G protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and also the targets of many drugs. Understanding of the functional significance of the wide structural ...diversity of GPCRs has been aided considerably in recent years by the sequencing of the human genome and by structural studies, and has important implications for the future therapeutic potential of targeting this receptor family. This article aims to provide a comprehensive overview of the five main human GPCR families--Rhodopsin, Secretin, Adhesion, Glutamate and Frizzled/Taste2--with a focus on gene repertoire, general ligand preference, common and unique structural features, and the potential for future drug discovery.
The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce ...principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.
Abstract
Motivation
Cell to cell communication is critical for all multicellular organisms, and single-cell sequencing facilitates the construction of full connectivity graphs between cell types in ...tissues. Such complex data structures demand novel analysis methods and tools for exploratory analysis.
Results
We propose a method to predict the putative ligand–receptor interactions between cell types from single-cell RNA-sequencing data. This is achieved by inferring and incorporating interactions in a multi-directional graph, thereby enabling contextual exploratory analysis. We demonstrate that our approach can detect common and specific interactions between cell types in mouse brain and human tumors, and that these interactions fit with expected outcomes. These interactions also include predictions made with molecular ligands integrating information from several types of genes necessary for ligand production and transport. Our implementation is general and can be appended to any transcriptome analysis pipeline to provide unbiased hypothesis generation regarding ligand to receptor interactions between cell populations or for network analysis in silico.
Availability and implementation
scConnect is open source and available as a Python package at https://github.com/JonETJakobsson/scConnect. scConnect is directly compatible with Scanpy scRNA-sequencing pipelines.
Supplementary information
Supplementary data are available at Bioinformatics online.
Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) ...have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox;Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch.
Primary afferents are known to use glutamate as their principal fast neurotransmitter. However, it has become increasingly clear that peptides have an influential role in both mediating and ...modulating sensory transmission. Here we describe the transmission accounting for different acute pain states and itch transmitted via the transient receptor potential cation channel subfamily V member 1 (TRPV1) population by either ablating Trpv1-Cre-expressing neurons or inducing vesicular glutamate transporter 2 (VGLUT2) deficiency in Trpv1-Cre-expressing neurons. Furthermore, by pharmacological inhibition of substance P or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we evaluated the contribution of substance P or CGRP to these sensory modulations, with or without the presence of VGLUT2-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that glutamate via VGLUT2 in the Trpv1-Cre population together with substance P mediate acute cold pain, whereas glutamate together with CGRP mediate noxious heat. Moreover, we demonstrate that glutamate together with both substance P and CGRP mediate tissue-injury associated pain. We further show that itch, regulated by the VGLUT2-mediated transmission via the Trpv1-Cre population, depends on CGRP and gastrin-releasing peptide receptor (GRPR) transmission because pharmacological blockade of the CGRP or GRPR pathway, or genetic ablation of Grpr, led to a drastically attenuated itch. Our study reveals how different neurotransmitters combined can cooperate with each other to transmit or regulate various acute sensations, including itch.
The superfamily of G-protein-coupled receptors (GPCRs) is very diverse in structure and function and its members are among
the most pursued targets for drug development. We identified more than 800 ...human GPCR sequences and simultaneously analyzed
342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. Our results show, with high bootstrap
support, five main families, named glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin, forming the GRAFS classification
system. The rhodopsin family is the largest and forms four main groups with 13 sub-branches. Positions of the GPCRs in chromosomal
paralogons regions indicate the importance of tetraploidizations or local gene duplication events for their creation. We
also searched for âfingerprintâ motifs using Hidden Markov Models delineating the putative inter-relationship of the GRAFS
families. We show several common structural features indicating that the human GPCRs in the GRAFS families share a common
ancestor. This study represents the first overall map of the GPCRs in a single mammalian genome. Our novel approach of analyzing
such large and diverse sequence sets may be useful for studies on GPCRs in other genomes and divergent protein families.
The glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the ...glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice.
Single-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas suggested from the single-cell data.
Glra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.
Itch is an unpleasant sensation that can be debilitating, especially if it is chronic and of non-histaminergic origin, as treatment options are limited. Endothelin-1 (ET-1) is a potent endogenous ...vasoconstrictor that also has the ability to induce a burning, non-histaminergic pruritus when exogenously administered, by activating the endothelin A receptor (ET
R) on primary afferents. ET-1 is released endogenously by several cell-types found in the skin, including macrophages and keratinocytes. Mast cells express ET
Rs and can thereby be degranulated by ET-1, and mast cell proteases chymase and carboxypeptidase A3 (CPA3) are known to either generate or degrade ET-1, respectively, suggesting a role for mast cell proteases in the regulation of ET-1-induced itch. The mouse mast cell proteases (mMCPs) mMCP4 (chymase), mMCP6 (tryptase), and CPA3 are found in connective tissue type mast cells and are the closest functional homologs to human mast cell proteases, but little is known about their role in endothelin-induced itch.
In this study, we evaluated the effects of mast cell protease deficiency on scratching behavior induced by ET-1. To investigate this, mMCP knock-out and transgenic mice were injected intradermally with ET-1 and their scratching behavior was recorded and analyzed.
CPA3-deficient mice and mice lacking all three proteases demonstrated highly elevated levels of scratching behavior compared with wild-type controls. A modest increase in the number of scratching bouts was also seen in mMCP6-deficient mice, while mMCP4-deficiency did not have any effect.
Altogether, these findings identify a prominent role for the mast cell proteases, in particular CPA3, in the protection against itch induced by ET-1.
Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized ...spinal glycine receptor alpha 3 (
) subunit-expressing neurons in
-Cre female and male mice.
-Cre(+) neurons express
, are located mainly in laminae III-VI, and respond to glycine. Chemogenetic activation of spinal
-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express
, further supporting the phenotype. Retrograde tracing revealed that spinal
-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal
(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.
The melanocortin system has a well-established role in the regulation of energy homeostasis, but there is growing evidence of its involvement in memory, nociception, mood disorders and addiction. In ...this Review, we focus on the role of the melanocortin 4 receptor and provide an integrative view of the molecular mechanisms that lead to melanocortin-induced changes in synaptic plasticity within these diverse physiological systems. We also highlight the importance of melanocortin peptides and receptors in chronic pain syndromes, memory impairments, depression and drug abuse, and the possibility of targeting them for therapeutic purposes.
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