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Background Intestinal epithelial barrier ( IEB) dysfunction plays a critical role in various intestinal disorders affecting infants and children, including the development of food allergies and colitis. Recent studies highlighted the role of probiotics in regulating IEB functions and behavior in adults, but their effects in the newborn remain largely unknown. We therefore characterized in rat pups, the impact of Lactobacillus fermentum CECT 5716 (L. fermentum) on stress-induced IEB dysfunction, systemic immune response and exploratory behavior. Methods Newborn rats received daily by gavage either L. fermentum or water. Intestinal permeability to fluorescein sulfonic acid ( FSA) and horseradish peroxidase ( HRP) was measured following maternal separation ( MS) and water avoidance stress ( WAS). Immunohistochemical, transcriptomic, and Western blot analysis of zonula occludens-1 ( ZO-1) distribution and expression were performed. Anxiety-like and exploratory behavior was assessed using the elevated plus maze test. Cytokine secretion of activated splenocytes was also evaluated. Key Results L. fermentum prevented MS and WAS-induced IEB dysfunction in vivo. L. fermentum reduced permeability to both FSA and HRP in the small intestine but not in the colon. L. fermentum increased expression of ZO-1 and prevented WAS-induced ZO-1 disorganization in ileal epithelial cells. L. fermentum also significantly reduced stress-induced increase in plasma corticosteronemia. In activated splenocytes, L. fermentum enhanced IFNγ secretion while it prevented IL-4 secretion. Finally, L. fermentum increased exploratory behavior. Conclusions & Inferences These results suggest that L. fermentum could provide a novel tool for the prevention and/or treatment of gastrointestinal disorders associated with altered IEB functions in the newborn. [ABSTRACT FROM AUTHOR]

The neural network of the enteric nervous system (ENS) underlies gastrointestinal functions. However, the molecular mechanisms involved in enteric neuronal connectivity are poorly characterized. Here, we studied the role of semaphorin 5A (Sema5A), previously characterized in the central nervous system, on ENS neuronal connectivity. Sema5A is linked to autism spectrum disorder (ASD), a neurodevelopmental disorder frequently associated with gastrointestinal comorbidities, and potentially associated with ENS impairments. This study investigated in rat enteric neuron cultures and gut explants the role of Sema5A on enteric neuron connectivity and the impact of ASD-associated mutations on Sema5A activity. Our findings demonstrated that Sema5A promoted axonal complexity and reduced functional connectivity in enteric neurons. Strikingly, the ASD-associated mutation S956G in Sema5A strongly affected these activities. This study identifies a critical role of Sema5A in the ENS as a regulator of neuronal connectivity that might be compromised in ASD., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Introduction: Gestational hypothyroxinemia (HTX) is a condition that occurs frequently at the beginning of pregnancy, and it correlates with cognitive impairment, autism, and attentional deficit in the offspring. Evidence in animal models suggests that gestational HTX can increase the susceptibility of the offspring to develop strong inflammation in immune-mediated inflammatory diseases. Ulcerative colitis (UC) is a frequent inflammatory bowel disease with unknown causes. Therefore, the intensity of ulcerative colitis-like disorder (UCLD) and the cellular and molecular factors involved in proinflammatory or anti-inflammatory responses were analyzed in the offspring gestated in HTX (HTX-offspring) and compared with the offspring gestated in euthyroidism (Control-offspring)., Methods: Gestational HTX was induced by the administration of 2-mercapto-1-methylimidazole in drinking water to pregnant mice during E10-E14. The HTX-offspring were induced with UCLD by the acute administration of dextran sodium sulfate (DSS). The score of UCLD symptomatology was registered every day, and colon histopathology, immune cells, and molecular factors involved in the inflammatory or anti-inflammatory response were analyzed on day 6 of DSS treatment., Results: The HTX-offspring displayed earlier UCLD pathological symptoms compared with the Control-offspring. After 6 days of DSS treatment, the HTX-offspring almost doubled the score of the Control-offspring. The histopathological analyses of the colon samples showed signs of inflammation at the distal and medial colon for both the HTX-offspring and Control-offspring. However, significantly more inflammatory features were detected in the proximal colon of the HTX-offspring induced with UCLD compared with the Control-offspring induced with UCLD. Significantly reduced mRNA contents encoding for protective molecules like glutamate-cysteine ligase catalytic subunit (GCLC) and mucin-2 (MUC-2) were found in the colon of the HTX-offspring as compared with the Control-offspring. Higher percentages of Th17 lymphocytes were detected in the colon tissues of the HTX-offspring induced or not with UCLD as compared with the Control-offspring., Discussion: Gestational HTX accelerates the onset and increases the intensity of UCLD in the offspring. The low expression of MUC-2 and GCLC together with high levels of Th17 Lymphocytes in the colon tissue suggests that the HTX-offspring has molecular and cellular features that favor inflammation and tissue damage. These results are important evidence to be aware of the impact of gestational HTX as a risk factor for UCLD development in offspring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rivera, Opazo, Hernández-Armengol, Álvarez, Mendoza-León, Caamaño, Gatica, Bohmwald, Bueno, González, Neunlist, Boudin, Kalergis and Riedel.)

