The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons.

The undisturbed development of the enteric nervous system depends on the supply of various neurotrophic factors during ontogenesis. Besides glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) take part in its development. CNTF and LIF belong to the interleukin-6 (IL-6) family of cytokines. The combination of IL-6 and the soluble IL-6 receptor accelerates peripheral nerve regeneration. In this study, we examined the effect of the fusion protein Hyper-IL-6, which consists of IL-6 and the soluble receptor sIL-6R, on neurite outgrowth and neuronal survival in vitro. Myenteric plexus of newborn rats was dissected and dissociated. Cells were grown in either serum-free chemically defined medium alone or medium supplemented with sIL-6R, IL-6, sIL-6+IL-6, Hyper-IL-6, CNTF, LIF, or GDNF. Average neurite outgrowth per neuron was highest in GDNF-treated and Hyper-IL-6-treated cultures. The number of neurite-bearing neurons was reduced in GDNF cultures compared with Hyper-IL-6-treated cells, so that the total neurite outgrowth was maximal after Hyper-IL-6 stimulation. Hyper-IL-6 furthermore stimulated neuronal survival and morphologic differentiation of the enteric glia.     

Differing effects of NT-3 and GDNF on dissociated enteric ganglion cells exposed to hydrogen peroxide in vitro

Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and ageing. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF), are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuroprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent the death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H(2)O(2)). H(2)O(2) treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, bis-benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H(2)O(2)-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress.     

Trophic actions of neurotrophin-3 on postnatal rat myenteric neurons in vitro

A number of neurotrophic factors have been implicated in the prenatal development of the enteric nervous system. Although several of these factors continue to be expressed in the gut during postnatal life, their actions on postnatal enteric neurons are not understood. One such factor is the neurotrophin, NT-3. Both NT-3 and its high affinity receptor, trk C, are expressed in the postnatal gut at a time when changes in the density of intestinal innervation are occurring. We have therefore examined the effects of NT-3 on postnatal myenteric neurons, using dissociated cell cultures of ganglia isolated from 6-8 day postnatal rat small intestine. Effects of NT-3 on neurite outgrowth and neuronal and glial cell numbers were measured after 2 days in vitro. The proportion of neurons was increased in NT-3 treated cultures, as was the proportion of neurons that extended processes. NT-3 treatment, at concentrations of between 0.1 ng and 10 ng/ml, also resulted in a significant increase in mean total neurite length. These results indicate that NT-3 may play a role in the postnatal development of the enteric nervous system.     

Recent progress in studies of neurotrophic factors and their clinical implications

 Neurotrophic factors are endogenous soluble proteins that regulate long-term survival and differentiation of neurons of the peripheral and central nervous systems. These factors play an important role in the structural integrity of the nervous system, and therefore are good candidates as therapeutic agents for neurodegenerative diseases. However, recent studies have revealed some unexpected, novel roles of neurotrophic factors. Of particular significance is the discovery of the new functions of brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). Physiological experiments indicate that BDNF may serve as regulatory factors for synaptic transmission as well as for learning and memory. Gene targeting studies demonstrate that GDNF may be essential for development of the enteric nervous system (ENS) and kidney organogenesis. These results not only provide new insights into our understanding of the function of neurotrophic factors but may also have significant implications in the therapeutic usages of neurotrophic factors. Received: 12 November 1996 / Accepted: 26 March 1997     

GDNF induces synaptic vesicle markers in enteric neurons

Regulation of intestinal motility depends on an intact synaptic vesicle apparatus. Thus, we investigated the expression of the synaptic vesicle markers synaptophysin and synaptobrevin in the human enteric nervous system (ENS) and their regulation by glial cell line-derived neurotrophic factor (GDNF) in cultured enteric neurons.     

NGF、CNTF和GDNF对感觉和运动神经元的协同作用研究

为探讨神经生长因子(NGF)、睫状神经营养因子(CNTF)和胶质细胞源性神经营养因子(GDNF)对感觉和运动神经元的协同作用机制,本研究采用免疫组织化学技术对脊髓背根节感觉神经元和脊髓运动神经元经特异性烯醇化酶(NSE)和胆碱乙酰转移酶(ChAT)染色后,通过图像分析测量阳性神经元数量、胞体直径、突起数量及长度。结果表明:NGF能明显促进感觉神经元的存活,对突起发育有轻微作用,对胞体发育的作用不显著,对运动神经元的存活无明显作用;CNTF对感觉和运动神经元的胞体发育均有很强的作用,对感觉和运动神经元的存活有一定的作用;GDNF对感觉和运动神经元的突起发育和延伸作用最强,对运动神经元的存活有很强的促进作用,对胞体发育的作用不如CNTF显著。本研究结果提示:联合应用上述三种神经营养因子,可克服单一因子功能的局限,全面促进感觉和运动神经元的存活和生长。     

GDNF family ligand immunoreactivity in the gut of teleostean fish

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRα, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.     

