Cu/Zn Superoxide Dismutase (Sod1) regulates the canonical Wnt signaling pathway.

Cu/Zn Superoxide Dismutase (Sod1) catalyzes the disproportionation of cytotoxic superoxide radicals (O2•-) into oxygen (O2) and hydrogen peroxide (H2O2), a key signaling molecule. In Saccharomyces cerevisiae, we previously discovered that Sod1 participates in an H2O2-mediated redox signaling circuit that links nutrient availability to the control of energy metabolism. In response to glucose and O2, Sod1-derived H2O2 stabilizes a pair of conserved plasma membrane kinases - yeast casein kinase 1 and 2 (Yck1/2) - that signal glycolytic growth and the repression of respiration. The Yck1/2 homolog in humans, casein kinase 1-γ (CK1γ), is an integral component of the Wingless and Int-1 (Wnt) signaling pathway, which is essential for regulating cell fate and proliferation in early development and adult tissue and is dysregulated in many cancers. Herein, we establish the conservation of the SOD1/YCK1 redox signaling axis in humans by finding that SOD1 regulates CK1γ expression in human embryonic kidney 293 (HEK293) cells and is required for canonical Wnt signaling and Wnt-dependent cell proliferation.

Interactions Between Commensal Bacteria and Enteric Neurons, via FPR1 Induction of ROS, Increase Gastrointestinal Motility in Mice.

BACKGROUND & AIMS: Reduced gastrointestinal (GI) motility is a feature of disorders associated with intestinal dysbiosis and loss of beneficial microbes. It is not clear how consumption of beneficial commensal microbes, marketed as probiotics, affects the enteric nervous system (ENS). We studied the effects of the widely used probiotic and the commensal Lactobacillus rhamnosus GG (LGG) on ENS and GI motility in mice. METHODS: Conventional and germ free C57B6 mice were gavaged with LGG and intestinal tissues were collected; changes in the enteric neuronal subtypes were assessed by real-time polymerase chain reaction, immunoblots, and immunostaining. Production of reactive oxygen species (ROS) in the jejunal myenteric plexi and phosphorylation (p) of mitogen-activated protein kinase 1 (MAPK1) in the enteric ganglia were assessed by immunoblots and immunostaining. Fluorescence in situ hybridization was performed on jejunal cryosections with probes to detect formyl peptide receptor 1 (FPR1). GI motility in conventional mice was assessed after daily gavage of LGG for 1 week. RESULTS: Feeding of LGG to mice stimulated myenteric production of ROS, increased levels of phosphorylated MAPK1, and increased expression of choline acetyl transferase by neurons (P < .001). These effects were not observed in mice given N-acetyl cysteine (a ROS inhibitor) or LGGΩSpaC (an adhesion-mutant strain of LGG) or FPR1-knockout mice. Gavage of mice with LGG for 1 week significantly increased stool frequency, reduced total GI transit time, and increased contractions of ileal circular muscle strips in ex vivo experiments (P < .05). CONCLUSIONS: Using mouse models, we found that LGG-mediated signaling in the ENS requires bacterial adhesion, redox mechanisms, and FPR1. This pathway might be activated to increase GI motility in patients.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.

Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However, the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSS) were used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY-/-) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 µM) on epithelial proliferation and apoptosis in vitro. DSS-WT mice exhibited enhanced intestinal inflammation, polyp size, and polyp number (7.5 ± 0.8) compared with DSS-NPY-/- mice (4 ± 0.5, P < 0.01). Accordingly, DSS-WT mice also showed increased colonic epithelial proliferation (PCNA, Ki67) and reduced apoptosis (TUNEL) compared with DSS-NPY-/- mice. The apoptosis regulating microRNA, miR-375, was significantly downregulated in the colon of DSS-WT (2-fold, P < 0.01) compared with DSS-NPY-/--mice. In vitro studies indicated that NPY promotes cell proliferation (increase in PCNA and ß-catenin, P < 0.05) via phosphatidyl-inositol-3-kinase (PI3-K)-ß-catenin signaling, suppressed miR-375 expression, and reduced apoptosis (increase in phospho-Bad). NPY-treated cells also displayed increased c-Myc and cyclin D1, and reduction in p21 (P < 0.05). Addition of miR-375 inhibitor to cells already treated with NPY did not further enhance the effects induced by NPY alone. Our findings demonstrate a novel regulation of inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and downregulation of miR-375. NEW & NOTEWORTHY: Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

Emerging neuropeptide targets in inflammation: NPY and VIP.

