Helicobacter pylori Activate and Expand Lgr5+ Stem Cells Through Direct Colonization of the Gastric Glands (check out Movie S4 when it gets out)
Gastroenterology. 2015 Feb 25.
Sigal M, Rothenberg ME, Logan CY, Lee JY, Honaker RW, Cooper RL, Passarelli B, Camorlinga M, Bouley DM, Alvarez G, Nusse R, Torres J, Amieva MR
Background & Aims Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. Methods We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from Lgr5+ stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. Results H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5+ stem cell proliferation, and upregulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. Moreover, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. Conclusions H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.
Xu J, Molinas AJR, Mukerjee S, Morgan DA, Rahmouni K, Zsombok A, Lazartigues E.
PMID: 31006330 | DOI: 10.1161/HYPERTENSIONAHA.119.12832
Chronic activation of the brain renin-angiotensin system contributes to the development of hypertension by altering autonomic balance. Beyond the essential role of Ang II (angiotensin II) type 1 receptors, ADAM17 (A disintegrin and metalloprotease 17) is also found to promote brain renin-angiotensin system overactivation. ADAM17 is robustly expressed in various cell types within the central nervous system. The aim of this study was to determine whether ADAM17 modulates presympathetic neuronal activity to promote autonomic dysregulation in salt-sensitive hypertension. To test our hypothesis, ADAM17 was selectively knocked down in glutamatergic neurons using Cre-loxP technology. In mice lacking ADAM17 in glutamatergic neurons, the blood pressure increase induced by deoxycorticosterone acetate-salt treatment was blunted. Deoxycorticosterone acetate-salt significantly elevated cardiac and vascular sympathetic drive in control mice, while such effects were reduced in mice with ADAM17 knockdown. This blunted sympathoexcitation was extended to the spleen, with a lesser activation of the peripheral immune system, translating into a sequestration of circulating T cells within this organ, compared with controls. Within the paraventricular nucleus, Ang II-induced activation of kidney-related presympathetic glutamatergic neurons was reduced in ADAM17 knockdown mice, with the majority of cells no longer responding to Ang II stimulation, confirming the supportive role of ADAM17 in increasing presympathetic neuronal activity. Overall, our data highlight the pivotal role of neuronal ADAM17 in regulating sympathetic activity and demonstrate that activation of ADAM17 in glutamatergic neurons leads to a selective increase of sympathetic output, but not vagal tone, to specific organs, ultimately contributing to dysautonomia and salt-sensitive hypertension.
Cell Mol Gastroenterol Hepatol.
Meijer BJ1, Giugliano FP, Baan B, van der Meer JHM, Meisner S, van Roest M, Koelink PJ, de Boer RJ, Jones N, Breitwieser W, van der Wel NN, Wildenberg ME, van den Brink GR, Heijmans J, Muncan V
PMID: 31958521 | DOI: 10.1016/j.jcmgh.2020.01.005
BACKGROUND & AIMS:
Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model.
METHODS:
We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage.
RESULTS:
Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-?-induced damage.
CONCLUSIONS:
ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.
Cellular and molecular gastroenterology and hepatology
Li, C;Zhou, Y;Wei, R;Napier, DL;Sengoku, T;Alstott, MC;Liu, J;Wang, C;Zaytseva, YY;Weiss, HL;Wang, Q;Evers, BM;
PMID: 36584817 | DOI: 10.1016/j.jcmgh.2022.12.012
The Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined.Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 MAPK, the transcription factor atonal homolog 1 (ATOH1), and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by western blot, qPCR or IF and IHC staining.HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout (KO) or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 KO resulted in activation of p38 MAPK and increased expression of ATOH1; inhibition of p38 MAPK signaling attenuated the phenotypes induced by HK2 KO in intestinal organoids. HK2 KO significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 KO.Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 MAPK/ATOH1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.
