The GDNF-induced neurite outgrowth and neuronal survival in dissociated myenteric plexus cultures of the rat small intestine decreases postnatally

 Glial cell-line-derived neurotrophic factor (GDNF), a member of the transforming growth-factor- (TGF-) β-family, is an essential factor for the development of the enteric nervous system (ENS) during embryogenesis. In the present study, the effects of GDNF on postnatal ENS development were investigated using cultures of myenteric plexus from the small intestine of newborn albino rats of different developmental phases (P1, P7, P14). Myenteric plexus was dissociated and cultivated as mixed cultures of enteric neurons and glial cells. After seeding, the cultures were kept for 24 h or 7 days in serum-free medium containing various doses (1, 10, 100 ng/ml) of GDNF. The effect of the neurotrophic factor was evaluated using parameters such as cell size, neuronal survival, or neurite elongation. While neither glial-cell nor neuronal size was influenced by GDNF, there was an observable effect upon neuronal survival and neurite elongation. The cultures treated with GDNF displayed increased neurite outgrowth. The promoting effect was dose- and age-dependent, decreasing clearly during the early postnatal period. Already after 24 h, neuronal survival was increased in P1 and P7, but not in P14 cultures. In long-term cultures, a marked tendency to form cell aggregates and dense fiber networks was observed when treated with GDNF. These observations suggest that GDNF plays an important role not only in pre-, but also in postnatal development of the enteric nervous system. Received: 29 May 1998 / Accepted: 10 December 1998     

Additive effect of glial cell line-derived neurotrophic factor and neurotrophin-4/5 on rat fetal nigral explant cultures.

Transplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson's disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic effects on dopaminergic neurons via different receptor systems. In this study, we investigated possible additive or synergistic effects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days. Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were significantly larger than controls (1.6- and 2.0-fold, respectively) and contained significantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 significantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7-fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin-D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron-specific enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was unaffected. The release of lactate dehydrogenase into the culture medium was significantly reduced for GDNF+NT-4/5-treated cultures only. These results indicate that combined treatment with GDNF and NT4/5 may be beneficial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson's disease.     

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.     

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.     

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.     

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.     

Depletion of glial cell line-derived neurotrophic factor in substantia nigra neurons of Parkinson''s disease brain

The distribution of nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in substantia nigra pars compacta (SNc) of Parkinson's disease (PD) brains was investigated by immunofluorescence. Cases studied included four 69–77 year old neurologically normal male controls and four 72–79 year old male PD patients. Integrated optical densities (IODs) of immunofluorescence over individual neuromelanin-containing neurons and in areas of neuropil and the number of neurons on H & E stained adjacent sections were quantitated with the use of the BioQuant Image Analyzer. Data were statistically analyzed by ANOVA, including the unpaired two-tailed Student t-test and the Mann–Whitney test. The results showed 55.8% (P<0.0001) dropout of SNc neurons in PD brains compared to age-matched controls. Despite considerable neuronal dropout, immunofluorescent NTFs in the PD brains showed differential reductions that were consistent within the group as compared to age-matched controls: reductions were GDNF, 19.4%/neuron (P<0.0001), 20.2%/neuropil (P<0.0001); CNTF, 11.1%/neuron (P<0.0001), 9.4%/neuropil (P<0.0001); BDNF, 8.6%/neuron (P<0.0001), 2.5%/neuropil. NGF, NT-3 and NT-4 showed no significant differences within surviving neurons or neuropil. Since the depletion of GDNF both within surviving neurons and neuropil was twice as great as that of CNTF and BDNF and since the other NTFs showed no changes, GDNF, of the tested NTFs, is probably the most susceptible and the earliest to decrease in the surviving neurons of SNc. These observations suggest a role for decreased availability of GDNF in the process of SNc neurodegeneration in PD.     

Glial cell line-derived neurotrophic factor family receptors are abnormally expressed in aganglionic bowel of a subpopulation of patients with Hirschsprung's disease

