Salinas CBG, Lu TT, Gabery S, Marstal K, Alanentalo T, Mercer AJ, Cornea A, Conradsen K, Hecksher-Sørensen J, Dahl AB, Knudsen LB, Secher A.
PMID: 29985439 | DOI: 10.1038/s41598-018-28496-6
Light Sheet Fluorescence Microscopy (LSFM) of whole organs, in particular the brain, offers a plethora of biological data imaged in 3D. This technique is however often hindered by cumbersome non-automated analysis methods. Here we describe an approach to fully automate the analysis by integrating with data from the Allen Institute of Brain Science (AIBS), to provide precise assessment of the distribution and action of peptide-based pharmaceuticals in the brain. To illustrate this approach, we examined the acute central nervous system effects of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide. Peripherally administered liraglutide accessed the hypothalamus and brainstem, and led to activation in several brain regions of which most were intersected by projections from neurons in the lateral parabrachial nucleus. Collectively, we provide a rapid and unbiased analytical framework for LSFM data which enables quantification and exploration based on data from AIBS to support basic and translational discovery.
Li C, Navarrete J, Liang-Guallpa J, Lu C, Funderburk SC, Chang RB, Liberles SD, Olson DP, Krashes MJ.
PMID: 30472090 | DOI: 10.1016/j.cmet.2018.10.016
Understanding the neural framework behind appetite control is fundamental to developing effective therapies to combat the obesity epidemic. The paraventricular hypothalamus (PVH) is critical for appetite regulation, yet, the real-time, physiological response properties of PVH neurons to nutrients are unknown. Using a combination of fiber photometry, electrophysiology, immunohistochemistry, and neural manipulation strategies, we determined the population dynamics of four molecularly delineated PVH subsets implicated in feeding behavior: glucagon-like peptide 1 receptor (PVHGlp1r), melanocortin-4 receptor (PVHMc4r), oxytocin (PVHOxt), and corticotropin-releasing hormone (PVHCrh). We identified both calorie- and state-dependent sustained activity increases and decreases in PVHGlp1r and PVHCrh populations, respectively, while observing transient bulk changes of PVHMc4r, but no response in PVHOxt, neurons to food. Furthermore, we highlight the role of PVHGlp1r neurons in orchestrating acute feeding behavior, independent of the anti-obesity drug liraglutide, and demonstrate the indispensability of PVHGlp1r and PVHMc4r, but not PVHOxt or PVHCrh neurons, in body weight maintenance.
Lin, M;Hartl, K;Heuberger, J;Beccaceci, G;Berger, H;Li, H;Liu, L;Müllerke, S;Conrad, T;Heymann, F;Woehler, A;Tacke, F;Rajewsky, N;Sigal, M;
PMID: 37230989 | DOI: 10.1038/s41467-023-38780-3
The cellular organization of gastrointestinal crypts is orchestrated by different cells of the stromal niche but available in vitro models fail to fully recapitulate the interplay between epithelium and stroma. Here, we establish a colon assembloid system comprising the epithelium and diverse stromal cell subtypes. These assembloids recapitulate the development of mature crypts resembling in vivo cellular diversity and organization, including maintenance of a stem/progenitor cell compartment in the base and their maturation into secretory/absorptive cell types. This process is supported by self-organizing stromal cells around the crypts that resemble in vivo organization, with cell types that support stem cell turnover adjacent to the stem cell compartment. Assembloids that lack BMP receptors either in epithelial or stromal cells fail to undergo proper crypt formation. Our data highlight the crucial role of bidirectional signaling between epithelium and stroma, with BMP as a central determinant of compartmentalization along the crypt axis.
bioRxiv : the preprint server for biology
Manieri, E;Tie, G;Seruggia, D;Madha, S;Maglieri, A;Huang, K;Fujiwara, Y;Zhang, K;Orkin, SH;He, R;McCarthy, N;Shivdasani, RA;
PMID: 36798304 | DOI: 10.1101/2023.02.11.527728
PDGFRA-expressing mesenchyme provides a niche for intestinal stem cells. Corresponding compartments are unknown in the stomach, where corpus and antral glandular epithelia have similar niche dependencies but are structurally distinct from the intestine and from each other. Previous studies considered antrum and corpus as a whole and did not assess niche functions. Using high-resolution imaging and sequencing, we identify regional subpopulations and niche properties of purified mouse corpus and antral PDGFRA + cells. PDGFRA Hi sub-epithelial myofibroblasts are principal sources of BMP ligands in both gastric segments; two molecularly distinct groups distribute asymmetrically along antral glands but together fail to support epithelial organoids in vitro . In contrast, strategically positioned PDGFRA Lo cells that express CD55 enable corpus and antral organoid growth in the absence of other cellular or soluble factors. Our study provides detailed insights into spatial, molecular, and functional organization of gastric mesenchyme and the spectrum of signaling sources for stem cell support.
Madissoon, E;Oliver, AJ;Kleshchevnikov, V;Wilbrey-Clark, A;Polanski, K;Richoz, N;Ribeiro Orsi, A;Mamanova, L;Bolt, L;Elmentaite, R;Pett, JP;Huang, N;Xu, C;He, P;Dabrowska, M;Pritchard, S;Tuck, L;Prigmore, E;Perera, S;Knights, A;Oszlanczi, A;Hunter, A;Vieira, SF;Patel, M;Lindeboom, RGH;Campos, LS;Matsuo, K;Nakayama, T;Yoshida, M;Worlock, KB;Nikolić, MZ;Georgakopoulos, N;Mahbubani, KT;Saeb-Parsy, K;Bayraktar, OA;Clatworthy, MR;Stegle, O;Kumasaka, N;Teichmann, SA;Meyer, KB;
PMID: 36543915 | DOI: 10.1038/s41588-022-01243-4
Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.
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.
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.
