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A new model of Parkinson’s disease (PD) pathogenesis is proposed, the α-Synuclein Origin site and Connectome (SOC) model, incorporating two aspects of α-synuclein pathobiology that impact the disease course for each patient: the anatomical location of the initial α-synuclein inclusion, and α-synuclein propagation dependent on the ipsilateral connections that dominate connectivity of the human brain. In some patients, initial α-synuclein pathology occurs within the CNS, leading to a brain-first subtype of PD. In others, pathology begins in the peripheral autonomic nervous system, leading to a body-first subtype. In brain-first cases, it is proposed that the first pathology appears unilaterally, often in the amygdala. If α-synuclein propagation depends on connection strength, a unilateral focus of pathology will disseminate more to the ipsilateral hemisphere. Thus, α-synuclein spreads mainly to ipsilateral structures including the substantia nigra. The asymmetric distribution of pathology leads to asymmetric dopaminergic degeneration and motor asymmetry. In body-first cases, the α-synuclein pathology ascends via the vagus to both the left and right dorsal motor nuclei of the vagus owing to the overlapping parasympathetic innervation of the gut. Consequently, the initial α-synuclein pathology inside the CNS is more symmetric, which promotes more symmetric propagation in the brainstem, leading to more symmetric dopaminergic degeneration and less motor asymmetry. At diagnosis, body-first patients already have a larger, more symmetric burden of α-synuclein pathology, which in turn promotes faster disease progression and accelerated cognitive decline. The SOC model is supported by a considerable body of existing evidence and may have improved explanatory power.

Background MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.

Realistic models to understand the developmental appearance of the pelvic nervous system in mammals are scarce. We visualized the development of the inferior hypogastric plexus and its preganglionic connections in human embryos at 4–8 weeks post‐fertilization, using Amira 3D reconstruction and Cinema 4D‐remodelling software. We defined the embryonic lesser pelvis as the pelvic area caudal to both umbilical arteries and containing the hindgut. Neural crest cells (NCCs) appeared dorsolateral to the median sacral artery near vertebra S1 at ~5 weeks and had extended to vertebra S5 1 day later. Once para‐arterial, NCCs either formed sympathetic ganglia or continued to migrate ventrally to the pre‐arterial region, where they formed large bilateral inferior hypogastric ganglionic cell clusters (IHGCs). Unlike more cranial pre‐aortic plexuses, both IHGCs did not merge because the 'pelvic pouch', a temporary caudal extension of the peritoneal cavity, interposed. Although NCCs in the sacral area started to migrate later, they reached their pre‐arterial position simultaneously with the NCCs in the thoracolumbar regions. Accordingly, the superior hypogastric nerve, a caudal extension of the lumbar splanchnic nerves along the superior rectal artery, contacted the IHGCs only 1 day later than the lumbar splanchnic nerves contacted the inferior mesenteric ganglion. The superior hypogastric nerve subsequently splits to become the superior hypogastric plexus. The IHGCs had two additional sources of preganglionic innervation, of which the pelvic splanchnic nerves arrived at ~6.5 weeks and the sacral splanchnic nerves only at ~8 weeks. After all preganglionic connections had formed, separate parts of the inferior hypogastric plexus formed at the bladder neck and distal hindgut., The umbilical arteries separate the lesser pelvis from the abdomen. Development of the inferior hypogastric plexus is divided into migration of neural crest cells (blue dots) to para‐ and pre‐arterial positions, followed by association of the ganglionic cell cluster with the superior hypogastric nerve, then the pelvic splanchnic nerves, and finally the sacral splanchnic nerves. Only thereafter nerves and ganglionic cells extend from this plexus along the bladder wall and the caudal hindgut.

Idiopathic Parkinson's disease (PD) may take decades to develop, during which many risk or protective factors may come into play to initiate the pathogenesis or modify its progression to clinical PD. The lack of understanding of this prodromal phase of PD and the factors involved has been a major hurdle in the study of PD etiology and preventive strategies. Although still controversial, the Braak and dual-hit hypotheses that PD may start peripherally in the olfactory structures and/or the gut provides a theoretical platform to identify the triggers and modifiers of PD prodromal development and progression. This is particularly true for the search of environmental causes of PD as the olfactory structures and gut are the major human mucosal interfaces with the environment. In this review, we lay out our personal views about how the Braak and dual-hit hypotheses may help us search for the environmental triggers and modifiers for PD, summarize available experimental and epidemiological evidence, and discuss research gaps and strategies.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest part of the peripheral nervous system; moreover, abnormal ENS development and function are associated with multiple human pathologies. Data from several groups suggest that under normal physiological conditions in adult animals, enteric nerve cells do not replicate. A study by Kulkarni et al in 2017 challenged this view and proposed that nearly 70% of enteric neurons in the myenteric ganglia are born in 1 week The authors of this study suggested that differences in DNA labelling times and DNA denaturation conditions might explain discrepancies with previous reports. Previous studies were carried out using different conditions and labelling techniques in various regions of the gastrointestinal tract; thus, conclusions have remained elusive. METHODS: Here, we have eliminated those variables by analyzing the whole small intestine using the reagents and conditions that Kulkarni et al used. To exclude variables related to immunohistochemistry, we carried out parallel experiments with "click chemistry"-based detection of DNA replication. RESULTS: Although proliferation was readily detected in the epithelium, we found no evidence of neuronal replication in the myenteric ganglia. CONCLUSIONS: We conclude that within 1 week under normal physiological conditions, myenteric neurons in the small intestine do not replicate.

Aggregation of alpha-synuclein into inclusion bodies, termed Lewy pathology, is a defining feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). In the majority of post mortem cases, the distribution of Lewy pathology seems to follow two overarching patterns: a caudo-rostral pattern with relatively more pathology in the brainstem than in the telencephalon, and an amygdala-centered pattern with the most abundant pathology in the "center of the brain", including the amygdala, entorhinal cortex, and substantia nigra, and relatively less pathology in the lower brainstem and spinal autonomic nuclei. The recent body-first versus brain-first model of Lewy Body Disorders proposes that the initial pathogenic alpha-synuclein in some patients originates in the enteric nervous system with secondary spreading to the brain; and in other patients originates inside the CNS with secondary spreading to the lower brainstem and peripheral autonomic nervous system. Here, we use two existing post mortem datasets to explore the possibility that clinical body-first and brain-first subtypes are equivalent to the caudo-rostral and amygdala-centered patterns of Lewy pathology seen at post mortem.

Background: Gastrointestinal (GI) complaints are frequently noted in aging dystrophinopathy patients, yet their underlying molecular mechanisms are largely unknown. As dystrophin protein isoform 71 (Dp71) is particularly implicated in the development of smooth muscle cells, we evaluated its distribution in the neonatal and adult rat intestine in this study. Methods: Dp71 expression levels were assessed in the proximal (duodenum, jejunum and ileum) and distal (caecum, colon and rectum) intestine by Western blotting and qPCR. In addition, the cellular distribution of total Dp was evaluated in the duodenum and colon by immunohistochemical colocalization studies with alpha-smooth muscle actin (aSMA), Hu RNA binding proteins C and D (HuC/HuD) for neurons and vimentin (VIM) for interstitial cells. Results: In neonatal and adult rats, the distal intestine expressed 2.5 times more Dp71 protein than the proximal part (p &lt, 0.01). This regional difference was not observed in Dp71 mRNA. During both stages, Dp-immunoreactivity was predominant in the muscularis propria, where it co-localized with aSMA and HuC/HuD. Conclusions: In neonatal and adult rats, Dp71 was expressed highest in the distal intestine. Together with the observation that Dp may be expressed by myenteric neurons, this warrants a paradigm shift in the treatment of GI comorbidities.

