Anti-Gephyrin Antibodies: A Novel Specificity in Patients With Systemic Sclerosis and Lower Bowel Dysfunction.

OBJECTIVE: Autoantibodies are clinically useful in phenotyping patients with systemic sclerosis (SSc). Gastrointestinal (GI) function is regulated by the enteric nervous system (ENS) and commonly impaired in SSc, suggesting that the SSc autoimmune response may target ENS antigens. We sought to identify novel anti-ENS autoantibodies with an aim to clinically phenotype SSc GI dysfunction. METHODS: Serum from a patient with SSc with GI dysfunction but without defined SSc-associated autoantibodies was used for autoantibody discovery. Immunoprecipitations performed with murine myenteric plexus lysates were on-bead digested, and autoantigens were identified by mass spectrometry. Prevalence was determined, and clinical features associated with novel autoantibodies were evaluated in a SSc cohort using regression analyses. The expression of gephyrin in human GI tract tissue was examined by immunohistochemistry. RESULTS: We identified gephyrin as a novel SSc autoantigen. Anti-gephyrin antibodies were present in 9% of patients with SSc (16/188) and absent in healthy controls (0/46). Anti-gephyrin antibody-positive patients had higher constipation scores (1.00 vs 0.50, P = 0.02) and were more likely to have severe constipation and severe distention/bloating (46% vs 15%, P = 0.005; 54% vs 25%, P = 0.023, respectively). Anti-gephyrin antibody levels were significantly higher among patients with severe constipation (0.04 vs 0.00; P = 0.001) and severe distention and bloating (0.03 vs 0.004; P = 0.010). Severe constipation was associated with anti-gephyrin antibodies even in the adjusted model. Importantly, gephyrin was expressed in the ENS, which regulates gut motility. CONCLUSION: Gephyrin is a novel ENS autoantigen that is expressed in human myenteric ganglia. Anti-gephyrin autoantibodies are associated with the presence and severity of constipation in patients with SSc.

CXCR4 and CXCL12 signaling regulates the development of extrinsic innervation to the colorectum.

The gastrointestinal tract is innervated by an intrinsic neuronal network, known as the enteric nervous system (ENS), and by extrinsic axons arising from peripheral ganglia. The nerve of Remak (NoR) is an avian-specific sacral neural crest-derived ganglionated structure that extends from the cloaca to the proximal midgut and, similar to the pelvic plexus, provides extrinsic innervation to the distal intestine. The molecular mechanisms controlling extrinsic nerve fiber growth into the gut is unknown. In vertebrates, CXCR4, a cell-surface receptor for the CXCL12 chemokine, regulates migration of neural crest cells and axon pathfinding. We have employed chimeric tissue recombinations and organ culture assays to study the role of CXCR4 and CXCL12 molecules in the development of colorectal innervation. CXCR4 is specifically expressed in nerve fibers arising from the NoR and pelvic plexus, while CXCL12 is localized to the hindgut mesenchyme and enteric ganglia. Overexpression of CXCL12 results in significantly enhanced axonal projections to the gut from the NoR, while CXCR4 inhibition disrupts nerve fiber extension, supporting a previously unreported role for CXCR4 and CXCL12 signaling in extrinsic innervation of the colorectum.

Optical Approaches to Understanding Enteric Circuits Along the Radial Axis.

The gastrointestinal tract operates in a highly dynamic environment. The gut is typically exposed to continually changing and highly convoluted luminal compositions comprising not only ingested content but also a multitude of resident microbes and microbial factors. It is therefore critical that the gut is capable of distinguishing between nutritious components from noxious substances. This is facilitated by specialized cellular sensory machinery that are in place in the intestinal epithelium and the ENS. However, the specific chemosensory processes and enteric neuronal pathways that enable the gut to discern and respond appropriately to different chemicals remain unclear. A major hurdle in studying the neural processing of luminal information has been the complex spatial organization of the mucosal structures and their innervation along the radial axis. Much of our current knowledge of enteric neuronal responses to luminal stimuli stems from studies that used semi-dissected guinea pig small intestine preparations with the mucosa and submucosa removed in one-half in order to record electrical activity from exposed myenteric neurons or in the circular muscle. Building on this, we ultimately strive to work towards integrated systems with all the gut layers intact. With advanced microscopy techniques including multiphoton intravital imaging, together with transgenic technologies utilizing cell-type specific activity-dependent reporters, we stand in good stead for studying the ENS in more intact preparations and even in live animals. In this chapter, we highlight recent contributions to the knowledge of sensory gut innervation by the developing and mature ENS. We also revisit established work examining the functional connectivity between the myenteric and submucosal plexus, and discuss the methodologies that can help advance our understanding of the enteric circuitry and signaling along the mucosa-serosa axis.