The enteric nervous system (ENS) is the intrinsic nervous system that innervates the entire digestive tract and regulates major digestive functions. Recent evidence has shown that functions of the ENS critically rely on enteric neuronal connectivity; however, experimental models to decipher the underlying mechanisms are limited. Compared to the central nervous system, for which pure neuronal cultures have been developed for decades and are recognized as a reference in the field of neuroscience, an equivalent model for enteric neurons is lacking. In this study, we developed a novel model of highly pure rat embryonic enteric neurons with dense and functional synaptic networks. The methodology is simple and relatively fast. We characterized enteric neurons using immunohistochemical, morphological, and electrophysiological approaches. In particular, we demonstrated the applicability of this culture model to multi-electrode array technology as a new approach for monitoring enteric neuronal network activity. This in vitro model of highly pure enteric neurons represents a valuable new tool for better understanding the mechanisms involved in the establishment and maintenance of enteric neuron synaptic connectivity and functional networks., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Caillaud, Le Dréan, De-Guilhem-de-Lataillade, Le Berre-Scoul, Montnach, Nedellec, Loussouarn, Paillé, Neunlist and Boudin.)

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders defined by impaired social interactions and communication with repetitive behaviors, activities, or interests. Gastrointestinal (GI) disturbances and gut microbiota dysbiosis are frequently associated with ASD in childhood. However, it is not known whether microbiota dysbiosis in ASD patients also occurs in adulthood. Further, the consequences of altered gut microbiota on digestive functions and the enteric nervous system (ENS) remain unexplored. Therefore, we studied, in mice, the ability offecal supernatant (FS) from adult ASD patients to induce GI dysfunctions and ENS remodeling. First, the analyses of the fecal microbiota composition in adult ASD patients indicated a reduced α-diversity and increased abundance of three bacterial 16S rRNA gene amplicon sequence variants compared to healthy controls (HC). The transfer of FS from ASD patients (FS-ASD) to mice decreased colonic barrier permeability by 29% and 58% compared to FS-HC for paracellular and transcellular permeability, respectively. These effects are associated with the reduced expression of the tight junction proteins JAM-A, ZO-2, cingulin, and proinflammatory cytokines TNFα and IL1β. In addition, the expression of glial and neuronal molecules was reduced by FS-ASD as compared to FS-HC in particular for those involved in neuronal connectivity (βIII-tubulin and synapsin decreased by 31% and 67%, respectively). Our data suggest that changes in microbiota composition in ASD may contribute to GI alterations, and in part, via ENS remodeling.

In Hirschsprung's disease (HSCR), postoperative course remains unpredictable. Our aim was to define predictive factors of the main postoperative complications: obstructive symptoms (OS) and Hirschsprung-associated enterocolitis (HAEC). In this prospective multicentre cohort study, samples of resected bowel were collected at time of surgery in 18 neonates with short-segment HSCR in tertiary care hospitals. OS and HAEC were noted during postoperative follow-up. We assessed the enteric nervous system and the intestinal epithelial barrier (IEB) in ganglionic segments by combining immunohistochemical, proteomic and transcriptomic approaches, with functional ex vivo analysis of motility and para/transcellular permeability. Ten HSCR patients presented postoperative complications (median follow-up 23.5 months): 6 OS, 4 HAEC (2 with OS), 2 diarrhoea (without OS/HAEC). Immunohistochemical analysis showed a significant 41% and 60% decrease in median number of nNOS-IR myenteric neurons per ganglion in HSCR with OS as compared to HSCR with HAEC/diarrhoea (without OS) and HSCR without complications (p = 0.0095; p = 0.002, respectively). Paracellular and transcellular permeability was significantly increased in HSCR with HAEC as compared to HSCR with OS/diarrhoea without HAEC (p = 0.016; p = 0.009) and HSCR without complications (p = 0.029; p = 0.017). This pilot study supports the hypothesis that modulating neuronal phenotype and enhancing IEB permeability may treat or prevent postoperative complications in HSCR.

