Neuroplasticity and neuroprotection in enteric neurons: Role of epithelial cells

Neurons of enteric nervous system (ENS) regulate intestinal epithelial cells (IEC) functions but whether IEC can impact upon the neurochemical coding and survival of enteric neurons remain unknown. Neuro-epithelial interactions were studied using a coculture model composed of IEC lines and primary culture of rat ENS or human neuroblastoma cells (SH-SY5Y). Neurochemical coding of enteric neurons was analysed by immunohistochemistry and quantitative PCR. Neuroprotective effects of IEC were tested by measuring neuron specific enolase (NSE) release or cell permeability to 7-amino-actinomycin D (7-AAD). Following coculture with IEC, the percentage of VIP-immunoreactive (IR) neurons but not NOS-IR and VIP mRNA expression were significantly increased. IEC significantly reduced dopamine-induced NSE release and 7-AAD permeability in culture of ENS and SH-SY5Y, respectively. Finally, we showed that NGF had neuroprotective effects but reduced VIP expression in enteric neurons. In conclusion, our study identified a novel role for IEC in the regulation of enteric neuronal properties.     

Ligand binding and calcium influx induce distinct ectodomain/gamma-secretase-processing pathways of EphB2 receptor

Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent gamma-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx- and N-methyl-d-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor.     

Activity‐dependent secretion of alpha‐synuclein by enteric neurons

There is growing evidence supporting a role of extracellular alpha‐synuclein in the spreading of Parkinson's disease (PD) pathology. Recent pathological studies have raised the possibility that the enteric nervous system (ENS) is one of the initial sites of alpha‐synuclein pathology in PD. We therefore undertook this survey to determine whether alpha‐synuclein can be secreted by enteric neurons. Alpha‐synuclein secretion was assessed by immunoblot analysis of the culture medium from primary culture of ENS. We show that alpha‐synuclein is physiologically secreted by enteric neurons via a conventional, endoplasmic reticulum/Golgi‐dependent exocytosis, in a neuronal activity‐regulated manner. Our study is the first to evidence that enteric neurons are capable of secreting alpha‐synuclein, thereby providing new insights into the role of the ENS in the pathophysiology of PD.     

Reelin induces EphB activation

The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the ''classical'' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.     

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca²⁺) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca²⁺ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca²⁺ indicator such as Fluo-4 permits measurements of Ca²⁺ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca²⁺ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca²⁺ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca²⁺ responses in enteric neurons and glial cells.     

Pleiotropic effects of the bone morphogenetic proteins on development of the enteric nervous system

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.     

EphB4 signaling is capable of mediating ephrinB2-induced inhibition of cell migration

Signaling between the ligand ephrinB2 and the respective receptors of the EphB class is known to play a vital role during vascular morphogenesis and angiogenesis. The relative contribution of each EphB receptor type present on endothelial cells to these processes remains to be determined. It has been shown that ephrinB2-EphB receptor signal transduction leads to a repulsive migratory behavior of endothelial cells. It remained unclear whether this anti-migratory effect can be mediated by EphB4 signaling alone or whether other EphB receptors are necessary. It also remained unclear whether the kinase activity of EphB4 is pivotal to its action. To answer these questions, we developed a cellular migration system solely dependent on ephrinB2-EphB4 signaling. Using this system, we could show that EphB4 activation leads to the inhibition of cell migration. Furthermore we identified PP2, a known inhibitor of kinases of the Src family, and PD 153035, a known inhibitor of EGF receptor kinase, as inhibitors of EphB4 kinase activity. Using PP2, the inhibition of cell migration by ephrinB2 could be relieved, demonstrating that the kinase function of EphB4 is of prominent importance in this process. These results show that EphB4 activation is not only accompanying ephrinB2 induced repulsive behavior of cells, but is capable of directly mediating this effect.     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.     