Enteric Neurogenesis During Life Span Under Physiological and Pathophysiological Conditions

The enteric nervous system (ENS) controls gastrointestinal key functions and is mainly characterized by two ganglionated plexus located in the gut wall: the myenteric plexus and the submucous plexus. The ENS harbors a high number and diversity of enteric neurons and glial cells, which generate neuronal circuitry to regulate intestinal physiology. In the past few years, the pivotal role of enteric neurons in the underlying mechanism of several intestinal diseases was revealed. Intestinal diseases are associated with neuronal death that could in turn compromise intestinal functionality. Enteric neurogenesis and regeneration is therefore a crucial aspect within the ENS and could be revealed not only during embryogenesis and early postnatal periods, but also in the adulthood. Enteric glia and/or enteric neural precursor/progenitor cells differentiate into enteric neurons, both under homeostatic and pathologic conditions beyond the perinatal period. The unique role of the intestinal microbiota and serotonin signaling in postnatal and adult neurogenesis has been shown by several studies in health and disease. In this review article, we will mainly focus on different recent studies, which advanced the concept of postnatal and adult ENS neurogenesis. Moreover, we will discuss the key factors and underlying mechanisms, which promote enteric neurogenesis. Finally, we will shortly describe neurogenesis of transplanted enteric neural progenitor cells. Anat Rec, 302:1345–1353, 2019. © 2019 Wiley Periodicals, Inc.     

GDNF and GFRα co-receptor family in the developing feline gut

Glial cell-line derived neurotrophic factor (GDNF) and the GFRα co-receptors play a role in the developing enteric nervous system. The co-receptors elicit their action by binding receptor tyrosine kinase RET.This immunohistochemical study reports the presence of GDNF and its specific co-receptor GFRα1 in the cat gastrointestinal apparatus during development, from stage 9 to 22. At stage 9 and 11, immunoreactivity (IR) to GDNF was observed in the cells of mesenchyme of the anterior gut. From stage 14 to 22, GDNF IR was detected in nervous plexuses; moreover, GDNF and GFRα1 IR appeared localized in gastrointestinal endocrine cells. The presence of GDNF in the enteric nervous system and in the endocrine cells suggests an involvement of this neurotrophic factor in the gastrointestinal development. Moreover, the presence of the co-receptor GFRα1 in endocrine cells and its absence in the enteric nervous system seems to indicate a different mode of transduction of GDNF signal. GFRα2 and GFRα3 co-receptors were not detected.     

Gdnf is mitogenic,neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon

Glial‐derived neurotrophic factor (Gdnf) is required for morphogenesis of the enteric nervous system (ENS) and it has been shown to regulate proliferation, differentiation, and survival of cultured enteric neural crest–derived cells (ENCCs). The goal of this study was to investigate its in vivo role in the colon, the site most commonly affected by intestinal neuropathies such as Hirschsprung's disease. Gdnf activity was modulated in ovo in the distal gut of avian embryos using targeted retrovirus‐mediated gene overexpression and retroviral vector‐based gene silencing. We find that Gdnf has a pleiotropic effect on colonic ENCCs, promoting proliferation, inducing neuronal differentiation, and acting as a chemoattractant. Down‐regulating Gdnf similarly induces premature neuronal differentiation, but also inhibits ENCC proliferation, leading to distal colorectal aganglionosis with severe proximal hypoganglionosis. These results indicate an important role for Gdnf signaling in colonic ENS formation and emphasize the critical balance between proliferation and differentiation in the developing ENS. Developmental Dynamics 240:1402–1411, 2011. © 2011 Wiley‐Liss, Inc.     

Olfactory ensheathing cells exert a trophic effect on the hypothalamic neurons in vitro

Olfactory ensheathing cells (OECs) constitute an usual population of glial cells sharing properties with both Schwann cells (SC) of peripheral nervous system (PNS) and astrocytes of the central nervous system (CNS). They express a high level of growth factors which play a very important role as neuronal support. Recent evidence in literature suggests that OECs may facilitate axonal regeneration in the injured nervous system. In this study, we developed an in vitro model to evaluate the neurotrophic effect of OECs on the survival and axonal outgrowth of hypothalamic neurons. Co-cultures of OECs and hypothalamus neuronal cells of postnatal rats were successfully established and cells were immunocytochemically characterized. Furthermore, some neuronal cultures were added with NGF, bFGF and GDNF to compare with the co-cultures. Our results indicate that in co-cultures of hypothalamic neurons and OECs, the number of neurons was significantly increased compared to control cultures exhibiting a dense axonal outgrowth. Moreover, we show that NGF promoted a major neuronal survival than bFGF and GDNF, while bFGF and GDNF exerted an evidence axonal and dendritic outgrowth compared to NGF. In conclusion, these data suggest that OECs have the capacity to promote the survival and axonal outgrowth of hypothalamic neurons in vitro and that bFGF, NGF and GDNF differentially support hypothalamic neurons promoting and enhancing the neuronal survival and outgrowth. Therefore, the OECs are a source of growth factors and might be considered a better approach for functional recovery and growth factors might exert a neuroprotective effect in neurodegenerative disorders.     

