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SFPQ is a ubiquitous nuclear RNA-binding protein implicated in many aspects of RNA biogenesis. Importantly, nuclear depletion and cytoplasmic accumulation of SFPQ has been linked to neuropathological conditions such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Here, we describe a molecular mechanism by which SFPQ is mislocalized to the cytoplasm. We report an unexpected discovery of the infinite polymerization of SFPQ that is induced by zinc binding to the protein. The crystal structure of human SFPQ in complex with zinc at 1.94 Å resolution reveals intermolecular interactions between SFPQ molecules that are mediated by zinc. As anticipated from the crystal structure, the application of zinc to primary cortical neurons induced the cytoplasmic accumulation and aggregation of SFPQ. Mutagenesis of the three zinc-coordinating histidine residues resulted in a significant reduction in the zinc-binding affinity of SFPQ in solution and the zinc-induced cytoplasmic aggregation of SFPQ in cultured neurons. Taken together, we propose that dysregulation of zinc availability and/or localization in neuronal cells may represent a mechanism for the imbalance in the nucleocytoplasmic distribution of SFPQ, which is an emerging hallmark of neurodegenerative diseases including AD and ALS.

Dietary organic selenium (Se) is commonly utilized to increase formation of selenoproteins, including the major antioxidant protein, glutathione peroxidase (GPx). Inorganic Se salts, such as sodium selenite, are also incorporated into selenoproteins, and there is evidence that nanoelemental Se added to the diet may also be effective. We conducted two trials, the first investigated inorganic Se (selenite), organic Se (L-selenomethionine) and nanoelemental Se, in conventional mice. Their bioavailability and effectiveness to increase GPx activity were examined. The second trial focused on determining the mechanism by which dietary Se is incorporated into tissue, utilising both conventional and germ-free (GF) mice. Mice were fed a diet with minimal Se, 0.018 parts per million (ppm), and diets with Se supplementation, to achieve 0.07, 0.15, 0.3 and 1.7 ppm Se, for 5 weeks (first trial). Mass spectrometry, Western blotting and enzymatic assays were used to investigate bioavailability, protein levels and GPx activity in fresh frozen tissue (liver, ileum, plasma, muscle and feces) from the Se fed animals. Inorganic, organic and nanoelemental Se were all effectively incorporated into tissues. The high Se diet (1.7 ppm) resulted in the highest Se levels in all tissues and plasma, independent of the Se source. Interestingly, despite being ~11 to ~25 times less concentrated than the high Se, the lower Se diets (0.07, 0.15) resulted in comparably high Se levels in liver, ileum and plasma for all Se sources. GPx protein levels and enzyme activity were significantly increased by each diet, relative to control. We hypothesised that bacteria may be a vector for the conversion of nanoelemental Se, perhaps in exchange for S in sulphate metabolising bacteria. We therefore investigated Se incorporation from low sulphate diets and in GF mice. All forms of selenium were bioavailable and similarly significantly increased the antioxidant capability of GPx in the intestine and liver of GF mice and mice with sulphate free diets. Se from nanoelemental Se resulted in similar tissue levels to inorganic and organic sources in germ free mice. Thus, endogenous mechanisms, not dependent on bacteria, reduce nanoelemental Se to the metabolite selenide that is then converted to selenophosphate, synthesised to selenocysteine, and incorporated into selenoproteins. In particular, the similar efficacy of nanoelemental Se in comparison to organic Se in both trials is important in the view of the currently limited cheap sources of Se.

BACKGROUND: Dopamine receptor 2 (DRD2) and ghrelin receptor (GHSR1a) agonists both stimulate defecation by actions at the lumbosacral defecation center. Dopamine is in nerve terminals surrounding autonomic neurons of the defecation center, whereas ghrelin is not present in the spinal cord. Dopamine at D2 receptors generally inhibits neurons, but at the defecation center, its effect is excitatory. METHODS: In vivo recording of defecation and colorectal propulsion was used to investigate interaction between DRD2 and GHSR1a. Localization studies were used to determine sites of receptor expression in rat and human spinal cord. KEY RESULTS: Dopamine, and the DRD2 agonist, quinpirole, directly applied to the lumbosacral cord, caused defecation. The effect of intrathecal dopamine was inhibited by the GHSR1a antagonist, YIL781, given systemically, but YIL781 was not an antagonist at DRD2. The DRD2 agonist, pramipexole, administered systemically caused colorectal propulsion that was prevented when the pelvic nerves were cut. Drd2 and Ghsr were expressed together in autonomic preganglionic neurons at the level of the defecation centers in rat and human. Behaviorally induced defecation (caused by water avoidance stress) was reduced by the DRD2 antagonist, sulpiride. We had previously shown it is reduced by YIL781. CONCLUSIONS AND INFERENCES: Our observations imply that dopamine is a transmitter of the defecation pathways whose actions are exerted through interacting dopamine (D2) and ghrelin receptors on lumbosacral autonomic neurons that project to the colorectum. The results explain the excitation by dopamine agonists and the conservation of GHSR1a in the absence of ghrelin.

Parkinson’s disease (PD) is associated with neuronal damage in the brain and gut. This work compares changes in the enteric nervous system (ENS) of commonly used mouse models of PD that exhibit central neuropathy and a gut phenotype. Enteric neuropathy was assessed in five mouse models: peripheral injection of MPTP; intracerebral injection of 6-OHDA; oral rotenone; and mice transgenic for A53T variant human α-synuclein with and without rotenone. Changes in the ENS of the colon were quantified using pan-neuronal marker, Hu, and neuronal nitric oxide synthase (nNOS) and were correlated with GI function. MPTP had no effect on the number of Hu+ neurons but was associated with an increase in Hu+ nuclear translocation (P 2 in WT mice (P

With increases in the frequency, intensity and duration of heat waves forecast plus expansion of tropical agriculture, heat stress (HS) is both a current and an emerging problem. As cinnamon has been shown to increase insulin sensitivity, which is part of the adaptive response to HS, the aim of this experiment was to determine if cinnamon could improve insulin sensitivity and ameliorate HS in grower pigs. In a 2 &times, 2 factorial design, 36 female Large White &times, Landrace pigs were fed control (0%) vs. cinnamon (1.5%) diets and housed for 7 day under thermoneutral (20 &deg, C, TN) vs. HS conditions (8 h 35 &deg, C/16 h 28 &deg, C, 35% relative humidity). At the completion of the challenge, insulin sensitivity was assessed by an intravenous glucose tolerance test (IVGTT). Heat stress increased parameters such as respiration rate and rectal temperature. Furthermore, biochemical changes in blood and urine indicated the pigs were experiencing respiratory alkalosis. Minimal modelling of parameters of insulin sensitivity showed that HS pigs had a lower insulin response to the IVGTT and improved insulin sensitivity. Cinnamon had additive effects with heat stress, reflected in lowering the insulin area under curve (AUC) and elevated insulin sensitivity compared to TN. However, this apparent improvement in insulin sensitivity did not ameliorate any of the other physiological symptoms of HS.

We use a monoclonal antibody against the C-terminal of oxyntomodulin (OXM) to investigate enteroendocrine cells (EEC) in mouse, rat, human and pig. This antibody has cross-reactivity with the OXM precursor, glicentin (Gli) but does not recognise glucagon. The antibody stained EEC in the jejunum and colon of each species. We compared OXM/Gli immunoreactivity with that revealed by antibodies against structurally related peptides, GLP-1 and glucagon and against GIP and PYY that are predicted to be in some EEC that express OXM/Gli. We used super-resolution to locate immunoreactive vesicles. In the pancreas, OXM/Gli was in glucagon cells but was located in separate storage vesicles to glucagon. In jejunal EEC, OXM/Gli and GIP were in many of the same cells but often in separate vesicles, whereas PYY and OXM/Gli were commonly colocalised in the same storage vesicles of colonic EEC. When binding of anti-GLP-1 to the structurally related GIP was removed by absorption with GIP peptide, GLP-1 and OXM/Gli immunoreactivities were contained in the same population of EEC in the intestine. We conclude that anti-OXM/Gli is a more reliable marker than anti-GLP-1 for EEC expressing preproglucagon products. Storage vesicles that were immunoreactive for OXM/Gli were almost always immunoreactive for GLP-1. OXM concentrations, measured by ELISA, were highest in the distal ileum and colon. Lesser concentrations were found in more proximal parts of small intestine and pancreas. Very little was in the stomach. In EEC containing GIP and OXM/Gli, these hormones are packaged in different secretory vesicles. Separate packaging also occurred for OXM and glucagon, whereas OXM/Gli and PYY and OXM/Gli and GLP-1 were commonly contained together in secretory vesicles.

Background Chronic stress exacerbates motor deficits and increases dopaminergic cell loss in several rodent models of Parkinson's disease (PD). However, little is known about effects of stress on gastrointestinal (GI) dysfunction, a common non-motor symptom of PD. We aimed to determine whether chronic stress exacerbates GI dysfunction in the A53T mouse model of PD and whether this relates to changes in α-synuclein distribution. Methods Chronic isolation stress was induced by single-housing WT and homozygote A53T mice between 5 and 15 months of age. GI and motor function were compared with mice that had been group-housed. Key results Chronic isolation stress increased plasma corticosterone and exacerbated deficits in colonic propulsion and whole-gut transit in A53T mice and also increased motor deficits. However, our results indicated that the novel environment-induced defecation response, a common method used to evaluate colorectal function, was not a useful test to measure exacerbation of GI dysfunction, most likely because of the reported reduced level of anxiety in A53T mice. A53T mice had lower corticosterone levels than WT mice under both housing conditions, but single-housing increased levels for both genotypes. Enteric neuropathy was observed in aging A53T mice and A53T mice had a greater accumulation of alpha-synuclein (αsyn) in myenteric ganglia under both housing conditions. Conclusions & inferences Chronic isolation stress exacerbates PD-associated GI dysfunction, in addition to increasing motor deficits. However, these changes in GI symptoms are not directly related to corticosterone levels, worsened enteric neuropathy, or enteric αsyn accumulation.

Progeny of primiparous sows (gilts) have poor production performance compared to those of multiparous sows. This has been attributed to underlying biological events that occur early in life. We specifically focused on the development of the gastrointestinal tract (GIT) and its function in sow vs gilt progeny (SP vs GP) reared under commercial conditions. Piglet tissues were harvested at birth, at 24 h, and pre- and post-weaning (28 v 29 d) and used to quantify GIT integrity. All data were analysed using Genstat V18 for the effects of parity (sow vs gilt) in birth (0 vs 24 h) and weaning (pre- vs post) cohorts. GIT integrity was quantified from freshly excised tissue in Ussing chambers for transepithelial electrical resistance (TER) and macromolecule permeability (FD4; labelled 4kD dextran). Permeability was measured in stomach, colon, jejunum and ileum. Over the 24-h neonatal period, TER was significantly reduced in the stomach and colon (P = 0.048 and 0.003 respectively) with GP having lower stomach TER than SP (P = 0.015). TER increased in the jejunum and ileum over the 24-h neonatal period (P = 0.004 and 0.002 respectively). An interactive effect of parity and time on ileum FD4 was observed (P = 0.043) where FD4 in SP ileum decreased over the 24-h neonatal period and FD4 in GP ileum increased over the 24-h period (P = 0.043). Jejunum FD4 was significantly higher in GP than SP over the 24-h neonatal period (P = 0.048). An interactive effect of parity and time was observed in the colon over the weaning period where TER increased in SP but decreased in GP (P = 0.005). Stomach TER decreased over the weaning period in both GP and SP (P = 0.013). Jejunum and ileum TER decreased over the weaning period (P = 0.003 and 0.057 respectively). Ileum FD4 was higher in GP than SP over the weaning period (P = 0.037). These findings reflect a reduction in GIT development in GP compared with SP, and gut leakiness may be an underlying reason for poorer production performance and increased morbidity of GP.

Background Because M1 muscarinic receptors are expressed by enteric neurons, we investigated whether positive allosteric modulators of these receptors (M1PAMs) would enhance colorectal propulsion and defecation in dogs, mice, and rats. Methods The potencies of the M1PAMs, T662 or T523, were investigated using M1 receptor-expressing CHO cells. Effectiveness of M1PAMs on defecation was investigated by oral administration in mice and rats, by recording propulsive contractions in anaesthetized rats and by recording high amplitude propagating contractions in dogs. Key results PAM EC50 values in M1 receptor-expressing CHO cells were 0.7-1.8 nmol/L for T662 and 8-10 nmol/L for T523. The compounds had 1000-fold lower potencies as agonists. In anesthetized rats, both compounds elicited propulsive colorectal contractions, and in dogs, mice, and rats, oral administration increased fecal output. No adverse effects were observed in conscious animals. M1PAMs triggered propagated high amplitude contractions and caused defecation in dogs. Nerve-mediated contractions were enhanced in the isolated mouse colon. M1PAMs were equi-effective in rats with or without the pelvic nerves being severed. In two models of constipation in mice, opiate-induced constipation and constipation of aging, defecation was induced and constipation was reversed. Conclusion and inferences M1PAMs act at targets sites in the colorectum to enhance colorectal propulsion. They are effective across species, and they reverse experimentally induced constipation. Previous studies have shown that they are safe in human. Because they provide an enhancement of physiological control rather than being direct agonists, they are predicted to provide effective treatment for constipation.

Recent studies have shown that patterns of colocalisation of hormones in enteroendocrine cells are more complex than previously appreciated and that the patterns differ substantially between species. In this study, the human sigmoid colon is investigated by immunohistochemistry for the presence of gastrointestinal hormones and their colocalisation. The segments of colon were distant from the pathology that led to colectomy and appeared structurally normal. Only four hormones, 5-hydroxytryptamine (5-HT), glucagon-like peptide 1 (GLP-1), peptide YY (PYY) and somatostatin, were common in enteroendocrine cells of the human colon. Cholecystokinin, present in the colon of some species, was absent, as were glucose-dependent insulinotropic peptide, ghrelin and motilin. Neurotensin cells were extremely rare. The most numerous cells were 5-HT cells, some of which also contained PYY or somatostatin and very rarely GLP-1. Almost all GLP-1 cells contained PYY. It is concluded that enteroendocrine cells of the human colon, like those of other regions and species, exhibit overlapping patterns of hormone colocalisation and that the hormones and their patterns of expression differ between human and other species.

Heat stress (HS) is an environmental stressor challenging poultry production and requires a strategy to cope with it. A total of 288-day-old male broiler chicks were fed with one of the following diets: basal diet, basal with betaine (BET), or with selenium and vitamin E (AOX), or with a combination of BET and AOX, under thermoneutral and cyclic HS. Results showed that HS reduced average daily feed intake (ADFI) (p = 0.01) and average daily gain (ADG) (p &lt, 0.001), and impaired feed conversion ratio (FCR) (p = 0.03) during rearing period (0&ndash, 42 day). BET increased ADG (p = 0.001) and decreased FCR (p = 0.02), whereas AOX had no effects. Breast muscle weight was decreased by HS (p &lt, 0.001) and increased by BET (p &lt, 0.001). Rectal temperature was increased by HS (p &lt, 0.001) and improved by BET overall. Respiration rate was increased by HS (p &lt, 0.001), but BET decreased it during HS (p = 0.04). Jejunum transepithelial resistance was reduced by HS and had no effect on permeability whereas BET increased jejunum permeability (p = 0.013). Overall, the reductions in ADG of broiler chickens during HS were ameliorated by supplementation with BET, with much of the increase in ADG being breast muscle.

Pain associated with skeletal pathology or disease is a significant clinical problem, but the mechanisms that generate and/or maintain it remain poorly understood. In this study, we explored roles for GDNF, neurturin, and artemin signaling in bone pain using male Sprague Dawley rats. We have shown that inflammatory bone pain involves activation and sensitization of peptidergic, NGF-sensitive neurons via artemin/GDNF family receptor α-3 (GFRα3) signaling pathways, and that sequestering artemin might be useful to prevent inflammatory bone pain derived from activation of NGF-sensitive bone afferent neurons. In addition, we have shown that inflammatory bone pain also involves activation and sensitization of nonpeptidergic neurons via GDNF/GFRα1 and neurturin/GFRα2 signaling pathways, and that sequestration of neurturin, but not GDNF, might be useful to treat inflammatory bone pain derived from activation of nonpeptidergic bone afferent neurons. Our findings suggest that GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.SIGNIFICANCE STATEMENTPain associated with skeletal pathology, including bone cancer, bone marrow edema syndromes, osteomyelitis, osteoarthritis, and fractures causes a major burden (both in terms of quality of life and cost) on individuals and health care systems worldwide. We have shown the first evidence of a role for GDNF, neurturin, and artemin in the activation and sensitization of bone afferent neurons, and that sequestering these ligands reduces pain behavior in a model of inflammatory bone pain. Thus, GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.

Background It has been recently demonstrated that the ghrelin receptor agonist, HM01, caused defecation in rats that were treated to provide a model for the constipation of Parkinson's disease. HM01 significantly increased fecal output and increased Fos activity in neurons of the hypothalamus and hindbrain, but not in the spinal defecation center. Other ghrelin agonists act on the defecation center. Methods Receptor pharmacology was examined in ghrelin receptor (GHSR1a) transfected cells. Anesthetized rats were used to investigate sites and mechanisms of action. Key Results HM01 activated rat GHSR1a at nanomolar concentrations and was antagonized by the GHSR1a antagonist, YIL781. HM01, intravenous, was potent to activate propulsive colorectal contractions. This was prevented by pelvic nerve section and by intravenous YIL781, but not by spinal cord section rostral to the defecation centers. Direct intrathecal application of HM01 to the defecation center at spinal level L6-S1 initiated propulsive contractions of the colorectum. Conclusions & Inferences HM01 stimulates GHSR1a receptors on neurons in the lumbosacral defecation centers to cause propulsive contractions and emptying of the colorectum. It has greater potency when given systemically, compared with other GHSR1a agonists.

The majority of 5-HT (serotonin) in the body is contained in enteroendocrine cells of the gastrointestinal mucosa. From the time of their discovery over 80 years ago, the 5-HT-containing cells have been regarded as a class of cell that is distinct from enteroendocrine cells that contain peptide hormones. However, recent studies have cast doubt on the concept of there being distinct classes of enteroendocrine cells, each containing a single hormone or occasionally more than one hormone. Instead, data are rapidly accumulating that there are complex patterns of colocalisation of hormones that identify multiple subclasses of enteroendocrine cells. In the present work, multiple labelling immunohistochemistry is used to investigate patterns of colocalisation of 5-HT with enteric peptide hormones. Over 95 % of 5-HT cells in the duodenum also contained cholecystokinin and about 40 % of them also contained secretin. In the jejunum, about 75 % of 5-HT cells contained cholecystokinin but not secretin and 25 % contained 5-HT plus both cholecystokinin and secretin. Small proportions of 5-HT cells contained gastrin or somatostatin in the stomach, PYY or GLP-1 in the small intestine and GLP-1 or somatostatin in the large intestine. Rare or very rare 5-HT cells contained ghrelin (stomach), neurotensin (small and large intestines), somatostatin (small intestine) and PYY (in the large intestine). It is concluded that 5-HT-containing enteroendocrine cells are heterogeneous in their chemical coding and by implication in their functions.

In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS. In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild‐type rats, WAS‐induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex.

Sequestration of nerve growth factor has been used successfully in the management of pain in animal models of bone disease and in human osteoarthritis. However, the mechanisms of nerve growth factor-induced bone pain and its role in modulating inflammatory bone pain remain to be determined. In this study, we show that nerve growth factor receptors (TrkA and p75) and some other nerve growth factor-signaling molecules (TRPV1 and Nav1.8, but not Nav1.9) are expressed in substantial proportions of rat bone nociceptors. We demonstrate that nerve growth factor injected directly into rat tibia rapidly activates and sensitizes bone nociceptors and produces acute behavioral responses with a similar time course. The nerve growth factor-induced changes in the activity and sensitivity of bone nociceptors we report are dependent on signaling through the TrkA receptor, but are not affected by mast cell stabilization. We failed to show evidence for longer term changes in expression of TrkA, TRPV1, Nav1.8 or Nav1.9 in the soma of bone nociceptors in a rat model of inflammatory bone pain. Thus, retrograde transport of NGF/TrkA and increased expression of some of the common nerve growth factor signaling molecules do not appear to be important for the maintenance of inflammatory bone pain. The findings are relevant to understand the basis of nerve growth factor sequestration and other therapies directed at nerve growth factor signaling, in managing pain in bone disease.