Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

Generation of CHOPi012-A iPSC line from a patient with visceral myopathy-related chronic intestinal pseudo-obstruction.

Visceral myopathies are debilitating conditions characterized by dysfunction of smooth muscle in visceral organs (bowel, bladder, and uterus). Individuals affected by visceral myopathy experience feeding difficulties, growth failure, life-threatening abdominal distension, and may depend on intravenous nutrition for survival. Unfortunately, our limited understanding of the pathophysiology of visceral myopathies means that current therapies remain supportive, with no mechanism-based treatments. We developed a patient-derived iPSC line with a c.769C > T p.R257C/+ mutation, the most common genetic cause of visceral myopathy. This cell line will facilitate studies of how the ACTG2 R257C heterozygous variant affects smooth muscle development and function.

Mini-review: Enteric glial cell reactions to inflammation and potential therapeutic implications for GI diseases, motility disorders, and abdominal pain.

Enteric glial cells are emerging as critical players in the regulation of intestinal motility, secretion, epithelial barrier function, and gut homeostasis in health and disease. Enteric glia react to intestinal inflammation by converting to a 'reactive glial phenotype' and enteric gliosis, contributing to neuroinflammation, enteric neuropathy, bowel motor dysfunction and dysmotility, diarrhea or constipation, 'leaky gut', and visceral pain. The focus of the minireview is on the impact of inflammation on enteric glia reactivity in response to diverse insults such as intestinal surgery, ischemia, infections (C. difficile infection, HIV-Tat-induced diarrhea, endotoxemia and paralytic ileus), GI diseases (inflammatory bowel diseases, diverticular disease, necrotizing enterocolitis, colorectal cancer) and functional GI disorders (postoperative ileus, chronic intestinal pseudo-obstruction, constipation, irritable bowel syndrome). Significant progress has been made in recent years on molecular pathogenic mechanisms of glial reactivity and enteric gliosis, resulting in enteric neuropathy, disruption of motility, diarrhea, visceral hypersensitivity and abdominal pain. There is a growing number of glial molecular targets with therapeutic implications that includes receptors for interleukin-1 (IL-1R), purines (P2X2R, A2BR), PPARα, lysophosphatidic acid (LPAR1), Toll-like receptor 4 (TLR4R), estrogen-ß receptor (ERß) adrenergic α-2 (α-2R) and endothelin B (ETBR), connexin-43 / Colony-stimulating factor 1 signaling (Cx43/CSF1) and the S100ß/RAGE signaling pathway. These exciting new developments are the subject of the minireview. Some of the findings in pre-clinical models may be translatable to humans, raising the possibility of designing future clinical trials to test therapeutic application(s). Overall, research on enteric glia has resulted in significant advances in our understanding of GI pathophysiology.

Patient's dermal fibroblasts as disease markers for visceral myopathy.

Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

Histopathological, Ultrastructural, and Immunohistochemical Findings in MYH11-Variant Visceral Myopathy.

BACKGROUND: Pathogenic mutations in the smooth muscle myosin heavy chain gene, MYH11, cause megacystis megacolon intestinal hypoperistalsis syndrome and other forms of chronic intestinal pseudo-obstruction. Evaluation of intestinal tissues from affected patients is often performed before mutational analysis, but the pathological findings of MYH11-variant visceral myopathy have not been well defined. METHODS: Light microscopic, immunohistochemical, and ultrastructural findings from multiple intestinal samples from 2 patients with MYH11-variant visceral myopathy were reviewed, including MYH11-specific immunohistochemistry. The findings were compared with intestinal samples from patients with gamma-smooth muscle actin (ACTG2)-variant visceral myopathy and non-pseudo-obstruction controls. RESULTS: Apart from non-specific changes (e.g., muscle hypertrophy and distension-related muscularis propria necrosis), no alterations were identified by routine histopathological evaluation or electron microscopy. Immunohistochemistry with antibodies against a battery of smooth muscle proteins, including MYH11, revealed indistinguishable patterns of immunoreactivity in the muscularis propria of both patients and controls. CONCLUSIONS: Myopathic morphological or immunohistochemical changes may not be present in intestinal specimens from patients with MYH11-variant visceral myopathy. Molecular genetic studies should be considered for patients with chronic intestinal pseudo-obstruction and normal or non-specific pathology findings.