A tissue-like neurotransmitter sensor for the brain and gut.

Neurotransmitters play essential roles in regulating neural circuit dynamics both in the central nervous system as well as at the peripheral, including the gastrointestinal tract1-3. Their real-time monitoring will offer critical information for understanding neural function and diagnosing disease1-3. However, bioelectronic tools to monitor the dynamics of neurotransmitters in vivo, especially in the enteric nervous systems, are underdeveloped. This is mainly owing to the limited availability of biosensing tools that are capable of examining soft, complex and actively moving organs. Here we introduce a tissue-mimicking, stretchable, neurochemical biological interface termed NeuroString, which is prepared by laser patterning of a metal-complexed polyimide into an interconnected graphene/nanoparticle network embedded in an elastomer. NeuroString sensors allow chronic in vivo real-time, multichannel and multiplexed monoamine sensing in the brain of behaving mouse, as well as measuring serotonin dynamics in the gut without undesired stimulations and perturbing peristaltic movements. The described elastic and conformable biosensing interface has broad potential for studying the impact of neurotransmitters on gut microbes, brain-gut communication and may ultimately be extended to biomolecular sensing in other soft organs across the body.

Engaging biological oscillators through second messenger pathways permits emergence of a robust gastric slow-wave during peristalsis.

Peristalsis, the coordinated contraction-relaxation of the muscles of the stomach is important for normal gastric motility and is impaired in motility disorders. Coordinated electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICC) and smooth muscle (SM) cells of the stomach wall as a slow-wave, underly peristalsis. Normally, the gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. Understanding of the integrative role of neurotransmission and intercellular coupling in the propagation of an entrained gastric slow-wave, important for understanding motility disorders, however, remains incomplete. Using a computational framework constituted of a novel gastric motility network (GMN) model we address the hypothesis that engaging biological oscillators (i.e., ICCs) by constitutive gap junction coupling mechanisms and enteric neural innervation activated signals can confer a robust entrained gastric slow-wave. We demonstrate that while a decreasing enteric neural innervation gradient that modulates the intracellular IP3 concentration in the ICCs can guide the aboral slow-wave propagation essential for peristalsis, engaging ICCs by recruiting the exchange of second messengers (inositol trisphosphate (IP3) and Ca2+) ensures a robust entrained longitudinal slow-wave, even in the presence of biological variability in electrical coupling strengths. Our GMN with the distinct intercellular coupling in conjunction with the intracellular feedback pathways and a rostrocaudal enteric neural innervation gradient allows gastric slow waves to oscillate with a moderate range of frequencies and to propagate with a broad range of velocities, thus preventing decoupling observed in motility disorders. Overall, the findings provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach, offer directions for future experiments and theoretical work, and can potentially aid in the design of new interventional pharmacological and neuromodulation device treatments for addressing gastric motility disorders.

Microbial influences on gut development and gut-brain communication.

The developmental programs that build and sustain animal forms also encode the capacity to sense and adapt to the microbial world within which they evolved. This is abundantly apparent in the development of the digestive tract, which typically harbors the densest microbial communities of the body. Here, we review studies in human, mouse, zebrafish and Drosophila that are revealing how the microbiota impacts the development of the gut and its communication with the nervous system, highlighting important implications for human and animal health.

Brain-gut communication: vagovagal reflexes interconnect the two "brains".

The gastrointestinal tract has its own "brain," the enteric nervous system or ENS, that executes routine housekeeping functions of digestion. The dorsal vagal complex in the central nervous system (CNS) brainstem, however, organizes vagovagal reflexes and establishes interconnections between the entire neuroaxis of the CNS and the gut. Thus, the dorsal vagal complex links the "CNS brain" to the "ENS brain." This brain-gut connectome provides reflex adjustments that optimize digestion and assimilation of nutrients and fluid. Vagovagal circuitry also generates the plasticity and adaptability needed to maintain homeostasis to coordinate among organs and to react to environmental situations. Arguably, this dynamic flexibility provided by the vagal circuitry may, in some circumstances, lead to or complicate maladaptive disorders.

