Fibroblast growth factor-20 promotes the differentiation of Nurr1-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons (original) (raw)
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Frontiers in Neuroscience, 2008
Human embryonic stem cells (hESCs) are a potential source of dopaminergic neurons for treatment of patients with Parkinson's disease (PD). Dopaminergic neurons can be derived from hESCs and display a characteristic midbrain phenotype. Once transplanted, they can induce partial behavioral recovery in animal models of PD. However, the potential research fi eld faces several challenges that need to be overcome before clinical application of hESCs in a transplantation therapy in PD can be considered. These include low survival of the hESC-derived, grafted dopaminergic neurons after transplantation; unclear functional integration of the grafted neurons in the host brain; and, the risk of teratoma/tumor formation from the transplanted cells. This review is focused on our recent efforts to improve the survival of hESC-dervied dopaminergic neurons. In a recent study, we examined the effect of fi broblast growth factor (FGF)-20 in the differentiation of hESCs into dopaminergic neurons. We supplemented cultures of hESCs with FGF-20 during differentiation on PA6 mouse stromal cells for 3 weeks. When we added FGF-20 the yield of neurons expressing tyrosine hydroxylase increased. We demonstrated that at least part of the effect is contributed by enhanced cell differentiation towards the dopaminergic phenotype as well as reduced cell death. We compare our results with those obtained in other published protocols using different sets of growth factors. Taken together, our data indicate that FGF-20 has potent effects to generate large number of dopaminergic neurons derived from hESCs, which may be useful for hESC-based therapy in PD.
Frontiers in Neuroanatomy, 2007
In the central nervous system, fibroblast growth factor (FGF)-20 has been reported to act preferentially on midbrain dopaminergic neurons. It also promotes the dopaminergic differentiation of stem cells. We have analyzed the effects of FGF-20 on human embryonic stem cells (hESCs) differentiation into dopaminergic neurons. We induced neuronal differentiation of hESCs by co-culturing those with PA6 mouse stromal cells for 3 weeks. When we supplemented the culture medium with FGF-20, the number of tyrosine hydroxylase (TH)expressing neurons increased fivefold, from 3% to 15% of the hESC-derived cells. The cultured cells also expressed other midbrain dopaminergic markers (PITX3, En1, Msx1, and Aldh1), suggesting that some had differentiated into midbrain dopaminergic neurons. We observed no effect of FGF-20 on the size of the soma area or neurite length of the TH-immunopositive neurons. Regardless of whether FGF-20 had been added or not, 17% of the hESC-derived cells expressed the pan-neuronal marker b-III-Tubulin. The proportion of proliferating cells positive for Ki-67 was also not affected by FGF-20 (7% of the hESC-derived cells). By contrast, after 3 weeks in culture FGF-20 significantly reduced the proportion of cells undergoing cell death, as revealed by immunoreactivity for cleaved caspase-8, Bcl-2 associated X protein (BAX) and cleaved caspase-3 (2.5% to 1.2% of cleaved caspase-3-positive cells out of the hESC-derived cells). Taken together, our results indicate that FGF-20 specifically increases the yield of dopaminergic neurons from hESCs grown on PA6 feeder cells and at least part of this effect is due to a reduction in cell death.
Fibroblast growth factor 20 is protective towards dopaminergic neurons in vivo in a paracrine manner
Neuropharmacology, 2018
Neuroprotective strategies are an unmet medical need for Parkinson's disease. Fibroblast growth factor 20 (FGF20) enhances survival of cultured dopaminergic neurons but little is known about its in vivo potential. We set out to examine whether manipulation of the FGF20 system affected nigrostriatal tract integrity in rats, to identify which fibroblast growth factor receptors (FGFRs) might reside on dopaminergic neurons and to discover the source of endogenous FGF20 in the substantia nigra (SN). Male Sprague Dawley rats were subject to a partial 6-OHDA lesion alongside treatment with exogenous FGF20 or an FGFR antagonist. Behavioural readouts and tyrosine-hydroxylase (TH) immunohistochemistry were used to evaluate nigrostriatal tract integrity. Fluorescent immunohistochemistry was used to examine FGFR subtype expression on TH-positive dopamine neurons and FGF20 cellular localisation within the SN. FGF20 (2.5 μg/day) significantly protected TH-positive cells in the SN and terminal...
