 might thus provide a useful means to treat certain genetic and environmental (neurotoxic) forms of human PD.

Nitric oxide synthase inhibition decreases l-DOPA-induced dyskinesia and the expression of striatal molecular markers in Pitx3(-/-) aphakia mice.

Nitric oxide (NO), a gaseous messenger molecule synthesized by nitric oxide synthase (NOS), plays a pivotal role in integrating dopamine transmission in the basal ganglia and has been implicated in the pathogenesis of Parkinson disease (PD). To study the role of the nitrergic system in l-DOPA-induced dyskinesia (LID), we assessed the effect of the pharmacological manipulation of NO levels and NO/cyclic guanosine monophosphate (cGMP) signaling on LID in the Pitx3(-/-) aphakia mouse, a genetic model of PD. To evaluate the effect of decreased NO signaling on the development of LID, Pitx3(-/-) mice were chronically treated with l-DOPA and 7-nitroindazole (7-NI, a neuronal NOS inhibitor). To evaluate its effect on the expression of established LID, 7-NI was administered acutely to dyskinetic mice. The chronic 7-NI treatment attenuated the development of LID in the Pitx3(-/-) mice, and the sub-acute 7-NI treatment attenuated established dyskinesia without affecting the beneficial therapeutic effect of l-DOPA. Moreover, 7-NI significantly reduced FosB and pAcH3 expression in the acutely and chronically l-DOPA-treated mice. We also examined how increasing NO/cGMP signaling affects LID expression by acutely administering molsidomine (an NO donor) or zaprinast (a cGMP phosphodiesterase 5-PDE5 inhibitor) before l-DOPA in mice with established dyskinesia. Paradoxically, the administration of either of these drugs also significantly diminished the expression of established LID; however, the effect occurred at the expense of the antiparkinsonian l-DOPA properties. We demonstrate that targeting the NO/cGMP signaling pathway reduces dyskinetic behaviors and molecular markers, but only the 7-NI treatment preserved the antiparkinsonian effect of l-DOPA, indicating that NOS inhibitors represent a potential therapy to reduce LID.

Time course and side-by-side analysis of mesodermal, pre-myogenic, myogenic and differentiated cell markers in the chicken model for skeletal muscle formation.

The chicken is a well-established model for amniote (including human) skeletal muscle formation because the developmental anatomy of chicken skeletal muscle matches that of mammals. The accessibility of the chicken in the egg as well as the sequencing of its genome and novel molecular techniques have raised the profile of this model. Over the years, a number of regulatory and marker genes have been identified that are suited to monitor the progress of skeletal myogenesis both in wildtype and in experimental embryos. However, in the various studies, differing markers at different stages of development have been used. Moreover, contradictory results on the hierarchy of regulatory factors are now emerging, and clearly, factors need to be able to cooperate. Thus, a reference paper describing in detail and side-by-side the time course of marker gene expression during avian myogenesis is needed. We comparatively analysed onset and expression patterns of the key markers for the chicken immature paraxial mesoderm, for muscle-competent cells, for cells committed to myogenesis and for cells entering terminal differentiation. We performed this analysis from stages when the first paraxial mesoderm is being laid down to the stage when mesoderm formation comes to a conclusion. Our data show that, although the sequence of marker gene expression is the same at the various stages of development, the timing of the expression onset is quite different. Moreover, marker gene expression in myogenic cells being deployed from the dorsomedial and ventrolateral lips of the dermomyotome is different from those being deployed from the rostrocaudal lips, suggesting different molecular programs. Furthermore, expression of Myosin Heavy Chain genes is overlapping but different along the length of a myotube. Finally, Mef2c is the most likely partner of Mrf proteins, and, in contrast to the mouse and more alike frog and zebrafish fish, chicken Mrf4 is co-expressed with MyoG as cells enter terminal differentiation.

Impaired motor coordination in Pitx3 overexpression mice.

Midbrain dopaminergic (DA) neurons are involved in the regulation of voluntary movement and in emotion-related behaviors and are affected in Parkinson's disease (PD). The homeodomain transcription factor Pitx3, which is uniquely expressed in midbrain DA neurons, plays a critical role in the development, function and maintenance of midbrain DA neurons. Pitx3 deficiency results in selective deficits of midbrain DA neurons in the substantia nigra pars compacta (SNc), reminiscent of the specific DA neuronal loss observed in PD. In this study, we found that selective overexpression of Pitx3 in intact midbrain DA neurons significantly affects the function of midbrain DA neurons. We observed changes in DA levels and gene expressions in mice overexpressing Pitx3. Furthermore, motor coordination and locomotion activities are significantly affected in mice overexpressing Pitx3, suggesting that the expression level of Pitx3 plays an important role in the function of midbrain DA neuron in vivo.