Most of the gut functions are controlled by the enteric nervous system (ENS), a complex network of enteric neurons located throughout the wall of the gastrointestinal tract. The formation of ENS connectivity during the perinatal period critically underlies the establishment of gastrointestinal motility, but the factors involved in this maturation process remain poorly characterized. Here, we examined the role of Semaphorin 3A (Sema3A) on ENS maturation and its potential implication in Hirschsprung disease (HSCR), a developmental disorder of the ENS with impaired colonic motility. We found that Sema3A and its receptor Neuropilin 1 (NRP1) are expressed in the rat gut during the early postnatal period. At the cellular level, NRP1 is expressed by enteric neurons, where it is particularly enriched at growth areas of developing axons. Treatment of primary ENS cultures and gut explants with Sema3A restricts axon elongation and synapse formation. Comparison of the ganglionic colon of HSCR patients to the colon of patients with anorectal malformation shows reduced expression of the synaptic molecule synapsin 1 in HSCR, which is inversely correlated with Sema3A expression. Our study identifies Sema3A as a critical regulator of ENS connectivity and provides a link between altered ENS connectivity and HSCR.

The accumulation of adverse events in utero and during childhood differentially increases the vulnerability to psychiatric diseases in men and women. Gut microbiota is highly sensitive to the early environment and has been recently hypothesized to affect brain development. However, the impact of early-life adversity on gut microbiota, notably with regards to sex differences, remains to be explored. We examined the effects of multifactorial early-life adversity on behavior and microbiota composition in C3H/HeN mice of both sexes exposed to a combination of maternal immune activation (lipopolysaccharide injection on embryonic day 17, 120 µg/kg, i.p.), maternal separation (3hr per day from postnatal day (PND)2 to PND14) and maternal unpredictable chronic mild stress. At adulthood, offspring exposed to multi-hit early adversity showed sex-specific behavioral phenotypes with males exhibiting deficits in social behavior and females showing increased anxiety in the elevated plus maze and increased compulsive behavior in the marble burying test. Early adversity also differentially regulated gene expression in the medial prefrontal cortex (mPFC) according to sex. Interestingly, several genes such as Arc, Btg2, Fosb, Egr4 or Klf2 were oppositely regulated by early adversity in males versus females. Finally, 16S-based microbiota profiling revealed sex-dependent gut dysbiosis. In males, abundance of taxa belonging to Lachnospiraceae and Porphyromonadaceae families or other unclassified Firmicutes, but also Bacteroides, Lactobacillus and Alloprevotella genera was regulated by early adversity. In females, the effects of early adversity were limited and mainly restricted to Lactobacillus and Mucispirillum genera. Our work reveals marked sex differences in a multifactorial model of early-life adversity, both on emotional behaviors and gut microbiota, suggesting that sex should systematically be considered in preclinical studies both in neurogastroenterology and psychiatric research., (Copyright © 2019. Published by Elsevier Inc.)

Early-life adversity is a major risk factor for the development of diseases later in life. Maternal protein restriction (MPR) is associated with morbidities in offspring affecting multiple organs, but its impact on the gastrointestinal (GI) tract remains poorly studied. Using a rat model, we examined the consequences of MPR on GI function and on the enteric nervous system (ENS) in the offspring at postnatal d 35 under basal state and following a water avoidance stress (WAS). Compared with control rats, MPR rats exhibited greater colonic motility, permeability, and corticosteronemia. In contrast to controls, MPR rats presented a blunted functional and corticosteronemic response to WAS. Furthermore, MPR rats showed an increased proportion of choline acetyltransferase-immunoreactive (ChAT-IR) neurons and a reduced level of autophagy in colonic myenteric neurons. In ENS cultures, corticosterone treatment increased the proportion of ChAT-IR neurons and reduced autophagy level in enteric neurons. Inhibition of autophagy in ENS cultures resulted in a higher vulnerability of enteric neurons to a cellular stress. Altogether, this study suggests that MPR induced GI dysfunction and ENS alterations in offspring rats and that MPR-induced increased corticosteronemia might be involved in ENS remodeling and altered responsiveness of the gut to stressors later in life.-Aubert, P., Oleynikova, E., Rizvi, H., Ndjim, M., Le Berre-Scoul, C., Grohard, P. A., Chevalier, J., Segain, J.-P., Le Drean, G., Neunlist, M., Boudin, H. Maternal protein restriction induces gastrointestinal dysfunction and enteric nervous system remodeling in rat offspring.

