The enteric nervous system (ENS) is unique among components of the peripheral nervous system due to its complexity in structure and neurotransmitter phenotype diversity. In this complexity, the ENS resembles the central nervous system (CNS). Although the ENS is derived from the neural crest rather than the neural tube, similar mechanisms may generate complex connectivity in both the ENS and the CNS. Neuroligins and neurexins are cell adhesion molecules that participate in regulating CNS synaptogenesis. We investigated whether these molecules also play a role in establishing enteric synapses. We found that neuroligins and neurexins were expressed in mouse, rat, and human gut. Transcripts of both types of molecule were extensively spliced in the bowel during fetal and adult life. When transfected into non-neuronal cells, neuroligins and neurexins were sufficient to recruit, respectively, presynaptic and postsynaptic elements. Engineered soluble neurexin, which interferes with endogenous neurexin-neuroligin binding, inhibited enteric synapse formation/stabilization and recruitment of neurotransmitter receptors. Finally, we demonstrated that alternative splicing of neuroligin and neurexin contributes to ENS synaptic specificity. Some isoforms preferentially induced cholinergic synapses, whereas others promoted serotonergic synaptogenesis.NEW & NOTEWORTHY We found that neuroligins and neurexins play roles in establishing ENS synapses. Both are extensively spliced in the fetal and adult gut. When expressed in non-neuronal cells, both neuroligins and neurexins are sufficient to recruit, respectively, presynaptic and postsynaptic elements. Soluble neurexin inhibits enteric synapse formation and recruitment of neurotransmitter receptors by interfering with endogenous neurexin/neuroligin binding. The neurexin/neuroligin splice code contributes physiologically to ENS synaptic specificity, promoting for example, cholinergic or serotonergic synaptogenesis.

In addition to the well-known degeneration of midbrain dopaminergic neurons, enteric neurons can also be affected in neurodegenerative disorders such as Parkinson’s disease (PD). Dopaminergic neurons have recently been identified in the enteric nervous system (ENS). While ENS dopaminergic neurons have been shown to degenerate in genetic mouse models of PD, analyses of their survival in enteric biopsies of PD patients have provided inconsistent results to date. In this context, this review seeks to highlight the distinctive and shared factors and properties that control the evolution of these two sets of dopaminergic neurons from neuronal precursors to aging neurons. Although their cellular sources and developmental times of origin differ, midbrain and ENS dopaminergic neurons express many transcription factors in common and their respective environments express similar neurotrophic molecules. For example, Foxa2 and Sox6 are expressed by both populations to promote the specification, differentiation, and long-term maintenance of the dopaminergic phenotype. Both populations exhibit sustained patterns of excitability that drive intrinsic vulnerability over time. In disorders such as PD, colon biopsies have revealed aggregation of alpha-synuclein in the submucosal plexus where dopaminergic neurons reside and lack blood barrier protection. Thus, these enteric neurons may be more susceptible to neurotoxic insults and aggregation of α-synuclein that spreads from gut to midbrain. Under sustained stress, inefficient autophagy leads to neurodegeneration, GI motility dysfunction, and PD symptoms. Recent findings suggest that novel neurotrophic factors such as CDNF have the potential to be used as neuroprotective agents to prevent and treat ENS symptoms of PD.

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf−/−), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf−/− mouse are much greater than in those of Cdnf+/+ mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf−/− mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf−/− male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf−/− mouse brain is altered. The deficiencies of Cdnf−/− mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) is expressed in the brain and is neuroprotective. We have previously shown that CDNF is also expressed in the bowel and that its absence leads to degeneration and autophagy in the enteric nervous system (ENS), particularly in the submucosal plexus. We now demonstrate that enteric CDNF immunoreactivity is restricted to neurons (submucosal > myenteric) and is not seen in glia, interstitial cells of Cajal, or smooth muscle. Expression of CDNF, moreover, is essential for the normal development and survival of enteric dopaminergic neurons; thus, expression of the dopaminergic neuronal markers, dopamine, tyrosine hydroxylase, and dopamine transporter are deficient in the ileum of Cdnf( -/-) mice. The normal age-related decline in proportions of submucosal dopaminergic neurons is exacerbated in Cdnf( -/-) animals. The defect in Cdnf( -/-) animals is not dopamine-restricted; proportions of other submucosal neurons (NOS-, GABA-, and CGRP-expressing), are also deficient. The deficits in submucosal neurons are reflected functionally in delayed gastric emptying, slowed colonic motility, and prolonged total gastrointestinal transit. CDNF is expressed selectively in isolated enteric neural crest-derived cells (ENCDC), which also express the dopamine-related transcription factor Foxa2. Addition of CDNF to ENCDC promotes development of dopaminergic neurons; moreover, survival of these neurons becomes CDNF-dependent after exposure to bone morphogenetic protein 4. The effects of neither glial cell-derived neurotrophic factor (GDNF) nor serotonin are additive with CDNF. We suggest that CDNF plays a critical role in development and long-term maintenance of dopaminergic and other sets of submucosal neurons.