胶质细胞源性神经营养因子对培养的背根神经节的影响

目的 探讨胶质细胞源性神经营养因子(GDNF)在体外促进正常胚胎大鼠背根神经节(DRGn)的存活及突起生长情况。方法 用原代分离培养法建立体外胚胎大鼠背根神经节单细胞培养体系，通过活体观察、MTT微量比色法、NSE免疫组织化学染色观察不同浓度GDNF对体外培养的正常感觉神经元的影响。结果 GDNF组培养的DRG神经元存活数量增加，神经元突起的长度比对照组明显增长。结论 GDNF能明显促进体外培养的正常大鼠胚胎背根神经节感觉神经元的存活及突起生长，表明GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

Glial cell line-derived neurotrophic factor (GDNF) is required for differentiation of pontine noradrenergic neurons and patterning of central respiratory output

Genetic mutations affecting signaling by glial cell line-derived neurotrophic factor (GDNF) perturb development of breathing in mice and are associated with congenital central hypoventilation syndrome in humans. However, the role of GDNF in development of brainstem neurons that control breathing is largely unknown. The present study demonstrates that genetic loss of GDNF decreases the number of tyrosine hydroxylase (TH) neurons in the pontine A5 noradrenergic cell group, a major source of inhibitory input to the medullary respiratory pattern generator. This phenotype is associated with a significant increase in the frequency of central respiratory output recorded from the fetal medulla-spinal cord in vitro. In dissociate cultures of the A5 region from rat embryos, GDNF increases TH cell number and neurite growth without affecting total neuronal survival or proliferation of TH neurons. These effects of GDNF are inhibited by function blocking antibodies against endogenous brain-derived neurotrophic factor (BDNF), indicating that GDNF requires BDNF as a cofactor to stimulate differentiation of A5 neurons. Our findings demonstrate that GDNF is required for development of pontine noradrenergic neurons in vivo and indicate that defects in the A5 cell group may contribute to the effects of genetic disruption of GDNF signaling on respiratory control.     

Developmental changes in neurite outgrowth responses of dorsal root and sympathetic ganglia to GDNF, neurturin, and artemin.

The ability of glial cell line-derived neurotrophic factor (GDNF), neurturin, and artemin to induce neurite outgrowth from dorsal root, superior cervical, and lumbar sympathetic ganglia from mice at a variety of development stages between embryonic day (E) 11.5 and postnatal day (P) 7 was examined by explanting ganglia onto collagen gels and growing them in the presence of agarose beads impregnated with the different GDNF family ligands. Artemin, GDNF, and neurturin were all capable of influencing neurite outgrowth from dorsal root and sympathetic ganglia, but the responses of each neuron type to the different ligands varied during development. Neurites from dorsal root ganglia responded to artemin at P0 and P7, to GDNF at E15.5 and P0, and to neurturin at E15.5, P0, and P6/7; thus, artemin, GDNF, and neurturin are all capable of influencing neurite outgrowth from dorsal root ganglion neurons. Neurites from superior cervical sympathetic ganglia responded significantly to artemin at E15.5, to GDNF at E15.5 and P0, and to neurturin at E15.5. Neurites from lumbar sympathetic ganglia responded to artemin at all stages from E11.5 to P7, to GDNF at P0 and P7 and to neurturin at E11.5 to P6/7. Combined with the data from previous studies that have examined the expression of GDNF family members, our data suggest that artemin plays a role in inducing neurite outgrowth from young sympathetic neurons in the early stages of sympathetic axon pathfinding, whereas GDNF and neurturin are likely to be important at later stages of sympathetic neuron development in inducing axons to enter particular target tissues once they are in the vicinity or to induce branching within target tissues. Superior cervical and lumbar sympathetic ganglia showed temporal differences in their responsiveness to artemin, GDNF, and neurturin, which probably partly reflects the rostrocaudal development of sympathetic ganglia and the tissues they innervate.     