The enteric nervous system (ENS), referred to as the "second brain," comprises a vast number of neurons that form an elegant network throughout the gastrointestinal tract. Neuropeptides produced by the ENS play a crucial role in the regulation of inflammatory processes via cross talk with the enteric immune system. In addition, neuropeptides have paracrine effects on epithelial secretion, thus regulating epithelial barrier functions and thereby susceptibility to inflammation. Ultimately the inflammatory response damages the enteric neurons themselves, resulting in deregulations in circuitry and gut motility. In this review, we have emphasized the concept of neurogenic inflammation and the interaction between the enteric immune system and enteric nervous system, focusing on neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). The alterations in the expression of NPY and VIP in inflammation and their significant roles in immunomodulation are discussed. We highlight the mechanism of action of these neuropeptides on immune cells, focusing on the key receptors as well as the intracellular signaling pathways that are activated to regulate the release of cytokines. In addition, we also examine the direct and indirect mechanisms of neuropeptide regulation of epithelial tight junctions and permeability, which are a crucial determinant of susceptibility to inflammation. Finally, we also discuss the potential of emerging neuropeptide-based therapies that utilize peptide agonists, antagonists, siRNA, oligonucleotides, and lentiviral vectors.

Intracolonic Neuropeptide Y Y1 Receptor Inhibition Attenuates Intestinal Inflammation in Murine Colitis and Cytokine Release in IBD Biopsies.

We have demonstrated that neuropeptide Y (NPY) can regulate pro-inflammatory signaling in the gut via cross-talk with the pro-inflammatory cytokine tumor necrosis factor (TNF). Here, we investigated if selective blocking of NPY receptors, NPY1R or NPY2R, using small molecule non-peptide antagonists (BIBP-3222 for NPY1R and BIIE-0246 for NPY2R) in the colon could attenuate intestinal inflammation by lowering TNF levels (BIBP - N-[(1R)]-4-[(Aminoiminomethyl)amino-1-[[[(4-hydroxyphenyl)methyl]amino]carbonyl]butyl-α-phenylbenzeneacetamide; BIIE - N-[(1S)-4-[(Aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide). Colitis was induced using dextran sodium sulfate in drinking water for 7 days, or by adoptive T-cell transfer in RAG-/- mice. Colonic biopsies from healthy subjects (n = 10) and IBD patients (n = 34, UC = 20, CD = 14) were cultured ex vivo in presence or absence of NPY antagonists (100 µM, 20 h), and cytokine release into culture supernatants was measured by ELISA. Intracolonic administration of BIBP (but not BIIE) significantly reduced clinical, endoscopic, and histological scores, and serum TNF, interleukin (IL)-6, and IL-12p70 in DSS colitis; it also significantly attenuated histological damage and serum IL-6 in T-cell colitis (P < .05). Intracolonic administration of BIBP significantly reduced TNF and interferon (IFN)-γ release from UC biopsies, whereas BIIE downregulated only IFN-γ (P < .05). BIBP significantly reduced TNF and interferon (IFN)-γ release from UC biopsies, whereas BIIE downregulated only IFN-γ (P < .05). Our data suggest a promising therapeutic value for NPY1R inhibition in alleviating intestinal inflammation in UC, possibly as enemas to IBD patients.

Imaging the Gut with "CLARITY".

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 °C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 °C. On day 3, the tissue-hydrogel solution is incubated at 37 °C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 °C for 2 days (days 4 & 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4°C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

Glial cell line-derived neurotrophic factor enhances neurogenin3 gene expression and beta-cell proliferation in the developing mouse pancreas.