Neurological disorders cause gastrointestinal (GI) symptoms that are debilitating and markedly diminish quality of life in patients. The enteric nervous system (ENS), the intrinsic nervous system of the GI tract that is often referred to as "the second brain", shares many features with the central nervous system. The ENS plays an essential role in regulating many GI functions including motility and fluid secretion. Enteric neuronal degeneration could therefore be responsible for the GI symptoms commonly observed in neurological conditions. Here we describe the organization and functions of the ENS and then review the evidence for ENS involvement in two common neurodegenerative disorders, Parkinson's disease (PD) and Alzheimer's disease (AD). Data from patients as well as animal models suggest that PD affects distinct subsets of neurons and glia in the ENS, and that the ENS may participate in the pathogenesis of this disorder. While there has been great enthusiasm for the possibility of sampling the ENS for diagnosis or therapeutic monitoring of PD, further work is needed to determine which enteric neurons are most affected and how ENS function could be modulated to ameliorate GI symptoms in patients. Although AD is far more common than PD and AD patients also experience GI symptoms, understanding of ENS dysfunction in AD is in its infancy. Much work remains to be done in both of these fields to determine how the ENS contributes to and/or is altered by these disorders, and how to target the ENS for more effective treatment of GI comorbidities.

Formation of the enteric nervous system (ENS) from migratory neural crest-derived cells that colonize the primordial gut involves a complex interplay among different signaling molecules. The bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, play a particularly important role in virtually every stage of gut and ENS development. BMP signaling helps to pattern both the anterior-posterior axis and the radial axis of the gut prior to colonization by migratory crest progenitor cells. BMP signaling then helps regulate the migration of enteric neural crest-derived precursors as they colonize the fetal gut and form ganglia. BMP2 and -4 promote differentiation of enteric neurons in early fetal ENS development and glia at later stages. A major role for BMP signaling in the ENS is regulation of responses to other growth factors. Thus BMP signaling first regulates neurogenesis by modulating responses to GDNF and later gliogenesis through its effects on GGF-2 responses. Furthermore, BMPs promote growth factor dependency for survival of ENS neurons (on NT-3) and glia (on GGF-2) by inducing TrkC (neurons) and ErbB3 (glia). BMP signaling limits total neuron numbers, favoring the differentiation of later born neuronal phenotypes at the expense of earlier born ones thus influencing the neuronal composition of the ENS and the glia/neuron ratio. BMP2 and -4 also promote gangliogenesis via modification of neural cell adhesion molecules and promote differentiation of the circular and then longitudinal smooth muscles. Disruption of BMP signaling leads to defects in the gut and in ENS function commensurate with these complex developmental roles.

This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung’s disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.

The gut contains a large 5-HT pool in enterochromaffin (EC) cells and a smaller 5-HT pool in the enteric nervous system (ENS). During development, enteric neurons are generated asynchronously. We tested hypotheses that serotonergic neurons, which arise early, affect development/survival of later-born dopaminergic, GABAergic, nitrergic, and calcitonin gene-related peptide-expressing neurons and are essential for gastrointestinal motility. 5-HT biosynthesis depends on tryptophan hydroxylase 1 (TPH1) in EC cells and on TPH2 in neurons; therefore, mice lacking TPH1 and/or TPH2 distinguish EC-derived from neuronal 5-HT. Deletion of TPH2, but not TPH1, decreased myenteric neuronal density and proportions of dopaminergic and GABAergic neurons but did not affect the extrinsic sympathetic innervation of the gut; intestinal transit slowed in mice lacking TPH2 mice, but gastric emptying accelerated. Isolated enteric crest-derived cells (ENCDCs) expressed the serotonin reuptake transporter (SERT) and 15 subtypes of 5-HT receptor. Addition of 5-HT to cultures of isolated ENCDCs promoted total and dopaminergic neuronal development. Rings of SERT-immunoreactive terminal axons surrounded myenteric dopaminergic neurons and SERT knock-out increased intestinal levels of dopamine metabolites, implying that enteric dopaminergic neurons receive a serotonergic innervation. Observations suggest that constitutive gastrointestinal motility depends more on neuronal than EC cell serotonin; moreover, serotonergic neurons promote development/survival of some classes of late-born enteric neurons, including dopaminergic neurons, which appear to innervate and activate in the adult ENS.