Kroeger, D;Thundercliffe, J;Phung, A;De Luca, R;Geraci, C;Bragg, S;McCafferty, KJ;Bandaru, SS;Arrigoni, E;Scammell, TE;
PMID: 36170177 | DOI: 10.1093/sleep/zsac242
The pedunculopontine tegmental nucleus (PPT) is implicated in many brain functions, ranging from sleep/wake control and locomotion, to reward mechanisms and learning. The PPT contains cholinergic, GABAergic and glutamatergic neurons with extensive ascending and descending axonal projections. Glutamatergic PPT (PPT vGlut2) neurons are thought to promote wakefulness, but the mechanisms through which this occurs are unknown. In addition, some researchers propose that PPT vGlut2 neurons promote locomotion, yet even though the PPT is a target for deep brain stimulation in Parkinson's disease, the role of the PPT in locomotion is debated. We hypothesized that PPT vGluT2 neurons drive arousal and specific waking behaviors via certain projections and modulate locomotion via others.We mapped the axonal projections of PPT vGlut2 neurons using conditional anterograde tracing and then photostimulated PPT vGlut2 soma or their axon terminal fields across sleep/wake states and analyzed sleep/wake behavior, muscle activity, and locomotion in transgenic mice.We found that stimulation of PPT vGlut2 soma and their axon terminals rapidly triggered arousals from NREM sleep, especially with activation of terminals in the basal forebrain (BF) and lateral hypothalamus (LH). With photoactivation of PPT vGlut2 terminals in the BF and LH, this wakefulness was accompanied by locomotion and other active behaviors, but stimulation of PPT vGlut2 soma and terminals in the substantia nigra triggered only quiet wakefulness without locomotion.These findings demonstrate the importance of the PPT vGluT2 neurons in driving various aspects of arousal and show that heterogeneous brain nuclei, such as the PPT, can promote a variety of behaviors via distinct axonal projections.
VGLUT2 is a determinant of dopamine neuron resilience in a rotenone model of dopamine neurodegeneration
The Journal of neuroscience : the official journal of the Society for Neuroscience
Buck, SA;Miranda, BR;Logan, RW;Fish, KN;Greenamyre, JT;Freyberg, Z;
PMID: 33893220 | DOI: 10.1523/JNEUROSCI.2770-20.2021
Parkinson's disease (PD) is characterized by progressive dopamine (DA) neuron loss in the substantia nigra pars compacta (SNc). In contrast, DA neurons in the ventral tegmental area (VTA) are relatively protected from neurodegeneration, but the underlying mechanisms for this resilience remain poorly understood. Recent work suggests that expression of the vesicular glutamate transporter 2 (VGLUT2) selectively impacts midbrain DA neuron vulnerability. We investigated whether altered DA neuron VGLUT2 expression determines neuronal resilience in rats exposed to rotenone, a mitochondrial complex I inhibitor and toxicant model of PD. We discovered that VTA/SNc DA neurons that expressed VGLUT2 are more resilient to rotenone-induced DA neurodegeneration. Surprisingly, the density of neurons with detectable VGLUT2 expression in the VTA and SNc increases in response to rotenone. Furthermore, dopaminergic terminals within the nucleus accumbens, where the majority of VGLUT2-expressing DA neurons project, exhibit greater resilience compared to DA terminals in the caudate/putamen. More broadly, VGLUT2-expressing terminals are protected throughout the striatum from rotenone-induced degeneration. Together, our data demonstrate that a distinct subpopulation of VGLUT2-expressing DA neurons are relatively protected from rotenone neurotoxicity. Rotenone-induced upregulation of the glutamatergic machinery in VTA and SNc neurons and their projections may be part of a broader neuroprotective mechanism. These findings offer a putative new target for neuronal resilience that can be manipulated to prevent toxicant-induced DA neurodegeneration in PD.SIGNIFICANCE STATEMENT:Environmental exposures to pesticides contribute significantly to pathological processes that culminate in Parkinson's disease (PD). The pesticide rotenone has been used to generate a PD model that replicates key features of the illness including dopamine neurodegeneration. To date, longstanding questions remain: are there dopamine neuron subpopulations resilient to rotenone, and if so, what are the molecular determinants of this resilience? Here we show that the subpopulation of midbrain dopaminergic neurons that express the vesicular glutamate transporter 2 (VGLUT2) are more resilient to rotenone-induced neurodegeneration. Rotenone also upregulates VGLUT2 more broadly in the midbrain, suggesting VGLUT2 expression generally confers increased resilience to rotenone. VGLUT2 may therefore be a new target for boosting neuronal resilience to prevent toxicant-induced DA neurodegeneration in PD.