Effect of Reactive EGCs on Intestinal Motility and Enteric Neurons During Endotoxemia.

Paralytic ileus is common in patients with septic shock, causing high morbidity and mortality. Enteric neurons and enteric glial cells (EGCs) regulate intestinal motility. However, little is known about their interaction in endotoxemia. This study aimed to investigate whether reactive EGCs had harmful effects on enteric neurons and participated in intestinal motility disorder in mice during endotoxemia. Endotoxemia was induced by the intraperitoneal injection of lipopolysaccharide (LPS) in mice. Fluorocitrate (FC) was administered before LPS injection to inhibit the reactive EGCs. The effects of reactive EGCs on intestinal motility were analyzed by motility assays in vivo and colonic migrating motor complexes ex vivo. The number of enteric neurons was evaluated by immunofluorescent staining of HuCD, nNOS, and ChAT in vivo. In addition, we stimulated EGCs with IL-1ß and TNF-α in vitro and cultured the primary enteric neurons in the conditioned medium, detecting the apoptosis and morphology of neurons through staining TUNEL, cleaved caspase-3 protein, and anti-ß-III tubulin. Intestinal motility and peristaltic reflex were improved by inhibiting reactive EGCs in vivo. The density of the neuronal population in the colonic myenteric plexus increased significantly, while the reactive EGCs were inhibited, especially the nitrergic neurons. In vitro, the enteric neurons cultured in the conditioned medium of reactive EGCs had a considerably higher apoptotic rate, less dendritic complexity, and fewer primary neurites. Reactive enteric glial cells probably participated in paralytic ileus by damaging enteric neurons during endotoxemia. They might provide a novel therapeutic strategy for intestinal motility disorders during endotoxemia or sepsis.

Loss-of-function variants within LMOD1 actin-binding site 2 cause pediatric intestinal pseudo-obstruction by impairing protein stability and actin nucleation.

The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo-obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin-binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG-132, indicating that there might be proteasome-independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1-mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1-mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability.

LPAR1 regulates enteric nervous system function through glial signaling and contributes to chronic intestinal pseudo-obstruction.

Gastrointestinal motility disorders involve alterations to the structure and/or function of the enteric nervous system (ENS) but the causal mechanisms remain unresolved in most cases. Homeostasis and disease in the ENS are processes that are regulated by enteric glia. Signaling mediated through type I lysophosphatidic acid receptors (LPAR1) has recently emerged as an important mechanism that contributes to disease, in part, through effects on peripheral glial survival and function. Enteric glia express LPAR1 but its role in ENS function and motility disorders is unknown. We used a combination of genetic, immunohistochemical, calcium imaging, and in vivo pharmacological approaches to investigate the role of LPAR1 in enteric glia. LPAR1 was enriched in enteric glia in mice and humans and LPA stimulated intracellular calcium responses in enteric glia, subsequently recruiting activity in a subpopulation of myenteric neurons. Blocking LPAR1 in vivo with AM966 attenuated gastrointestinal motility in mice and produced marked enteric neuro- and gliopathy. Samples from humans with chronic intestinal pseudo-obstruction (CIPO), a severe motility disorder, showed reduced glial LPAR1 expression in the colon and ileum. These data suggest that enteric glial LPAR1 signaling regulates gastrointestinal motility through enteric glia and could contribute to severe motility disorders in humans such as CIPO.

Loss function of Bcr mutation causes gastrointestinal dysmotility and brain developmental defects.