Increased glial‐derived neurotrophic factor in the small intestine of rats infected with the tapeworm,Hymenolepis diminuta

The neurotrophin, glial‐derived neurotrophic factor (GDNF), is essential for the development of the enteric nervous system (ENS) in both the embryo and neonate and may be important for maintenance and plasticity of ENS. The tapeworm, Hymenolepis diminuta, altered the number of cells containing GNDF in the host’s jejunum and ileum. Numbers and locations of GDNF‐containing cells were determined by applying monoclonal anti‐GDNF antibody to intestinal segments collected from infected and uninfected age‐matched rats during the initial 34 days post‐infection (dpi). Most cells staining positive for GDNF were present in the lamina propria of the jejunum and ileum from both infected and uninfected rats. The co‐localization of staining by the antibodies, anti‐GDNF and anti‐ED2 (a nuclear specific antibody for resident macrophages) indicated that at least 74% of the cells staining for GDNF were macrophages. Mast cells did not stain with the anti‐GDNF antibody. The increased number of GDNF+ cells in the infected rat intestine suggests that this neurotrophin may play a role in the neural and mucosal responses to lumenal tapeworm infection.     

Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development

The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS.     

Regulation of neural development by glial cell line-derived neurotrophic factor family ligands

Glial cell line-derived neurotrophic factor (GDNF) and its three relatives constitute a novel family of neurotrophic factors, the GDNF family ligands. These factors signal through a multicomponent receptor complex comprising a glycosylphosphatidylinositol-anchored cell surface molecule (GDNF family receptor (GFR) alpha) and RET tyrosine kinase, triggering the activation of multiple signaling pathways in responsive cells. Recent gene-targeting studies have demonstrated that GDNF family ligands are essential for the development of a diverse set of neuronal populations and we have now started to understand how these ligands uniquely regulate the formation and sculpting of the nervous system. Recent studies have also revealed interactions by multiple extracellular signals during neural development. The deciphering of GDNF family ligand signaling in neural cells promises to provide vital new insights into the development and pathology of the nervous system.     

GDNF mRNA expression in normal postnatal development, aging, and in weaver mutant mice

Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to enhance the survival and process outgrowth of mesencephalic dopamine neurons. A nuclease protection assay was utilized to determine whether GDNF mRNA is expressed in the ventral mesencephalon and/or striatum during normal mouse postnatal development. While no GDNF mRNA was detected in the ventral mesencephalon expression was detected in the striatum throughout postnatal development and maturity with the peak of expression being in the second postnatal week. In the process of normal aging no change in the levels of GDNF mRNA was observed in the striatum while a 10-fold increase in glial fibrillary acid protein (GFAP) mRNA was detected in 24-month-old relative to either 4.5- or 11-month-old mice. Further analysis addressed whether there are changes in GDNF gene expression associated with the neurodegeneration of dopamine neurons that occurs in the weaver mutant mouse. A transient 65% increase in the expression of GDNF mRNA was observed in weaver mutant striatum on postnatal day 22. The results of this study suggest that GDNF could provide target derived dopaminergic neurotrophic support and stimulate fiber outgrowth during development and that decreased levels of GDNF expression are not responsible for either aging-associated decreases in dopaminergic neuronal plasticity or neurodegeneration in the weaver mutant mouse     

Differential survival response of neurons to exogenous GDNF depends on the presence of skeletal muscle

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent survival factor for neurons in vitro and in vivo. The current study investigated the effects of a single in utero injection with GDNF in both wild-type and Myf5-/-:MyoD-/- embryos. The embryos in the latter group, denoted double mutants (DM), do not contain skeletal muscle and associated neurotrophic factors due to lack of myogenesis and, therefore, neurons of the central and peripheral nervous system undergo excessively occurring programmed cell death (EPCD). We found that treatment with GDNF had no effect on wild type neuronal numbers in any of the anatomic locations investigated. However, GDNF rescued the neurons of the facial motor nucleus, the mesencephalic nucleus and the median motor column in the absence of skeletal muscle. The findings of the current study agree with previous reports that compromised mouse neurons have increased survival response to GDNF.     

Neurotropic growth factors and glycosaminoglycan based matrices to induce dopaminergic tissue formation

Current cell replacement therapies in Parkinson's disease (PD) are limited by low survival of transplanted cell and lacking regeneration of neuronal circuitries. Therefore, bioartificial cell carriers and growth/differentiation factors are applied to improve the integration of transplants and maximize newly generated and/or residual dopaminergic function. In this work, biohybrid poly(ethylene glycol) (starPEG)-heparin hydrogels releasing fibroblast growth factor 2 (FGF-2) and glial-derived neurotrophic factor (GDNF) were used to trigger dopaminergic tissue formation by primary murine midbrain cells in vitro. Matrix-delivered FGF-2 enhanced cell viability while release of GDNF had a pro-neuronal/dopaminergic effect. Combined delivery of both factors from the glycosaminoglycan-based matrices resulted in a tremendous improvement in survival and maturation capacity of dopaminergic neurons as obvious from tyrosine hydroxylase expression and neurite outgrowth. The reported data demonstrate that glycosaminoglycan-based hydrogels can facilitate the administration of neurotrophic factors and are therefore instrumental in potential future treatments of PD.