Cloft, S;Uni, Z;Wong, E;
| DOI: 10.1016/j.psj.2023.102495
Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5 and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.
Stanisavljević L, Myklebust MP, Leh S, Dahl O.
PMID: 27435662 | DOI: 10.1080/0284186X.2016.1201215
Abstract
BACKGROUND:
Expression of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) gene is associated with a metastatic phenotype and poor prognosis in colorectal cancer (CRC). CD133 expression is a putative cancer stem cell marker and a proposed prognostic marker in CRC, whereas the predictive value of CD133 expression for effect of adjuvant chemotherapy in CRC is unclear.
MATERIAL AND METHODS:
For the study of LGR5 mRNA and CD133 expression, tissue microarrays from 409 primary CRC stage II and III tumors, where patients had been randomized to adjuvant chemotherapy or surgery only, were available. LGR5 mRNA and CD133 expression were assessed by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. LGR5 mRNA and CD133 expression as prognostic and predictive markers were evaluated by univariate and multivariate analyses.
RESULTS:
For all CRC patients, positive LGR5 mRNA and CD133 expression were associated with classic adenocarcinoma histology type (p = 0.001 and p = 0.014, respectively). Positive LGR5 mRNA expression was also associated with smaller tumor diameter for CRC stage II (p = 0.005), but not for CRC stage III (p = 0.054). For CRC stage II, lack of LGR5 mRNA expression was associated with longer time to recurrence (TTR) in Kaplan-Meier (p = 0.045) and in multivariate Cox analysis (HR 0.27, 95% CI 0.08-0.95, p = 0.041). For colon cancer stage III patients, lack of CD133 expression was associated with better effect of adjuvant chemotherapy (p = 0.016) in Kaplan-Meier univariate analysis, but the interaction between CD133 and adjuvant chemotherapy was not statistically significant in multivariate analysis (HR 0.59, 95% CI 0.18-1.89, p = 0.374).
CONCLUSION:
LGR5 mRNA expression is a prognostic factor for CRC stage II patients, whereas the value of CD133 expression as prognostic and predictive biomarker is inconclusive.
Journal of chemical neuroanatomy
Beebe, NL;Silveira, MA;Goyer, D;Noftz, WA;Roberts, MT;Schofield, BR;
PMID: 36375740 | DOI: 10.1016/j.jchemneu.2022.102189
Neurons in the inferior colliculus (IC), the midbrain hub of the central auditory pathway, send ascending and descending projections to other auditory brain regions, as well as projections to other sensory and non-sensory brain regions. However, the axonal projection patterns of individual classes of IC neurons remain largely unknown. Vasoactive intestinal polypeptide (VIP) is a neuropeptide expressed by subsets of neurons in many brain regions. We recently identified a class of IC stellate neurons that we called VIP neurons because they are labeled by tdTomato (tdT) expression in VIP-IRES-Cre x Ai14 mice. Here, using fluorescence in situ hybridization, we found that tdT+ neurons in VIP-IRES-Cre x Ai14 mice express Vglut2, a marker of glutamatergic neurons, and VIP, suggesting that VIP neurons use both glutamatergic and VIPergic signaling to influence their postsynaptic targets. Next, using viral transfections with a Cre-dependent eGFP construct, we labeled the axonal projections of VIP neurons. As a group, VIP neurons project intrinsically, within the ipsilateral and contralateral IC, and extrinsically to all the major targets of the IC. Within the auditory system, VIP neurons sent axons and formed axonal boutons in higher centers, including the medial geniculate nucleus and the nucleus of the brachium of the IC. Less dense projections terminated in lower centers, including the nuclei of the lateral lemniscus, superior olivary complex, and dorsal cochlear nucleus. VIP neurons also project to several non-auditory brain regions, including the superior colliculus, periaqueductal gray, and cuneiform nucleus. The diversity of VIP projections compared to the homogeneity of VIP neuron intrinsic properties suggests that VIP neurons play a conserved role at the microcircuit level, likely involving neuromodulation through glutamatergic and VIPergic signaling, but support diverse functions at the systems level through their participation in different projection pathways.
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology
You, ZB;Galaj, E;Alén, F;Wang, B;Bi, GH;Moore, AR;Buck, T;Crissman, M;Pari, S;Xi, ZX;Leggio, L;Wise, RA;Gardner, EL;
PMID: 34923576 | DOI: 10.1038/s41386-021-01249-2
Cocaine addiction is a significant medical and public concern. Despite decades of research effort, development of pharmacotherapy for cocaine use disorder remains largely unsuccessful. This may be partially due to insufficient understanding of the complex biological mechanisms involved in the pathophysiology of this disorder. In the present study, we show that: (1) elevation of ghrelin by cocaine plays a critical role in maintenance of cocaine self-administration and cocaine-seeking motivated by cocaine-conditioned stimuli; (2) acquisition of cocaine-taking behavior is associated with the acquisition of stimulatory effects of cocaine by cocaine-conditioned stimuli on ghrelin secretion, and with an upregulation of ghrelin receptor mRNA levels in the ventral tegmental area (VTA); (3) blockade of ghrelin signaling by pretreatment with JMV2959, a selective ghrelin receptor antagonist, dose-dependently inhibits reinstatement of cocaine-seeking triggered by either cocaine or yohimbine in behaviorally extinguished animals with a history of cocaine self-administration; (4) JMV2959 pretreatment also inhibits brain stimulation reward (BSR) and cocaine-potentiated BSR maintained by optogenetic stimulation of VTA dopamine neurons in DAT-Cre mice; (5) blockade of peripheral adrenergic β1 receptors by atenolol potently attenuates the elevation in circulating ghrelin induced by cocaine and inhibits cocaine self-administration and cocaine reinstatement triggered by cocaine. These findings demonstrate that the endogenous ghrelin system plays an important role in cocaine-related addictive behaviors and suggest that manipulating and targeting this system may be viable for mitigating cocaine use disorder.
Cellular and Molecular Gastroenterology and Hepatology
Tsai YH, Hill DR, Kumar N, Huang S, Chin AM, Dye BR, Nagy MS, Verzi MP, Spence JR.
PMID: - | DOI: 10.1016/j.jcmgh.2016.06.002
Background & Aims
The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic of contexts in mice. However, the function ofLGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail.
Methods
We interrogated the function and expression of LGR family members using human pluripotent stem cell–derived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5–creER–eGFP mice.
Results
We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human–mouse species-specific differences at later time points of embryonic development.
Conclusions
Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.
Toxicol Sci. 2018 Oct 26.
Peters MF, Landry T, Pin C, Maratea K, Dick C, Wagoner MP, Choy AL, Barthlow H, Snow D, Stevens Z, Armento A, Scott CW, Ayehunie S.
PMID: 30364994 | DOI: 10.1093/toxsci/kfy268
Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro assays. Recent breakthroughs in in vitro culture methods support intestinal stem cell maintenance and continual differentiation into the epithelial cell types resident in the intestine. These diverse cells self-assemble into microtissues with in vivo-like architecture. Here, we evaluate human GI microtissues grown in transwell plates that allow apical and/or basolateral drug treatment and 96-well throughput. Evaluation of assay utility focused on predictivity for diarrhea since this adverse effect correlates with intestinal barrier dysfunction which can be measured in GI microtissues using transepithelial electrical resistance (TEER). A validation set of widely prescribed drugs was assembled and tested for effects on TEER. When the resulting TEER inhibition potencies were adjusted for clinical exposure, a threshold was identified that distinguished drugs that induced clinical diarrhea from those that lack this liability. Microtissue TEER assay predictivity was further challenged with a smaller set of drugs whose clinical development was limited by diarrhea that was unexpected based on one-month animal studies. Microtissue TEER accurately predicted diarrhea for each of these drugs. The label-free nature of TEER enabled repeated quantitation with sufficient precision to develop a mathematical model describing the temporal dynamics of barrier damage and recovery. This human 3D GI microtissue is the first in vitro assay with validated predictivity for diarrhea-inducing drugs. It should provide a platform for lead optimization and offers potential for dose schedule exploration.
