Nobiletin restores HFD-induced enteric nerve injury by regulating enteric glial activation and the GDNF/AKT/FOXO3a/P21 pathway.

BACKGROUND: To explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism. METHODS: An obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee's index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, ß-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis. RESULTS: HFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, ß-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs. CONCLUSIONS: Nobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.

[The gut, a whistleblower, in the early stages of Parkinson's disease]. / L'intestin, lanceur d'alerte, dans les prémices de la maladie de Parkinson.

The enteric nervous system (ENS), often called the "second brain", plays a crucial role in regulating digestive functions. Dysfunctions of the ENS are associated with several diseases such as Parkinson's disease. Recent studies suggest that early digestive disorders, notably chronic constipation, may be early signs of this neurodegenerative disease. Three-dimensional imaging of the ENS offers new insights into early diagnosis, in particular through the analysis of intestinal biopsies. This new research axis raises questions about the intestinal cause of Parkinson's disease, and opens the door to a better understanding and earlier treatment of this disease.

Title: L'intestin, lanceur d'alerte, dans les prémices de la maladie de Parkinson. Abstract: Le système nerveux entérique (SNE), souvent qualifié de « deuxième cerveau ¼, joue un rôle crucial dans la régulation des fonctions digestives. Des dysfonctionnements du SNE sont associés à diverses maladies telles que la maladie de Parkinson. Des études récentes suggèrent que les troubles digestifs précoces, notamment la constipation chronique, pourraient être des signes avant-coureurs de cette maladie neurodégénérative. L'imagerie tridimensionnelle du SNE offre de nouvelles perspectives pour un diagnostic précoce via notamment l'analyse de biopsies intestinales. Ce nouvel axe de recherche soulève des questions sur l'origine intestinale de la maladie de Parkinson et ouvre la porte à une meilleure compréhension et une prise en charge anticipée de cette maladie.

Comparison of the Influence of Bisphenol A and Bisphenol S on the Enteric Nervous System of the Mouse Jejunum.

Bisphenols are dangerous endocrine disruptors that pollute the environment. Due to their chemical properties, they are globally used to produce plastics. Structural similarities to oestrogen allow bisphenols to bind to oestrogen receptors and affect internal body systems. Most commonly used in the plastic industry is bisphenol A (BPA), which also has negative effects on the nervous, immune, endocrine, and cardiovascular systems. A popular analogue of BPA-bisphenol S (BPS) also seems to have harmful effects similar to BPA on living organisms. Therefore, with the use of double immunofluorescence labelling, this study aimed to compare the effect of BPA and BPS on the enteric nervous system (ENS) in mouse jejunum. The study showed that both studied toxins impact the number of nerve cells immunoreactive to substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), the neuronal isoform of nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VAChT). The observed changes were similar in the case of both tested bisphenols. However, the influence of BPA showed stronger changes in neurochemical coding. The results also showed that long-term exposure to BPS significantly affects the ENS.

Moniezia benedeni drives the SNAP-25 expression of the enteric nerves in sheep's small intestine.

BACKGROUND: The neuroimmune network plays a crucial role in regulating mucosal immune homeostasis within the digestive tract. Synaptosome-associated protein 25 (SNAP-25) is a presynaptic membrane-binding protein that activates ILC2s, initiating the host's anti-parasitic immune response. METHODS: To investigate the effect of Moniezia benedeni (M. benedeni) infection on the distribution of SNAP-25 in the sheep's small intestine, the recombinant plasmid pET-28a-SNAP-25 was constructed and expressed in BL21, yielding the recombinant protein. Then, the rabbit anti-sheep SNAP-25 polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of SNAP-25 in the intestines of normal and M. benedeni-infected sheep were detected by ELISA. RESULTS: The results showed that the SNAP-25 recombinant protein was 29.3 KDa, the titer of the prepared immune serum reached 1:128,000. It was demonstrated that the rabbit anti-sheep SNAP-25 polyclonal antibody could bind to the natural protein of sheep SNAP-25 specifically. The expression levels of SNAP-25 in the sheep's small intestine revealed its primary presence in the muscular layer and lamina propria, particularly around nerve fibers surrounding the intestinal glands. Average expression levels in the duodenum, jejunum, and ileum were 130.32 pg/mg, 185.71 pg/mg, and 172.68 pg/mg, respectively. Under conditions of M. benedeni infection, the spatial distribution of SNAP-25-expressing nerve fibers remained consistent, but its expression level in each intestine segment was increased significantly (P < 0.05), up to 262.02 pg/mg, 276.84 pg/mg, and 326.65 pg/mg in the duodenum, jejunum, and ileum, and it was increased by 101.06%, 49.07%, and 89.16% respectively. CONCLUSIONS: These findings suggest that M. benedeni could induce the SNAP-25 expression levels in sheep's intestinal nerves significantly. The results lay a foundation for further exploration of the molecular mechanism by which the gastrointestinal nerve-mucosal immune network perceives parasites in sheep.

