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.
Frontiers in molecular neuroscience
Koga, K;Kobayashi, K;Tsuda, M;Kubota, K;Kitano, Y;Furue, H;
PMID: 37033377 | DOI: 10.3389/fnmol.2023.1099925
Neuropathic pain, an intractable pain symptom that occurs after nerve damage, is caused by the aberrant excitability of spinal dorsal horn (SDH) neurons. Gabapentinoids, the most commonly used drugs for neuropathic pain, inhibit spinal calcium-mediated neurotransmitter release by binding to α2δ-1, a subunit of voltage-gated calcium channels, and alleviate neuropathic pain. However, the exact contribution of α2δ-1 expressed in SDH neurons to the altered synaptic transmission and mechanical hypersensitivity following nerve injury is not fully understood. In this study, we investigated which types of SDH neurons express α2δ-1 and how α2δ-1 in SDH neurons contributes to the mechanical hypersensitivity and altered spinal synaptic transmission after nerve injury. Using in situ hybridization technique, we found that Cacna2d1, mRNA coding α2δ-1, was mainly colocalized with Slc17a6, an excitatory neuronal marker, but not with Slc32a1, an inhibitory neuronal marker in the SDH. To investigate the role of α2δ-1 in SDH neurons, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and showed that SDH neuron-specific ablation of Cacna2d1 alleviated mechanical hypersensitivity following nerve injury. We further found that excitatory post-synaptic responses evoked by electrical stimulation applied to the SDH were significantly enhanced after nerve injury, and that these enhanced responses were significantly decreased by application of mirogabalin, a potent α2δ-1 inhibitor, and by SDH neuron-specific ablation of Cacna2d1. These results suggest that α2δ-1 expressed in SDH excitatory neurons facilitates spinal nociceptive synaptic transmission and contributes to the development of mechanical hypersensitivity after nerve injury.
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.
Frontiers in neuroscience
Liu, A;Cheng, Y;Huang, J;
PMID: 37214399 | DOI: 10.3389/fnins.2023.1178693
Mammals are frequently exposed to various environmental stimuli, and to determine whether to approach or avoid these stimuli, the brain must assign emotional valence to them. Therefore, it is crucial to investigate the neural circuitry mechanisms involved in the mammalian brain's processing of emotional valence. Although the central amygdala (CeA) and the ventral tegmental area (VTA) individually encode different or even opposing emotional valences, it is unclear whether there are common upstream input neurons that innervate and control both these regions, and it is interesting to know what emotional valences of these common upstream neurons. In this study, we identify three major brain regions containing neurons that project to both the CeA and the VTA, including the posterior bed nucleus of the stria terminalis (pBNST), the pedunculopontine tegmental nucleus (PPTg), and the anterior part of the basomedial amygdala (BMA). We discover that these neural populations encode distinct emotional valences. Activating neurons in the pBNST produces positive valence, enabling mice to overcome their innate avoidance behavior. Conversely, activating neurons in the PPTg produces negative valence and induces anxiety-like behaviors in mice. Neuronal activity in the BMA, on the other hand, does not influence valence processing. Thus, our study has discovered three neural populations that project to both the CeA and the VTA and has revealed the distinct emotional valences these populations encode. These results provide new insights into the neurological mechanisms involved in emotional regulation.
Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
Lim, HYG;Yada, S;Barker, N;
PMID: 35620071 | DOI: 10.1016/j.xpro.2022.101411
Intestinal cells marked by Lgr5 function as tissue-resident stem cells that sustain the homeostatic replenishment of the epithelium. By incorporating a diphtheria toxin receptor (DTR) cassette linked to the Lgr5 coding region, native Lgr5-expressing cells are susceptible to ablation upon DT administration in vivo. A similar strategy can be used for Lgr5-expressing cells within organoids established from DTR models. Together, these in vivo and in vitro approaches will facilitate dissection of the roles of Lgr5-expressing cells residing in different tissue compartments. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021).
Topilko, T;Diaz, SL;Pacheco, CM;Verny, F;Rousseau, CV;Kirst, C;Deleuze, C;Gaspar, P;Renier, N;
PMID: 35123655 | DOI: 10.1016/j.neuron.2022.01.012
Optimizing reproductive fitness in mammalians requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Our study deciphers the role of midbrain CART+ neurons in behavioral adaptations during pregnancy vital for reproductive fitness.
Gerling M, Büller NV, Kirn LM, Joost S, Frings O, Englert B, Bergström Å, Kuiper RV, Blaas L, Wielenga MC, Almer S, Kühl AA, Fredlund E, van den Brink GR, Toftgård R.
PMID: 27492255 | DOI: 10.1038/ncomms12321
A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor.
Leushacke M, Tan SH, Wong A, Swathi Y, Hajamohideen A, Tan LT, Goh J, Wong E, Denil SLIJ, Murakami K, Barker N.
PMID: 28581476 | DOI: 10.1038/ncb3541
The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and 'reserve' stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.
Biochemical and Biophysical Research Communications
Choi Y, Park J, Ko YS, Kim Y, Pyo JS, Jange BG, Kim MA, Leef JS, Chang MS, Lee BL.
PMID: - | DOI: 10.1016/j.bbrc.2017.09.163
Gastric cancer (GC) is a major of cause of cancer-related death and is characterized by its heterogeneity and molecular complexity. FOXO1 is a transcription factor that plays a key role in GC growth and metastasis. However, the implication of FOXO1 in GC cell stemness has been elusive. This study, for the first time, demonstrates that FOXO1 regulates GC cell stemness in association with LGR5. FOXO1 expression was significantly lower in GC tumorsphere cells than in adherent GC cells. FOXO1 silencing and overexpression promoted and inhibited the tumorsphere formation capacity of GC cells, respectively. Additionally, there was an inverse correlation between FOXO1 and GC stem cell marker LGR5 in human GC specimens. Further in vitro and in vivo experiments showed that negative crosstalk between these two molecules exists and that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation even without FOXO1 restoration. Taken together, our results suggest that FOXO1 inhibits the self-renewal capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC.
