ABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.
Subject(s)
Dopaminergic Neurons/drug effects , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Neuroprotective Agents/pharmacology , Parkinson Disease/drug therapy , Proto-Oncogene Proteins c-ret/genetics , Animals , Disease Models, Animal , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Gene Expression , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Humans , Injections, Intraventricular , Male , Mesencephalon/drug effects , Mesencephalon/metabolism , Mesencephalon/pathology , Mice , Mice, Knockout , Neostriatum/drug effects , Neostriatum/metabolism , Neostriatum/pathology , Neuroprotective Agents/metabolism , Parkinson Disease/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Proto-Oncogene Proteins c-ret/deficiency , Signal Transduction , Stereotaxic Techniques , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Substantia Nigra/pathologyABSTRACT
Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed, but how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glialILC3epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably, ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit, revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals.
Subject(s)
Immunity, Innate , Intestines/immunology , Lymphocytes/immunology , Neuroglia/metabolism , Neurotransmitter Agents/metabolism , Animals , Cellular Microenvironment/immunology , Epithelial Cells/cytology , Epithelial Cells/immunology , Female , Gastrointestinal Microbiome/immunology , Immunity, Mucosal , Inflammation/immunology , Inflammation/metabolism , Interleukins/biosynthesis , Interleukins/immunology , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Intestines/cytology , Lymphocytes/cytology , Lymphocytes/metabolism , MAP Kinase Signaling System , Male , Mice , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/metabolism , Neuroglia/immunology , Neurotransmitter Agents/immunology , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/metabolism , STAT3 Transcription Factor/metabolism , Interleukin-22ABSTRACT
RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.
Subject(s)
Ganglia, Spinal/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Mechanoreceptors/metabolism , Mechanotransduction, Cellular , Proto-Oncogene Proteins c-ret/genetics , Animals , Cell Survival , Embryo, Mammalian , Ganglia, Spinal/cytology , Ganglia, Spinal/growth & development , Gene Expression Regulation, Developmental , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Mechanoreceptors/cytology , Mice , Mice, Knockout , Morphogenesis/genetics , Neurturin/deficiency , Neurturin/genetics , Proto-Oncogene Proteins c-ret/deficiencyABSTRACT
Parkin and the glial cell line-derived neurotrophic factor (GDNF) receptor RET have both been independently linked to the dopaminergic neuron degeneration that underlies Parkinson's disease (PD). In the present study, we demonstrate that there is genetic crosstalk between parkin and the receptor tyrosine kinase RET in two different mouse models of PD. Mice lacking both parkin and RET exhibited accelerated dopaminergic cell and axonal loss compared with parkin-deficient animals, which showed none, and RET-deficient mice, in which we found moderate degeneration. Transgenic expression of parkin protected the dopaminergic systems of aged RET-deficient mice. Downregulation of either parkin or RET in neuronal cells impaired mitochondrial function and morphology. Parkin expression restored mitochondrial function in GDNF/RET-deficient cells, while GDNF stimulation rescued mitochondrial defects in parkin-deficient cells. In both cases, improved mitochondrial function was the result of activation of the prosurvival NF-κB pathway, which was mediated by RET through the phosphoinositide-3-kinase (PI3K) pathway. Taken together, these observations indicate that parkin and the RET signaling cascade converge to control mitochondrial integrity and thereby properly maintain substantia nigra pars compacta dopaminergic neurons and their innervation in the striatum. The demonstration of crosstalk between parkin and RET highlights the interplay in the protein network that is altered in PD and suggests potential therapeutic targets and strategies to treat PD.