BACKGROUND: The breakpoint cluster region (BCR) is a protein that originally forms a fusion protein with c-Abl tyrosine kinase and induces leukemia. Researchers have shown that BCR is enriched in the central nervous system and may contribute to neurological disorders. We aimed to investigate the physiological function of BCR in neural development in the gastrointestinal (GI) tract and brain. METHODS: Whole-exome sequencing was used to screen for mutations in the BCR. Bcr knockout mice (Bcr-/- , ΔExon 2-22) were generated using the CRISPR/Cas9 system. Transit of carmine red dye and glass bead expulsion assays were used to record total and proximal GI transit and distal colonic transit. KEY RESULTS: In an infant with pediatric intestinal pseudo-obstruction, we found a heterozygous de novo mutation (NM_004327.3:c.3072+1G>A) in BCR. Bcr deficiency mice (Bcr-/- ) exhibited growth retardation and impaired gastrointestinal motility. Bcr-/- mice had a prolonged average total GI transit time with increased distal colonic transit and proximal GI transit in isolation. Morphology analysis indicated that Bcr-/- mice had a less number of neurons in the submucosal plexus and myenteric plexus. Bcr-/- mice exhibited apparent structural defects in the brain, particularly in the cortex. Additionally, Bcr- depletion in the mouse cortex altered the expression of Ras homologous (Rho) family small GTPases. CONCLUSIONS AND INFERENCES: BCR mutations are associated with intestinal obstruction in children. Loss of Bcr can cause intestinal dysmotility and brain developmental defects may via regulation of Rho GTPases.

Evidence of enteric angiopathy and neuromuscular hypoxia in patients with mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by thymidine phosphorylase (TP) enzyme defect. As gastrointestinal changes do not revert in patients undergone TP replacement therapy, one can postulate that other unexplored mechanisms contribute to MNGIE pathophysiology. Hence, we focused on the local TP angiogenic potential that has never been considered in MNGIE. In this study, we investigated the enteric submucosal microvasculature and the effect of hypoxia on fibrosis and enteric neurons density in jejunal full-thickness biopsies collected from patients with MNGIE. Orcein staining was used to count blood vessels based on their size. Fibrosis was assessed using the Sirius Red and Fast Green method. Hypoxia and neoangiogenesis were determined via hypoxia-inducible-factor-1α (HIF-1α) and vascular endothelial cell growth factor (VEGF) protein expression, respectively. Neuron-specific enolase was used to label enteric neurons. Compared with controls, patients with MNGIE showed a decreased area of vascular tissue, but a twofold increase of submucosal vessels/mm2 with increased small size and decreased medium and large size vessels. VEGF positive vessels, fibrosis index, and HIF-1α protein expression were increased, whereas there was a diminished thickness of the longitudinal muscle layer with an increased interganglionic distance and reduced number of myenteric neurons. We demonstrated the occurrence of an angiopathy in the GI tract of patients with MNGIE. Neoangiogenetic changes, as detected by the abundance of small size vessels in the jejunal submucosa, along with hypoxia provide a morphological basis to explain neuromuscular alterations, vasculature breakdown, and ischemic abnormalities in MNGIE.NEW & NOTEWORTHY Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is characterized by a genetically driven defect of thymidine phosphorylase, a multitask enzyme playing a role also in angiogenesis. Indeed, major gastrointestinal bleedings are life-threatening complications of MNGIE. Thus, we focused on jejunal submucosal vasculature and showed intestinal microangiopathy as a novel feature occurring in this disease. Notably, vascular changes were associated with neuromuscular abnormalities, which may explain gut dysfunction and help to develop future therapeutic approaches in MNGIE.

Phenotypic switch of smooth muscle cells in paediatric chronic intestinal pseudo-obstruction syndrome.

Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo-obstruction (CIPO) syndrome, a life-threatening intestinal motility disorder. We showed that CIPO-SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet-Derived Growth Factor Receptor-alpha (PDGFRA) expression. We showed that this modulation occurs without origin-related differences in CIPO circular and longitudinal-derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO-SMC towards an undifferentiated stage. The development of CIPO-SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.

Burn-Induced Impairment of Ileal Muscle Contractility Is Associated with Increased Extracellular Matrix Components.