Background There is increasing evidence that Parkinson’s disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest. Methods Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS. Results A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model. Conclusions These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

Simple SummaryThe influence of nerves on different types of cancers, including colorectal cancer, is increasingly recognized. The intestines are highly innervated, both from outside the intestines (extrinsic innervation) and by a nervous system of their own; the enteric nervous system (intrinsic innervation). Nerves and cancer cells have been described to communicate with each other, although the exact mechanism in colorectal cancer is not yet explored. Nerves can enhance cancer progression by secreting signaling molecules, and cancer cells are capable of stimulating nerve growth. This review summarizes the innervation of the intestines and current knowledge on the role of the nervous system in colorectal cancer. Additionally, the therapeutic potential of these new insights is discussed.The role of the nervous system as a contributor in the tumor microenvironment has been recognized in different cancer types, including colorectal cancer (CRC). The gastrointestinal tract is a highly innervated organ system, which is not only innervated by the autonomic nervous system, but also contains an extensive nervous system of its own; the enteric nervous system (ENS). The ENS is important for gut function and homeostasis by regulating processes such as fluid absorption, blood flow, and gut motility. Dysfunction of the ENS has been linked with multiple gastrointestinal diseases, such as Hirschsprung disease and inflammatory bowel disease, and even with neurodegenerative disorders. How the extrinsic and intrinsic innervation of the gut contributes to CRC is not fully understood, although a mutual relationship between cancer cells and nerves has been described. Nerves enhance cancer progression through the secretion of neurotransmitters and neuropeptides, and cancer cells are capable of stimulating nerve growth. This review summarizes and discusses the nervous system innervation of the gastrointestinal tract and how it can influence carcinogenesis, and vice versa. Lastly, the therapeutic potential of these novel insights is discussed.

Compared to the intrinsic enteric nervous system (ENS), development of the extrinsic ENS is poorly documented, even though its presence is easily detectable with histological techniques. We visualised its development in human embryos and foetuses of 4–9.5 weeks post‐fertilisation using Amira 3D‐reconstruction and Cinema 4D‐remodelling software. The extrinsic ENS originated from small, basophilic neural crest cells (NCCs) that migrated to the para‐aortic region and then continued ventrally to the pre‐aortic region, where they formed autonomic pre‐aortic plexuses. From here, nerve fibres extended along the ventral abdominal arteries and finally connected to the intrinsic system. Schwann cell precursors (SCPs), a subgroup of NCCs that migrate on nerve fibres, showed region‐specific differences in differentiation. SCPs developed into scattered chromaffin cells of the adrenal medulla dorsolateral to the coeliac artery (CA) and into more tightly packed chromaffin cells of the para‐aortic bodies ventrolateral to the inferior mesenteric artery (IMA), with reciprocal topographic gradients between both fates. The extrinsic ENS first extended along the CA and then along the superior mesenteric artery (SMA) and IMA 5 days later. Apart from the branch to the caecum, extrinsic nerves did not extend along SMA branches in the herniated parts of the midgut until the gut loops had returned in the abdominal cavity, suggesting a permissive role of the intraperitoneal environment. Accordingly, extrinsic innervation had not yet reached the distal (colonic) loop of the midgut at 9.5 weeks development. Based on intrinsic ENS‐dependent architectural remodelling of the gut layers, extrinsic innervation followed intrinsic innervation 3–4 Carnegie stages later., The neural crest cells that form the extrinsic enteric nervous system migrate to the para‐ and then the pre‐aortic regions. Concomitantly, the thoracic (left) and lumbar (right) splanchnic nerves extend ventrally to the pre‐aortic plexuses. Pre‐aortic nerve fibres from the coeliac and inferior mesenteric plexus extend further ventrally to connect to the intrinsic nerve fibres inside the gut wall. The herniated portions of the midgut become innervated only after return into the abdominal cavity.

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which cause the majority of chronic kidney diseases in children. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including renal aplasia, hypodysplasia, hypoplasia, ectopia, and different forms of ureter abnormalities. The majority of the genetic causes of CAKUT remain unknown. Research on mutant mice has identified multiple genes that critically regulate renal differentiation. The data generated from this research have served as an excellent resource to identify the genetic bases of human kidney defects and have led to significantly improved diagnostics. Furthermore, genetic data from human CAKUT studies have also revealed novel genes regulating kidney differentiation.

Optogenetics and chemogenetics comprise a wide variety of applications in which genetically encoded actuators and indicators are used to modulate and monitor activity with high cellular specificity. Over the past 10 years, development of these genetically encoded tools has contributed tremendously to our understanding of integrated physiology. In concert with the continued refinement of probes, strategies to target transgene expression to specific cell types have also made much progress in the past 20 years. In addition, the successful implementation of optogenetic and chemogenetic techniques thrives thanks to ongoing advances in live imaging microscopy and optical technology. Although innovation of optogenetic and chemogenetic methods has been primarily driven by researchers studying the central nervous system, these techniques also hold great promise to boost research in neurogastroenterology. In this Review, we describe the different classes of tools that are currently available and give an overview of the strategies to target them to specific cell types in the gut wall. We discuss the possibilities and limitations of optogenetic and chemogenetic technology in the gut and provide an overview of their current use, with a focus on the enteric nervous system. Furthermore, we suggest some experiments that can advance our understanding of how the intrinsic and extrinsic neural networks of the gut control gastrointestinal function.

Optogenetics and chemogenetics comprise a wide variety of applications in which genetically encoded actuators and indicators are used to modulate and monitor activity with high cellular specificity. Over the past 10 years, development of these genetically encoded tools has contributed tremendously to our understanding of integrated physiology. In concert with the continued refinement of probes, strategies to target transgene expression to specific cell types have also made much progress in the past 20 years. In addition, the successful implementation of optogenetic and chemogenetic techniques thrives thanks to ongoing advances in live imaging microscopy and optical technology. Although innovation of optogenetic and chemogenetic methods has been primarily driven by researchers studying the central nervous system, these techniques also hold great promise to boost research in neurogastroenterology. In this Review, we describe the different classes of tools that are currently available and give an overview of the strategies to target them to specific cell types in the gut wall. We discuss the possibilities and limitations of optogenetic and chemogenetic technology in the gut and provide an overview of their current use, with a focus on the enteric nervous system. Furthermore, we suggest some experiments that can advance our understanding of how the intrinsic and extrinsic neural networks of the gut control gastrointestinal function.

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease. Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.

Alterations in colonic motility are implicated in the pathophysiology of bowel disorders, but high-resolution manometry of human colonic motor function has revealed that our knowledge of normal motor patterns is limited. Furthermore, various terminologies and definitions have been used to describe colonic motor patterns in children, adults and animals. An example is the distinction between the high-amplitude propagating contractions in humans and giant contractions in animals. Harmonized terminology and definitions are required that are applicable to the study of colonic motility performed by basic scientists and clinicians, as well as adult and paediatric gastroenterologists. As clinical studies increasingly require adequate animal models to develop and test new therapies, there is a need for rational use of terminology to describe those motor patterns that are equivalent between animals and humans. This Consensus Statement provides the first harmonized interpretation of commonly used terminology to describe colonic motor function and delineates possible similarities between motor patterns observed in animal models and humans in vitro (ex vivo) and in vivo. The consolidated terminology can be an impetus for new research that will considerably improve our understanding of colonic motor function and will facilitate the development and testing of new therapies for colonic motility disorders., This Consensus Statement provides a conceptual and methodological framework to expand research on colonic motility in experimental animals and humans. The work is intended to facilitate the development of new drugs for common colonic motility disorders and of appropriate diagnostic and therapeutic algorithms for the management of paediatric and adult patients.

Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the “death by a thousand cuts” profile of α-synucleinopathies. Electronic supplementary material The online version of this article (10.1007/s00401-019-02010-2) contains supplementary material, which is available to authorized users.

Background & Aims RET, the receptor for the glial cell line–derived neurotrophic factor (GDNF) family ligands, is the most frequently mutated gene in congenital aganglionic megacolon or Hirschsprung’s disease (HSCR). The leading cause of mortality in HSCR is HSCR-associated enterocolitis (HAEC), which is characterized by altered mucin composition, mucin retention, bacterial adhesion to enterocytes, and epithelial damage, although the order of these events is obscure. In mice, loss of GDNF signaling leads to a severely underdeveloped enteric nervous system and neonatally fatal kidney agenesis, thereby precluding the use of these mice for modeling postnatal HSCR and HAEC. Our aim was to generate a postnatally viable mouse model for HSCR/HAEC and analyze HAEC etiology. Methods GDNF family receptor alpha-1 (GFRa1) hypomorphic mice were generated by placing a selectable marker gene in the sixth intron of the Gfra1 locus using gene targeting in mouse embryonic stem cells. Results We report that 70%–80% reduction in GDNF co-receptor GFRa1 expression levels in mice results in HSCR and HAEC, leading to death within the first 25 postnatal days. These mice mirror the disease progression and histopathologic findings in children with untreated HSCR/HAEC. Conclusions In GFRa1 hypomorphic mice, HAEC proceeds from goblet cell dysplasia, with abnormal mucin production and retention, to epithelial damage. Microbial enterocyte adherence and tissue invasion are late events and therefore unlikely to be the primary cause of HAEC. These results suggest that goblet cells may be a potential target for preventative treatment and that reduced expression of GFRa1 may contribute to HSCR susceptibility., Graphical abstract

