Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers

Scientific reports

2021 Sep 07

Lee, HJ;Myung, JK;Kim, HS;Lee, DH;Go, HS;Choi, JH;Koh, HM;Lee, SJ;Jang, B;
PMID: 34493772 | DOI: 10.1038/s41598-021-97351-y

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

Cannabinoid CB2 receptors are expressed in glutamate neurons in the red nucleus and functionally modulate motor behavior in mice

Neuropharmacology

2021 Mar 28

Zhang, HY;Shen, H;Gao, M;Ma, Z;Hempel, B;Bi, GH;Gardner, EL;Wu, J;Xi, ZX;
PMID: 33789118 | DOI: 10.1016/j.neuropharm.2021.108538

Cannabinoids produce a number of central nervous system effects via the CB2 receptor (CB2R), including analgesia, antianxiety, anti-reward, hypoactivity and attenuation of opioid-induced respiratory depression. However, the cellular distributions of the CB2Rs in the brain remain unclear. We have reported that CB2Rs are expressed in midbrain dopamine (DA) neurons and functionally regulate DA-mediated behavior(s). Unexpectedly, high densities of CB2-like signaling were also found in a neighboring motor structure - the red nucleus (RN) of the midbrain. In the present study, we systematically explored CB2R expression and function in the RN. Immunohistochemistry and in situ hybridization assays showed high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in wildtype and CB1-knockout, but not CB2-knockout, mice. Ex vivo electrophysiological recordings in midbrain slices demonstrated that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in wildtype, but not CB2-knockout, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. In addition, microinjection of JWH133 into the RN produced robust ipsilateral rotations in wildtype, but not CB2-knockout mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Finally, fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the ventral tegmental area, nucleus accumbens, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders.

IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies

Mucosal immunology

2022 Aug 24

Yamazaki, S;Inohara, N;Ohmuraya, M;Tsuneoka, Y;Yagita, H;Katagiri, T;Nishina, T;Mikami, T;Funato, H;Araki, K;Nakano, H;
PMID: 35999460 | DOI: 10.1038/s41385-022-00554-3

Control of gut microbes is crucial for not only local defense in the intestine but also proper systemic immune responses. Although intestinal epithelial cells (IECs) play important roles in cytokine-mediated control of enterobacteria, the underlying mechanisms are not fully understood. Here we show that deletion of IκBζ in IECs in mice leads to dysbiosis with marked expansion of segmented filamentous bacteria (SFB), thereby enhancing Th17 cell development and exacerbating inflammatory diseases. Mechanistically, the IκBζ deficiency results in decrease in the number of Paneth cells and impairment in expression of IL-17-inducible genes involved in IgA production. The decrease in Paneth cells is caused by aberrant activation of IFN-γ signaling and a failure of IL-17-dependent recovery from IFN-γ-induced damage. Thus, the IL-17R-IκBζ axis in IECs contributes to the maintenance of intestinal homeostasis by serving as a key component in a regulatory loop between the gut microbiota and immune cells.

Dopamine Neurons That Cotransmit Glutamate, From Synapses to Circuits to Behavior

Frontiers in Neural Circuits

2021 May 19

Eskenazi, D;Malave, L;Mingote, S;Yetnikoff, L;Ztaou, S;Velicu, V;Rayport, S;Chuhma, N;
| DOI: 10.3389/fncir.2021.665386

Discovered just over 20 years ago, dopamine neurons have the ability to cotransmit both dopamine and glutamate. Yet, the functional roles of dopamine neuron glutamate cotransmission and their implications for therapeutic use are just emerging. This review article encompasses the current body of evidence investigating the functions of dopamine neurons of the ventral midbrain that cotransmit glutamate. Since its discovery in dopamine neuron cultures, further work in vivo confirmed dopamine neuron glutamate cotransmission across species. From there, growing interest has led to research related to neural functioning including roles in synaptic signaling, development, and behavior. Functional connectome mapping reveals robust connections in multiple forebrain regions to various cell types, most notably to cholinergic interneurons in both the medial shell of the nucleus accumbens and the lateral dorsal striatum. Glutamate markers in dopamine neurons reach peak levels during embryonic development and increase in response to various toxins, suggesting dopamine neuron glutamate cotransmission may serve neuroprotective roles. Findings from behavioral analyses reveal prominent roles for dopamine neuron glutamate cotransmission in responses to psychostimulants, in positive valence and cognitive systems and for subtle roles in negative valence systems. Insight into dopamine neuron glutamate cotransmission informs the pathophysiology of neuropsychiatric disorders such as addiction, schizophrenia and Parkinson Disease, with therapeutic implications.