Hirschsprung's disease (HSCR), a congenital disease, is characterized by the absence of ganglion cells in the ganglion plexuses of the caudal most gut. In the aganglionic colon, the plexus remnants are replaced by aggregates of glial cells and hypertrophied nerve fibers. Signaling of glial cell line-derived neurotrophic factor (GDNF)-GFRAs-receptor tyrosine kinase (RET) is crucial for the development and maintenance of ganglion cells. Mutations of genes such as GDNF and RET lead to the perturbation of this signaling pathway, which causes HSCR. To understand the role of GFRAs in ganglion cells and the pathogenesis of HSCR, we intended to determine the specific cell lineages in the enteric nervous system that normally express GFRAs but are affected in HSCR. We studied colon biopsy specimens from 13 patients with HSCR (aged 1 day to 38 months) and 6 age-matched patients without HSCR as normal controls. RT-PCR, in situ hybridization, and immunohistochemistry were performed to examine the expression and cellular distributions of GFRAs in resected bowel segments of normal infants and those with HSCR. In normal infants and normoganglionic colon of patients with HSCR, the expression of GFRA1 was restricted to the glial cells and neurones of the ganglion plexuses. GFRAs expression was found to be markedly reduced in the aganglionic colons of 3 infants with HSCR but was unaffected in the aganglionic colons of 10 other infants with HSCR. Residual GFRA expression was restricted to enteric glial cells in the plexus remnants of the aganglionic colons. Hypertrophied nerve fibers were not found to express GFRA1. We provide the first evidence that abnormal expression of GFRAs in the enteric nervous system may be involved in the pathogenesis of HSCR in a subpopulation of patients.     

胶质细胞源性神经营养因子在成年大鼠三叉神经节及三叉神经核团中的分布

目的 观察胶质细胞源性神经营养因子(GDNF)在成年大鼠三叉神经节及三叉神经核团内的分布，探讨其对三叉神经感觉神经元及运动神经元的作用。方法 抗GDNF多克隆抗体免疫组织化学ABC法。结果 成年大鼠三叉神经运动核、三叉神经感觉核簇及三叉神经节中出现GDNF免疫反应阳性。结论 成年大鼠三叉神经运动核、三叉神经感觉核簇及三叉神经节中存在GDNF神经元。     

A neuronal subpopulation in the mammalian enteric nervous system expresses TrkA and TrkC neurotrophin receptor-like proteins

Increasing evidence suggests that, in addition to peripheral sensory and sympathetic neurons, the enteric neurons are also under the control of neurotrophins. Recently, neurotrophin receptors have been detected in the developing and adult mammalian enteric nervous system (ENS). Nevertheless, it remains to be established whether neurotrophin receptors are expressed in all enteric neurons and/or in glial cells and whether expression is a common feature in the enteric nervous system of all mammals or if interspecific differences exist. Rabbit polyclonal antibodies against Trk proteins (regarded as essential constituents of the high-affinity signal-transducing neurotrophin receptors) and p75 protein (considered as a low-affinity pan-neurotrophin receptor) were used to investigate the cell localization of these proteins in the ENS of adult man, horse, cow, sheep, pig, rabbit, and rat. Moreover, the percentage of neurons displaying immunoreactivity (IR) for each neurotrophin receptor protein was determined. TrkA-like IR and TrkC-like IR were observed in a neuronal subpopulation in both the myenteric and submucous plexuses, from esophagus to rectum in humans, and in the jejunum-ileum of the other species. Many neurons, and apparently all glial cells, in the human and rat enteric nervous system also displayed p75 IR. TrkB-like IR was found restricted to the glial cells of all species studied, with the exception of humans, in whom IR was mainly in glial cells and a small percentage of enteric neurons (about 5%). These findings indicate that the ENS of adult mammals express neuronal TrkA and TrkC, glial TrkB, and neuronal-glial p75, this pattern of distribution being similar in all examined species. Thus, influence of specific neurotrophins on their cognate receptors may be considered in the physiology and/or pathology of the adult ENS. Anat. Rec. 251:360–370, 1998. © 1998 Wiley-Liss, Inc.     

Peripheral nerve regeneration and neurotrophic factors

The role of neurotrophic factors in the maintenance and survival of peripheral neuronal cells has been the subject of numerous studies. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ-derived trophic factors on neuronal cells. After axotomy and during peripheral nerve regeneration, the neurotrophins NGF, NT-3 and BDNF show a well defined and selective beneficial effect on the survival and phenotypic expression of primary sensory neurons in dorsal root ganglia and of motoneurons in spinal cord. Other neurotrophic factors such as CNTF, GDNF and LIF also exert a variety of actions on neuronal cells, which appear to overlap and complement those of the neurotrophins. In addition, there is an indirect contribution of GGF to nerve regeneration. GGF is produced by neurons and stimulates proliferation of Schwann cells, underlining the close interaction between neuronal and glial cells during peripheral nerve regeneration. Different possibilities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. The studies reviewed in this article show the therapeutic potential of neurotrophic factors for the treatment of peripheral nerve injury and for neuropathies.     

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.     