Genome-wide association studies have discovered hundreds of loci associated with complex brain disorders, but it remains unclear in which cell types these loci are active. Here we integrate genome-wide association study results with single-cell transcriptomic data from the entire mouse nervous system to systematically identify cell types underlying brain complex traits. We show that psychiatric disorders are predominantly associated with projecting excitatory and inhibitory neurons. Neurological diseases were associated with different cell types, which is consistent with other lines of evidence. Notably, Parkinson’s disease was genetically associated not only with cholinergic and monoaminergic neurons (which include dopaminergic neurons) but also with enteric neurons and oligodendrocytes. Using post-mortem brain transcriptomic data, we confirmed alterations in these cells, even at the earliest stages of disease progression. Our study provides an important framework for understanding the cellular basis of complex brain maladies, and reveals an unexpected role of oligodendrocytes in Parkinson’s disease. Eating Disorders Working Group of the Psychiatric Genomics Consortium Roger Adan17,18,19, Lars Alfredsson20, Tetsuya Ando21, Ole Andreassen22, Jessica Baker9, Andrew Bergen23,24, Wade Berrettini25, Andreas Birgegård26,27, Joseph Boden28, Ilka Boehm29, Claudette Boni30, Vesna Boraska Perica31,32, Harry Brandt33, Gerome Breen13,14, Julien Bryois1, Katharina Buehren34, Cynthia Bulik1,9,15, Roland Burghardt35, Matteo Cassina36, Sven Cichon37, Maurizio Clementi36, Jonathan Coleman13,14, Roger Cone38, Philippe Courtet39, Steven Crawford33, Scott Crow40, James Crowley16,26, unna Danner18, Oliver Davis41,42, Martina de Zwaan43, George Dedoussis44, Daniela Degortes45, Janiece DeSocio46, Danielle Dick47, Dimitris Dikeos48, Christian Dina49,50, Monika Dmitrzak-Weglarz51, Elisa Docampo Martinez52,53,54, Laramie Duncan55, Karin Egberts56, Stefan Ehrlich29, Geòrgia Escaramís52,53,54, Tõnu Esko57,58, Xavier Estivill52,53,54,59, Anne Farmer13, Angela Favaro45, Fernando Fernández-Aranda60,61, Manfred Fichter62,63, Krista Fischer57, Manuel Föcker64, Lenka Foretova65, Andreas Forstner37,66,67,68,69, Monica Forzan36, Christopher Franklin31, Steven Gallinger70, Héléna Gaspar13,14, Ina Giegling71, Johanna Giuranna64, Paola Giusti-Rodríquez16, Fragiskos Gonidakis72, Scott Gordon73, Philip Gorwood30,74, Monica Gratacos Mayora52,53,54, Jakob Grove75,76,77,78, Sébastien Guillaume39, Yiran Guo79, Hakon Hakonarson79,80, Katherine Halmi81, Ken Hanscombe82, Konstantinos Hatzikotoulas31, Joanna Hauser83, Johannes Hebebrand64, Sietske Helder13,84, Anjali Henders85, Stefan Herms37,69, Beate Herpertz-Dahlmann34, Wolfgang Herzog86, Anke Hinney64, L. John Horwood28, Christopher Hübel1,13, Laura Huckins31,87, James Hudson88, Hartmut Imgart89, Hidetoshi Inoko90, Vladimir Janout91, Susana Jiménez-Murcia60,61, Craig Johnson92, Jennifer Jordan93,94, Antonio Julià95, Anders Juréus1, Gursharan Kalsi13, Deborah Kaminská96, Allan Kaplan97, Jaakko Kaprio98,99, Leila Karhunen100, Andreas Karwautz101, Martien Kas17,102, Walter Kaye103, James Kennedy97, Martin Kennedy104, Anna Keski-Rahkonen98, Kirsty Kiezebrink105, Youl-Ri Kim106, Katherine Kirk73, Lars Klareskog107, Kelly Klump108, Gun Peggy Knudsen109, Maria La Via9, Mikael Landén1,19, Janne Larsen76,110,111, Stephanie Le Hellard112,113,114, Virpi Leppä1, Robert Levitan115, Dong Li79, Paul Lichtenstein1, Lisa Lilenfeld116, Bochao Danae Lin17, Jolanta Lissowska117, Jurjen Luykx17, Pierre Magistretti118,119, Mario Maj120, Katrin Mannik57,121, Sara Marsal95, Christian Marshall122, Nicholas Martin73, Manuel Mattheisen26,27,75,123, Morten Mattingsdal22, Sara McDevitt124,125, Peter McGuffin13, Sarah Medland73, Andres Metspalu57,126, Ingrid Meulenbelt127, Nadia Micali128,129, James Mitchell130, Karen Mitchell131, Palmiero Monteleone132, Alessio Maria Monteleone120, Grant Montgomery73,85,133, Preben Bo Mortensen76,110,111, Melissa Munn-Chernoff9, Benedetta Nacmias134, Marie Navratilova65, Claes Norring26,27, Ioanna Ntalla44, Catherine Olsen73, Roel Ophoff17,135, Julie O’Toole136, Leonid Padyukov107, Aarno Palotie58,99,137, Jacques Pantel30, Hana Papezova96, Richard Parker73, John Pearson138, Nancy Pedersen1, Liselotte Petersen76,110,111, Dalila Pinto87, Kirstin Purves13, Raquel Rabionet139,140,141, Anu Raevuori98, Nicolas Ramoz30, Ted Reichborn-Kjennerud109,142, Valdo Ricca134,143, Samuli Ripatti144, Stephan Ripke145,146,147, Franziska Ritschel29,148, Marion Roberts13, Alessandro Rotondo149, Dan Rujescu62,71, Filip Rybakowski150, Paolo Santonastaso151, André Scherag152, Stephen Scherer153, ulrike Schmidt13, Nicholas Schork154, Alexandra Schosser155, Jochen Seitz34, Lenka Slachtova156, P. Eline Slagboom127, Margarita Slof-Op ‘t Landt157,158, Agnieszka Slopien159, Sandro Sorbi134,160, Michael Strober161,162, Garret Stuber9,163, Patrick Sullivan1,16, Beata Świątkowska164, Jin Szatkiewicz16, Ioanna Tachmazidou31, Elena Tenconi45, Laura Thornton9, Alfonso Tortorella165,166, Federica Tozzi167, Janet Treasure13, Artemis Tsitsika168, Marta Tyszkiewicz-Nwafor150, Konstantinos Tziouvas169, Annemarie van Elburg18,170, Eric van Furth157,158, Tracey Wade171, Gudrun Wagner101, Esther Walton29, Hunna Watson9,10,11, Thomas Werge172, David Whiteman73, Elisabeth Widen99, D. Blake Woodside173,174, Shuyang Yao1, Zeynep Yilmaz9,16, Eleftheria Zeggini31,175, Stephanie Zerwas9 and Stephan Zipfel176 17Brain Center Rudolf Magnus, Department of Translational Neuroscience, University Medical Center Utrecht, Utrecht, the Netherlands. 18Center for Eating Disorders Rintveld, Altrecht Mental Health Institute, Zeist, the Netherlands. 19Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. 20Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 21Department of Behavioral Medicine, National Institute of Mental Health, National Center of Neurology and Psychiatry, Tokyo, Japan. 22NORMENT KG Jebsen Centre, Division of Mental Health and Addiction, University of Oslo, Oslo University Hospital, Oslo, Norway. 23BioRealm, LLC, Walnut, CA, USA. 24Oregon Research Institute, Eugene, OR, USA. 25Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. 26Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. 27Center for Psychiatry Research, Stockholm Health Care Services, Stockholm City Council, Stockholm, Sweden. 28Christchurch Health and Development Study, University of Otago, Christchurch, New Zealand. 29Division of Psychological and Social Medicine and Developmental Neurosciences, Faculty of Medicine, Technische Universität Dresden, Dresden, Germany. 30INSERM U894, Centre of Psychiatry and Neuroscience, Paris, France. 31Wellcome Sanger Institute, Hinxton, Cambridge, UK. 32Department of Medical Biology, School of Medicine, University of Split, Split, Croatia. 33The Center for Eating Disorders at Sheppard Pratt, Baltimore, MD, USA. 34Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, RWTH Aachen University, Aachen, Germany. 35Klinikum Frankfurt/Oder, Frankfurt, Germany. 36Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Padua, Italy. 37Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland. 38Life Sciences Institute and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA. 39Department of Emergency Psychiatry and Post-Acute Care, CHRU Montpellier, University of Montpellier, Montpellier, France. 40Department of Psychiatry, University of Minnesota, Minneapolis, MN, USA. 41MRC Integrative Epidemiology Unit, University of Bristol, Bristol, UK. 42School of Social and Community Medicine, University of Bristol, Bristol, UK. 43Department of Psychosomatic Medicine and Psychotherapy, Hannover Medical School, Hannover, Germany. 44Department of Nutrition and Dietetics, Harokopio University, Athens, Greece. 45Department of Neurosciences, University of Padova, Padua, Italy. 46College of Nursing, Seattle University, Seattle, WA, USA. 47Department of Psychology, Virginia Commonwealth University, Richmond, VA, USA. 48Department of Psychiatry, Athens University Medical School, Athens University, Athens, Greece. 49L’institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes, France. 50L’institut du thorax, CHU Nantes, Nantes, France. 51Department of Psychiatric Genetics, Poznań University of Medical Sciences, Poznań, Poland. 52Barcelona Institute of Science and Technology, Barcelona, Spain. 53Universitat Pompeu Fabra, Barcelona, Spain. 54Centro de Investigación Biomédica en Red en Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain. 55Department of Psychiatry and Behavioral Sciences, Stanford University Stanford, CA, USA. 56Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Würzburg, Centre for Mental Health, Würzburg, Germany. 57Estonian Genome Center, University of Tartu, Tartu, Estonia. 58Program in Medical and Population Genetics, Broad Institute of the Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA. 59Genomics and Disease, Bioinformatics and Genomics Programme, Centre for Genomic Regulation, Barcelona, Spain. 60Department of Psychiatry, University Hospital of Bellvitge –IDIBELL and CIBERobn, Barcelona, Spain. 61Department of Clinical Sciences, School of Medicine, University of Barcelona, Barcelona, Spain. 62Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University (LMU), Munich, Germany. 63Schön Klinik Roseneck affiliated with the Medical Faculty of the University of Munich (LMU), Munich, Germany. 64Department of Child and Adolescent Psychiatry, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. 65Department of Cancer, Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Brno, Czech Republic. 66Institute of Human Genetics, University of Bonn School of Medicine & University Hospital Bonn, Bonn, Germany. 67Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany. 68Department of Psychiatry (UPK), University of Basel, Basel, Switzerland. 69Department of Biomedicine, University of Basel, Basel, Switzerland. 70Department of Surgery, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. 71Department of Psychiatry, Psychotherapy and Psychosomatics, Martin Luther University of Halle-Wittenberg, Halle, Germany. 721st Psychiatric Department, National and Kapodistrian University of Athens, Medical School, Eginition Hospital, Athens, Greece. 73QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. 74CMME (Groupe Hospitalier Sainte-Anne), Paris Descartes University, Paris, France. 75Department of Biomedicine, Aarhus University, Aarhus, Denmark. 76The Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSyCH), Aarhus, Denmark. 77Centre for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, Denmark. 78Bioinformatics Research Centre, Aarhus University, Aarhus, Denmark. 79Center for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 80Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 81Department of Psychiatry, Weill Cornell Medical College, New york, Ny, USA. 82Department of Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London, UK. 83Department of Adult Psychiatry, Poznań University of Medical Sciences, Poznań, Poland. 84Zorg op Orde, Leidschendam, the Netherlands. 85Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia. 86Department of General Internal Medicine and Psychosomatics, Heidelberg University Hospital, Heidelberg University, Heidelberg, Germany. 87Department of Psychiatry, and Genetics and Genomics Sciences, Division of Psychiatric Genomics, Icahn School of Medicine at Mount Sinai, New york, Ny, USA. 88Biological Psychiatry Laboratory, McLean Hospital/Harvard Medical School, Boston, MA, USA. 89Eating Disorders Unit, Parklandklinik, Bad Wildungen, Germany. 90Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Isehara, Japan. 