Trophic factor signaling is important for the migration, differentiation, and survival of enteric neurons during development. The mechanisms that regulate the maturation of enteric neurons in postnatal life, however, are poorly understood. Here, we show that transcriptional cofactor HIPK2 (homeodomain interacting protein kinase 2) is required for the maturation of enteric neurons and for regulating gliogenesis during postnatal development. Mice lacking HIPK2 display a spectrum of gastrointestinal (GI) phenotypes, including distention of colon and slowed GI transit time. Although loss of HIPK2 does not affect the enteric neurons in prenatal development, a progressive loss of enteric neurons occurs during postnatal life in Hipk2 −/− mutant mice that preferentially affects the dopaminergic population of neurons in the caudal region of the intestine. The mechanism by which HIPK2 regulates postnatal enteric neuron development appears to involve the response of enteric neurons to bone morphogenetic proteins (BMPs). Specifically, compared to wild type mice, a larger proportion of enteric neurons in Hipk2 −/− mutants have an abnormally high level of phosphorylated Smad1/5/8. Consistent with the ability of BMP signaling to promote gliogenesis, Hipk2 −/− mutants show a significant increase in glia in the enteric nervous system. In addition, numbers of autophagosomes are increased in enteric neurons in Hipk2 −/− mutants, and synaptic maturation is arrested. These results reveal a new role for HIPK2 as an important transcriptional cofactor that regulates the BMP signaling pathway in the maintenance of enteric neurons and glia, and further suggest that HIPK2 and its associated signaling mechanisms may be therapeutically altered to promote postnatal neuronal maturation.

BACKGROUND & AIMSEnteric neurons have been reported to be increased in inflamed regions of the bowel in patients with inflammatory bowel disease or intestinal neurogangliomatosis. It is impossible to determine whether this hyperinnervation predates intestinal inflammation, results from it, or contributes to its severity in humans, so we studied this process in mice.METHODSTo determine whether the density of enteric neurons determines the severity of inflammation, we studied transgenic mice that have greater than normal (NSE-noggin mice, which overexpress noggin under the control of the neuron-specific enolase promoter) or fewer than normal (Hand2(+/-) mice) numbers of neurons in the enteric nervous system. Colitis was induced with trinitrobenzene sulfonic acid or dextran sulfate sodium, and the intensity of the resulting inflammation in Hand2(+/-) and NSE-noggin mice was compared with that of wild-type littermates.RESULTSSeverity of each form of colitis (based on survival, symptom, and histologic scores; intestinal expression of genes that encode proinflammatory molecules; and levels of neutrophil elastase and p50 nuclear factor κB) were significantly reduced in Hand2(+/-) mice and significantly increased in NSE-noggin animals. Neither mouse differed from wild-type in the severity of delayed-type hypersensitivity (edema, T-cell and neutrophil infiltration, or expression of interleukin-1β, interferon-γ, or tumor necrosis factor-α) induced in the ears using 2,4-dinitro-1-fluorobenzene. Transgene effects on inflammation were therefore restricted to the gastrointestinal tract.CONCLUSIONSThe severity of intestinal inflammation is associated with the density of the enteric innervation in mice. Abnormalities in development of the enteric nervous system might therefore contribute to the pathogenesis of inflammatory bowel disease.

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.

The neural crest-derived cell population that colonizes the bowel (ENCDC) contains proliferating neural/glial progenitors. We tested the hypothesis that bone morphogenetic proteins (BMPs 2 and 4), which are known to promote enteric neuronal differentiation at the expense of proliferation, function similarly in gliogenesis. Enteric gliogenesis was analyzed in mice that overexpress the BMP antagonist, noggin, or BMP4 in the primordial ENS. Noggin-induced loss-of-function decreased, while BMP4-induced gain-of-function increased the glial density and glia/neuron ratio. When added to immunoisolated ENCDC, BMPs provoked nuclear translocation of phosphorylated SMAD proteins and enhanced both glial differentiation and expression of the neuregulin receptor ErbB3. ErbB3 transcripts were detected in E12 rat gut, before glial markers are expressed; moreover, expression of the ErbB3 ligand, glial growth factor 2 (GGF2) escalated rapidly after its first detection at E14. ErbB3-immunoreactive cells were located in the ENS of fetal and adult mice. GGF2 stimulated gliogenesis and proliferation and inhibited glial cell derived neurotrophic factor (GDNF)-promoted neurogenesis. Enhanced glial apoptosis occurred following GGF2 withdrawal; BMPs intensified this GGF2-dependence and reduced GGF2-stimulated proliferation. These observations support the hypotheses that BMPs are required for enteric gliogenesis and act by promoting responsiveness of ENCDC to ErbB3 ligands such as GGF2.