Growth factors stimulate expression of neuronal and glial miR-132

Brain-specific microRNAs (miRs) and brain-derived neurotrophic factor (BDNF) are both involved in synaptic function. We previously reported that upregulation of miR-132 is involved in BDNF-increased synaptic proteins, including glutamate receptors (NR2A, NR2B, and GluR1) in mature cortical neurons [7]. However, the potential role of other growth factors in miR-132 induction has not been clarified. Here, we examined the effect of growth factors including basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), and epidermal growth factor (EGF), on expression of miR-132 and glutamate receptors in immature cortical neurons. We found that BDNF and bFGF upregulated levels of miR-132 in cortical cultures, though bFGF failed to increase glutamate receptors such as NR2A, NR2B, and GluR1. IGF-1, GDNF, and EGF did not have a positive influence on miR-132 and glutamate receptors in neuronal cultures. Furthermore, bFGF significantly upregulated miR-132 in cultured astroglial cells, while other growth factors failed to elicit such a response. It is possible that the growth factor-stimulated neuronal and glial action of miR-132 plays a critical role in brain function.     

Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system

The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON). In this immunohistochemical study, we localized GDNF, neurturin, GFRα1, GFRα2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRα1, whereas progenitors and immature neurons more avidly expressed GFRα2. In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRα2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.     

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.     

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.     

Programmed Cell Death in the Developing Nervous System

Virtually all cell populations in the vertebrate nervous system undergo massive "naturally-occurring" or "programmed" cell death (PCD) early in development. Initially neurons and glia are overproduced followed by the demise of approximately one-half of the original cell population. In this review we highlight current hypotheses regarding how large-scale PCD contributes to the construction of the developing nervous system. More germane to the theme of this symposium, we emphasize that the survival of cells during PCD depends critically on their ability to access "trophic" molecular signals derived primarily from interactions with other cells. Here we review the cell-cell interactions and molecular mechanisms that control neuronal and glial cell survival during PCD, and how the inability of such signals to suppress PCD may contribute to cell death in some diseases such as spinal muscular atrophy. Finally, by using neurotrophic factors (e.g. CNTF, GDNF) and genes that control the cell death cascade (e.g. Bcl-2) as examples, we underscore the importance of studying the mechanisms that control neuronal and glial cell survival during normal development as a means of identifying molecules that prevent pathology-induced cell death. Ultimately this line of investigation could reveal effective strategies for arresting neuronal and glial cell death induced by injury, disease, and/or aging in humans.     

Expression profile of some neuronal and glial cell markers in the ovine ileal enteric nervous system during prenatal development

The enteric nervous system (ENS) is a network of neurons and glia found in the gut wall and governs this gastrointestinal function independently from the central nervous system (CNS). ENS comprises the myenteric plexus (MP) and the submucous plexus (SP). In this study, we examined the expression profile of neurofilament heavy chain (NF-H), neuron-specific enolase (NSE), calcyclin (S100A6), vimentin and glial fibril acidic protein (GFAP) in ovine ileal enteric neurons and enteric glia cells (EGCs) during prenatal development using an immunohistochemical method. The material of the study consisted of 15 different fetal ileum tissues obtained between days 60 and 150 of pregnancy. NF-H was observed in the majority of ganglion cells in SP and MP throughout the fetal period. It was determined that there was no NF-H reaction in some ganglion cells in Peyer’s patches of internal submucosal plexus (ISPF). In the early stage of pregnancy (60–90 days), there was no expression of NSE and S1006 in ileum. After this period, NSE and S1006 were expressed in the ganglion cells of the plexus, indicating an increase in the amount of expression towards the end of pregnancy. In the early period, vimentin expression was only detected in intramuscular interstitial cells (ICs) (60–90 days), but later (90–150 days) it was also seen in the cells around the ganglion cells in the plexus. On days 60–90 of gestation, GFAP expression only occurred in MP, but in later stages, staining was also detected in SP. In the plexus, an immunoreactivity was present in EGCs forming a network around the ganglion cell. During the last period of gestation (120–150 days), the number of GFAP-positive plexus increased, with the majority of these stained cells being observed in MP. Interestingly, weak staining or reaction did not occur in ISPF, unlike other plexuses. In conclusion, this is the first study that demonstrated the expression of NF-H, vimentin, S100A6, NSE and glial fibril acidic protein (GFAP) in ovine ileal ENS in the prenatal period. In the last period of gestation (120–150 days), the expression profile of ENS was similar to that of adult animals. The expression of the used markers increased toward the end of pregnancy. Our results suggest that neurons and EGCs show heterogeneity, and GFAP and NF-H cannot be used as panenteric glial or panneuronal markers, respectively. We also demonstrated, for the first time, the prenatal expression of S100A6 in enteric neurons and the possibility of using this protein for the identification of enteric neurons.