The human body is colonized by millions of microorganisms named microbiota that interact with our tissues in a cooperative and non-pathogenic manner. These microorganisms are present in the skin, gut, nasal, oral cavities, and genital tract. In fact, it has been described that the microbiota contributes to balancing the immune system to maintain host homeostasis. The gut is a vital organ where microbiota can influence and determine the function of cells of the immune system and contributes to preserve the wellbeing of the individual. Several articles have emphasized the connection between intestinal autoimmune diseases, such as Crohn's disease with dysbiosis or an imbalance in the microbiota composition in the gut. However, little is known about the role of the microbiota in autoimmune pathologies affecting other tissues than the intestine. This article focuses on what is known about the role that gut microbiota can play in the pathogenesis of non-intestinal autoimmune diseases, such as Grave's diseases, multiple sclerosis, type-1 diabetes, systemic lupus erythematosus, psoriasis, schizophrenia, and autism spectrum disorders. Furthermore, we discuss as to how metabolites derived from bacteria could be used as potential therapies for non-intestinal autoimmune diseases.

Background & Aims: In several types of cancers, tumor cells invade adjacent tissues by migrating along the resident nerves of the tumor microenvironment. This process, called perineural invasion , typically occurs along extrinsic nerves, with Schwann cells providing physical guidance for the tumor cells. However, in the colorectal cancer microenvironment, the most abundant nervous structures belong to the nonmyelinated intrinsic enteric nervous system (ENS). In this study, we investigated whether colon cancer cells interact with the ENS., Methods: Tumor epithelial cells (TECs) from human primary colon adenocarcinomas and cell lines were cocultured with primary cultures of ENS and cultures of human ENS plexus explants. By combining confocal and atomic force microscopy, as well as video microscopy, we assessed tumor cell adhesion and migration on the ENS. We identified the adhesion proteins involved using a proteomics approach based on biotin/streptavidin interaction, and their implication was confirmed further using selective blocking antibodies., Results: TEC adhered preferentially and with stronger adhesion forces to enteric nervous structures than to mesenchymal cells. TEC adhesion to ENS involved direct interactions with enteric neurons. Enteric neuron removal from ENS cultures led to a significant decrease in tumor cell adhesion. TECs migrated significantly longer and further when adherent on ENS compared with on mesenchymal cells, and their trajectory faithfully followed ENS structures. Blocking N-cadherin and L1CAM decreased TEC migration along ENS structures., Conclusions: Our data show that the enteric neuronal network guides tumor cell migration, partly via L1CAM and N-cadherin. These results open a new avenue of research on the underlying mechanisms and consequences of perineural invasion in colorectal cancer.

Thyroid hormones (THs) during pregnancy contribute significantly to cellular differentiation and development in several tissues of the offspring, principally the central nervous system (CNS). TH deficiencies, such as hypothyroidism or hypothyroxinemia, are highly frequent during pregnancy worldwide and known to be detrimental for the development of the fetus. The function of CNS in the offspring gestated under TH deficiency will be irreversible impaired, causing low intellectual quotient, attention deficit, and mental retardation. On the other hand, little is known about the effects of TH deficiency in the offspring immune system, being the prevalent notion that the effects are reversible and only for a while will affect the number of B and T cells. Recent studies have shown that maternal hypothyroidism can altered the function of immune system in the offspring, rendering the female offspring more susceptible to suffer autoimmune-inflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) and to be more resistant to a bacterial infection. In this article we discuss these recent findings, as well as the possible mechanisms underlying these effects and the potential implications for human health.

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN -associated FTD and GRN -associated NCL., (Copyright © 2017, American Association for the Advancement of Science.)

Key Points: Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown. To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron-enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs. We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation., Abstract: In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron-enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial-derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y 1 receptor- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Gestational hypothyroxinemia, characterized by low levels of maternal thyroxine (T 4 ) during gestation, is closely associated with cognitive impairment in offspring. Studies in animal models have shown that this condition alters neuronal glutamatergic synapses in the hippocampus. Given that astrocytes critically contribute to the establishment and functioning of synapses, the aim of this study was to determine the effects of gestational hypothyroxinemia on the capacity of astrocytes to regulate glutamatergic synapses. In an in vitro co-culture model of astrocytes and hippocampal neurons, gestational hypothyroxinemia profoundly affected the synaptic patterns of GluN1 and CD3ζ in an astrocyte-dependent manner. These effects were associated with impaired plasticity that was dependent on both neuronal and astrocyte contributions. These results highlight the importance of neuron-astrocyte interplay in the deleterious effects of gestational hypothyroxinemia and the timely diagnosis and treatment of this condition during gestation to ensure proper central nervous system development in offspring.