Glial cell line-derived neurotrophic factor (GDNF) is a factor produced by glial cells that is required for the development of the enteric nervous system. In transgenic mice that overexpress GDNF in the pancreas, GDNF has been shown to enhance beta-cell mass and improve glucose control, but the transcriptional and cellular processes involved are not known. In this study we examined the influence of GDNF on the expression of neurogenin3 (Ngn3) and other transcription factors implicated in early beta-cell development, as well as on beta-cell proliferation during embryonic and early postnatal mouse pancreas development. Embryonic day 15.5 (E15.5) mouse pancreatic tissue when exposed to GDNF for 24 h showed higher Ngn3, pancreatic and duodenal homeobox gene 1 (Pdx1), neuroD1/beta(2), paired homeobox gene 4 (Pax4), and insulin mRNA expression than tissue exposed to vehicle only. Transgenic expression of GDNF in mouse pancreata was associated with increased numbers of Ngn3-expressing pancreatic cells and higher beta-cell mass at embryonic day 18 (E18), as well as higher beta-cell proliferation and Pdx1 expression in beta-cells at E18 and postnatal day 1. In the HIT-T15 beta-cell line, GDNF enhanced the expression of Pax6. This response was, however, blocked in the presence of Pdx1 small interfering RNA (siRNA). Chromatin immunoprecipitation studies using the HIT-T15 beta-cell line demonstrated that GDNF can influence Pdx1 gene expression by enhancing the binding of Sox9 and neuroD1/beta(2) to the Pdx1 promoter. Our data provide evidence of a mechanism by which GDNF influences beta-cell development. GDNF could be a potential therapeutic target for the treatment and prevention of diabetes.

Adenosine 2B receptors (A(2B)AR) on enteric neurons regulate murine distal colonic motility.

Delayed colonic emptying leading to constipation is a significant health concern. We investigated the role of adenosine 2B receptor (A(2B)AR) in modulating distal colonic motility using wild-type and A(2B)AR-knockout (A(2B)AR(-/-)) mice. Colon motility was assessed using stool characteristics and colonic transit. Distal colonic ganglia, isolated by laser capture microdissection, were tested for A(2B)AR expression by RT-PCR. The distal colon contraction and relaxation responses were assessed by electrical field stimulation (EFS) in presence of A(2B)AR agonists, antagonists or inhibitors of nitric oxide (NO) and guanylate cyclase. Nitrite levels were measured in enteric neuronal cultures exposed to A(2B)AR agonists/antagonists. A(2B)AR(-/-) mice display increased stool retention, decreased stool frequency, delayed colonic emptying, and decreased circular muscle relaxation. RT-PCR identified A(2B)AR expression in distal colonic ganglia. EFS studies revealed that enteric neuronal A(2B)AR is essential for distal colonic relaxation, and A(2B)AR antagonists can inhibit relaxation. Enteric neurons stimulated with A(2B)AR agonists produced more nitrite than cultures treated with antagonists. We demonstrate an essential role of A(2B)AR in regulating distal colon relaxation, as A(2B)AR activation is linked to NO signaling. Hence targeting the colonic A(2B)AR could represent a novel therapeutic strategy to treat constipation.

Initiation of Parkinson's disease from gut to brain by δ-secretase.

Lewy pathology, composed of α-Synuclein (α-Syn) inclusions, a hallmark of Parkinson's disease (PD), progressively spreads from the enteric nervous system (ENS) to the central nervous system (CNS). However, it remains unclear how this process is regulated at a molecular level. Here we show that δ-secretase (asparagine endopeptidase, AEP) cleaves both α-Syn at N103 and Tau at N368, and mediates their fibrillization and retrograde propagation from the gut to the brain, triggering nigra dopaminergic neuronal loss associated with Lewy bodies and motor dysfunction. α-Syn N103 and Tau N368 robustly interact with each other and are highly elevated in PD patients' gut and brain. Chronic oral administration of the neurotoxin rotenone induces AEP activation and α-Syn N103/Tau N368 complex formation in the gut, eliciting constipation and dopaminergic neuronal death in an AEP-dependent manner. Preformed fibrils (PFFs) of α-Syn N103/Tau N368 are more neurotoxic and compact, and aggregate more quickly along the vagus nerve than their FL/FL counterparts or the individual fragments' fibrils. Colonic injection of PFFs induces PD pathologies, motor dysfunctions, and cognitive impairments. Thus, δ-secretase plays a crucial role in initiating PD pathology progression from the ENS to the CNS.

Gut-Resident Lactobacilli Activate Hepatic Nrf2 and Protect Against Oxidative Liver Injury.