Receiving an M.D. from the Université Libre de Bruxelles (ULB), Belgium which was followed by his internship and residency in Neurology and Psychiatry at the ULB-Erasme Medical Center, Belgium; Dr. Serge Przedborski received his Ph.D. degree in Neurological Sciences from, ULB School of Medicine, Belgium. Subsequently, his postdoctoral research was done at Columbia University, where he became Assistant Professor of Neurology in 1991 and presently resides as the Page and William Black Professor of Neurology holding a joint appointment in the Departments of Neurology, Pathology, and Cell Biology.

The vagal sensory innervation of the gut is established during fetal life. The guidance of vagal sensory axons is, in part, mediated by enteric netrins and by their receptor, deleted in colorectal cancer (DCC), which is expressed on the growth cones of extending vagal fibers. Within the developing bowel, netrin immunoreactivity has been found in the endoderm, outer gut mesenchyme, and primordial ganglia. We tested the hypotheses that the presence of netrin in developing enteric neurons is due to biosynthesis, rather than uptake, and that neuronal netrin expression is essential for the establishment of the close relationship of vagal sensory axons and enteric ganglia. Crest- and non-crest-derived cells were isolated, respectively, by positive and negative immunoselection from the E15 rat gut with antibodies to p75NTR, which bind selectively to crest-derived cells in the fetal bowel. Transcripts encoding netrin-1 and -3 were detected in the non-crest-derived cells but not in the crest-derived precursors. In contrast, transcripts encoding netrin-1 and -3 were detected after the crest-derived precursors were cultured for 6 days to allow neurons to develop in vitro. To determine whether neurons or their precursors take up netrin, crest-derived cells were cultured for 6 days with stably transfected 293-EBNA cells expressing c-Myc-tagged netrin-1. Although cultured neurons displayed netrin-1 immunoreactivity, they could not be immunostained with antibodies to c-Myc. Enteric neurons, but not their precursors thus express, but do not take up netrin-1. To evaluate the role of neuronal netrin expression in the development of the vagal sensory innervation, we analyzed the intestines of Ret −/− mice, which lack enteric neurons. Vagal sensory axons were traced by applying DiI to fetal nodose ganglia. Vagal axons failed to innervate the bowel distal to the esophagus in the Ret −/− animals. These data support the concept that netrins, produced by intrinsic neurons, are essential for the establishment of the vagal sensory innervation of the gut.

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice overexpressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (gamma-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene-related peptide, TrkC). The numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin-overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development.

International Journal of Developmental NeuroscienceVolume 26, Issue 8 p. 862-862 Article [P1.62]: Essential role of TGFβ-HIPK2 signaling pathway in survival and synapse formation in enteric neurons A. Tang, Corresponding Author A. Tang n/[email protected] University of California, San Francisco, USACorresponding author.Search for more papers by this authorA. Chalazonitis, A. Chalazonitis Columbia University, USASearch for more papers by this authorT. Pham, T. Pham Columbia University, USASearch for more papers by this authorI. Hsieh, I. Hsieh University of California, San Francisco, USASearch for more papers by this authorM. Gershon, M. Gershon Columbia University, USASearch for more papers by this authorE. Huang, E. Huang University of California, San Francisco, USASearch for more papers by this author A. Tang, Corresponding Author A. Tang n/[email protected] University of California, San Francisco, USACorresponding author.Search for more papers by this authorA. Chalazonitis, A. Chalazonitis Columbia University, USASearch for more papers by this authorT. Pham, T. Pham Columbia University, USASearch for more papers by this authorI. Hsieh, I. Hsieh University of California, San Francisco, USASearch for more papers by this authorM. Gershon, M. Gershon Columbia University, USASearch for more papers by this authorE. Huang, E. Huang University of California, San Francisco, USASearch for more papers by this author First published: 25 November 2008 https://doi.org/10.1016/j.ijdevneu.2008.09.112Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume26, Issue8Abstracts to the 17th Biennial Meeting of the International Society for Developmental Neuroscience, 1‐5 June 2008, Asilomar, USADecember 2008Pages 862-862 RelatedInformation