91Faculty of Health Sciences, Palacky University, Olomouc, Czech Republic. 92Eating Recovery Center, Denver, CO, USA. 93Department of Psychological Medicine, University of Otago, Christchurch, New Zealand. 94Canterbury District Health Board, Christchurch, New Zealand. 95Rheumatology Research Group, Vall d’Hebron Research Institute, Barcelona, Spain. 96Department of Psychiatry, First Faculty of Medicine, Charles University, Prague, Czech Republic. 97Center for Addiction and Mental Health, Department of Psychiatry, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. 98Department of Public Health, University of Helsinki, Helsinki, Finland. 99Institute for Molecular Medicine Finland, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland. 100Institute of Public Health and Clinical Nutrition, Department of Clinical Nutrition, University of Eastern Finland, Kuopio, Finland. 101Eating Disorders Unit, Department of Child and Adolescent Psychiatry, Medical University of Vienna, Vienna, Austria. 102Groningen Institute for Evolutionary Life Sciences, University of Groningen, Groningen, the Netherlands. 103Department of Psychiatry, University of California San Diego, San Diego, CA, USA. 104Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand. 105Health Services Research Unit, University of Aberdeen, Aberdeen, UK. 106Department of Psychiatry, Seoul Paik Hospital, Inje University, Seoul, Korea. 107Rheumatology Unit, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden. 108Department of Psychology, Michigan State University, East Lansing, MI, USA. 109Department of Mental Disorders, Norwegian Institute of Public Health, Oslo, Norway. 110National Centre for Register-Based Research, Aarhus BSS, Aarhus University, Aarhus, Denmark. 111Centre for Integrated Register-based Research (CIRRAU), Aarhus University, Aarhus, Denmark. 112Department of Clinical Science, K.G. Jebsen Centre for Psychosis Research, Norwegian Centre for Mental Disorders Research (NORMENT), University of Bergen, Bergen, Norway. 113Dr. Einar Martens Research Group for Biological Psychiatry, Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway. 114Department of Clinical Medicine, Laboratory Building, Haukeland University Hospital, Bergen, Norway. 115Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. 116American School of Professional Psychology, Argosy University, Northern Virginia, Arlington, VA, USA. 117Department of Cancer Epidemiology and Prevention, M Skłodowska-Curie Cancer Center - Oncology Center, Warsaw, Poland. 118BESE Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia. 119Department of Psychiatry, University of Lausanne-University Hospital of Lausanne (UNIL-CHUV), Lausanne, Switzerland. 120Department of Psychiatry, University of Campania ‘Luigi Vanvitelli’, Naples, Italy. 121Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland. 122Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. 123Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Würzburg, Germany. 124Department of Psychiatry, University College Cork, Cork, Ireland. 125Eist Linn Adolescent Unit, Bessborough, Health Service Executive South, Cork, Ireland. 126Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. 127Molecular Epidemiology Section (Department of Medical Statistics), Leiden University Medical Centre, Leiden, the Netherlands. 128Department of Psychiatry, Faculty of Medicine, University of Geneva, Geneva, Switzerland. 129Division of Child and Adolescent Psychiatry, Geneva University Hospital, Geneva, Switzerland. 130Department of Psychiatry and Behavioral Science, University of North Dakota School of Medicine and Health Sciences, Fargo, ND, USA. 131National Center for PTSD, VA Boston Healthcare System, Department of Psychiatry, Boston University School of Medicine, Boston, MA, USA. 132Department of Medicine, Surgery and Dentistry ‘Scuola Medica Salernitana’, University of Salerno, Salerno, Italy. 133Queensland Brain Institute, University of Queensland, Brisbane, Queensland, Australia. 134Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Florence, Italy. 135Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA, USA. 136Kartini Clinic, Portland, OR, USA. 137Center for Human Genome Research at the Massachusetts General Hospital, Boston, MA, USA. 138Biostatistics and Computational Biology Unit, University of Otago, Christchurch, New Zealand. 139Saint Joan de Déu Research Institute, Saint Joan de Déu Barcelona Children’s Hospital, Barcelona, Spain. 140Institute of Biomedicine (IBUB), University of Barcelona, Barcelona, Spain. 141Department of Genetics, Microbiology and Statistics, University of Barcelona, Barcelona, Spain. 142Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 143Department of Health Science, University of Florence, Florence, Italy. 144Department of Biometry, University of Helsinki, Helsinki, Finland. 145Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA. 146Stanley Center for Psychiatric Research, Broad Institute of the Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA. 147Department of Psychiatry and Psychotherapy, Charité - Universitätsmedizin, Berlin, Germany. 148Eating Disorders Research and Treatment Center, Department of Child and Adolescent Psychiatry, Faculty of Medicine, Technische Universität Dresden, Dresden, Germany. 149Department of Psychiatry, Neurobiology, Pharmacology, and Biotechnologies, University of Pisa, Pisa, Italy. 150Department of Psychiatry, Poznań University of Medical Sciences, Poznań, Poland. 151Department of Neurosciences, Padua Neuroscience Center, University of Padova, Padua, Italy. 152Institute of Medical Statistics, Computer and Data Sciences, Jena University Hospital, Jena, Germany. 153Department of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario, Canada. 154J. Craig Venter Institute (JCVI), La Jolla, CA, USA. 155Department of Psychiatry and Psychotherapy, Medical University of Vienna, Vienna, Austria. 156Department of Pediatrics and Center of Applied Genomics, First Faculty of Medicine, Charles University, Prague, Czech Republic. 157Center for Eating Disorders Ursula, Rivierduinen, Leiden, the Netherlands. 158Department of Psychiatry, Leiden University Medical Centre, Leiden, the Netherlands. 159Department of Child and Adolescent Psychiatry, Poznań University of Medical Sciences, Poznań, Poland. 160IRCCS Fondazione Don Carlo Gnocchi, Florence, Italy. 161Department of Psychiatry and Biobehavioral Science, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA, USA. 162David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. 163Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 164Department of Environmental Epidemiology, Nofer Institute of Occupational Medicine, Lodz, Poland. 165Department of Psychiatry, University of Naples SUN, Naples, Italy. 166Department of Psychiatry, University of Perugia, Perugia, Italy. 167Brain Sciences Department, Stremble Ventures, Limassol, Cyprus. 168Adolescent Health Unit, Second Department of Pediatrics, ‘P. & A. Kyriakou’ Children’s Hospital, University of Athens, Athens, Greece. 169Pediatric Intensive Care Unit, ‘P. & A. Kyriakou’ Children’s Hospital, University of Athens, Athens, Greece. 170Faculty of Social and Behavioral Sciences, Utrecht University, Utrecht, the Netherlands. 171School of Psychology, Flinders University, Adelaide, South Australia, Australia. 172Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark. 173Department of Psychiatry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. 174Toronto General Hospital, Toronto, Ontario, Canada. 175Institute of Translational Genomics, Helmholtz Zentrum München, Neuherberg, Germany. 176Department of Internal Medicine VI, Psychosomatic Medicine and Psychotherapy, University Medical Hospital Tübingen, Tübingen, Germany International Headache Genetics Consortium Verneri Anttila177, Ville Artto178, Andrea Carmine Belin179, Irene de Boer180, Dorret I. Boomsma181, Sigrid Børte182, Daniel I. Chasman183, Lynn Cherkas184, Anne Francke Christensen185, Bru Cormand186, Ester Cuenca-Leon177, George Davey-Smith187, Martin Dichgans188, Cornelia van Duijn189, Tonu Esko57, Ann Louise Esserlind190, Michel Ferrari180, Rune R. Frants180, Tobias Freilinger191, Nick Furlotte192, Padhraig Gormley177, Lyn Griffiths193, Eija Hamalainen194, Thomas Folkmann Hansen6, Marjo Hiekkala195, M. Arfan Ikram189, Andres Ingason196, Marjo-Riitta Järvelin197, Risto Kajanne194, Mikko Kallela178, Jaakko Kaprio98,99, Mari Kaunisto195, Lisette J. A. Kogelman6, Christian Kubisch198, Mitja Kurki177, Tobias Kurth199, Lenore Launer200, Terho Lehtimaki201, Davor Lessel198, Lannie Ligthart181, Nadia Litterman192, Arn van den Maagdenberg180, Alfons Macaya202, Rainer Malik188, Massimo Mangino184, George McMahon187, Bertram Muller-Myhsok203, Benjamin M. Neale177, Carrie Northover192, Dale R. Nyholt193, Jes Olesen190, Aarno Palotie58,99,137, Priit Palta194, Linda Pedersen182, Nancy Pedersen1, Danielle Posthuma181, Patricia Pozo-Rosich204, Alice Pressman205, Olli Raitakari206, Markus Schürks199, Celia Sintas186, Kari Stefansson196, Hreinn Stefansson196, Stacy Steinberg196, David Strachan207, Gisela Terwindt180, Marta Vila-Pueyo202, Maija Wessman195, Bendik S. Winsvold182, Huiying Zhao193 and John Anker Zwart182 177Broad Institute of MIT and Harvard, Cambridge, MA, USA. 178Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland. 179Karolinska Institutet, Stockholm, Sweden. 180Leiden University Medical Centre, Leiden, the Netherlands. 181VU University, Amsterdam, the Netherlands. 182Oslo University Hospital and University of Oslo, Oslo, Norway. 183Harvard Medical School, Cambridge, MA, USA. 184Department of Twin Research and Genetic Epidemiology, King’s College London, London, UK. 185Danish Headache Center, Copenhagen University Hospital, Copenhagen, Denmark. 186University of Barcelona, Barcelona, Spain. 187Medical Research Council (MRC) Integrative Epidemiology Unit, University of Bristol, Bristol, UK. 188Institute for Stroke and Dementia Research, Munich, Germany. 189Erasmus University Medical Centre, Rotterdam, the Netherlands. 190Danish Headache Center, Department of Neurology, Rigshospitalet, Glostrup, Denmark. 191University of Tübingen, Tübingen, Germany. 19223&Me Inc., Mountain View, CA, USA. 193Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. 194Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland. 195Folkhälsan Institute of Genetics, Helsinki, Finland. 196Decode genetics Inc., Reykjavik, Iceland. 197University of Oulu, Biocenter Oulu, Finland. 198University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 199Harvard Medical School, Boston, MA, USA. 200National Institute on Aging, Bethesda, MD, USA. 201School of Medicine, University of Tampere, Tampere, Finland. 202Vall d’Hebron Research Institute, Barcelona, Spain. 203Max Planck Institute of Psychiatry, Munich, Germany. 204Headache Research Group, Universitat Autònoma de Barcelona, Barcelona, Spain. 205Sutter Health, Sacramento, CA, USA. 206Department of Medicine, University of Turku, Turku, Finland. 207Population Health Research Institute, St George’s University of London, London, UK. 23andMe Research Team Michelle Agee208, Babak Alipanahi208, Adam Auton208, Robert Bell208, Katarzyna Bryc208, Sarah Elson208, Pierre Fontanillas208, Nicholas Furlotte208, Karl Heilbron208, David Hinds208, Karen Huber208, Aaron Kleinman208, Nadia Litterman208, Jennifer McCreight208, Matthew McIntyre208, Joanna Mountain208, Elizabeth Noblin208, Carrie Northover208, Steven Pitts208, J. Sathirapongsasuti208, Olga Sazonova208, Janie Shelton208, Suyash Shringarpure208, Chao Tian208, Joyce Tung208, Vladimir Vacic208 and Catherine Wilson208 20823andMe, Inc., Mountain View, CA, US

Gastrointestinal (GI) alpha-synuclein (ASN) detection may represent a clinically useful biomarker of Parkinson’s disease (PD), but this has been challenged by conflicting results of recent studies employing different immunohistochemical (IHC) methods and reporting diverse morphological patterns with variable biological interpretation. To increase sensitivity and specificity, we applied three different techniques to detect different possible conformations of ASN in GI tissue derived from biopsies of the GI tract, which were obtained from a longitudinally followed, clinically well-characterized cohort of PD subjects and healthy controls (HC) (Oxford Discovery study). With IHC, we used antibodies reactive for total (T-ASN-Abs), phosphorylated (P-ASN-Abs) and oligomeric (O-ASN-Abs) ASN; with the ASN Proximity Ligation Assay (AS-PLA), we targeted oligomeric ASN species specifically; finally, with the Paraffin-Embedded Tissue Blot (PET-Blot) we aimed to detect fibrillary conformations of ASN specifically. Optimisation and validation of the PET-Blot and PLA techniques was carried out with studies on brain tissue from subjects with ASN pathology, and these experiments were used to gain insight into morphology and distribution of different conformational variants of ASN in the brain of subjects with Lewy pathology. We specified all the detected morphological staining patterns with each technique interpreting them as pathologic or non-specific. Correlation to clinical symptoms was assessed to investigate the potential predictive or diagnostic value of specific staining patterns as biomarkers. A total of 163 GI tissue blocks were collected from 51 PD patients (113 blocks) and 21 healthy controls (50 blocks). In 31 PD patients, GI biopsies had been taken before PD diagnosis (Prodromal PD group); while in 20 PD patients biopsies were obtained after PD diagnosis (Manifest PD group). The majority of these tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four ASN staining patterns were detected in GI tissue (Neuritic, Ganglionic, Epithelial, and Cellular), while two distinct staining patterns were detected with AS-PLA (cellular and diffuse signal) and with AS-PET-Blot (ASN-localised and peri-crypt signal). The level of agreement between different techniques was generally low, and no single technique or staining pattern was able to reliably distinguish PD patients (Prodromal or Manifest) from HC. Overall, our study suggests that even specific detection of ASN conformational variants currently considered pathologic was not adequate for the prediction of PD. Future studies with these or other novel techniques focusing on the upper part of the GI tract could overcome current limitations in sensitivity and specificity.

The human gastrointestinal tract is inhabited by trillions of commensal bacteria collectively known as the gut microbiota. Our recognition of the significance of the complex interaction between the microbiota, and its host has grown dramatically over the past years. A balanced microbial community is a key regulator of the immune response, and metabolism of dietary components, which in turn, modulates several brain processes impacting mood and behavior. Consequently, it is likely that disruptions within the composition of the microbiota would remotely affect the mental state of the host. Here, we discuss how intestinal bacteria and their metabolites can orchestrate gut-associated neuroimmune mechanisms that influence mood and behavior leading to depression. In particular, we focus on microbiota-triggered gut inflammation and its implications in shifting the tryptophan metabolism towards kynurenine biosynthesis while disrupting the serotonergic signaling. We further investigate the gaps to be bridged in this exciting field of research in order to clarify our understanding of the multifaceted crosstalk in the microbiota-gut-brain interphase, bringing about novel, microbiota-targeted therapeutics for mental illnesses.

The human gastrointestinal tract is inhabited by trillions of commensal bacteria collectively known as the gut microbiota. Our recognition of the significance of the complex interaction between the microbiota, and its host has grown dramatically over the past years. A balanced microbial community is a key regulator of the immune response, and metabolism of dietary components, which in turn, modulates several brain processes impacting mood and behavior. Consequently, it is likely that disruptions within the composition of the microbiota would remotely affect the mental state of the host. Here, we discuss how intestinal bacteria and their metabolites can orchestrate gut-associated neuroimmune mechanisms that influence mood and behavior leading to depression. In particular, we focus on microbiota-triggered gut inflammation and its implications in shifting the tryptophan metabolism towards kynurenine biosynthesis while disrupting the serotonergic signaling. We further investigate the gaps to be bridged in this exciting field of research in order to clarify our understanding of the multifaceted crosstalk in the microbiota-gut-brain interphase, bringing about a novel microbiota-targeted therapeutics for mental illnesses.

γ-Aminobutyric Acid (GABA) and its receptors, GABAA,B,C, are expressed in several locations along the gastrointestinal tract. Nevertheless, a role for GABA in enteric synaptic transmission remains elusive. In this study, we characterized the expression and function of GABA in the myenteric plexus of the mouse ileum. About 8% of all myenteric neurons were found to be GABA-immunoreactive (GABA+) including some Calretinin+ and some neuronal nitric oxide synthase (nNOS+) neurons. We used Wnt1-Cre;R26R-GCaMP3 mice, which express a genetically encoded fluorescent calcium indicator in all enteric neurons and glia. Exogenous GABA increased the intracellular calcium concentration, [Ca2+]i of some myenteric neurons including many that did not express GABA or nNOS (the majority), some GABA+, Calretinin+ or Neurofilament-M (NFM)+ but rarely nNOS+ neurons. GABA+ terminals contacted a significantly larger proportion of the cell body surface area of Calretinin+ neurons than of nNOS+ neurons. Numbers of neurons with GABA-induced [Ca2+]i transients were reduced by GABAA,B,C and nicotinic receptor blockade. Electrical stimulation of interganglionic fiber tracts was used to examine possible effects of endogenous GABA release. [Ca2+]i transients evoked by single pulses were unaffected by specific antagonists for each of the 3 GABA receptor subtypes. [Ca2+]i transients evoked by 20 pulse trains were significantly amplified by GABAC receptor blockade. These data suggest that GABAA and GABAB receptors are not involved in synaptic transmission, but suggest a novel role for GABAC receptors in modulating slow synaptic transmission, as indicated by changes in [Ca2+]i transients, within the ENS. ispartof: FRONTIERS IN PHYSIOLOGY vol:9 issue:FEB ispartof: location:Switzerland status: published

Background Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1174-6) contains supplementary material, which is available to authorized users.

Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common 'low penetrant' variants in combination with rare or private 'high penetrant' variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development. (C) 2013 Elsevier Inc. All rights reserved.

Leptin ameliorates the prosecretory and prokinetic effects of the pro-inflammatory cytokine interleukin-6 on rat colon. Leptin also suppresses the neurostimulatory effects of irritable bowel syndrome plasma, which has elevated concentrations of interleukin-6, on enteric neurons. This may indicate a regulatory role for leptin in immune-mediated bowel dysfunction. In addition to its role in regulating energy homeostasis, the adipokine leptin modifies gastrointestinal (GI) function. Indeed, leptin-resistant obese humans and leptin-deficient obese mice exhibit altered GI motility. In the functional GI disorder irritable bowel syndrome (IBS), circulating leptin concentrations are reported to differ from those of healthy control subjects. Additionally, IBS patients display altered cytokine profiles, including elevated circulating concentrations of the pro-inflammatory cytokine interleukin-6 (IL-6), which bears structural homology and similarities in intracellular signalling to leptin. This study aimed to investigate interactions between leptin and IL-6 in colonic neurons and their possible contribution to IBS pathophysiology. The functional effects of leptin and IL-6 on colonic contractility and absorptosecretory function were assessed in organ baths and Ussing chambers in Sprague–Dawley rat colon. Calcium imaging and immunohistochemical techniques were used to investigate the neural regulation of GI function by these signalling molecules. Our findings provide a neuromodulatory role for leptin in submucosal neurons, where it inhibited the stimulatory effects of IL-6. Functionally, this translated to suppression of IL-6-evoked potentiation of veratridine-induced secretory currents. Leptin also attenuated IL-6-induced colonic contractions, although it had little direct effect on myenteric neurons. Calcium responses evoked by IBS plasma in both myenteric and submucosal neurons were also suppressed by leptin, possibly through interactions with IL-6, which is elevated in IBS plasma. As leptin has the capacity to ameliorate the neurostimulatory effects of soluble mediators in IBS plasma and modulated IL-6-evoked changes in bowel function, leptin may have a role in immune-mediated bowel dysfunction in IBS patients.

Embryonic neural crest cells travel in discrete streams to precise locations throughout the head and body. We previously showed that cranial neural crest cells respond chemotactically to vascular endothelial growth factor (VEGF) and that cells within the migratory front have distinct behaviors and gene expression. We proposed a cell-induced gradient model in which lead neural crest cells read out directional information from a chemoattractant profile and instruct trailers to follow. In this study, we show that migrating chick neural crest cells do not display distinct lead and trailer gene expression profiles in culture. However, exposure to VEGF in vitro results in the upregulation of a small subset of genes associated with an in vivo lead cell signature. Timed addition and removal of VEGF in culture reveals the changes in neural crest cell gene expression are rapid. A computational model incorporating an integrate-and-switch mechanism between cellular phenotypes predicts migration efficiency is influenced by the timescale of cell behavior switching. To test the model hypothesis that neural crest cellular phenotypes respond to changes in the VEGF chemoattractant profile, we presented ectopic sources of VEGF to the trailer neural crest cell subpopulation and show diverted cell trajectories and stream alterations consistent with model predictions. Gene profiling of trailer cells that diverted and encountered VEGF revealed upregulation of a subset of 'lead' genes. Injection of neuropilin1 (Np1)-Fc into the trailer subpopulation or electroporation of VEGF morpholino to reduce VEGF signaling failed to alter trailer neural crest cell trajectories, suggesting trailers do not require VEGF to maintain coordinated migration. These results indicate that VEGF is one of the signals that establishes lead cell identity and its chemoattractant profile is critical to neural crest cell migration.

The gut microbiome plays a crucial role in host physiology. Disruption of its community structure and function can have wide-ranging effects making it critical to understand exactly how the interactive dialogue between the host and its microbiota is regulated to maintain homeostasis. An array of multidirectional signalling molecules is clearly involved in the host-microbiome communication. This interactive signalling not only impacts the gastrointestinal tract, where the majority of microbiota resides, but also extends to affect other host systems including the brain and liver as well as the microbiome itself. Understanding the mechanistic principles of this inter-kingdom signalling is fundamental to unravelling how our supraorganism function to maintain wellbeing, subsequently opening up new avenues for microbiome manipulation to favour desirable mental health outcome.