2013
We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson’s disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote
Fibroblast growth factor 2 regulates adequate nigrostriatal pathway formation in mice
Journal of Comparative Neurology, 2012
Fibroblast growth factor 2 (FGF-2) is an important neurotrophic factor that promotes survival of adult mesencephalic dopaminergic (mDA) neurons and regulates their adequate development. Since mDA neurons degenerate in Parkinson's disease, a comprehensive understanding of their development and maintenance might contribute to the development of causative therapeutic approaches. The current analysis addressed the role of FGF-2 in mDA axonal outgrowth, pathway formation, and innervation of respective forebrain targets using organotypic explant cocultures of ventral midbrain (VM) and forebrain (FB). An enhanced green fluorescent protein (EGFP) transgenic mouse strain was used for the VM explants, which allowed combining and distinguishing of individual VM and FB tissue from wildtype and FGF-2-deficient embryonic day (E)14.5 embryos, respectively. These cocultures provided a suitable
Stem Cells, 2008
Infusion of TGFα into the adult dopamine (DA)-depleted striatum generates a local population of nestin + /PCNA + newborn cells . The precise origin and fate of these new striatal cells are unknown, making it difficult to direct them for neural repair in Parkinson's disease (PD). Experiments in rats using BrdU to label neural progenitor cells (NPCs) showed that during TGFα infusion in the DAdepleted striatum, newborn striatal cells formed a homogenous population of precursors, with the majority coexpressing nestin, Mash1, Olig2 and EGFR, consistent with the phenotype of multipotent C cells. Upon TGFα pump withdrawal, the subventricular zone (SVZ) was repopulated by neuroblasts. Strikingly, during this period, numerous clusters of DCX + /PSANCAM + neuroblasts were also produced in the ipsilateral medial striatum. In parallel, striatal BrdU + /GFAP + astrocytes were generated, but no BrdU + /O4 + /CNPase + oligodendrocytes. Infusion of the neuralizing BMP antagonist noggin after TGFα pump withdrawal increased the neuroblast to astrocyte ratio among new striatal cells by blocking glial differentiation, but did not alter striatal neurogenesis. At no time or no treatment condition were differentiated neurons generated, including DA neurons. Using 6-OHDA lesioned nestin-CreER T2 /R26R-YFP mice that allow genetic fate-mapping of SVZ nestin + cells, we show that TGFα-generated striatal cells originate from SVZ nestin + precursors that confirmed data from the rats on the phenotype and fate of striatal nestin + /PCNA + cells upon TGFα withdrawal. This work demonstrates that a large population of multipotent striatal C-like cells can be generated in the DA-depleted striatum that do not spontaneously differentiate into DA neurons.
Journal of Biological Chemistry, 2012
Background: Nurr1 and FGFR1 are integrative nuclear factors participating in postmitotic dopaminergic neuron development. Results: Both nuclear receptors show a functional interaction in co-immunoprecipitation, FRAP, ChIP, and luciferase gene reporter assay. Conclusion: Cooperation of nuclear FGFR1 and Nurr1 offers a new mechanism in transcriptional regulation and integration. Significance: This mechanism may channel diverse stimuli in developing and mature dopaminergic neurons, providing a potential therapeutic target. Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation. Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1-EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23-kDa FGF-2 ligand (FGF-2 23) enhances Nurr1-dependent activation of the TH gene promoter. Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.
Experimental Neurology, 2004
Dopaminergic (DA) micrografts were co-transplanted with Schwann cells (SC) overexpressing 18 kDa and 21/23 kDa FGF-2 into the caudate-putamen unit (CPu) of unilaterally 6-hydroxydopamine-lesioned rats. We report here that SC engineered to overexpress FGF-2 promoted DA-graft-induced restoration, whether co-transplanted at the same site or grafted at a second more distant site within the CPu. In addition, the 21/23 kDa FGF-2 isoforms resulted in a significantly better reinnervation and survival of dopaminergic micrografts when compared to the 18-kDa FGF-2 isoform. However, this effect was not that distinct on functional recovery due to, for example, ceiling effects. One main finding of this study was the influence of the gene promotor on DA survival, respectively, vector-mediated trophism. Therefore, comparisons in terms of survival between 18 kDa and higher molecular weight (HMW) FGF-2 are complicated in the mixed grafted experiments. Furthermore, the first demonstration of the presence of the 21/23 kDa FGF-2 isoforms in the nigrostriatal system and their potent neurotrophic in vivo activities, as shown in the present study, suggest (I) a physiological role of these proteins for dopaminergic neurons and (II) a restorative potential under normal as well as regenerative processes. However, FGF-2-mediated effects are more pronounced after co-transplantation with SC/DA cells mixed in one suspension at the same implantation side than in the side-by-side approach with a spatially and temporally separated transplantation of SC (day 1) and DA-cells (day 3). These findings indicate the necessity of direct contact between FGF-2 and DA-neurons, further elucidate the neurotrophic role of FGF-2 for DA-neurons and highlight the differential restorative potentials of its respective isoforms. We propose that administration of HMW FGF-2 may be used to improve function in the rat Parkinson's disease model. D 2004 Elsevier Inc. All rights reserved.
Neuroscience, 2005
The effects of i.c.v. infused platelet-derived growth factor and brain-derived neurotrophic factor on cell genesis, as assessed with bromodeoxyuridine (BrdU) incorporation, were studied in adult rats with unilateral 6-hydroxydopamine lesions. Both growth factors increased the numbers of newly formed cells in the striatum and substantia nigra to an equal extent following 10 days of treatment. At 3 weeks after termination of growth factor treatment, immunostaining of BrdUlabeled cells with the neuronal marker NeuN revealed a significant increase in newly generated neurons in the striatum. In correspondence, many doublecortin-labeled neuroblasts were also observed in the denervated striatum following growth factor treatment. Further evaluation suggested that a subset of these new neurons expresses the early marker for striatal neurons Pbx. However, no BrdU-positive cells were co-labeled with DARPP-32, a protein expressed by mature striatal projection neurons. Both in the striatum and in the substantia nigra there were no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment, such that BrdU-labeled cells never co-expressed tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In conclusion, our results suggest that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats.