RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis.

Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Piezo1 axis as a potential prophylactic target for treatment of bone and gut disorders.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

The 5-HT3 Receptor Affects Rotavirus-Induced Motility.

Rotavirus infection is highly prevalent in children, and the most severe effects are diarrhea and vomiting. It is well accepted that the enteric nervous system (ENS) is activated and plays an important role, but knowledge of how rotavirus activates nerves within ENS and to the vomiting center is lacking. Serotonin is released during rotavirus infection, and antagonists to the serotonin receptor subtype 3 (5-HT3 receptor) can attenuate rotavirus-induced diarrhea. In this study, we used a 5-HT3 receptor knockout (KO) mouse model to investigate the role of this receptor in rotavirus-induced diarrhea, motility, electrolyte secretion, inflammatory response, and vomiting reflex. The number of diarrhea days (P = 0.03) and the number of mice with diarrhea were lower in infected 5-HT3 receptor KO than wild-type pups. In vivo investigation of fluorescein isothiocyanate (FITC)-dextran transit time showed that intestinal motility was lower in the infected 5-HT3 receptor KO compared to wild-type mice (P = 0.0023). Ex vivo Ussing chamber measurements of potential difference across the intestinal epithelia showed no significant difference in electrolyte secretion between the two groups. Immediate early gene cFos expression level showed no difference in activation of the vomiting center in the brain. Cytokine analysis of the intestine indicated a low effect of inflammatory response in rotavirus-infected mice lacking the 5-HT3 receptor. Our findings indicate that the 5-HT3 receptor is involved in rotavirus-induced diarrhea via its effect on intestinal motility and that the vagus nerve signaling to the vomiting center occurs also in the absence of the 5-HT3 receptor. IMPORTANCE The mechanisms underlying rotavirus-induced diarrhea and vomiting are not yet fully understood. To better understand rotavirus pathophysiology, characterization of nerve signaling within the ENS and through vagal efferent nerves to the brain, which have been shown to be of great importance to the disease, is necessary. Serotonin (5-HT), a mediator of both diarrhea and vomiting, has been shown to be released from enterochromaffin cells in response to rotavirus infection and the rotavirus enterotoxin NSP4. Here, we investigated the role of the serotonin receptor 5-HT3, which is known to be involved in the nerve signals that regulate gut motility, intestinal secretion, and signal transduction through the vagus nerve to the brain. We show that the 5-HT3 receptor is involved in rotavirus-induced diarrhea by promoting intestinal motility. The findings shed light on new treatment possibilities for rotavirus diarrhea.

Biallelic variants in LIG3 cause a novel mitochondrial neurogastrointestinal encephalomyopathy.

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.

Clinical and Pathological Features of Severe Gut Dysmotility.

Severe gut motility disorders are characterized by ineffective propulsion of intestinal contents. As a result, patients often develop extremely uncomfortable symptoms, ranging from nausea and vomiting along with alterations of bowel habits, up to radiologically confirmed subobstructive episodes. Chronic intestinal pseudo-obstruction (CIPO) is a typical clinical phenotype of severe gut dysmotility due to morphological and functional alterations of the intrinsic (enteric) innervation and extrinsic nerve supply (hence neuropathy), interstitial cells of Cajal (ICCs) (mesenchymopathy), and smooth muscle cells (myopathy). In this chapter, we highlight some molecular mechanisms of CIPO and review the clinical phenotypes and the genetics of the different types of CIPO. Specifically, we will detail the role of some of the most representative genetic mutations involving RAD21, LIG3, and ACTG2 to provide a better understanding of CIPO and related underlying neuropathic or myopathic histopathological abnormalities. This knowledge may unveil targeted strategies to better manage patients with such severe disease.

Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility in mouse ileum.

Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca2+/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca2+ requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOSS1412A knock-in mouse and evaluated its enteric neurotransmission and gastrointestinal (GI) motility to understand the physiologic significance of nNOS S1412 phosphorylation. Electrical field stimulation (EFS) of wild-type (WT) mouse ileum induced nNOS S1412 phosphorylation that was blocked by tetrodotoxin and by inhibitors of the protein kinase Akt but not by PKA inhibitors. Low-frequency depolarization increased nNOS S1412 phosphorylation and relaxed WT ileum but only partially relaxed nNOSS1412A ileum. At higher frequencies, nNOS S1412 had no effect. nNOSS1412A ileum expressed less phosphodiesterase-5 and was more sensitive to relaxation by exogenous NO. Under non-NANC conditions, peristalsis and segmentation were faster in the nNOSS1412A ileum. Together these findings show that neuronal depolarization stimulates enteric nNOS phosphorylation by Akt to promote normal GI motility. Thus, phosphorylation of nNOS S1412 is a significant regulatory mechanism for nitrergic neurotransmission in the gut.

Moxibustion Regulates Gastrointestinal Motility via HCN1 in Functional Dyspepsia Rats.

BACKGROUND Moxibustion therapy has been found to ameliorate clinical symptoms of functional dyspepsia (FD). We aimed to examine the regulatory effect of moxibustion on the gastrointestinal (GI) motility in FD and explore the underlying mechanism based on the hyperpolarization-activated cyclic nucleotide-gated cation channel 1 (HCN1). MATERIAL AND METHODS Moxibustion therapy was used in FD rats induced by using classic tail-pinch and irregular feeding. Weight gain and food intake were recorded weekly, followed by detecting gastric residual rate (GRR) and small intestine propulsion rate (IPR). Next, western blotting was performed to determine the expression levels of HCN1 in the gastric antrum. qRT-PCR was used to detect HCN1 in the small intestine and hypothalamic satiety center. Double immunolabeling was used for HCN1 and ICCs in gastric antrum and small intestine. RESULTS The obtained results suggested that moxibustion treatment could increase weight gain and food intake in FD rats. The GRR and IPR were compared among the groups, which showed that moxibustion treatment could decrease GRR and increase IPR. Moxibustion increased the expression of HCN1 in the gastric antrum, small intestine, and hypothalamic satiety center. Histologically, the co-expressions of HCN1 and ICCs tended to increase in gastric antrum and small intestine. Meanwhile, HCN channel inhibitor ZD7288 prevented the above-mentioned therapeutic effects of moxibustion. CONCLUSIONS The results of the present study suggest that moxibustion can effectively improve the GI motility of FD rats, which may be related to the upregulation of HCN1 expression in gastric antrum, small intestine, and satiety center.

Angiotensin-Converting Enzyme Gene Polymorphism Influences Gastrointestinal Motility in Type 2 Diabetes Mellitus.

Uncontrolled diabetes mellitus may affect any part of the gastrointestinal tract (GIT) and impact negatively the quality of life. Angiotensin-converting enzyme (ACE) gene polymorphism can have direct effect on circulating level of ACE which further modifies the degradation of substance P and thus may influence the gut motility. Hence, it could be hypothesised that ACE gene polymorphism would influence the gut motility. An observational analytical study was conducted at PGIMER, Chandigarh. 300 Type2 diabetes mellitus (T2DM) and 200 age and sex matched healthy individuals were enrolled. After taking written consent, 5 ml blood sample was collected for measurement of substance P by ELISA method and for ACE gene polymorphism (insertion[I]/deletion[D]) by polymerase chain reaction. Orocecal transit time (OCTT) was measured using non-invasive lactulose breath test. Out of 300 diabetic patients, 32.7%, 44% and 23.3% belonged to II, ID and DD genotypes, respectively. The frequency of D allele (OR = 1.39) and DD genotype (OR = 2.17) was significantly higher in patients than in controls and was associated with increased risk. Moreover, more number of diabetes patients with constipation (90%) belonged to DD genotype and their OCTT was significantly delayed (166.7 ± 7.3 min) as compared to ID (143.5 ± 4.2 min) or II (121.8 ± 4.9 min) genotype. From this study, it could be concluded that ACE gene polymorphism could be an important contributing factor to influence the gut motility and thus giving rise to the GI symptoms for T2DM patients.

microRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility.

OBJECTIVE: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). DESIGN: All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. RESULTS: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. CONCLUSIONS: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Enteric Murine Ganglionitis Induced by Autoimmune CD8 T Cells Mimics Human Gastrointestinal Dysmotility.

Inflammatory bowel diseases frequently cause gastrointestinal dysmotility, suggesting that they may also affect the enteric nervous system. So far, the precise mechanisms that lead to gastrointestinal dysmotility in inflammatory bowel diseases have not been elucidated. To determine the effect of CD8 T cells on gastrointestinal motility, transgenic mice expressing ovalbumin on enteric neurons were generated. In these mice, adoptive transfer of ovalbumin-specific OT-I CD8 T cells induced severe enteric ganglionitis. CD8 T cells homed to submucosal and myenteric plexus neurons, 60% of which were lost, clinically resulting in severely impaired gastrointestinal transition. Anti-interferon-γ treatment rescued neurons by preventing their up-regulation of major histocompatibility complex class I antigen, thus preserving gut motility. These preclinical murine data translated well into human gastrointestinal dysmotility. In a series of 30 colonic biopsy specimens from patients with gastrointestinal dysmotility, CD8 T cell-mediated ganglionitis was detected that was followed by severe loss of enteric neurons (74.8%). Together, the preclinical and clinical data support the concept that autoimmune CD8 T cells play an important pathogenetic role in gastrointestinal dysmotility and may destroy enteric neurons.

Molecular Characterization of Constipation Disease as Novel Phenotypes in CRISPR-Cas9-Generated Leptin Knockout Mice with Obesity.

(1) Background: We characterized a novel animal model with obesity-induced constipation because constipation is rarely known in genetically engineered mice (GEM); (2) Methods: The changes in the constipation parameters and mechanisms were analyzed in CRISPR-Cas9-mediated leptin (Lep) knockout (KO) mice from eight to 24 weeks; (3) Results: Significant constipation phenotypes were observed in the Lep KO mice since 16 weeks old. These mice showed a significant decrease in the gastrointestinal motility, mucosal layer thickness and ability for mucin secretion as well as the abnormal ultrastructure of Lieberkühn crypts in the transverse colon. The density or function of the enteric neurons, intestinal Cajal cells (ICC), smooth muscle cells, and the concentration of gastrointestinal (GI) hormones for the GI motility were remarkably changed in Lep KO mice. The downstream signaling pathway of muscarinic acetylcholine receptors (mAChRs) were activated in Lep KO mice, while the expression of adipogenesis-regulating genes were alternatively reduced in the transverse colon of the same mice; (4) Conclusions: These results provide the first strong evidence that Lep KO mice can represent constipation successfully through dysregulation of the GI motility mediated by myenteric neurons, ICC, and smooth muscle cells in the transverse colon during an abnormal function of the lipid metabolism.

Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Acetylcholine (ACh)-synthesizing neurons are major components of the enteric nervous system (ENS). They release ACh and peptidergic neurotransmitters onto enteric neurons and muscle. However, pharmacological interrogation has proven inadequate to demonstrate an essential role for ACh. Our objective was to determine whether elimination of ACh synthesis during embryogenesis alters prenatal viability, intestinal function, the neurotransmitter complement, and the microbiome. Conditional deletion of choline acetyltransferase ( ChAT), the ACh synthetic enzyme, in neural crest-derived neurons ( ChAT-Null) was performed. Survival, ChAT activity, gut motility, and the microbiome were studied. ChAT was conditionally deleted in ENS neural crest-derived cells. Despite ChAT absence, mice were born live and survived the first 2 wk. They failed to gain significant weight in the third postnatal week, dying between postnatal d 18 and 30. Small intestinal transit of carmine red was 50% slower in ChAT-Nulls vs. WT and ChAT- Het. The colons of many neonatal ChAT-Null mice contained compacted feces, suggesting dysmotility. Microbiome analysis revealed dysbiosis in ChAT-Null mice. Developmental deletion of ChAT activity in enteric neurons results in proximal gastrointestinal tract dysmotility, critically diminished colonic transit, failure to thrive, intestinal dysbiosis, and death. ACh is necessary for sustained gut motility and survival of neonatal mice after weaning.-Johnson, C. D., Barlow-Anacker, A. J., Pierre, J. F., Touw, K., Erickson, C. S., Furness, J. B., Epstein, M. L., Gosain, A. Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

A novel 86-bp indel of the motilin receptor gene is significantly associated with growth and carcass traits in Gushi-Anka F2 reciprocal cross chickens.

1. A previous whole-genome association analysis has identified the motilin receptor gene (MLNR), which regulates gastrointestinal motility and gastric emptying, as a candidate gene related to chicken growth.2. MLNR mRNA was expressed in all tissues tested, and the expression level in digestive tissues was greater than in other tissues. Expression levels in the pancreas, duodenum and glandular stomach at day old and one, two and three weeks of age indicated a possible correlation with the digestive system. This suggested that the MLNR gene plays a central role in gastrointestinal tract function and affects the growth and development of chickens. Moreover, there was a significant difference in expression in the glandular stomach tissue between Ross 308 and Gushi chickens at six weeks of age.3. Re-sequencing revealed an 86-bp insertion/deletion polymorphism in the downstream region of the MLNR gene. The mutation locus was genotyped in 2,261 individuals from nine different chicken breeds. MLNR expression levels in the glandular stomach of chickens with DD genotypes were greater than those in chickens with the ID and II genotypes. The DD genotype was the most dominant genotype in commercial broiler's (Ross 308 and Arbor Acres broilers), and the D allele frequency in these breeds exceeded 91%. The deletion mutation tended towards fixation in commercial broilers.4. Association with growth and carcass traits analysed in a Gushi-Anka F2 intercrossed population, showed that the DD genotype was significantly associated with the greatest growth and carcass trait values, whereas values associated with the II genotype were the lowest in the F2 reciprocal cross chickens.5. The results suggest that the mutation is strongly associated with growth related traits and it is likely to be useful for marker-assisted selection of chickens.

Metal Transporter Zip14 (Slc39a14) Deletion in Mice Increases Manganese Deposition and Produces Neurotoxic Signatures and Diminished Motor Activity.

Mutations in human ZIP14 have been linked to symptoms of the early onset of Parkinsonism and Dystonia. This phenotype is likely related to excess manganese accumulation in the CNS. The metal transporter ZIP14 (SLC39A14) is viewed primarily as a zinc transporter that is inducible via proinflammatory stimuli. In vitro evidence shows that ZIP14 can also transport manganese. To examine a role for ZIP14 in manganese homeostasis, we used Zip14 knock-out (KO) male and female mice to conduct comparative metabolic, imaging, and functional studies. Manganese accumulation was fourfold to fivefold higher in brains of Zip14 KO mice compared with young adult wild-type mice. There was less accumulation of subcutaneously administered 54Mn in the liver, gallbladder, and gastrointestinal tract of the KO mice, suggesting that manganese elimination is impaired with Zip14 ablation. Impaired elimination creates the opportunity for atypical manganese accumulation in tissues, including the brain. The intensity of MR images from brains of the Zip14 KO mice is indicative of major manganese accumulation. In agreement with excessive manganese accumulation was the impaired motor function observed in the Zip14 KO mice. These results also demonstrate that ZIP14 is not essential for manganese uptake by the brain. Nevertheless, the upregulation of signatures of brain injury observed in the Zip14 KO mice demonstrates that normal ZIP14 function is an essential factor required to prevent manganese-linked neurodegeneration.SIGNIFICANCE STATEMENT Manganese is an essential micronutrient. When acquired in excess, manganese accumulates in tissues of the CNS and is associated with neurodegenerative disease, particularly Parkinson-like syndrome and dystonia. Some members of the ZIP metal transporter family transport manganese. Using mutant mice deficient in the ZIP14 metal transporter, we have discovered that ZIP14 is essential for manganese elimination via the gastrointestinal tract, and a lack of ZIP14 results in manganese accumulation in critical tissues such as the brain, as measured by MRI, and produces signatures of brain injury and impaired motor function. Humans with altered ZIP14 function would lack this gatekeeper function of ZIP14 and therefore would be prone to manganese-related neurological diseases.

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

BACKGROUND & AIMS: The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. METHODS: We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (GsaSMKO and SM22-CreERT2, induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. RESULTS: Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. CONCLUSIONS: Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.

Genetic Polymorphisms Associated With Constipation and Anticholinergic Symptoms in Patients Receiving Clozapine.

BACKGROUND: Clozapine impairs gastrointestinal motility owing to its anticholinergic and antiserotonergic properties. This commonly leads to constipation and potentially to more severe complications such as bowel obstruction and ischemia. The aim of this study was to determine whether genetic variations in the genes encoding muscarinic and serotonergic receptors (CHRM2, CHRM3, HTR2, HTR3, HTR4, and HTR7) explain the variations in incidence of constipation and anticholinergic symptoms during clozapine treatment. Genes associated with opiate-induced constipation were also included in this analysis (TPH1, OPRM1, ABCB1, and COMT). PROCEDURES: Blood samples from 176 clozapine-treated, Finnish, white patients with schizophrenia were genotyped. Constipation and anticholinergic symptoms were rated using the Liverpool University Neuroleptic Side Effect Rating Scale self-report questionnaire. In total, 192 single-nucleotide polymorphisms (SNPs) were detected and grouped to formulate a weighted genetic-risk score (GRS). RESULTS: No significant associations between individual SNPs or GRSs and constipation or laxative use were observed. A GRS of 19 SNPs in CHRM2, CHRM3, HTR3C, HTR7, ABCB1, OPRM1, and TPH1 was associated with anticholinergic symptoms in a generalized linear univariate model, with body mass index, clozapine monotherapy, and GRS as explaining variables (permuted P = 0.014). Generalized linear univariate model analysis performed on the opiate-induced constipation-associated SNPs and a single CHRM3 SNP revealed an association between anticholinergic symptoms and a score of 8 SNPs (adjusted P = 0.038, permuted P = 0.002). CONCLUSIONS: Two GRSs are able to predict the risk of anticholinergic symptoms in patients receiving clozapine and possibly an increased risk of gastrointestinal hypomotility.

Anti-inflammatory Effects of Enhanced Recovery Programs on Early-Stage Colorectal Cancer Surgery.

BACKGROUND: Postoperative ileus (POI) is observed in 20-30% of patients undergoing colorectal cancer surgery, despite enhanced recovery programs (ERPs). Cyclooxygenase (COX)-2 is identified as a key enzyme in POI, but other arachidonic acid pathway enzymes have received little attention despite their potential as selective targets to prevent POI. The objectives were to compare the expression of arachidonic acid metabolism (AAM) enzymes (1) between patients who underwent colorectal cancer surgery and followed an ERP or not (NERP), (2) and between ERP patients who experimented POI or not and (3) to determine the ability of antagonists of these pathways to modulate contractile activity of colonic muscle. METHODS: This was a translational study. Main outcome measures were gastrointestinal motility recovery data, mRNA expressions of key enzymes involved in AAM (RT-qPCR) and ex vivo motility values of the circular colon muscle. Twenty-eight prospectively included ERP patients were compared to eleven retrospectively included NERP patients that underwent colorectal cancer surgery. RESULTS: ERP reduced colonic mucosal COX-2, microsomal prostaglandin E synthase (mPGES1) and hematopoietic prostaglandin D synthase (HPGDS) mRNA expression. mPGES1 and HPGDS mRNA expression were significantly associated with ERP compliance (respectively, r2 = 0.25, p = 0.002 and r2 = 0.6, p < 0.001). In muscularis propria, HPGDS mRNA expression was correlated with GI motility recovery (p = 0.002). The pharmacological inhibition of mPGES1 increased spontaneous ex vivo contractile activity in circular muscle (p = 0.03). CONCLUSION: The effects of ERP on GI recovery are correlated with the compliance of ERP and could be mediated at least in part by mPGES1, HPGDS and COX-2. Furthermore, mPGES1 shows promise as a therapeutic target to further reduce POI duration among ERP patients.

Genetics of enteric neuropathies.

Abnormal development or disturbed functioning of the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is associated with the development of neuropathic gastrointestinal motility disorders. Here, we review the underlying molecular basis of these disorders and hypothesize that many of them have a common defective biological mechanism. Genetic burden and environmental components affecting this common mechanism are ultimately responsible for disease severity and symptom heterogeneity. We believe that they act together as the fulcrum in a seesaw balanced with harmful and protective factors, and are responsible for a continuum of symptoms ranging from neuronal hyperplasia to absence of neurons.

FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF.

Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.