In the hematopoietic system, Syk family tyrosine kinases are essential components of immunoreceptor ITAM-based signaling. While there is increasing data indicating the involvement of immunoreceptors in neural functions, the contribution of Syk kinases remains obscure. Previously, we identified phosphorylated forms of Syk kinases in specialized populations of migrating neurons or projecting axons. Moreover, we identified ephrin/Eph as guidance molecules utilizing the ITAM-bearing CD3zeta (Cd247) and associated Syk kinases for the growth cone collapse response induced in vitro Here, we show that in the developing spinal cord, Syk is phosphorylated in navigating commissural axons. By analyzing axon trajectories in open-book preparations of Syk(-/-); Zap70(-/-) mouse embryos, we show that Syk kinases are dispensable for attraction towards the midline but confer growth cone responsiveness to repulsive signals that expel commissural axons from the midline. Known to serve a repulsive function at the midline, ephrin B3/EphB2 are obvious candidates for driving the Syk-dependent repulsive response. Indeed, Syk kinases were found to be required for ephrin B3-induced growth cone collapse in cultured commissural neurons. In fragments of commissural neuron-enriched tissues, Syk is in a constitutively phosphorylated state and ephrin B3 decreased its level of phosphorylation. Direct pharmacological inhibition of Syk kinase activity was sufficient to induce growth cone collapse. In conclusion, Syk kinases act as a molecular switch of growth cone adhesive and repulsive responses., (© 2016. Published by The Company of Biologists Ltd.)

Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia., (© 2015 Wiley Periodicals, Inc.)

Background: Recent works provide evidence of the importance of the prostaglandin D2 (PGD2) metabolic pathway in inflammatory bowel diseases. We investigated the expression of PGD2 metabolic pathway actors in Crohn's disease (CD) and the ability of the enteric nervous system (ENS) to produce PGD2 in inflammatory conditions., Methods: Expression of key actors involved in the PGD2 metabolic pathway and its receptors was analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in colonic mucosal biopsies of patients from three groups: controls, quiescent and active CD patients. To determine the ability of the ENS to secrete PGD2 in proinflammatory conditions, Lipocalin-type prostaglandin D synthase (L-PGDS) expression by neurons and glial cells was analyzed by immunostaining. PGD2 levels were determined in a medium of primary culture of ENS and neuro-glial coculture model treated by lipopolysaccharide (LPS)., Results: In patients with active CD, inflamed colonic mucosa showed significantly higher COX2 and L-PGDS mRNA expression, and significantly higher PGD2 levels than healthy colonic mucosa. On the contrary, peroxysome proliferator-activated receptor Gamma (PPARG) expression was reduced in inflamed colonic mucosa of CD patients with active disease. Immunostaining showed that L-PGDS was expressed in the neurons of human myenteric and submucosal plexi. A rat ENS primary culture model confirmed this expression. PGD2 levels were significantly increased on primary culture of ENS treated with LPS. This production was abolished by AT-56, a specific competitive L-PGDS inhibitor. The neuro-glial coculture model revealed that each component of the ENS, ECG and neurons, could contribute to PGD2 production., Conclusions: Our results highlight the activation of the PGD2 metabolic pathway in Crohn's disease. This study supports the hypothesis that in Crohn's disease, enteric neurons and glial cells form a functional unit reacting to inflammation by producing PGD2.