Subject(s)
Dopaminergic Neurons/pathology , Glial Cell Line-Derived Neurotrophic Factor/physiology , Nerve Degeneration/pathology , Parkinsonian Disorders/genetics , Proto-Oncogene Proteins c-ret/physiology , Ubiquitin-Protein Ligases/physiology , Adenosine Triphosphate/biosynthesis , Animals , Anxiety/genetics , Cell Line , Cell Size , Disease Progression , Exploratory Behavior , Glial Cell Line-Derived Neurotrophic Factor/deficiency , Glial Cell Line-Derived Neurotrophic Factor/genetics , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria/pathology , NF-kappa B/physiology , Parkinsonian Disorders/pathology , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Recombinant Fusion Proteins/metabolism , Rotarod Performance Test , Signal Transduction , Substantia Nigra/pathology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/geneticsABSTRACT
Enteric neurons and blood vessels form intricate networks throughout the gastrointestinal tract. To support the hypothesis of a possible interaction of both networks, we investigated whether primary mesenteric vascular cells (MVCs) and enteric nervous system (ENS)-derived cells (ENSc) depend on each other using two- and three-dimensional in vitro assays. In a confrontation assay, both cell types migrated in a target-oriented manner towards each other. The migration of MVCs was significantly increased when cultured in ENSc-conditioned medium. Co-cultures of ENSc with MVCs resulted in an improved ENSc proliferation and differentiation. Moreover, we analysed the formation of the vascular and nervous system in developing mice guts. It was found that the patterning of newly formed microvessels and neural stem cells, as confirmed by nestin and SOX2 stainings, is highly correlated in all parts of the developing gut. In particular in the distal colon, nestin/SOX2-positive cells were found in the tissues adjacent to the capillaries and in the capillaries themselves. Finally, in order to provide evidences for a mutual interaction between endothelial and neural cells, the vascular patterns of a RET((-/-)) knockout mouse model as well as human Hirschsprung's cases were analysed. In the distal colon of postnatal RET((-/-)) knockout mice, the vascular and neural networks were similarly disrupted. In aganglionic zones of Hirschsprung's patients, the microvascular density was significantly increased compared with the ganglionic zone within the submucosa. Taken together, these findings indicate a strong interaction between the enteric nervous and vascular system.
Subject(s)
Cell Communication , Enteric Nervous System/physiology , Intestines/blood supply , Intestines/innervation , Microvessels/physiology , Neural Stem Cells/physiology , Animals , Cell Proliferation , Cells, Cultured , Chemotaxis , Coculture Techniques , Culture Media, Conditioned/metabolism , Enteric Nervous System/cytology , Enteric Nervous System/metabolism , Female , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Hirschsprung Disease/pathology , Hirschsprung Disease/physiopathology , Humans , Infant , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Microvessels/cytology , Microvessels/metabolism , Neovascularization, Physiologic , Nestin/genetics , Nestin/metabolism , Neural Stem Cells/metabolism , Neurogenesis , Paracrine Communication , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Haematopoiesis is a developmental cascade that generates all blood cell lineages in health and disease. This process relies on quiescent haematopoietic stem cells capable of differentiating, self renewing and expanding upon physiological demand. However, the mechanisms that regulate haematopoietic stem cell homeostasis and function remain largely unknown. Here we show that the neurotrophic factor receptor RET (rearranged during transfection) drives haematopoietic stem cell survival, expansion and function. We find that haematopoietic stem cells express RET and that its neurotrophic factor partners are produced in the haematopoietic stem cell environment. Ablation of Ret leads to impaired survival and reduced numbers of haematopoietic stem cells with normal differentiation potential, but loss of cell-autonomous stress response and reconstitution potential. Strikingly, RET signals provide haematopoietic stem cells with critical Bcl2 and Bcl2l1 surviving cues, downstream of p38 mitogen-activated protein (MAP) kinase and cyclic-AMP-response element binding protein (CREB) activation. Accordingly, enforced expression of RET downstream targets, Bcl2 or Bcl2l1, is sufficient to restore the activity of Ret null progenitors in vivo. Activation of RET results in improved haematopoietic stem cell survival, expansion and in vivo transplantation efficiency. Remarkably, human cord-blood progenitor expansion and transplantation is also improved by neurotrophic factors, opening the way for exploration of RET agonists in human haematopoietic stem cell transplantation. Our work shows that neurotrophic factors are novel components of the haematopoietic stem cell microenvironment, revealing that haematopoietic stem cells and neurons are regulated by similar signals.
Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Nerve Growth Factors/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Animals , Cell Survival , Cyclic AMP Response Element-Binding Protein/metabolism , Enzyme Activation , Female , Hematopoiesis , Hematopoietic Stem Cell Transplantation , Humans , Male , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Signal Transduction , Stem Cell Niche , bcl-X Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismSubject(s)
Melanoma/physiopathology , Microfilament Proteins/physiology , Skin Neoplasms/physiopathology , Animals , Cell Cycle/physiology , Cell Proliferation/physiology , Disease Models, Animal , Humans , Melanoma/genetics , Mice, Nude , Mice, Transgenic , Microfilament Proteins/genetics , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Skin Neoplasms/genetics , Tumor Cells, CulturedABSTRACT
c-Ret has been shown to be crucial for neural development and survival. We have recently shown that complete impairment of tyrosine 1062 (Y1062)-phosphorylation in c-Ret causes congenital hearing loss with neurodegeneration of spiral ganglion neurons (SGNs) in homozygous c-Ret knockin mice (c-Ret-KI(Y1062F/Y1062F)-mice). However, there is no information to link c-Ret and age-related hearing loss. Here we show that partial impairment of Y1062-phosphorylation in c-Ret accelerates age-related hearing loss in heterozygous c-Ret Y1062F knockin mice (c-Ret-KI(Y1062F/+)-mice). In contrast, complete impairment of serine 697 (S697)-phosphorylation in c-Ret did not affect hearing levels in 10-month-old homozygous c-Ret S697A knockin mice (c-Ret-KI(S697A/S697A)-mice). The hearing loss involved late-onset neurodegeneration of spiral ganglion neurons in c-Ret-KI(Y1062F/+)-mice. Morphological abnormalities in inner- and outer-hair cells and the stria vascularis in c-Ret-KI(Y1062F/+)-mice were undetectable. The acceleration of age-related hearing loss in c-Ret-KI(Y1062F/+)-mice was rescued by introducing constitutively activated RET. Thus, our results suggest that c-Ret is a novel age-related hearing loss-related molecule in mice. Our results suggest that these hearing losses partially share a common pathogenesis that is monogenetically caused by a single point mutation (Y1062F) in c-Ret.
Subject(s)
Mutation , Presbycusis/genetics , Presbycusis/physiopathology , Proto-Oncogene Proteins c-ret/genetics , Tyrosine/genetics , Animals , Disease Models, Animal , Genetic Carrier Screening , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Phenylalanine/genetics , Phosphorylation/genetics , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-ret/deficiency , Spiral Ganglion/metabolism , Spiral Ganglion/pathology , Spiral Ganglion/physiopathology , Up-Regulation/geneticsABSTRACT
An appropriate organisation of muscles is crucial for their function, yet it is not known how functionally related muscles are coordinated with each other during development. In this study, we show that the development of a subset of functionally related head muscles in the zebrafish is regulated by Ret tyrosine kinase signalling. Three genes in the Ret pathway (gfra3, artemin2 and ret) are required specifically for the development of muscles attaching to the opercular bone (gill cover), but not other adjacent muscles. In animals lacking Ret or Gfra3 function, myogenic gene expression is reduced in forming opercular muscles, but not in non-opercular muscles derived from the same muscle anlagen. These animals have a normal skeleton with small or missing opercular muscles and tightly closed mouths. Myogenic defects correlate with a highly restricted expression of artn2, gfra3 and ret in mesenchymal cells in and around the forming opercular muscles. ret(+) cells become restricted to the forming opercular muscles and a loss of Ret signalling results in reductions of only these, but not adjacent, muscles, revealing a specific role of Ret in a subset of head muscles. We propose that Ret signalling regulates myogenesis in head muscles in a modular manner and that this is achieved by restricting Ret function to a subset of muscle precursors.
Subject(s)
Muscle Development/physiology , Proto-Oncogene Proteins c-ret/physiology , Zebrafish Proteins/physiology , Zebrafish/embryology , Zebrafish/physiology , Animals , Animals, Genetically Modified , Base Sequence , Biological Evolution , DNA Primers/genetics , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/physiology , Head , Muscle Development/genetics , Muscle, Skeletal/embryology , Mutation , Phenotype , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Signal Transduction , Zebrafish/genetics , Zebrafish Proteins/deficiency , Zebrafish Proteins/geneticsABSTRACT
During fetal life, vagal sensory fibers establish a reproducible distribution in the gut that includes an association with myenteric ganglia. Previous work has shown that netrin is expressed in the bowel wall and, by acting on its receptor, deleted in colorectal cancer (DCC), mediates the guidance of vagal sensory axons to the developing gut. Because the highest concentration of netrins in fetal bowel is in the endoderm, we tested the hypothesis that the ingrowth of vagal afferents to the gut would be independent of the presence of enteric neurons, although enteric neurons might influence the internal distribution of these fibers. Surprisingly, experiments indicated that the vagal sensory innervation is intrinsic neuron-dependent. To examine the vagal innervation in the absence of enteric ganglia, fetal Ret -/- mice were labeled by applying DiI bilaterally to nodose ganglia. In Ret -/- mice, DiI-labeled vagal sensory axons descended in paraesophageal trunks as far as the proximal stomach, which contains neurons, but did not enter the aganglionic bowel. To determine whether neurons produce netrins, enteric neural-crest-derived cells (ENCDCs) were immunoselected from E15 rat gut. Transcripts encoding netrin-1 and -3 were not detected in the ENCDCs, but appeared after they had given rise to neurons. When these neurons were cocultured with cells expressing c-Myc-tagged netrin-1, the neurons displayed netrin-1, but not c-Myc, immunoreactivity. Enteric neurons thus synthesize netrins. The extent to which neuronal netrin accounts for the dependence of the vagal sensory innervation on intrinsic neurons, remains to be determined.