Many studies have suggested a role for gut-resident microbes (the "gut microbiome") in modulating host health; however, the mechanisms by which they impact systemic physiology remain largely unknown. In this study, metabolomic and transcriptional profiling of germ-free and conventionalized mouse liver revealed an upregulation of the Nrf2 antioxidant and xenobiotic response in microbiome-replete animals. Using a Drosophila-based screening assay, we identified members of the genus Lactobacillus capable of stimulating Nrf2. Indeed, the human commensal Lactobacillus rhamnosus GG (LGG) potently activated Nrf2 in the Drosophila liver analog and the murine liver. This activation was sufficient to protect against two models of oxidative liver injury, acetaminophen overdose and acute ethanol toxicity. Characterization of the portal circulation of LGG-treated mice by tandem mass spectrometry identified a small molecule activator of Nrf2, 5-methoxyindoleacetic acid, produced by LGG. Taken together, these data demonstrate a mechanism by which intestinal microbes modulate hepatic susceptibility to oxidative injury.

Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis.

BACKGROUND & AIMS: The intestinal epithelium must be resilient to physiochemical stress to uphold the physiological barrier separating the systemic compartment from the microbial and antigenic components of the gut lumen. Identifying proteins that mediate protection and enhancing their expression is therefore a clear approach to promote intestinal health. We previously reported that oral ingestion of the probiotic Lactobacillus rhamnosus GG not only induced the expression of several recognized cytoprotective factors in the murine colon, but also many genes with no previously described function, including the gene encoding proline-rich acidic protein 1 (PRAP1). PRAP1 is a highly expressed protein in the epithelium of the gastrointestinal tract and we sought to define its function in this tissue. METHODS: Purified preparations of recombinant PRAP1 were analyzed biochemically and PRAP1 antisera were used to visualize localization in tissues. Prap1-/- mice were characterized at baseline and challenged with total body irradiation, then enteroids were generated to recapitulate the irradiation challenge ex vivo. RESULTS: PRAP1 is a 17-kilodalton intrinsically disordered protein with no recognizable sequence homology. PRAP1 expression levels were high in the epithelia of the small intestine. Although Prap1-/- mice presented only mild phenotypes at baseline, they were highly susceptible to intestinal injury upon challenge. After irradiation, the Prap1-/- mice showed accelerated death with a significant increase in apoptosis and p21 expression in the small intestinal epithelium. CONCLUSIONS: PRAP1 is an intrinsically disordered protein highly expressed by the gastrointestinal epithelium and functions at exposed surfaces to protect the barrier from oxidative insult.

Hydro-Cy3-Mediated Detection of Reactive Oxygen Species In Vitro and In Vivo.

Reactive oxygen species (ROS) are potent signaling molecules with critical roles in cellular pathology and homeostasis. They are produced in all cell types via a diverse array of cellular machinery, giving rise to an equally diverse repertoire of molecular effects. These range from cytotoxic killing of microbes to alteration of the cellular transcriptional response to stress. Despite their importance, research into ROS has been difficult given their inherent instability and transient signaling properties. Herein we describe methods for the use of the redox-sensitive probe hydro-Cy3 for the detection and quantification of ROS both in vitro and in vivo.

Extra-mitochondrial Cu/Zn superoxide dismutase (Sod1) is dispensable for protection against oxidative stress but mediates peroxide signaling in Saccharomyces cerevisiae.

Cu/Zn Superoxide Dismutase (Sod1) is a highly conserved and abundant metalloenzyme that catalyzes the disproportionation of superoxide radicals into hydrogen peroxide and molecular oxygen. As a consequence, Sod1 serves dual roles in oxidative stress protection and redox signaling by both scavenging cytotoxic superoxide radicals and producing hydrogen peroxide that can be used to oxidize and regulate the activity of downstream targets. However, the relative contributions of Sod1 to protection against oxidative stress and redox signaling are poorly understood. Using the model unicellular eukaryote, Baker's yeast, we found that only a small fraction of the total Sod1 pool is required for protection against superoxide toxicity and that this pool is localized to the mitochondrial intermembrane space. On the contrary, we find that much larger amounts of extra-mitochondrial Sod1 are critical for peroxide-mediated redox signaling. Altogether, our results force the re-evaluation of the physiological role of bulk Sod1 in redox biology; namely, we propose that the vast majority of Sod1 in yeast is utilized for peroxide-mediated signaling rather than superoxide scavenging.