Background & Aims: Mediators released by the intestinal mucosa of patients with irritable bowel syndrome (IBS) affect the function of enteric and extrinsic sensory nerves, which can contribute to the development of symptoms. Little is known about the effects of mucosal mediators on intestinal neuroplasticity. We investigated how these mediators affect the phenotypes of colonic mucosa nerve fibers, neuron differentiation, and fiber outgrowth., Methods: We analyzed mucosal biopsy samples collected from 101 patients with IBS and 23 asymptomatic healthy individuals (controls). We measured levels of neuronal-specific enolase, growth-associated protein 43, nerve growth factor (NGF), and tyrosine kinase receptor A (NTRK1) by immunohistochemistry and enzyme-linked immunosorbent assay. Primary rat enteric neurons and human SH-SY5Y cells were incubated with supernatants from the mucosal biopsies and analyzed by morphometric and polymerase chain reaction analyses., Results: Compared with mucosal tissues of controls, mucosa from patients with IBS had a significant increase in the area of lamina propria occupied by neuronal-specific enolase-positive (57.7% increase) and growth-associated protein 43-positive fibers (56.1% increase) and staining density of NGF (89.3% increase) (P < .05 for all). Levels of NGF protein were also increased in tissues from patients with IBS vs controls (18% increase; P = .16) along with levels of NTRK1 (64% increase; P < .05). Mucosal supernatants from tissues of patients with IBS induced higher levels of neuritogenesis in primary culture of enteric neurons, compared with controls, and more NGF-dependent neuronal sprouting in SH-SY5Y cells., Conclusions: Nerve fiber density and sprouting, as well as expression of NGF and NTRK1, are significantly increased in mucosal tissues of patients with IBS. Mucosal mediators participate to these neuroplastic changes., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The immunoreceptor-associated protein CD3ζ is known for its role in immunity and has also been implicated in neuronal development and synaptic plasticity. However, the mechanism by which CD3ζ regulates synaptic transmission remains unclear. In this study, we showed that mice lacking CD3ζ exhibited defects in spatial learning and memory as examined by the Barnes maze and object location memory tasks. Given that peripheral T cells have been shown to support cognitive functions and neural plasticity, we generated CD3ζ(-/-) mice in which the peripheral T cells were repopulated to a normal level by syngeneic bone marrow transplantation. Using this approach, we showed that T-cell replenishment in CD3ζ(-/-) mice did not restore spatial memory defects, suggesting that the cognitive deficits in CD3ζ(-/-) mice were most likely mediated through a T-cell-independent mechanism. In support of this idea, we showed that CD3ζ proteins were localized to glutamatergic postsynaptic sites, where they interacted with the NMDAR subunit GluN2A. Loss of CD3ζ in brain decreased GluN2A-PSD95 association and GluN2A synaptic localization. This effect was accompanied by a reduced interaction of GluN2A with the key NMDAR downstream signaling protein calcium/calmodulin-dependent protein kinase II (CaMKII). Using the glycine-induced, NMDA-dependent form of chemical long-term potentiation (LTP) in cultured cortical neurons, we showed that CD3ζ was required for activity-dependent CaMKII autophosphorylation and for the synaptic recruitment of the AMPAR subunit GluA1. Together, these results support the model that the procognitive function of CD3ζ may be mediated through its involvement in the NMDAR downstream signaling pathway leading to CaMKII-dependent LTP induction.

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.

Recent studies have highlighted the key role of the immune protein CD3ζ in the maturation of neuronal circuits in the CNS. Yet, the upstream signals that might recruit and activate CD3ζ in neurons are still unknown. In this study, we show that CD3ζ functions early in neuronal development and we identify ephrinA1-dependent EphA4 receptor activation as an upstream regulator of CD3ζ. When newly born neurons are still spherical, before neurite extension, we found a transient CD3ζ aggregation at the cell periphery matching the initiation site of the future neurite. This accumulation of CD3ζ correlated with a stimulatory effect on filopodia extension via a Rho-GEF Vav2 pathway and a repression of neurite outgrowth. Conversely, cultured neurons lacking CD3ζ isolated from CD3ζ(-/-) mice showed a decreased number of filopodia and an enhanced neurite number. Stimulation with ephrinA1 induces the translocation of both CD3ζ and its activated effector molecules, ZAP-70/Syk tyrosine kinases, to EphA4 receptor clusters. EphrinA1-induced growth cone collapse was abrogated in CD3ζ(-/-) neurons and was markedly reduced by ZAP-70/Syk inhibition. Moreover, ephrinA1-induced ZAP-70/Syk activation was inhibited in CD3ζ(-/-) neurons. Altogether, our data suggest that CD3ζ mediates the ZAP-70/Syk kinase activation triggered by ephrinA-activated pathway to regulate early neuronal morphogenesis., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