Gastrointestinal disorders in hyperkinetic movement disorders and ataxia.

BACKGROUND: Gastrointestinal (GI) disorders have been thoroughly investigated in hypokinetic disorders such as Parkinson's disease, but much less is known about GI disorders in hyperkinetic movement disorders and ataxia. The aim of this review is to draw attention to the GI disorders that are associated with these movement disorders. METHODS: References for this systematic review were identified by searches of PubMed through May 2020. Only publications in English were reviewed. RESULTS: Data from 249 articles were critically reviewed, compared, and integrated. The most frequently reported GI symptoms overall in hyperkinetic movement disorders and ataxia are dysphagia, sialorrhea, weight changes, esophago-gastritis, gastroparesis, constipation, diarrhea, and malabsorption. We report in detail on the frequency, characteristics, pathophysiology, and management of GI symptoms in essential tremor, restless legs syndrome, chorea, and spinocerebellar ataxias. The limited available data on GI disorders in dystonias, paroxysmal movement disorders, tardive dyskinesias, myoclonus, and non-SCA ataxias are also summarized. CONCLUSION: The purpose of our systematic review is to draw attention that, although primarily motor disorders, hyperkinetic movement disorders and ataxia can involve the GI system. Raising awareness about the GI symptom burden in hyperkinetic movement disorders and ataxia could contribute to a new research interest in that field, as well as improved patient care.

Nutritional, Gastrointestinal and Endo-Metabolic Challenges in the Management of Children with Spinal Muscular Atrophy Type 1.

The management of patients with spinal muscular atrophy type 1 (SMA1) is constantly evolving. In just a few decades, the medical approach has switched from an exclusively palliative therapy to a targeted therapy, transforming the natural history of the disease, improving survival time and quality of life and creating new challenges and goals. Many nutritional problems, gastrointestinal disorders and metabolic and endocrine alterations are commonly identified in patients affected by SMA1 during childhood and adolescence. For this reason, a proper pediatric multidisciplinary approach is then required in the clinical care of these patients, with a specific focus on the prevention of most common complications. The purpose of this narrative review is to provide the clinician with a practical and usable tool about SMA1 patients care, through a comprehensive insight into the nutritional, gastroenterological, metabolic and endocrine management of SMA1. Considering the possible horizons opened thanks to new therapeutic frontiers, a nutritional and endo-metabolic surveillance is a crucial element to be considered for a proper clinical care of these patients.

Non-zero-sum microbiome immune system interactions.

Fundamental asymmetries between the host and its microbiome in enzymatic activities and nutrient storage capabilities have promoted mutualistic adaptations on both sides. As a result, the enteric immune system has evolved so as not to cause a zero-sum sterilization of non-self, but rather achieve a non-zero-sum self-reinforcing cooperation with its evolutionary partner the microbiome. In this review, we attempt to integrate the accumulated knowledge of immune-microbiome interactions into an evolutionary framework and trace the pattern of positive immune-microbiome feedback loops across epithelial, enteric nervous system, innate, and adaptive immune circuits. Indeed, the immune system requires commensal signals for its development and function, and reciprocally protects the microbiome from nutrient shortage and pathogen outgrowth. In turn, a healthy microbiome is the result of immune system curatorship as well as microbial ecology. The paradigms of host-microbiome asymmetry and the cooperative nature of their interactions identified in the gut are applicable across all tissues influenced by microbial activities. Incorporation of immune system influences into models of microbiome ecology will be a step forward toward defining what constitutes a healthy human microbiome and guide discoveries of novel host-microbiome mutualistic adaptations that may be harnessed for the promotion of human health.

Selective stimulation of the ferret abdominal vagus nerve with multi-contact nerve cuff electrodes.

