Costa, A;Ai, M;Nunn, N;Culotta, I;Hunter, J;Boudjadja, MB;Valencia-Torres, L;Aviello, G;Hodson, DJ;Snider, BM;Coskun, T;Emmerson, PJ;Luckman, SM;D'Agostino, G;
PMID: 34844019 | DOI: 10.1016/j.molmet.2021.101407
Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective medications to reduce appetite and body weight. These actions are centrally mediated, however the neuronal substrates involved are poorly understood.We employed a combination of neuroanatomical, genetic and behavioral approaches in the mouse to investigate the involvement of caudal brainstem cholecystokinin-expressing neurons in the effect of the GLP-1RA exendin-4. We further confirmed key neuroanatomical findings in the non-human primate brain.We found that cholecystokinin-expressing neurons in the caudal brainstem are required for the anorectic and body weight-lowering effects of GLP-1RAs, as well as for induction of GLP-1RA-induced conditioned taste avoidance. We further show that, while cholecystokinin-expressing neurons are not a direct target for glucose-dependent insulinotropic peptide (GIP), GIP receptor activation results in a reduced recruitment of these GLP-1RA-responsive neurons and a selective reduction of conditioned taste avoidance.In addition to disclosing a neuronal population that is necessary for the full appetite- and body weight-lowering effect of GLP-1RAs, our data also provide a novel framework for understanding and ameliorating GLP-1RA-induced nausea - a major factor for withdrawal from treatment.
Long-term functional alterations following prenatal GLP-1R activation
Neurotoxicology and teratology
Graham, DL;Madkour, HS;Noble, BL;Schatschneider, C;Stanwood, GD;
PMID: 33864929 | DOI: 10.1016/j.ntt.2021.106984
Evidence supporting the use of glucagon-like peptide-1 (GLP-1) analogues to pharmacologically treat disorders beyond type 2 diabetes and obesity is increasing. However, little is known about how activation of the GLP-1 receptor (GLP-1R) during pregnancy affects maternal and offspring outcomes. We treated female C57Bl/6 J mice prior to conception and throughout gestation with a long-lasting GLP-1R agonist, Exendin-4. While GLP-1R activation has significant effects on food and drug reward, depression, locomotor activity, and cognition in adults, we found few changes in these domains in exendin-4-exposed offspring. Repeated injections of Exendin-4 had minimal effects on the dams and may have enhanced maternal care. Offspring exposed to the drug weighed significantly more than their control counterparts during the preweaning period and demonstrated alterations in anxiety-like outcomes, which indicate a developmental role for GLP-1R modulation in the stress response that may be sex-specific.
A genetic map of the mouse dorsal vagal complex and its role in obesity
Ludwig, MQ;Cheng, W;Gordian, D;Lee, J;Paulsen, SJ;Hansen, SN;Egerod, KL;Barkholt, P;Rhodes, CJ;Secher, A;Knudsen, LB;Pyke, C;Myers, MG;Pers, TH;
PMID: 33767443 | DOI: 10.1038/s42255-021-00363-1
The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.
Lowenstein, ED;Ruffault, PL;Misios, A;Osman, KL;Li, H;Greenberg, RS;Thompson, R;Song, K;Dietrich, S;Li, X;Vladimirov, N;Woehler, A;Brunet, JF;Zampieri, N;Kühn, R;Liberles, SD;Jia, S;Lewin, GR;Rajewsky, N;Lever, TE;Birchmeier, C;
PMID: 37192624 | DOI: 10.1016/j.neuron.2023.04.025
Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.
Steuernagel, L;Lam, BYH;Klemm, P;Dowsett, GKC;Bauder, CA;Tadross, JA;Hitschfeld, TS;Del Rio Martin, A;Chen, W;de Solis, AJ;Fenselau, H;Davidsen, P;Cimino, I;Kohnke, SN;Rimmington, D;Coll, AP;Beyer, A;Yeo, GSH;Brüning, JC;
PMID: 36266547 | DOI: 10.1038/s42255-022-00657-y
The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.
Ali Marandi Ghoddousi, R;Magalong, VM;Kamitakahara, AK;Levitt, P;
PMID: 36313803 | DOI: 10.1016/j.crmeth.2022.100316
Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.
Wang, X;Ramos, R;Phan, AQ;Yamaga, K;Flesher, JL;Jiang, S;Oh, JW;Jin, S;Jahid, S;Kuan, CH;Nguyen, TK;Liang, HY;Shettigar, NU;Hou, R;Tran, KH;Nguyen, A;Vu, KN;Phung, JL;Ingal, JP;Levitt, KM;Cao, X;Liu, Y;Deng, Z;Taguchi, N;Scarfone, VM;Wang, G;Paolilli, KN;Wang, X;Guerrero-Juarez, CF;Davis, RT;Greenberg, EN;Ruiz-Vega, R;Vasudeva, P;Murad, R;Widyastuti, LHP;Lee, HL;McElwee, KJ;Gadeau, AP;Lawson, DA;Andersen, B;Mortazavi, A;Yu, Z;Nie, Q;Kunisada, T;Karin, M;Tuckermann, J;Esko, JD;Ganesan, AK;Li, J;Plikus, MV;
PMID: 37344645 | DOI: 10.1038/s41586-023-06172-8
Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.