INTRODUCTION: Severe burns lead to marked impairment of gastrointestinal motility, such as delayed gastric emptying and small and large intestinal ileus. However, the cellular mechanism of these pathologic changes remains largely unknown. METHODS: Male Sprague Dawley rats approximately 3 months old and weighing 300-350 g were randomized to either a 60% total body surface area full-thickness scald burn or sham procedure and were sacrificed 24 h after the procedure. Gastric emptying, gastric antrum contractility ileal smooth muscle contractility, and colonic contractility were measured. Muscularis externa was isolated from the ileal segment to prepare smooth muscle protein extracts for Western blot analysis. RESULTS: Compared with sham controls, the baseline rhythmic contractile activities of the antral, ileal, and colonic smooth muscle strips were impaired in the burned rats. Simultaneously, our data showed that ileal muscularis ECM proteins fibronectin and laminin were significantly up-regulated in burned rats compared with sham rats. TGF-ß signaling is an important stimulating factor for ECM protein expression. Our results revealed that TGF-ß signaling was activated in the ileal muscle of burned rats evidenced by the activation of Smad2/3 expression and phosphorylation. In addition, the total and phosphorylated AKT, which is an important downstream factor of ECM signaling in smooth muscle cells, was also up-regulated in burned rats' ileal muscle. Notably, these changes were not seen in the colonic or gastric tissues. CONCLUSION: Deposition of fibrosis-related proteins after severe burn is contributors to decreased small intestinal motility.

Gut epithelial and vascular barrier abnormalities in patients with chronic intestinal pseudo-obstruction.

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a rare condition due to severe impairment of gut motility responsible for recurrent subocclusive episodes. Although neuromuscular-glial-ICC abnormalities represent the main pathogenetic mechanism, the pathophysiology of CIPO remains poorly understood. Intestinal epithelial and vascular endothelial barrier (IEVB) abnormalities can contribute to neuroepithelial changes by allowing passage of harmful substances. METHODS: To test retrospectively whether IEVB defects occur in patients with CIPO, we measured the jejunal protein expression of the major tight junction (TJ) components. CIPO patients were subdivided according to gut neuromuscular histopathology: apparently normal (AN); with inflammation (INF); or with degenerative alterations (DEG). The presence of occludin/claudin oligomers (index of TJ assembly), the amount of occludin, claudin-4, and zonula occludens-1 (ZO-1), and the expression of vasoactive intestinal polypeptide (VIP) and glial fibrillary acidic protein (GFAP) immunoreactivities were evaluated on jejunal full-thickness biopsies using Western blot. KEY RESULTS: Oligomers were absent in the 73% of CIPO. Total occludin decreased in CIPO with AN and INF changes. Claudin-4 was upregulated in CIPO with INF and DEG features. ZO-1 and VIP expression decreased selectively in DEG group. GFAP increased in CIPO regardless the histopathological phenotype. CONCLUSIONS & INFERENCES: The absence of oligomers demonstrated in our study suggests that IEBV is altered in CIPO. The mechanism leading to oligomerization is occludin-dependent in AN and INF, whereas is ZO-1-dependent in DEG. Our study provides support to IEVB abnormalities contributing to CIPO clinical and histopathological features.

Altered enteric expression of the homeobox transcription factor Phox2b in patients with diverticular disease.

Background: Diverticular disease, a major gastrointestinal disorder, is associated with modifications of the enteric nervous system, encompassing alterations of neurochemical coding and of the tyrosine receptor kinase Ret/GDNF pathway. However, molecular factors underlying these changes remain to be determined. Objectives: We aimed to characterise the expression of Phox2b, an essential regulator of Ret and of neuronal subtype development, in the adult human enteric nervous system, and to evaluate its potential involvement in acute diverticulitis. Methods: Site-specific gene expression of Phox2b in the adult colon was analysed by quantitative polymerase chain reaction. Colonic specimens of adult controls and patients with diverticulitis were subjected to quantitative polymerase chain reaction for Phox2b and dual-label immunochemistry for Phox2b and the neuronal markers RET and tyrosine hydroxylase or the glial marker S100ß. Results: The results indicate that Phox2b is physiologically expressed in myenteric neuronal and glial subpopulations in the adult enteric nervous system. Messenger RNA expression of Phox2b was increased in patients with diverticulitis and both neuronal, and glial protein expression of Phox2b were altered in these patients. Conclusions: Alterations of Phox2b expression may contribute to the enteric neuropathy observed in diverticular disease. Future studies are required to characterise the functions of Phox2b in the adult enteric nervous system and to determine its potential as a therapeutic target in gastrointestinal disorders.

Gastrointestinal Dysmotility in MNGIE: from thymidine phosphorylase enzyme deficiency to altered interstitial cells of Cajal.

BACKGROUND: MNGIE is a rare and fatal disease in which absence of the enzyme thymidine phosphorylase induces systemic accumulation of thymidine and deoxyuridine and secondary mitochondrial DNA alterations. Gastrointestinal (GI) symptoms are frequently reported in MNGIE patients, however, they are not resolved with the current treatment interventions. Recently, our understanding of the GI pathology has increased, which rationalizes the pursuit of more targeted therapeutic strategies. In particular, interstitial cells of Cajal (ICC) play key roles in GI physiology and are involved in the pathogenesis of the GI dysmotility. However, understanding of the triggers of ICC deficits in MNGIE is lacking. Herein, we review the current knowledge about the pathology of GI dysmotility in MNGIE, discuss potential mechanisms in relation to ICC loss/dysfunction, remark on the limited contribution of the current treatments, and propose intervention strategies to overcome ICC deficits. Finally, we address the advances and new research avenues offered by organoids and tissue engineering technologies, and propose schemes to implement to further our understanding of the GI pathology and utility in regenerative and personalized medicine in MNGIE. CONCLUSION: Interstitial cells of Cajal play key roles in the physiology of the gastrointestinal motility. Evaluation of their status in the GI dysmotility related to MNGIE would be valuable for diagnosis of MNGIE. Understanding the underlying pathological and molecular mechanisms affecting ICC is an asset for the development of targeted prevention and treatment strategies for the GI dysmotility related to MNGIE.

Oxaliplatin-induced enteric neuronal loss and intestinal dysfunction is prevented by co-treatment with BGP-15.

BACKGROUND AND PURPOSE: Gastrointestinal side effects of chemotherapy are an under-recognized clinical problem, leading to dose reduction, delays and cessation of treatment, presenting a constant challenge for efficient and tolerated anti-cancer treatment. We have found that oxaliplatin treatment results in intestinal dysfunction, oxidative stress and loss of enteric neurons. BGP-15 is a novel cytoprotective compound with potential HSP72 co-inducing and PARP inhibiting properties. In this study, we investigated the potential of BGP-15 to alleviate oxaliplatin-induced enteric neuropathy and intestinal dysfunction. EXPERIMENTAL APPROACH: Balb/c mice received oxaliplatin (3 mg·kg-1 ·day-1 ) with and without BGP-15 (15 mg·kg-1 ·day-1 : i.p.) tri-weekly for 14 days. Gastrointestinal transit was analysed via in vivo X-ray imaging, before and after treatment. Colons were collected to assess ex vivo motility, neuronal mitochondrial superoxide and cytochrome c levels and for immunohistochemical analysis of myenteric neurons. KEY RESULTS: Oxaliplatin-induced neuronal loss increased the proportion of neuronal NO synthase-immunoreactive neurons and increased levels of mitochondrial superoxide and cytochrome c in the myenteric plexus. These changes were correlated with an increase in PARP-2 immunoreactivity in the colonic mucosa and were attenuated by BGP-15 co-treatment. Significant delays in gastrointestinal transit, intestinal emptying and pellet formation, impaired colonic motor activity, reduced faecal water content and lack of weight gain associated with oxaliplatin treatment were restored to sham levels in mice co-treated with BGP-15. CONCLUSION AND IMPLICATIONS: Our results showed that BGP-15 ameliorated oxidative stress, increased enteric neuronal survival and alleviated oxaliplatin-induced intestinal dysfunction, suggesting that BGP-15 may relieve the gastrointestinal side effects of chemotherapy.

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.

Long­term follow­up of buserelin­induced enteric neuropathy in rats.