Tumor necrosis factor-neuropeptide Y cross talk regulates inflammation, epithelial barrier functions, and colonic motility.

BACKGROUND: Neuro-immune interactions play a significant role in regulating the severity of inflammation. Our previous work demonstrated that neuropeptide Y (NPY) is upregulated in the enteric nervous system during murine colitis and that NPY knockout mice exhibit reduced inflammation. Here, we investigated if NPY expression during inflammation is induced by tumor necrosis factor (TNF), the main proinflammatory cytokine. METHODS: Using primary enteric neurons and colon explant cultures from wild type and NPY knockout (NPY(-/-)) mice, we determined if NPY knockdown modulates TNF release and epithelial permeability. Further, we assessed if NPY expression is inducible by TNF in enteric neuronal cells and mouse model of experimental colitis, using the TNF inhibitors-etanercept (blocks transmembrane and soluble TNF) and XPro1595 (blocks soluble TNF only). RESULTS: We found that enteric neurons express TNF receptors (TNFR1 and R2). Primary enteric neurons from NPY(-/-) mice produced less TNF compared with wild type. Further, TNF activated NPY promoter in enteric neurons through phospho-c-Jun. NPY(-/-) mice had decreased intestinal permeability. In vitro, NPY increased epithelial permeability through phosphatidyl inositol-3-kinase (PI3-K)-induced pore-forming claudin-2. TNF inhibitors attenuated NPY expression in vitro and in vivo. TNF inhibitor-treated colitic mice exhibited reduced NPY expression and inflammation, reduced oxidative stress, enhanced neuronal survival, and improved colonic motility. XPro1595 had more protective effects on neuronal survival and motility compared with etanercept. CONCLUSIONS: We demonstrate a novel TNF-NPY cross talk that modulates inflammation, barrier functions, and colonic motility during inflammation. It is also suggested that selective blocking of soluble TNF may be a better therapeutic option than using anti-TNF antibodies.

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis.

BACKGROUND: Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis. METHODS: Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY(-/-)) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice. RESULTS: DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY(-/-) as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS(-/-) and NPY(-/-)/nNOS(-/-) mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY -treated rat enteric neurons in vitro exhibited increased nitrite and TNF-alpha production. CONCLUSIONS: NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.

Glial-derived neurotrophic factor modulates enteric neuronal survival and proliferation through neuropeptide Y.

BACKGROUND & AIMS: Glial-derived neurotrophic factor (GDNF) promotes the survival and proliferation of enteric neurons. Neuropeptide Y (NPY) is an important peptide regulating gastrointestinal motility. The role of NPY on the survival and proliferation of enteric neurons is not known. We examined the effects of GDNF on the expression and release of NPY from enteric neurons and the role of NPY in promoting enteric neuronal proliferation and survival. METHODS: Studies were performed in primary enteric neuronal cultures and NPY knockout mice (NPY(-/-)). GDNF-induced expression of NPY was assessed by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay. Using NPY-siRNA and NPY-Y1 receptor antagonist, we examined the role of NPY in mediating the survival and proliferation effects of GDNF. Gastrointestinal motility was assessed by measuring gastric emptying, intestinal transit, and isometric muscle recording from intestinal muscle strips. RESULTS: GDNF induced a significant increase in NPY messenger RNA and protein expression in primary enteric neurons and the release of NPY into the culture medium. NPY (1 mumol/L) significantly increased proliferation of neurons and reduced apoptosis. In the presence of NPY-siRNA and NPY-Y1 receptor antagonist or in enteric neurons cultured from NPY(-/-) mice, GDNF-mediated neuronal proliferation and survival was reduced. NPY increased the phosphorylation of Akt, a downstream target of the PI-3-kinase pathway. In NPY(-/-) mice, there were significantly fewer nNOS-containing enteric neurons compared with wild-type (WT) mice. NPY(-/-) mice had accelerated gastric emptying and delayed intestinal transit compared with WT mice. CONCLUSIONS: We demonstrate that NPY acts as an autocrine neurotrophic factor for enteric neurons.