Background Cellular changes associated with diabetic (DG) and idiopathic gastroparesis (IG) have recently been described from patients enrolled in the Gastroparesis Clinical Research Consortium. The association of these cellular changes with gastroparesis symptoms and gastric emptying is unknown. The aim of this study was to relate cellular changes to symptoms and gastric emptying in patients with gastroparesis. Methods Earlier, using full thickness gastric body biopsies from 20 DG, 20 IG, and 20 matched controls, we found decreased interstitial cells of Cajal (ICC) and enteric nerves and an increase in immune cells in both DG and IG. Here, demographic, symptoms [gastroparesis cardinal symptom index score (GCSI)], and gastric emptying were related to cellular alterations using Pearsons correlation coefficients. Key Results Interstitial cells of Cajal counts inversely correlated with 4 h gastric retention in DG but not in IG (r = -0.6, P = 0.008, DG, r = 0.2, P = 0.4, IG). There was also a significant correlation between loss of ICC and enteric nerves in DG but not in IG (r = 0.5, P = 0.03 for DG, r = 0.3, P = 0.16, IG). Idiopathic gastroparesis with a myenteric immune infiltrate scored higher on the average GCSI (3.6 +/- 0.7 vs 2.7 +/- 0.9, P = 0.05) and nausea score (3.8 +/- 0.9 vs 2.6 +/- 1.0, P = 0.02) as compared to those without an infiltrate. Conclusions & Inferences In DG, loss of ICC is associated with delayed gastric emptying. Interstitial cells of Cajal or enteric nerve loss did not correlate with symptom severity. Overall clinical severity and nausea in IG is associated with a myenteric immune infiltrate. Thus, full thickness gastric biopsies can help define specific cellular abnormalities in gastroparesis, some of which are associated with physiological and clinical characteristics of gastroparesis.

The gastro-intestinal tract is well known for its largest neural network outside the central nervous system and for the most extensive immune system in the body. Research in neurogastroenterology implicates the involvement of both enteric nervous system and immune system in symptoms of inflammatory bowel disease and irritable bowel syndrome. Since both disorders are associated with increased immune cell numbers, nerve growth and activation of both immune cells and nerves, we focus in this review on the involvement of immune cell-nerve interactions in inflammatory bowel disease and irritable bowel syndrome. Firstly, the possible effects of enteric nerves, especially of the nonadrenergic and noncholinergic nerves, on the intestinal immune system and their possible role in the pathogenesis of chronic intestinal inflammatory diseases are described. Secondly, the possible effects of immunological factors, from the innate (chemokines and Toll-like receptors) as well as the adaptive (cytokines and immunoglobulins) immune system, on gastro-intestinal nerves and its potential role in the development of inflammatory bowel disease and irritable bowel syndrome are reviewed. investigations of receptor-mediated and intracellular signal pathways in neuro-immune interactions might help to develop more effective therapeutic approaches for chronic inflammatory intestinal diseases.

Hirschsprung disease ( HSCR, aganglionic megacolon) represents the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births. This developmental disorder is a neurocristopathy and is characterised by the absence of the enteric ganglia along a variable length of the intestine. In the last decades, the development of surgical approaches has importantly decreased mortality and morbidity which allowed the emergence of familial cases. Isolated HSCR appears to be a non-Mendelian malformation with low, sex-dependent penetrance, and variable expression according to the length of the aganglionic segment. While all Mendelian modes of inheritance have been described in syndromic HSCR, isolated HSCR stands as a model for genetic disorders with complex patterns of inheritance. The tyrosine kinase receptor RET is the major gene with both rare coding sequence mutations and/or a frequent variant located in an enhancer element predisposing to the disease. Hitherto, 10 genes and five loci have been found to be involved in HSCR development.

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.

Background and aims: Achalasia is a disease of unknown aetiology. An immune mechanism has been suggested on the basis of previous morphological observations. The objective of this study was to test whether the serum of achalasia patients could reproduce the phenotype and functional changes that occur with disease progression in an ex vivo human model.Methods: Specimens of normal human fundus were maintained in culture in the presence of serum from patients with achalasia, gastro-oesophageal reflux disease (GORD), or healthy subjects (controls). Immunohistochemical detection of choline acetyltransferase (ChAT), neurone specific enolase (NSE), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), and substance P was carried out in whole mounts of gastric fundus myenteric plexus. In addition, the effects of achalasia serum on electrical field stimulation (EFS) induced contractions were measured in circular muscle preparations.Results: Serum from achalasia patients did not affect the number of myenteric neurones. Tissues incubated with serum from achalasia patients showed a decrease in the proportion of NOS (-26% of NSE positive neurones; p = 0.016) and VIP (-54%; p = 0.09) neurones, and a concomitant increase in ChAT neurones (+ 16%; p Conclusions: Serum from achalasia patients can induce phenotypic and functional changes which reproduce the characteristics of the disease. Further identification of putative seric factors and mechanisms involved could lead to the development of novel diagnostic and/or therapeutic strategies in achalasia.

The receptor tyrosine kinase RET is expressed in cell lineages derived from the neural crest and has a key role in regulating cell proliferation, migration, differentiation and survival during embryogenesis. Germline and somatic mutations in RET that produce constitutively activated receptors cause the cancer syndrome multiple endocrine neoplasia type 2 and several endocrine and neural-crest-derived tumors, whereas mutations resulting in nonfunctional RET or lower expression of RET are found in individuals affected with Hirschsprung disease. This review focuses on the genetics and molecular mechanisms underlying the different inherited human neural-crest-related disorders in which RET dysfunction has a crucial role and discusses RET as a potential therapeutic target.

The MEN2A oncogene encodes for a constitutive active member of the RET receptor tyrosine kinase family. Here, we report that MEN2A-RET activates Signal Transducer and Activator of Transcription 3 (STAT3) via two YxxV/Q STAT3 docking sites, Tyr752 and Tyr928. MEN2A-RET induces both Tyr705 and Ser727 phosphorylation of STAT3, and STAT3 serine phosphorylation is required for its maximal transcriptional activity. Stable NIH3T3 cell lines expressing both MEN2A-RET and STAT3 alpha but not STAT3 beta, are characterized by enhanced proliferation and cyclin-D1 promoter activity, and enhanced growth in soft agar. These data indicate that malignant cell growth induced by MEN2A-RET involves its activation of STAT3.

This article appeared in a journal published by Wiley-Blackwell. Under Wiley's copyright, mandated authors are not permitted to make work available in an institutional repository., Parkinson's disease is a neurodegenerative disorder characterized by motor and nonmotor impairments, including constipation. The hallmark pathological features of Parkinson's disease are Lewy bodies and neurites, of which aggregated α-synuclein is a major constituent. Frequently, Lewy pathology is identified in the distal gut of constipated Parkinson's disease patients. The neurons that innervate the distal gut that express α-synuclein have not been identified. We used multiple-labeling immunohistochemistry and anterograde tracing to quantify which neurons projecting to the guinea pig rectum and human colon expressed α-synuclein in their axons. α-Synuclein-immunoreactivity was present in 24 ± 0.7% of somatostatin (SOM)-immunoreactive (IR) varicosities; 20 ± 4.3% of substance P (SP)-IR varicosities and 9 ± 1.3% vasoactive intestinal polypeptide (VIP)-IR varicosities in guinea pig rectal myenteric ganglia. However, α-synuclein-immunoreactivity was localized in significantly more vesicular acetylcholine transporter (VAChT)-IR varicosities (88 ± 3%, P < 0.001). Of SOM-IR, SP-IR, and VIP-IR varicosities that lacked VAChT-immunoreactivity, only 1 ± 0.3%, 0 ± 0.3%, and 0% contained α-synuclein-immunoreactivity, respectively. 71 ± 0.8% of VAChT-IR varicosities in myenteric ganglia of human colon were α-synuclein-IR. In guinea pig rectal myenteric ganglia, α-synuclein- and VAChT-immunoreactivity coexisted in 15 ± 1.4% of biotinamide-labeled extrinsic varicosities; only 1 ± 0.3% of biotinamide-labeled extrinsic varicosities contained α-synuclein-immunoreactivity without VAChT-immunoreactivity. α-Synuclein expression in axons to the distal gut correlates closely with expression of the cholinergic marker, VAChT. This is the first report of cell-selective α-synuclein expression in the nervous system. Our results suggest cholinergic neurons in the gut may be vulnerable in Parkinson's disease., Australian National Health & Medical Research Council