Oral Exposure to Microplastics Affects the Neurochemical Plasticity of Reactive Neurons in the Porcine Jejunum.

Plastics are present in almost every aspect of our lives. Polyethylene terephthalate (PET) is commonly used in the food industry. Microparticles can contaminate food and drinks, posing a threat to consumers. The presented study aims to determine the effect of microparticles of PET on the population of neurons positive for selected neurotransmitters in the enteric nervous system of the jejunum and histological structure. An amount of 15 pigs were divided into three groups (control, receiving 0.1 g, and 1 g/day/animal orally). After 28 days, fragments of the jejunum were collected for immunofluorescence and histological examination. The obtained results show that histological changes (injury of the apical parts of the villi, accumulations of cellular debris and mucus, eosinophil infiltration, and hyperaemia) were more pronounced in pigs receiving a higher dose of microparticles. The effect on neuronal nitric oxide synthase-, and substance P-positive neurons, depends on the examined plexus and the dose of microparticles. An increase in the percentage of galanin-positive neurons and a decrease in cocaine and amphetamine-regulated transcript-, vesicular acetylcholine transporter-, and vasoactive intestinal peptide-positive neurons do not show such relationships. The present study shows that microparticles can potentially have neurotoxic and pro-inflammatory effects, but there is a need for further research to determine the mechanism of this process and possible further effects.

Enteric neuropathy in diabetes: Implications for gastrointestinal function.

Diabetes, commonly known for its metabolic effects, also critically affects the enteric nervous system (ENS), which is essential in regulating gastrointestinal (GI) motility, secretion, and absorption. The development of diabetes-induced enteric neuropathy can lead to various GI dysfunctions, such as gastroparesis and irregular bowel habits, primarily due to disruptions in the function of neuronal and glial cells within the ENS, as well as oxidative stress and inflammation. This editorial explores the pathophysiological mechanisms underlying the development of enteric neuropathy in diabetic patients. Additionally, it discusses the latest advances in diagnostic approaches, emphasizing the need for early detection and intervention to mitigate GI complications in diabetic individuals. The editorial also reviews current and emerging therapeutic strategies, focusing on pharmacological treatments, dietary management, and potential neuromodulatory interventions. Ultimately, this editorial highlights the necessity of a multidisciplinary approach in managing enteric neuropathy in diabetes, aiming to enhance patient quality of life and address a frequently overlooked complication of this widespread disease.

From diversity to disease: unravelling the role of enteric glial cells.

Enteric glial cells (EGCs) are an essential component of the enteric nervous system (ENS) and play key roles in gastrointestinal development, homeostasis, and disease. Derived from neural crest cells, EGCs undergo complex differentiation processes regulated by various signalling pathways. Being among the most dynamic cells of the digestive system, EGCs react to cues in their surrounding microenvironment and communicate with various cell types and systems within the gut. Morphological studies and recent single cell RNA sequencing studies have unveiled heterogeneity among EGC populations with implications for regional functions and roles in diseases. In gastrointestinal disorders, including inflammatory bowel disease (IBD), infections and cancer, EGCs modulate neuroplasticity, immune responses and tumorigenesis. Recent evidence suggests that EGCs respond plastically to the microenvironmental cues, adapting their phenotype and functions in disease states and taking on a crucial role. They exhibit molecular abnormalities and alter communication with other intestinal cell types, underscoring their therapeutic potential as targets. This review delves into the multifaceted roles of EGCs, particularly emphasizing their interactions with various cell types in the gut and their significant contributions to gastrointestinal disorders. Understanding the complex roles of EGCs in gastrointestinal physiology and pathology will be crucial for the development of novel therapeutic strategies for gastrointestinal disorders.

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells.

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Is the enteric nervous system a lost piece of the gut-kidney axis puzzle linked to chronic kidney disease?

The enteric nervous system (ENS) regulates numerous functional and immunological attributes of the gastrointestinal tract. Alterations in ENS cell function have been linked to intestinal outcomes in various metabolic, intestinal, and neurological disorders. Chronic kidney disease (CKD) is associated with a challenging intestinal environment due to gut dysbiosis, which further affects patient quality of life. Although the gut-related repercussions of CKD have been thoroughly investigated, the involvement of the ENS in this puzzle remains unclear. ENS cell dysfunction, such as glial reactivity and alterations in cholinergic signaling in the small intestine and colon, in CKD are associated with a wide range of intestinal pathways and responses in affected patients. This review discusses how the ENS is affected in CKD and how it is involved in gut-related outcomes, including intestinal permeability, inflammation, oxidative stress, and dysmotility.