El Eid, L;Reynolds, CA;Tomas, A;Ben Jones, ;
PMID: 36007775 | DOI: 10.1016/j.phrs.2022.106411
Glucagon-like peptide-1 receptor (GLP-1R) is a well-studied incretin hormone receptor and target of several therapeutic drugs for type 2 diabetes (T2D), obesity and, more recently, cardiovascular disease. Some signalling pathways downstream of GLP-1R may be responsible for drug adverse effects such as nausea, while others mediate therapeutic outcomes of incretin-based T2D therapeutics. Understanding the interplay between different factors that alter signalling, trafficking, and receptor activity, including biased agonism, single nucleotide polymorphisms and structural modifications is key to develop the next-generation of personalised GLP-1R agonists. However, these interactions remain poorly described, especially for novel therapeutics such as dual and tri-agonists that target more than one incretin receptor. Comparison of GLP-1R structures in complex with G proteins and different peptide and non-peptide agonists has revealed novel insights into important agonist-residue interactions and networks crucial for receptor activation, recruitment of G proteins and engagement of specific signalling pathways. Here, we review the latest knowledge on GLP-1R structure and activation, providing structural evidence for biased agonism and delineating important networks associated with this phenomenon. We survey current biased agonists and multi-agonists at different stages of development, highlighting possible challenges in their translational potential. Lastly, we discuss findings related to non-synonymous genomic variants of GLP1R and the functional importance of specific residues involved in GLP-1R function. We propose that studies of GLP-1R polymorphisms, and specifically their effect on receptor dynamics and pharmacology in response to biased agonists, could have a significant impact in delineating precision medicine approaches and development of novel therapeutics.
Abstract LB190: DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations
Molecular and Cellular Biology/Genetics
Wang, L;Tondnevis, F;Todorov, C;Gaspar, J;Sahajan, A;Murlidhar, V;Zhang, B;Ma, X;
| DOI: 10.1158/1538-7445.am2021-lb190
Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications.
Augustine V, Gokce SK, Lee S, Wang B, Davidson TJ, Reimann F, Gribble F, Deisseroth K, Lois C, Oka Y.
PMID: 29489747 | DOI: 10.1038/nature25488
Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.
Imbernon, M;Saponaro, C;Helms, HCC;Duquenne, M;Fernandois, D;Deligia, E;Denis, RGP;Chao, DHM;Rasika, S;Staels, B;Pattou, F;Pfrieger, FW;Brodin, B;Luquet, S;Bonner, C;Prevot, V;
PMID: 35716660 | DOI: 10.1016/j.cmet.2022.06.002
Liraglutide, an anti-diabetic drug and agonist of the glucagon-like peptide one receptor (GLP1R), has recently been approved to treat obesity in individuals with or without type 2 diabetes. Despite its extensive metabolic benefits, the mechanism and site of action of liraglutide remain unclear. Here, we demonstrate that liraglutide is shuttled to target cells in the mouse hypothalamus by specialized ependymoglial cells called tanycytes, bypassing the blood-brain barrier. Selectively silencing GLP1R in tanycytes or inhibiting tanycytic transcytosis by botulinum neurotoxin expression not only hampers liraglutide transport into the brain and its activation of target hypothalamic neurons, but also blocks its anti-obesity effects on food intake, body weight and fat mass, and fatty acid oxidation. Collectively, these striking data indicate that the liraglutide-induced activation of hypothalamic neurons and its downstream metabolic effects are mediated by its tanycytic transport into the mediobasal hypothalamus, strengthening the notion of tanycytes as key regulators of metabolic homeostasis.
Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting
Biglari, N;Gaziano, I;Schumacher, J;Radermacher, J;Paeger, L;Klemm, P;Chen, W;Corneliussen, S;Wunderlich, CM;Sue, M;Vollmar, S;Klöckener, T;Sotelo-Hitschfeld, T;Abbasloo, A;Edenhofer, F;Reimann, F;Gribble, FM;Fenselau, H;Kloppenburg, P;Wunderlich, FT;Brüning, JC;
PMID: 34002087 | DOI: 10.1038/s41593-021-00854-0
Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus represent key regulators of metabolic homeostasis. Electrophysiological and single-cell sequencing experiments have revealed a remarkable degree of heterogeneity of these neurons. However, the exact molecular basis and functional consequences of this heterogeneity have not yet been addressed. Here, we have developed new mouse models in which intersectional Cre/Dre-dependent recombination allowed for successful labeling, translational profiling and functional characterization of distinct POMC neurons expressing the leptin receptor (Lepr) and glucagon like peptide 1 receptor (Glp1r). Our experiments reveal that POMCLepr+ and POMCGlp1r+ neurons represent largely nonoverlapping subpopulations with distinct basic electrophysiological properties. They exhibit a specific anatomical distribution within the arcuate nucleus and differentially express receptors for energy-state communicating hormones and neurotransmitters. Finally, we identify a differential ability of these subpopulations to suppress feeding. Collectively, we reveal a notably distinct functional microarchitecture of critical metabolism-regulatory neurons.