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon

Enteric glial cells (EGC) actively mediate acute and chronic inflammation in the gut; EGC proliferate and release neurotrophins, growth factors, and pro-inflammatory cytokines which, in turn, may amplify the immune response, representing a very important link between the nervous and immune systems in the intestine. Cannabidiol (CBD) is an interesting compound because of its ability to control reactive gliosis in the CNS, without any unwanted psychotropic effects. Therefore the rationale of our study was to investigate the effect of CBD on intestinal biopsies from patients with ulcerative colitis (UC) and from intestinal segments of mice with LPS-induced intestinal inflammation. CBD markedly counteracted reactive enteric gliosis in LPS-mice trough the massive reduction of astroglial signalling neurotrophin S100B. Histological, biochemical and immunohistochemical data demonstrated that S100B decrease was associated with a considerable decrease in mast cell and macrophages in the intestine of LPS-treated mice after CBD treatment. Moreover the treatment of LPS-mice with CBD reduced TNF-α expression and the presence of cleaved caspase-3. Similar results were obtained in ex vivo cultured human derived colonic biopsies. In biopsies of UC patients, both during active inflammation and in remission stimulated with LPS+INF-γ, an increased glial cell activation and intestinal damage were evidenced. CBD reduced the expression of S100B and iNOS proteins in the human biopsies confirming its well documented effect in septic mice. The activity of CBD is, at least partly, mediated via the selective PPAR-gamma receptor pathway. CBD targets enteric reactive gliosis, counteracts the inflammatory environment induced by LPS in mice and in human colonic cultures derived from UC patients. These actions lead to a reduction of intestinal damage mediated by PPARgamma receptor pathway. Our results therefore indicate that CBD indeed unravels a new therapeutic strategy to treat inflammatory bowel diseases. ispartof: PLoS One vol:6 issue:12 ispartof: location:United States status: published

To establish bovine spongiform encephalopathy (BSE) public health protection measures it is important to precisely define the cattle tissues considered as specified risk materials (SRM). To date, in pre-clinical BSE infected cattle, no evidence of the BSE agent had been found in the gut outside of the ileal Peyer's Patches. This study was undertaken to determine when and where the pathological prion protein (PrPSc) and/or BSE infectivity can be found in the small intestine of cattle 4 to 6 months of age, orally challenged with BSE. Samples of the jejunum, the ileum and the ileocaecal junction from 46 BSE infected cattle, culled from 1 up to 44 months post infection (mpi) were examined by immunohistochemistry. Samples from cattle 8 mpi to 20 mpi were additionally studied by PTA Western blot, rapid tests, and by mouse (TgbovXV) bioassay. In doing so nearly all of the cattle, from 4 up to 44 mpi, had detectable amounts of PrPSc and/or infectivity in the distal ileum. In the distal ileum clear time-dependent variations were visible concerning the amount of PrPSc, the tissue structures affected, and the cells involved. BSE infectivity was found not only in the ileum and ileocaecal junction but also in the jejunum. The systematic approach of this study provides new data for qualitative and quantitative risk assessments and allows defining bovine SRM more precisely.

Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed immunocytochemistry to locate the 5-HT3A as well as the 5-HT3C, 5-HT3D, and 5-HT3E subunits within the human colon. Western blot analysis was used to confirm subunit expression, and RT-PCR was employed to detect transcripts encoding 5-HT3 receptor subunits in microdissected tissue samples. This investigation revealed, for the first time, that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons. Furthermore, 5-HT3A and 5-HT3D were found to be expressed in submucosal plexus of the human large intestine. These data provide a strong basis for future studies of the roles that specific 5-HT3 receptor subtypes play in the function of the enteric and central nervous systems and the contribution that specific 5-HT3 receptors make to the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome and dyspepsia. J. Comp. Neurol. 519:420-432, 2011. (C) 2010 Wiley-Liss, Inc.

Hirschsprung disease ( HSCR, aganglionic megacolon) represents the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births. This developmental disorder is a neurocristopathy and is characterised by the absence of the enteric ganglia along a variable length of the intestine. In the last decades, the development of surgical approaches has importantly decreased mortality and morbidity which allowed the emergence of familial cases. Isolated HSCR appears to be a non-Mendelian malformation with low, sex-dependent penetrance, and variable expression according to the length of the aganglionic segment. While all Mendelian modes of inheritance have been described in syndromic HSCR, isolated HSCR stands as a model for genetic disorders with complex patterns of inheritance. The tyrosine kinase receptor RET is the major gene with both rare coding sequence mutations and/or a frequent variant located in an enhancer element predisposing to the disease. Hitherto, 10 genes and five loci have been found to be involved in HSCR development.

The MEN2A oncogene encodes for a constitutive active member of the RET receptor tyrosine kinase family. Here, we report that MEN2A-RET activates Signal Transducer and Activator of Transcription 3 (STAT3) via two YxxV/Q STAT3 docking sites, Tyr752 and Tyr928. MEN2A-RET induces both Tyr705 and Ser727 phosphorylation of STAT3, and STAT3 serine phosphorylation is required for its maximal transcriptional activity. Stable NIH3T3 cell lines expressing both MEN2A-RET and STAT3 alpha but not STAT3 beta, are characterized by enhanced proliferation and cyclin-D1 promoter activity, and enhanced growth in soft agar. These data indicate that malignant cell growth induced by MEN2A-RET involves its activation of STAT3.

A reduction of gallbladder emptying in response to neural or hormonal stimulation has been reported in patients with diabetes mellitus. Decreased gallbladder emptying may be a key factor in the pathogenesis of gallbladder stones. Few drugs, if any, are able to stimulate gallbladder emptying. However, in a previous study we demonstrated that erythromycin, a macrolide antibiotic, stimulates gallbladder emptying and motilin release in healthy human subjects by an atropine-sensitive pathway. Therefore, the present study was designed to evaluate the effect of erythromycin on gallbladder emptying and motilin release in diabetic patients with or without cardiac autonomic neuropathy (AN). Thirteen diabetic patients, six with AN, and 10 healthy subjects were enrolled in the study protocol. Gallbladder emptying was determined by sonography after ingestion of a standard meal and during infusion of erythromycin alone or together with 6 μg/kg/hr atropine. We found that 100 mg/hr erythromycin caused a significant reduction in gallbladder volume in both healthy subjects and diabetic patients. The ejection fraction (mean ±se) of 45.3±8.2% and 37.3±5.0% was similar. The presence of AN had no influence on gallbladder emptying induced by erythromycin. Basal motilin plasma levels were 111.5±14.5 pmol/liter in diabetic patients and 63.3 ±6.0 pmol/liter in healthy subjects (P

Background Cellular changes associated with diabetic (DG) and idiopathic gastroparesis (IG) have recently been described from patients enrolled in the Gastroparesis Clinical Research Consortium. The association of these cellular changes with gastroparesis symptoms and gastric emptying is unknown. The aim of this study was to relate cellular changes to symptoms and gastric emptying in patients with gastroparesis. Methods Earlier, using full thickness gastric body biopsies from 20 DG, 20 IG, and 20 matched controls, we found decreased interstitial cells of Cajal (ICC) and enteric nerves and an increase in immune cells in both DG and IG. Here, demographic, symptoms [gastroparesis cardinal symptom index score (GCSI)], and gastric emptying were related to cellular alterations using Pearsons correlation coefficients. Key Results Interstitial cells of Cajal counts inversely correlated with 4 h gastric retention in DG but not in IG (r = -0.6, P = 0.008, DG, r = 0.2, P = 0.4, IG). There was also a significant correlation between loss of ICC and enteric nerves in DG but not in IG (r = 0.5, P = 0.03 for DG, r = 0.3, P = 0.16, IG). Idiopathic gastroparesis with a myenteric immune infiltrate scored higher on the average GCSI (3.6 +/- 0.7 vs 2.7 +/- 0.9, P = 0.05) and nausea score (3.8 +/- 0.9 vs 2.6 +/- 1.0, P = 0.02) as compared to those without an infiltrate. Conclusions & Inferences In DG, loss of ICC is associated with delayed gastric emptying. Interstitial cells of Cajal or enteric nerve loss did not correlate with symptom severity. Overall clinical severity and nausea in IG is associated with a myenteric immune infiltrate. Thus, full thickness gastric biopsies can help define specific cellular abnormalities in gastroparesis, some of which are associated with physiological and clinical characteristics of gastroparesis.