Subject(s)
Enteric Nervous System/physiology , Gastrointestinal Tract/innervation , Nerve Growth Factors/biosynthesis , Nerve Tissue Proteins/biosynthesis , Neurogenesis/physiology , Tumor Suppressor Proteins/biosynthesis , Vagus Nerve/metabolism , Animals , Blotting, Western , Enteric Nervous System/cytology , Enteric Nervous System/embryology , Gastrointestinal Tract/embryology , Gastrointestinal Tract/metabolism , Immunohistochemistry , Mice , Mice, Knockout , Netrin-1 , Netrins , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Sensory Receptor Cells/cytology , Vagus Nerve/cytologyABSTRACT
Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.
Subject(s)
Aldehyde Oxidoreductases/metabolism , GATA3 Transcription Factor/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Urinary Tract/embryology , Urinary Tract/metabolism , Aldehyde Oxidoreductases/deficiency , Aldehyde Oxidoreductases/genetics , Animals , Base Sequence , Cell Surface Extensions/metabolism , Cell Surface Extensions/ultrastructure , Cloaca/abnormalities , Cloaca/embryology , Cloaca/metabolism , DNA Primers/genetics , Female , GATA3 Transcription Factor/deficiency , GATA3 Transcription Factor/genetics , Gene Expression Regulation, Developmental , Hydronephrosis/embryology , Hydronephrosis/genetics , Hydronephrosis/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Morphogenesis , Pregnancy , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Signal Transduction , Urinary Tract/abnormalitiesABSTRACT
Embryonic kidney development begins with the outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) into the adjacent metanephric mesenchyme (MM). Both a GDNF-dependent and GDNF-independent (Maeshima et al., 2007) pathway have been identified. In vivo and in vitro, the GDNF-dependent pathway is inhibited by BMPs, one of the factors invoked to explain the limitation of UB formation in the unbudded regions of the WD surrounding the UB. However, the exact mechanism remains unknown. Here a previously described in vitro system that models UB budding from the WD was utilized to study this process. Because Protein kinase A (PKA) activation has been shown to prevent migration, morphogenesis and tubulogenesis of epithelial cells (Santos et al., 1993), its activity in budded and non-budded portions of the GDNF-induced WD was analyzed. The level of PKA activity was 15-fold higher in the unbudded portions of the WD compared to budded portions, suggesting that PKA activity plays a key role in controlling the site of UB emergence. Using well-characterized PKA agonists and antagonists, we demonstrated that at various levels of the PKA-signaling hierarchy, PKA regulates UB outgrowth from the WD by suppressing budding events. This process appeared to be PKA-2 isoform specific, and mediated by changes in the duct rather than the surrounding mesenchyme. In addition, it was not due to changes in either the sorting of junctional proteins, cell death, or cell proliferation. Furthermore, the suppressive effect of cAMP on budding did not appear to be mediated by spread to adjacent cells via gap junctions. Conversely, antagonism of PKA activity stimulated UB outgrowth from the WD and resulted in both an increase in the number of buds per unit length of WD as well as a larger surface area per bud. Using microarrays, analysis of gene expression in GDNF-treated WDs in which the PKA pathway had been activated revealed a nearly 14-fold decrease in Ret, a receptor for GDNF. A smaller decrease in GFRα1. a co-receptor for GDNF, was also observed. Using Ret-null WDs, we were able to demonstrate that PKA regulated GDNF-dependent budding but not GDNF-independent pathway for WD budding. We also found that BMP2 was higher in unbudded regions of the GDNF-stimulated WD. Treatment of isolated WDs with BMP2 suppressed budding and resulted in a 3-fold increase in PKA activity. The data suggests that the suppression of budding by BMPs and possibly other factors in non-budded zones of the WD may be regulated in part by increased PKA activity, probably partially through downregulation of Ret/GFRα1 coreceptor expression.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Kidney/embryology , Proto-Oncogene Proteins c-ret/metabolism , Ureter/embryology , Wolffian Ducts/embryology , Animals , Base Sequence , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 2/pharmacology , Cell Proliferation , DNA Primers/genetics , Embryonic Development/drug effects , Embryonic Development/genetics , Embryonic Development/physiology , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Male , Mesoderm/embryology , Mice , Mice, Knockout , Models, Biological , Pregnancy , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Signal TransductionABSTRACT
Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences, including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA, and half express the GDNF family ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied, and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance, and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Na(v)1.8 Cre and Ret conditional mice to produce Ret-Na(v)1.8 conditional knock-out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size, indicating that Ret signaling is important for the survival and trophic support of these cells. Ret-Na(v)1.8 CKO mice exhibit reduced epidermal innervation but normal central projections. In addition, Ret-Na(v)1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased prostatic acid phosphatase (PAP), as PAP levels are markedly reduced in Ret-Na(v)1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.