Neuroimmune Interactions in the Intestine.

Recent advances have contributed to a mechanistic understanding of neuroimmune interactions in the intestine and revealed an essential role of this cross talk for gut homeostasis and modulation of inflammatory and infectious intestinal diseases. In this review, we describe the innervation of the intestine by intrinsic and extrinsic neurons and then focus on the bidirectional communication between neurons and immune cells. First, we highlight the contribution of neuronal subtypes to the development of colitis and discuss the different immune and epithelial cell types that are regulated by neurons via the release of neuropeptides and neurotransmitters. Next, we review the role of intestinal inflammation in the development of visceral hypersensitivity and summarize how inflammatory mediators induce peripheral and central sensitization of gut-innervating sensory neurons. Finally, we outline the importance of immune cells and gut microbiota for the survival and function of different neuronal populations at homeostasis and during bacterial and helminth infection.

Inhibiting the activation of enteric glial cells alleviates intestinal inflammation and comorbid anxiety- and depressive-like behaviors in the ulcerative colitis mice.

Ulcerative colitis (UC) is a common inflammatory bowel disease with a complex origin in clinical settings. It is frequently accompanied by negative emotional responses, including anxiety and depression. Enteric glial cells (EGCs) are important components of the gut-brain axis and are involved in the development of the enteric nervous system (ENS), intestinal neuroimmune, and regulation of intestinal motor functions. Since there is limited research encompassing the regulatory function of EGCs in anxiety- and depression-like behaviors induced by UC, this study aims to reveal their regulatory role in such behaviors and associated intestinal inflammation. This study applied morphological, molecular biological, and behavioral methods to observe the morphological and functional changes of EGCs in UC mice. The results indicated a significant activation of EGCs in the ENS of dextran sodium sulfate -induced UC mice. This activation was evidenced by morphological alterations, such as elongation or terminal swelling of processes. Besides EGCs activation, UC mice exhibited significantly elevated expression levels of pro-inflammatory cytokines in the peripheral blood, accompanied by anxiety- and depression-like behaviors. The inhibition of EGCs activity within the ENS can ameliorate the anxiety- and depression-like behaviors caused by UC. Our data suggest that UC and its resulting behaviors may be related to the activation of EGCs within the ENS. Moreover, the modulation of intestinal inflammation through inhibition of EGCs activation emerges as a promising clinical approach for alleviating UC-induced anxiety- and depression-like behaviors.

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system.

The vertebrate enteric nervous system (ENS) is a crucial network of enteric neurons and glia resident within the entire gastrointestinal tract (GI). Overseeing essential GI functions such as gut motility and water balance, the ENS serves as a pivotal bidirectional link in the gut-brain axis. During early development, the ENS is primarily derived from enteric neural crest cells (ENCCs). Disruptions to ENCC development, as seen in conditions like Hirschsprung disease (HSCR), lead to the absence of ENS in the GI, particularly in the colon. In this study, using zebrafish, we devised an in vivo F0 CRISPR-based screen employing a robust, rapid pipeline integrating single-cell RNA sequencing, CRISPR reverse genetics, and high-content imaging. Our findings unveil various genes, including those encoding opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in the neurochemical coding of the larval ENS. In summary, our work presents a novel, efficient CRISPR screen targeting ENS development, facilitating the discovery of previously unknown genes, and increasing knowledge of nervous system construction.

The Imperative for Innovative Enteric Nervous System-Intestinal Organoid Co-Culture Models: Transforming GI Disease Modeling and Treatment.

This review addresses the need for innovative co-culture systems integrating the enteric nervous system (ENS) with intestinal organoids. The breakthroughs achieved through these techniques will pave the way for a transformative era in gastrointestinal (GI) disease modeling and treatment strategies. This review serves as an introduction to the companion protocol paper featured in this journal. The protocol outlines the isolation and co-culture of myenteric and submucosal neurons with small intestinal organoids. This review provides an overview of the intestinal organoid culture field to establish a solid foundation for effective protocol application. Remarkably, the ENS surpasses the number of neurons in the spinal cord. Referred to as the "second brain", the ENS orchestrates pivotal roles in GI functions, including motility, blood flow, and secretion. The ENS is organized into myenteric and submucosal plexuses. These plexuses house diverse subtypes of neurons. Due to its proximity to the gut musculature and its cell type complexity, there are methodological intricacies in studying the ENS. Diverse approaches such as primary cell cultures, three-dimensional (3D) neurospheres, and induced ENS cells offer diverse insights into the multifaceted functionality of the ENS. The ENS exhibits dynamic interactions with the intestinal epithelium, the muscle layer, and the immune system, influencing epithelial physiology, motility, immune responses, and the microbiome. Neurotransmitters, including acetylcholine (ACh), serotonin (5-HT), and vasoactive intestinal peptide (VIP), play pivotal roles in these intricate interactions. Understanding these dynamics is imperative, as the ENS is implicated in various diseases, ranging from neuropathies to GI disorders and neurodegenerative diseases. The emergence of organoid technology presents an unprecedented opportunity to study ENS interactions within the complex milieu of the small and large intestines. This manuscript underscores the urgent need for standardized protocols and advanced techniques to unravel the complexities of the ENS and its dynamic relationship with the gut ecosystem. The insights gleaned from such endeavors hold the potential to revolutionize GI disease modeling and treatment paradigms.