USP7 inactivation suppresses APC-mutant intestinal hyperproliferation and tumor development

Stem cell reports

2022 Dec 29

Novellasdemunt, L;Kucharska, A;Baulies, A;Hutton, C;Vlachogiannis, G;Repana, D;Rowan, A;Suárez-Bonnet, A;Ciccarelli, F;Valeri, N;Li, VSW;
PMID: 36669491 | DOI: 10.1016/j.stemcr.2022.12.013

Adenomatous polyposis coli (APC) mutation is the hallmark of colorectal cancer (CRC), resulting in constitutive WNT activation. Despite decades of research, targeting WNT signaling in cancer remains challenging due to its on-target toxicity. We have previously shown that the deubiquitinating enzyme USP7 is a tumor-specific WNT activator in APC-truncated cells by deubiquitinating and stabilizing β-catenin, but its role in gut tumorigenesis is unknown. Here, we show in vivo that deletion of Usp7 in Apc-truncated mice inhibits crypt hyperproliferation and intestinal tumor development. Loss of Usp7 prolongs the survival of the sporadic intestinal tumor model. Genetic deletion, but not pharmacological inhibition, of Usp7 in Apc+/- intestine induces colitis and enteritis. USP7 inhibitor treatment suppresses growth of patient-derived cancer organoids carrying APC truncations in vitro and in xenografts. Our findings provide direct evidence that USP7 inhibition may offer a safe and efficacious tumor-specific therapy for both sporadic and germline APC-mutated CRC.

CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis

The Journal of pathology

2022 Nov 14

Cui, Y;Wu, H;Liu, Z;Ma, T;Liang, W;Zeng, Q;Chen, D;Qin, Q;Huang, B;Wang, MH;Huang, X;He, Y;Kuang, Y;Sugimoto, S;Sato, T;Wang, L;
PMID: 36373877 | DOI: 10.1002/path.6031

Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease and so far, there is no specific targeted therapy. Here, we report that CXCL16 is up-regulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis, and meanwhile to transcriptionally modulate the levels of BMP4 and HGF in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE, and suggest that CXCL16 signaling could be a potential therapeutic target for RE. This article is protected by

Brainstem ADCYAP1+ neurons control multiple aspects of sickness behaviour

Nature

2022 Sep 01

Ilanges, A;Shiao, R;Shaked, J;Luo, JD;Yu, X;Friedman, JM;
PMID: 36071158 | DOI: 10.1038/s41586-022-05161-7

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells

Molecular nutrition & food research

2022 Oct 01

May, S;Greenow, KR;Higgins, AT;Derrick, AV;Taylor, E;Pan, P;Konstantinou, M;Nixon, C;Wooley, TE;Sansom, OJ;Wang, LS;Parry, L;
PMID: 36045438 | DOI: 10.1002/mnfr.202200234

Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs.Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel.BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

Whole-brain monosynaptic inputs and outputs of leptin receptor b neurons of the nucleus tractus solitarii in mice

Brain research bulletin

2023 Jun 20

Sun, L;Zhu, M;Wang, M;Hao, Y;Hao, Y;Jing, X;Yu, H;Shi, Y;Zhang, X;Wang, S;Yuan, F;Yuan, XS;
PMID: 37348822 | DOI: 10.1016/j.brainresbull.2023.110693

The nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation-i.e., the ventrolateral periaqueductal gray and locus coeruleus-also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.

Glutamatergic PPT neurons control wakefulness and locomotion via distinct axonal projections

Sleep

2022 Sep 28

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.

Green light analgesia in mice is mediated by visual activation of enkephalinergic neurons in the ventrolateral geniculate nucleus

Science translational medicine

2022 Dec 07

Tang, YL;Liu, AL;Lv, SS;Zhou, ZR;Cao, H;Weng, SJ;Zhang, YQ;
PMID: 36475906 | DOI: 10.1126/scitranslmed.abq6474

Green light exposure has been shown to reduce pain in animal models. Here, we report a vision-associated enkephalinergic neural circuit responsible for green light-mediated analgesia. Full-field green light exposure at an intensity of 10 lux produced analgesic effects in healthy mice and in a model of arthrosis. Ablation of cone photoreceptors completely inhibited the analgesic effect, whereas rod ablation only partially reduced pain relief. The analgesic effect was not modulated by the ablation of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are atypical photoreceptors that control various nonvisual effects of light. Inhibition of the retino-ventrolateral geniculate nucleus (vLGN) pathway completely abolished the analgesic effects. Activation of this pathway reduced nociceptive behavioral responses; such activation was blocked by the inhibition of proenkephalin (Penk)-positive neurons in the vLGN (vLGNPenk). Moreover, green light analgesia was prevented by knockdown of Penk in the vLGN or by ablation of vLGNPenk neurons. In addition, activation of the projections from vLGNPenk neurons to the dorsal raphe nucleus (DRN) was sufficient to suppress nociceptive behaviors, whereas its inhibition abolished the green light analgesia. Our findings indicate that cone-dominated retinal inputs mediated green light analgesia through the vLGNPenk-DRN pathway and suggest that this signaling pathway could be exploited for reducing pain.

The circadian clock gene, Bmal1, regulates intestinal stem cell signaling and represses tumor initiation

Cellular and molecular gastroenterology and hepatology

2021 Sep 14

Stokes, K;Nunes, M;Trombley, C;Flôres, DEFL;Wu, G;Taleb, Z;Alkhateeb, A;Banskota, S;Harris, C;Love, OP;Khan, WI;Rueda, L;Hogenesch, JB;Karpowicz, P;
PMID: 34534703 | DOI: 10.1016/j.jcmgh.2021.08.001

Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
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tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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