An increasing number of data involve immunoreceptors in brain development, synaptic plasticity and behavior. However it has yet to be determined whether these proteins in fact transmit an immunoreceptor-like signal in non-hematopoietic neuronal cells. The recruitment and activation of the Syk family tyrosine kinases, Syk and ZAP-70, being a critical step in this process, we conducted a thorough analysis of Syk/ZAP-70 expression pattern in nervous tissues. Syk/ZAP-70 is present in neurons of different structures including the cerebellum, the hippocampus, the visual system and the olfactory system. During the olfactory system ontogeny the protein is detected from the 16th embryonic day and persists in adulthood. Importantly, Syk was phosphorylated on tyrosine residues representative of an active form of the kinase in specialized neuronal subpopulations comprising rostral migratory stream neuronal progenitor cells, hippocampal pyramidal cells, retinal ganglion cells and cerebellar granular cells. Phospho-Syk staining was also observed in synapse-rich regions such as the olfactory bulb glomeruli and the retina inner plexiform layer. Furthermore, our work on cultured primary hippoccampal neurons indicates that as for hematopoietic cells, Syk phosphorylation is readily induced upon pervanadate treatment. Therefore, Syk appears to be a serious candidate in connecting immunoreceptors to downstream adaptor/effector molecules in neurons., (Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

Posttranscriptional events such as RNA stabilization are important for cell differentiation, but little is known about the impact of AU-rich binding proteins (AUBPs) on the fate of neural cells. Expression of destabilizing AUBPs such as AUF1 and neuronal-specific stabilizing proteins such as HuB, HuC and HuD was therefore analyzed in the developing central nervous system. Real-time RT-PCR indicated a specific developmental pattern in the postnatal cerebellum, with a progressive down-regulation of AUF1 from P1, whereas HuB was strongly up-regulated at about P7. These changes were accompanied by a progressive increase in AUF1p45 and the disappearance of one HuB isoform from P15, suggesting particular roles for these AUBPs in the developing cerebellum. AUF1 was detected in the three main cerebellar layers, whereas Hu proteins were found only in postmitotic neurons. A role for Hu proteins in the early stages of neuronal differentiation is further supported by arrest of cell proliferation following induction of HuB or HuD expression in a neural stem cell line. The decrease in nestin expression suggest that HuD, but not HuB, favors the transition of neural progenitors into early neuroblasts, but other factors are most probably required for their full differentiation into neurons, insofar as GAP-43 was not detected in HuD-transfected cells. These data suggest critical roles for HuB at the very earliest stages of neuronal differentiation, such as cell cycle exit, and HuD might also be involved in the transition of neural progenitors into early neuroblasts. Taken together, the present results strengthen the importance of AUBPs in brain ontogenesis.

A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3zeta, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3zeta is mainly expressed in neurons. Distribution of CD3zeta in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3zeta is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3zeta was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3zeta in cultured neurons or overexpression of a loss-of-function CD3zeta mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3zeta by a CD3zeta antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3zeta in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.

While the hematopoietic lineage has been extensively studied using cluster of differentiation (CD) antibodies, very few data are available on the extracellular epitopes expressed by rat neural progenitors (rNPC) and their derivatives. In the present study, we used flow cytometry to screen 47 cell surface antigens, initially known as immune markers. The quantitative analyses were performed on rat neurospheres and compared with primary cultures of astroglial cells or cerebellar neurons. Several antigens such as CD80 or CD86 were clearly undetectable while others, like CD26 or CD161, showed a weak expression. Interestingly, 10% and 15% of the cells were immunopositive for CD172a and CD200, two immunoglobulin superfamily members preferentially expressed by glial or neuronal cells, respectively. Over 40% of the cells were immunopositive for CD3, CD71, or MHCI. The biological significance of the latter markers in rNPC remains to be determined but analyses of the CD3(-)/CD3(+) populations isolated by magnetic cell separation revealed differences in their cell fate. Indeed, CD3(+) cells did not establish neurospheres and differentiated mostly into GFAP(+) cells while CD3(-) cells were able to generate neurospheres upon mitogen treatment and gave rise to GFAP(+), A2B5(+), Tuj-1(+), and RIP(+) cells under differentiating conditions. In contrast, CD71(-)/CD71(+) cells did not show any significant difference in their proliferating and differentiating potentials. Finally, it is worth noting that an subpopulation of cells in rat neurospheres exhibit an immunoreactivity against anti-CD25 (IL2 receptor) and anti-CD62L (L-selectin) antibodies. The results reveal particular surface antigen profiles, giving new perspectives on the properties of rat brain-derived cells.