A few patients have been shown to develop severe abdominal pain and gastrointestinal dysmotility during treatment with gonadotropin­releasing hormone (GnRH) analogs. A rat model of enteric neuropathy has been developed by administration of the GnRH analog buserelin to rats. Loss of enteric neurons and ganglioneuritis throughout the gastrointestinal tract has been described, without other histopathological changes. The aim of the present study was to investigate the long­term effects of this rat model on body weight, and on morphology and inflammatory changes in the gastrointestinal tract. Rats were administered subcutaneous injections of buserelin or saline once daily for 5 days and allowed to recover for 3 weeks. This regimen was repeated four times. The rats were weighed weekly and were sacrificed 16 weeks after the fourth treatment. The bowel wall was measured by morphometry, and the presence of enteric neurons, mast cells, eosinophils and T­lymphocytes was evaluated. Buserelin­treated rats were shown to have a lower body weight at sacrifice, as compared with the controls (P<0.05). Compared with controls, buserelin treatment caused loss of myenteric neurons in the ileum and colon (P<0.01), a thinner circular muscle layer in ileum (P<0.05) and longitudinal muscle layer in colon (P<0.05), increased number of eosinophils in the submucosa of the ileum (P<0.05), and an increased number of T­lymphocytes in the submucosa and circular muscle layer of the fundus (P<0.01 and P<0.05, respectively) and circular muscle layer of the colon (P<0.05). Mast cells were equally distributed in the two groups. Thus, long­term follow­up of buserelin­induced enteric neuropathy reveals reduced body weight, loss of myenteric neurons, thinning of muscle layers, and increased numbers of eosinophils and T­lymphocytes in the gastrointestinal tract.

Two Cases of Chronic Intestinal Pseudo-obstruction: A Comparison of Staining Characteristics of Enteric Visceral Myopathy With Hirschsprung Disease.

Chronic intestinal pseudo-obstruction (CIPO), a rare, debilitating disorder of bowel motility dysfunction, is largely a clinical diagnosis, without any universally accepted diagnostic criteria. Three subgroups are generally acknowledged based on the cell-type affected: enteric visceral myopathy (the most common subgroup), neuropathy, and mesenchymopathy. A fourth subgroup includes abnormalities of neurohormonal peptides. Although immunohistochemical staining is reportedly useful for identifying the mesenchymopathic type, its role in diagnosing enteric visceral myopathy and neuropathy has been fraught with difficulties. We present two cases of chronic intestinal pseudo-obstruction that are clinically and histopathologically suggestive of type III visceral enteric myopathy, aiming to expound upon the diagnostic and pathogenic features. We found that the outer-longitudinal layer of the muscularis propria was more severely affected as compared with the inner circular layer. To investigate the value of this finding, we performed immunostains in the one case in which a paraffin block was available. We found increased peripherin and calretinin immunopositive nerve fibers in the outer layer as compared with inner, but without any significant increase in S-100 positivity or alteration in neuronal morphology of myenteric plexus, a novel finding. This differential staining pattern was completely different from Hirschsprung disease, in which we found rare to absent peripherin and calretinin staining. It is unclear if this increase in the outer layer in visceral myopathy reflects a reactive change or dysfunctional axons. In addition, the history of volvulus in one patient and transmural inflammatory changes in the second raise concerns about the higher propensity of clinical complications secondary to the attenuated outer muscular layer. This study suggests that enteric visceral myopathy has histologic and staining characteristics different from Hirschsprung disease, a finding of diagnostic significance in the differential diagnosis of bowel obstruction. Moreover, these features may have pathogenic value and need further confirmation.

Potentially diagnostic electron paramagnetic resonance spectra elucidate the underlying mechanism of mitochondrial dysfunction in the deoxyguanosine kinase deficient rat model of a genetic mitochondrial DNA depletion syndrome.

A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated deoxyguanosine kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result from depressed expression and/or diminished specific activity of the distinct respiratory chain complexes. As EPR requires no sample preparation or non-physiological reagents, it provides information on the status of the mitochondrion as it was in the functioning state. On its own, this information is of use in identifying respiratory chain dysfunction; in conjunction with other techniques, the information from EPR shows how the respiratory chain is affected at the molecular level by the dysfunction. It is proposed that EPR has a role in mechanistic pathophysiological studies of mitochondrial disease and could be used to study the impact of new treatment modalities or as an additional diagnostic tool.