Subject(s)
Nociceptors/physiology , Proto-Oncogene Proteins c-ret/physiology , Signal Transduction/physiology , Animals , Cell Survival/genetics , Cell Survival/physiology , Female , Formaldehyde/administration & dosage , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , NAV1.8 Voltage-Gated Sodium Channel , Nociceptors/cytology , Pain Measurement/methods , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Signal Transduction/genetics , Sodium Channels/deficiency , Sodium Channels/geneticsABSTRACT
The enteric nervous system is the largest subdivision of the peripheral nervous system that plays a critical role in digestive functions. Despite considerable progress over the last 15 years in understanding the molecular and cellular mechanisms that control the development of the enteric nervous system, several questions remain unanswered. The present review will focus on recent progress on understanding the development of the mammalian enteric nervous system and highlight interesting directions of future research.
Subject(s)
Enteric Nervous System/embryology , Animals , Apoptosis , Cell Lineage , Cell Movement , Enteric Nervous System/cytology , Enteric Nervous System/growth & development , Enteric Nervous System/physiology , Forecasting , Hirschsprung Disease/embryology , Hirschsprung Disease/pathology , Homeodomain Proteins/physiology , Humans , Insecta/embryology , Insecta/physiology , Mice , Mice, Knockout , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Nerve Tissue Proteins/physiology , Neural Crest/cytology , Neurogenesis/physiology , Neuroglia/cytology , Neuroglia/metabolism , Neurons/cytology , Neurons/metabolism , Peristalsis/physiology , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/physiology , Transcription Factors/physiology , Vertebrates/embryology , Vertebrates/physiologyABSTRACT
This review summarizes the current understanding of neurotransmitter phenotype specification of postganglionic sympathetic neurons, focusing, in particular, on the cellular processes of induction versus trans-differentiation. The emerging evidence is discussed that the noradrenergic and cholinergic neurotransmitter phenotypes are co-induced during early development and that the mature phenotypes develop by positive and negative selection of cellular properties in initially bimodal neurons, depending on extracellular signals during migration and after target contact.
Subject(s)
Gene Expression Regulation, Developmental , Neurons/metabolism , Neurotransmitter Agents/biosynthesis , Sympathetic Nervous System/cytology , Adrenergic Fibers/metabolism , Animals , Cell Transdifferentiation , Chick Embryo , Cholinergic Fibers/metabolism , Intercellular Signaling Peptides and Proteins/physiology , Mice , Mice, Knockout , Mice, Neurologic Mutants , Models, Neurological , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neural Crest/cytology , Neural Crest/metabolism , Neurogenesis , Neurons/cytology , Neurotransmitter Agents/genetics , Phenotype , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/physiology , Signal Transduction , Sympathetic Nervous System/embryology , Transcription Factors/physiology , Transcription, GeneticABSTRACT
The receptor tyrosine kinase RET is expressed in a number of neuroblastoma tissues and cell lines, but its role in neuroblastoma remains to be determined. In this study, we examined the roles of RET protein in neuroblastoma by the RNA interference technique using the NB-39-nu neuroblastoma cell line. NB-39-nu neuroblastoma cells show high expression and elevated tyrosine phosphorylation of RET, although short interfering RNA against RET (RET siRNA) did not significantly inhibit cell proliferation or suppression of basal levels of phosphorylation of extracellular regulated kinase (ERK)1/2 or protein kinase B (AKT). By the addition of glial cell line-derived neurotrophic factor (GDNF), both the expression and phosphorylation of RET and the phosphorylation of ERK1/2 and AKT were further increased, whereas cell proliferation was not stimulated under normal culture conditions. However, proliferation of cells cultured under non-adherent conditions was significantly increased by GDNF. The increased proliferation was suppressed by RET siRNA, which also caused inhibition of the phosphorylation of ERK1/2 and AKT. These results suggest that RET signaling plays an important role in GDNF-induced enhancement of non-adherent proliferation of NB-39-nu cells, which might contribute to the metastasis of neuroblastoma.