Dysfunction and diseases of the gastrointestinal (GI) tract are a major driver of medical care. The vagus nerve innervates and controls multiple organs of the GI tract and vagus nerve stimulation (VNS) could provide a means for affecting GI function and treating disease. However, the vagus nerve also innervates many other organs throughout the body, and off-target effects of VNS could cause major side effects such as changes in blood pressure. In this study, we aimed to achieve selective stimulation of populations of vagal afferents using a multi-contact cuff electrode wrapped around the abdominal trunks of the vagus nerve. Four-contact nerve cuff electrodes were implanted around the dorsal (N = 3) or ventral (N = 3) abdominal vagus nerve in six ferrets, and the response to stimulation was measured via a 32-channel microelectrode array (MEA) inserted into the left or right nodose ganglion. Selectivity was characterized by the ability to evoke responses in MEA channels through one bipolar pair of cuff contacts but not through the other bipolar pair. We demonstrated that it was possible to selectively activate subpopulations of vagal neurons using abdominal VNS. Additionally, we quantified the conduction velocity of evoked responses to determine what types of nerve fibers (i.e., Aδ vs. C) responded to stimulation. We also quantified the spatial organization of evoked responses in the nodose MEA to determine if there is somatotopic organization of the neurons in that ganglion. Finally, we demonstrated in a separate set of three ferrets that stimulation of the abdominal vagus via a four-contact cuff could selectively alter gastric myoelectric activity, suggesting that abdominal VNS can potentially be used to control GI function.

A neural crest cell isotropic-to-nematic phase transition in the developing mammalian gut.

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of ß1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

Roles for the gut microbiota in regulating neuronal feeding circuits.

The gut microbiota has the capacity to affect host appetite via intestinal satiety pathways, as well as complex feeding behaviors. In this Review, we highlight recent evidence that the gut microbiota can modulate food preference across model organisms. We discuss effects of the gut microbiota on the vagus nerve and brain regions including the hypothalamus, mesolimbic system, and prefrontal cortex, which play key roles in regulating feeding behavior. Crosstalk between commensal bacteria and the central and peripheral nervous systems is associated with alterations in signaling of neurotransmitters and neuropeptides such as dopamine, brain-derived neurotrophic factor (BDNF), and glucagon-like peptide-1 (GLP-1). We further consider areas for future research on mechanisms by which gut microbes may influence feeding behavior involving these neural pathways. Understanding roles for the gut microbiota in feeding regulation will be important for informing therapeutic strategies to treat metabolic and eating disorders.

Exploiting unique features of the gut-brain interface to combat gastrointestinal cancer.

The gastrointestinal tract comprises a complex ecosystem with extensive opportunities for functional interactions between neoplastic epithelial cells and stromal, immune, neuronal, glial, and other cell types, as well as microorganisms and metabolites within the gut lumen. In this Review, we focus on interactions between gastrointestinal cancers and elements of the central and enteric nervous systems. This previously understudied but rapidly emerging area of investigation has blossomed in recent years, particularly with respect to improved understanding of neural contributions to the development and progression of esophageal, gastric, pancreatic, and colon neoplasia. Cancer neuroscience offers great promise to advance our understanding of how neural-cancer interactions promote alimentary tract neoplasia. The resulting mechanistic insights can be leveraged to identify diagnostic and prognostic biomarkers, and to develop novel therapeutic interventions.

Periphery signals generated by Piezo-mediated stomach stretch and Neuromedin-mediated glucose load regulate the Drosophila brain nutrient sensor.

Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.

Central Neurocircuits Regulating Food Intake in Response to Gut Inputs-Preclinical Evidence.

The regulation of energy balance requires the complex integration of homeostatic and hedonic pathways, but sensory inputs from the gastrointestinal (GI) tract are increasingly recognized as playing critical roles. The stomach and small intestine relay sensory information to the central nervous system (CNS) via the sensory afferent vagus nerve. This vast volume of complex sensory information is received by neurons of the nucleus of the tractus solitarius (NTS) and is integrated with responses to circulating factors as well as descending inputs from the brainstem, midbrain, and forebrain nuclei involved in autonomic regulation. The integrated signal is relayed to the adjacent dorsal motor nucleus of the vagus (DMV), which supplies the motor output response via the efferent vagus nerve to regulate and modulate gastric motility, tone, secretion, and emptying, as well as intestinal motility and transit; the precise coordination of these responses is essential for the control of meal size, meal termination, and nutrient absorption. The interconnectivity of the NTS implies that many other CNS areas are capable of modulating vagal efferent output, emphasized by the many CNS disorders associated with dysregulated GI functions including feeding. This review will summarize the role of major CNS centers to gut-related inputs in the regulation of gastric function with specific reference to the regulation of food intake.