The chemokine SDF-1 is a secreted protein that plays a critical role in several aspects of neuron development through interaction with its unique receptor CXCR4. A key mechanism that controls neuron responsiveness to extracellular signals during neuronal growth is receptor endocytosis. Since we previously reported that SDF-1 regulates axon development without affecting the other neurites, we asked whether this could correlate with a compartment-selective trafficking of CXCR4. We thus studied CXCR4 behavior upon SDF-1 exposure in rat hippocampus slices and in transfected neuron cultures. A massive agonist-induced redistribution of CXCR4 in endosomes was observed in dendrites whereas no modification was evidenced in axons. Our data suggest that CXCR4 trafficking may play a role in mediating selective effects of SDF-1 on distinct neuronal membrane subdomains.

The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.

Recent data have shown that the chemokine SDF-1 plays a critical role in several aspects of brain development such as cell migration and axon pathfinding. However, its potential function in the generation of axons and dendrites is poorly characterized. In order to better understand the role of SDF-1 in the development of central neurons, we studied the cellular distribution of the SDF-1 receptor CXCR4 by immunocytochemistry of developing hippocampal neurons and tested the effect of SDF-1 in process patterning at the early stages of neuronal development. We found that CXCR4 immunoreactivity undergoes a striking redistribution during development. At the early stages, from day 2 to day 4 in culture, CXCR4 is particularly concentrated at the leading edge of growing neurites. As the cells mature, staining declines at the tip of the processes and becomes more broadly distributed along axons and, to a lesser extent, dendrites. SDF-1 stimulation of neurons at day 1-2 in culture triggers several effects on neuronal morphogenesis. SDF-1 reduces growth cone number and axonal outgrowth but stimulates axonal branching. These latter two effects are not observed in other neurites. This study unravels a new role for SDF-1/CXCR4 in specifying hippocampal neuron morphology by regulating axonal patterning at an early stage of neuronal development.

The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophrenia and in psychostimulant-induced drug-seeking behavior, both of which are affected by activation of the functionally opposed high-affinity neurotensin receptor (NTS1). To determine the functionally relevant sites, we examined the dual electron microscopic immunocytochemical localization of D2R and NTS1 in the NAc shell of rat brain. Immunolabeling for each receptor was seen in association with cytoplasmic organelles, or more rarely, on the plasma membrane of both axonal and somatodendritic profiles. Some of the axonal and many of the dendritic processes colocalized the two receptors. The dually labeled axon terminals often formed symmetric synapses or appositional contacts with unlabeled dendritic profiles. The morphology of these terminals suggests that they contain either inhibitory amino acids or dopamine. Other axonal profiles expressing exclusively NTS1 or D2R were without synaptic specializations or formed asymmetric, excitatory-type synapses mainly on unlabeled dendritic spines. In addition, however, several D2R-immunoreactive terminals were observed presynaptic to dendrites containing NTS1. The somatodendritic profiles immunolabeled for NTS1 and/or D2R had morphological features typical of inhibitory spiny projection neurons in the NAc. These results suggest that activation of NTS1 and D2R can dually modulate transmitter release from the same or separate phenotypically distinct axon terminals in the NAc shell. These presynaptic receptors as well as the postsynaptic NTS1 distribution in neurons that also contain or receive input from terminals containing D2R may mediate the opposing actions of neurotensin and dopamine in the NAc., (Copyright 2004 Wiley-Liss, Inc.)

PSD-95/Disc-large/ZO-1 (PDZ) domain-containing proteins play a central role in synaptic organization by their involvement in neurotransmitter receptor clustering and signaling complex assembly. The protein interacting with protein kinase C (PICK1), a synaptic PDZ domain protein that also contains a coiled-coil and acidic domain, binds to several synaptic components including the metabotropic glutamate receptor mGluR7a. Coexpression of PICK1 and mGluR7a in heterologous cells induces coclustering of these two proteins. To examine the role of the different structural motifs of PICK1 in synaptic aggregation of PICK1 and mGluR7a coclustering, several PICK1 mutants were generated to analyze their distribution in transfected hippocampal cultured neurons and to test their ability to induce coclusters with mGluR7a when coexpressed in fibroblast cells. The PDZ and coiled-coil domains are both required, whereas the acidic region plays an inhibitory role in these processes. Our data suggest that synaptic aggregation and receptor coclustering depend on PICK1 binding to a target membrane receptor, e.g. mGluR7a, by a PDZ-mediated interaction and on PICK1 oligomerization through the coiled-coil domain. This study defined three structural signals within PICK1 regulating its synaptic localization and receptor coclustering activity, which could represent molecular substrates involved in synaptic development and plasticity.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor-associated NAc core. We examined the electron microscopic immunolabeling of the high-affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR-labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR-immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR-labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter-specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR., (Copyright 2001 Wiley-Liss, Inc.)