Subject(s)
Neuroblastoma/pathology , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , RNA, Small Interfering/genetics , Cell Adhesion , Cell Division/drug effects , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Neoplastic , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Humans , Neuroglia/cytology , Phosphorylation , Proto-Oncogene Proteins c-ret/deficiency , RNA Interference , RNA, Neoplasm/geneticsABSTRACT
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) [GDNF, NRTN (neurturin), ARTN (artemin), and PSPN (persephin)] interact with GDNF family receptors (GFRalphas) and activate intracellular signaling through the Ret receptor tyrosine kinase. To characterize the role of Ret signaling in retinal activity, we examined Ret hypomorphic and Ret conditional mice using electroretinography. We found that aberrant Ret function resulted in markedly diminished scotopic and photopic responses. Using mice deficient in individual GFLs, we found that only NRTN deficiency led to reduced retinal activity. To determine the potential target cell type for NRTN, we examined the retinal expression of its coreceptors (GFRalpha1 and GFRalpha2) and Ret using mice expressing fluorescence reporter enhanced green fluorescent protein from their respective loci. We found robust GFRalpha1 and Ret expression in horizontal, amacrine, and ganglion cells, whereas GFRalpha2 expression was only detected in a subset of amacrine and ganglion cells. In contrast to previous studies, no expression of GFRalpha1, GFRalpha2, or Ret was detected in photoreceptors or Müller cells, suggesting that these cells are not directly affected by Ret. Finally, detailed morphologic analyses of retinas from NRTN- and Ret-deficient mice demonstrated a reduction in normal horizontal cell dendrites and axons, abnormal extensions of horizontal cell and bipolar cell processes into the outer nuclear layer, and mislocalized synaptic complexes. These anatomic abnormalities indicate a possible basis for the abnormal retinal activity in the Ret and NRTN mutant mice.
Subject(s)
Neurturin/physiology , Proto-Oncogene Proteins c-ret/metabolism , Retina/physiology , Animals , Mice , Mice, Knockout , Neurturin/genetics , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Retina/cytology , Retina/growth & development , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor/physiology , Motor Neurons/classification , Motor Neurons/physiology , Spinal Cord/cytology , Animals , Animals, Newborn , Caspase 3/metabolism , Embryo, Mammalian , Gene Expression Regulation, Developmental/genetics , Glial Cell Line-Derived Neurotrophic Factor/deficiency , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hindlimb/embryology , Hindlimb/growth & development , Hindlimb/innervation , Mice , Mice, Knockout , Muscle Spindles/embryology , Muscle Spindles/growth & development , Muscle Spindles/metabolism , Muscle, Skeletal/metabolism , Point Mutation , Proto-Oncogene Proteins c-ret/deficiency , Proto-Oncogene Proteins c-ret/genetics , Vesicular Acetylcholine Transport Proteins/genetics , Vesicular Acetylcholine Transport Proteins/metabolismABSTRACT
The establishment of synaptic connections requires precise alignment of pre- and postsynaptic terminals. The glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 is enriched at pre- and postsynaptic compartments in hippocampal neurons, suggesting that it has a function in synapse formation. GDNF triggered trans-homophilic binding between GFRalpha1 molecules and cell adhesion between GFRalpha1-expressing cells. This represents the first example of a cell-cell interaction being mediated by a ligand-induced cell adhesion molecule (LICAM). In the presence of GDNF, ectopic GFRalpha1 induced localized presynaptic differentiation in hippocampal neurons, as visualized by clustering of vesicular proteins and neurotransmitter transporters, and by activity-dependent vesicle recycling. Presynaptic differentiation induced by GDNF was markedly reduced in neurons lacking GFRalpha1. Gdnf mutant mice showed reduced synaptic localization of presynaptic proteins and a marked decrease in the density of presynaptic puncta, indicating a role for GDNF signaling in hippocampal synaptogenesis in vivo. We propose that GFRalpha1 functions as a LICAM to establish precise synaptic contacts and induce presynaptic differentiation.