Cellular changes in the enteric nervous system during ageing

The intrinsic neurons of the gut, enteric neurons, have an essential role in gastrointestinal functions. The enteric nervous system is plastic and continues to undergo changes throughout life, as the gut grows and responds to dietary and other environmental changes. Detailed analysis of changes in the ENS during ageing suggests that enteric neurons are more vulnerable to age-related degeneration and cell death than neurons in other parts of the nervous system, although there is considerable variation in the extent and time course of age-related enteric neuronal loss reported in different studies. Specific neuronal subpopulations, particularly cholinergic myenteric neurons, may be more vulnerable than others to age-associated loss or damage. Enteric degeneration and other age-related neuronal changes may contribute to gastrointestinal dysfunction that is common in the elderly population. Evidence suggests that caloric restriction protects against age-associated loss of enteric neurons, but recent advances in the understanding of the effects of the microbiota and the complex interactions between enteric ganglion cells, mucosal immune system and intestinal epithelium indicate that other factors may well influence ageing of enteric neurons. Much remains to be understood about the mechanisms of neuronal loss and damage in the gut, although there is evidence that reactive oxygen species, neurotrophic factor dysregulation and/or activation of a senescence associated phenotype may be involved. To date, there is no evidence for ongoing neurogenesis that might replace dying neurons in the ageing gut, although small local sites of neurogenesis would be difficult to detect. Finally, despite the considerable evidence for enteric neurodegeneration during ageing, and evidence for some physiological changes in animal models, the ageing gut appears to maintain its function remarkably well in animals that exhibit major neuronal loss, indicating that the ENS has considerable functional reserve.     

Development of the enteric nervous system

The mature enteric nervous system (ENS) is characterized by a degree of neuronal phenotypic diversity and independence of central nervous system control unequaled by any other region of the peripheral nervous system. Studies that have utilized the immunocytochemical demonstration of neurofilament protein and explanation of primordial gut with subsequent growth in culture have indicated that the neural crest precursors of enteric neurons are already committed to the neuronal lineage when they colonize the bowel; however, neuronal phenotypic expression occurs within the gut itself. It is likely that precursors able to give rise to each type of neuron found in the mature ENS are present among the earliest neural crest émigrés to reach the bowel. In mice a proximodistal wave of neuronal phenotypic expression occurs that does not appear to reflect the descent of neuronal precursors. This observation, the known plasticity of developing neural crest-derived neurons, and the demonstration of a persistent population of proliferating neuroblasts in the gut raise the possibility that enteric neuronal phenotypic expression is influenced by the enteric microenvironment.     

Phactr4

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

EphB signaling regulates target innervation in the developing and deafferented auditory brainstem

Precision in auditory brainstem connectivity underlies sound localization. Cochlear activity is transmitted to the ventral cochlear nucleus (VCN) in the mammalian brainstem via the auditory nerve. VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB), where specialized axons terminals, the calyces of Held, encapsulate MNTB principal neurons. The VCN-MNTB pathway is an essential component of the circuitry used to compute interaural intensity differences that are used for localizing sounds. When input from one ear is removed during early postnatal development, auditory brainstem circuitry displays robust anatomical plasticity. The molecular mechanisms that control the development of auditory brainstem circuitry and the developmental plasticity of these pathways are poorly understood. In this study we examined the role of EphB signaling in the development of the VCN-MNTB projection and in the reorganization of this pathway after unilateral deafferentation. We found that EphB2 and EphB3 reverse signaling are critical for the normal development of the projection from VCN to MNTB, but that successful circuit assembly most likely relies upon the coordinated function of many EphB proteins. We have also found that ephrin-B reverse signaling repels induced projections to the ipsilateral MNTB after unilateral deafferentation, suggesting that similar mechanisms regulate these two processes.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Cell death and the developing enteric nervous system

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.     

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.Eph/ephrin work in the laboratory of the authors is supported by a grant from the Deutsche Forschungsgemeinschaft (Au83/3–2 within the SPP1069 "Angiogenesis")     

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.     

EphrinB2 induces pelvic-urethra reflex potentiation via Src kinase-dependent tyrosine phosphorylation of NR2B

Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in pain-related neural plasticity at the spinal cord level have been identified. To test whether Src-family tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate pelvic-urethra reflex potentiation, we recorded external urethra sphincter electromyogram reflex activity and analyzed protein expression in the lumbosacral (L(6)-S(2)) dorsal horn in response to intrathecal ephrinB2 injections. When compared with vehicle solution, exogenous ephrinB2 (5 μg/rat it)-induced reflex potentiation, in associated with phosphorylation of EphB1/2, Src-family kinase, NR2B Y1336 and Y1472 tyrosine residues. Both intrathecal EphB1 and EphB2 immunoglobulin fusion protein (both 10 μg/rat it) prevented ephrinB2-dependent reflex potentiation, as well as protein phosphorylation. Pretreatment with PP2 (50 μM, 10 μl it), an Src-family kinase antagonist, reversed the reflex potentiation, as well as Src kinase and NR2B phosphorylation. Together, these results suggest the ephrinB2-dependent EphBR activation, which subsequently provokes Src kinase-mediated N-methyl-d-aspartate receptor NR2B phosphorylation in the lumbosacral dorsal horn, is crucial for the induction of spinal reflex potentiation contributing to the development of visceral pain and/or hyperalgesia in the pelvic area.