Enteric nervous system regeneration and functional cure of experimental digestive Chagas disease with trypanocidal chemotherapy.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a female C3H/HeN mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology and the impact of treatment with the front-line, anti-parasitic drug benznidazole. Curative treatment 6 weeks post-infection resulted in sustained recovery of gastrointestinal transit function, whereas treatment failure led to infection relapse and gradual return of DCD symptoms. Neuro/immune gene expression patterns shifted from chronic inflammation to a tissue repair profile after cure, accompanied by increased cellular proliferation, glial cell marker expression and recovery of neuronal density in the myenteric plexus. Delaying treatment until 24 weeks post-infection led to partial reversal of DCD, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk of irreversible enteric neuromuscular tissue damage and dysfunction developing highlights the importance of prompt diagnosis and treatment. These findings support the concept of treating asymptomatic, T. cruzi-infected individuals with benznidazole to prevent DCD development.

Satiety controls behavior in Hydra through an interplay of pre-enteric and central nervous system-like neuron populations.

Hunger and satiety can have an influence on decision-making, sensory processing, and motor behavior by altering the internal state of the brain. This process necessitates the integration of peripheral sensory stimuli into the central nervous system. Here, we show how animals without a central nervous system such as the cnidarian Hydra measure and integrate satiety into neuronal circuits and which specific neuronal populations are involved. We demonstrate that this simple nervous system, previously referred to as diffuse, has an endodermal subpopulation (N4) similar to the enteric nervous system (feeding-associated behavior) and an ectodermal population (N3) that performs central nervous system-like functions (physiology/motor). This view of a supposedly simple nervous system could open an important window into the origin of more complex nervous systems.

Remote optogenetic control of the enteric nervous system and brain-gut axis in freely-behaving mice enabled by a wireless, battery-free optoelectronic device.

Wireless activation of the enteric nervous system (ENS) in freely moving animals with implantable optogenetic devices offers a unique and exciting opportunity to selectively control gastrointestinal (GI) transit in vivo, including the gut-brain axis. Programmed delivery of light to targeted locations in the GI-tract, however, poses many challenges not encountered within the central nervous system (CNS). We report here the development of a fully implantable, battery-free wireless device specifically designed for optogenetic control of the GI-tract, capable of generating sufficient light over large areas to robustly activate the ENS, potently inducing colonic motility ex vivo and increased propulsion in vivo. Use in in vivo studies reveals unique stimulation patterns that increase expulsion of colonic content, likely mediated in part by activation of an extrinsic brain-gut motor pathway, via pelvic nerves. This technology overcomes major limitations of conventional wireless optogenetic hardware designed for the CNS, providing targeted control of specific neurochemical classes of neurons in the ENS and brain-gut axis, for direct modulation of GI-transit and associated behaviours in freely moving animals.

BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

The gut-brain and gut-macrophage contribution to gastrointestinal dysfunction with systemic inflammation.

The gastrointestinal tract is one of the main organs affected during systemic inflammation and disrupted gastrointestinal motility is a major clinical manifestation. Many studies have investigated the involvement of neuroimmune interactions in regulating colonic motility during localized colonic inflammation, i.e., colitis. However, little is known about how the enteric nervous system and intestinal macrophages contribute to dysregulated motility during systemic inflammation. Given that systemic inflammation commonly results from the innate immune response against bacterial infection, we mimicked bacterial infection by administering lipopolysaccharide (LPS) to rats and assessed colonic motility using ex vivo video imaging techniques. We utilized the Cx3cr1-Dtr rat model of transient depletion of macrophages to investigate the role of intestinal macrophages in regulating colonic motility during LPS infection. To investigate the role of inhibitory enteric neurotransmission on colonic motility following LPS, we applied the nitric oxide synthase inhibitor, Nω-nitro-L-arginine (NOLA). Our results confirmed an increase in colonic contraction frequency during LPS-induced systemic inflammation. However, neither the depletion of intestinal macrophages, nor the suppression of inhibitory enteric nervous system activity impacted colonic motility disruption during inflammation. This implies that the interplay between the enteric nervous system and intestinal macrophages is nuanced, and complex, and further investigation is needed to clarify their joint roles in colonic motility.