Gastrointestinal Vagal Afferents and Food Intake: Relevance of Circadian Rhythms.

Gastrointestinal vagal afferents (VAs) play an important role in food intake regulation, providing the brain with information on the amount and nutrient composition of a meal. This is processed, eventually leading to meal termination. The response of gastric VAs, to food-related stimuli, is under circadian control and fluctuates depending on the time of day. These rhythms are highly correlated with meal size, with a nadir in VA sensitivity and increase in meal size during the dark phase and a peak in sensitivity and decrease in meal size during the light phase in mice. These rhythms are disrupted in diet-induced obesity and simulated shift work conditions and associated with disrupted food intake patterns. In diet-induced obesity the dampened responses during the light phase are not simply reversed by reverting back to a normal diet. However, time restricted feeding prevents loss of diurnal rhythms in VA signalling in high fat diet-fed mice and, therefore, provides a potential strategy to reset diurnal rhythms in VA signalling to a pre-obese phenotype. This review discusses the role of the circadian system in the regulation of gastrointestinal VA signals and the impact of factors, such as diet-induced obesity and shift work, on these rhythms.

Microbial signalling in colonic motility.

Sensory nerve endings within the wall of the gastrointestinal (GI) tract may respond to bacterial signalling, providing the basis for key biological processes that underlie intestinal motility and microbial homeostasis. Enteric neurons and smooth muscle cells are well known to express an array of receptors, including G-protein coupled receptors and ligand-gated ion channels, that can sense chemical ligands and other bacterially-derived substances. These include short chain fatty acids, secondary bile acids and lipopolysaccharide. For neural detection of microbial activators to occur, luminal substances must first interact with enterocytes for direct signalling or cross paracellularly. Recent studies indicate that bacterial-derived microvesicles can cross the gut epithelial barrier and affect motility. This suggests a possible intercellular communication pathway between the GI tract and the ENS. We explore the idea that bacterial microvesicles can behave as a delivery package for communication between microbe and host.

Central and peripheral GLP-1 systems independently suppress eating.

The anorexigenic peptide glucagon-like peptide-1 (GLP-1) is secreted from gut enteroendocrine cells and brain preproglucagon (PPG) neurons, which, respectively, define the peripheral and central GLP-1 systems. PPG neurons in the nucleus tractus solitarii (NTS) are widely assumed to link the peripheral and central GLP-1 systems in a unified gut-brain satiation circuit. However, direct evidence for this hypothesis is lacking, and the necessary circuitry remains to be demonstrated. Here we show that PPGNTS neurons encode satiation in mice, consistent with vagal signalling of gastrointestinal distension. However, PPGNTS neurons predominantly receive vagal input from oxytocin-receptor-expressing vagal neurons, rather than those expressing GLP-1 receptors. PPGNTS neurons are not necessary for eating suppression by GLP-1 receptor agonists, and concurrent PPGNTS neuron activation suppresses eating more potently than semaglutide alone. We conclude that central and peripheral GLP-1 systems suppress eating via independent gut-brain circuits, providing a rationale for pharmacological activation of PPGNTS neurons in combination with GLP-1 receptor agonists as an obesity treatment strategy.

Gut innervation and enteric nervous system development: a spatial, temporal and molecular tour de force.

During embryonic development, the gut is innervated by intrinsic (enteric) and extrinsic nerves. Focusing on mammalian ENS development, in this Review we highlight how important the different compartments of this innervation are to assure proper gut function. We specifically address the three-dimensional architecture of the innervation, paying special attention to the differences in development along the longitudinal and circumferential axes of the gut. We review recent information about the formation of both intrinsic innervation, which is fairly well-known, as well as the establishment of the extrinsic innervation, which, despite its importance in gut-brain signaling, has received much less attention. We further discuss how external microbial and nutritional cues or neuroimmune interactions may influence development of gut innervation. Finally, we provide summary tables, describing the location and function of several well-known molecules, along with some newer factors that have more recently been implicated in the development of gut innervation.