A Cold-Sensing Receptor Encoded by a Glutamate Receptor Gene.

In search of the molecular identities of cold-sensing receptors, we carried out an unbiased genetic screen for cold-sensing mutants in C. elegans and isolated a mutant allele of glr-3 gene that encodes a kainate-type glutamate receptor. While glutamate receptors are best known to transmit chemical synaptic signals in the CNS, we show that GLR-3 senses cold in the peripheral sensory neuron ASER to trigger cold-avoidance behavior. GLR-3 transmits cold signals via G protein signaling independently of its glutamate-gated channel function, suggesting GLR-3 as a metabotropic cold receptor. The vertebrate GLR-3 homolog GluK2 from zebrafish, mouse, and human can all function as a cold receptor in heterologous systems. Mouse DRG sensory neurons express GluK2, and GluK2 knockdown in these neurons suppresses their sensitivity to cold but not cool temperatures. Our study identifies an evolutionarily conserved cold receptor, revealing that a central chemical receptor unexpectedly functions as a thermal receptor in the periphery.

The C. elegans Taste Receptor Homolog LITE-1 Is a Photoreceptor.

Many animal tissues/cells are photosensitive, yet only two types of photoreceptors (i.e., opsins and cryptochromes) have been discovered in metazoans. The question arises as to whether unknown types of photoreceptors exist in the animal kingdom. LITE-1, a seven-transmembrane gustatory receptor (GR) homolog, mediates UV-light-induced avoidance behavior in C. elegans. However, it is not known whether LITE-1 functions as a chemoreceptor or photoreceptor. Here, we show that LITE-1 directly absorbs both UVA and UVB light with an extinction coefficient 10-100 times that of opsins and cryptochromes, indicating that LITE-1 is highly efficient in capturing photons. Unlike typical photoreceptors employing a prosthetic chromophore to capture photons, LITE-1 strictly depends on its protein conformation for photon absorption. We have further identified two tryptophan residues critical for LITE-1 function. Interestingly, unlike GPCRs, LITE-1 adopts a reversed membrane topology. Thus, LITE-1, a taste receptor homolog, represents a distinct type of photoreceptor in the animal kingdom.

The metabolic ER stress sensor IRE1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity.

Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1f/f; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1f/f; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.

A genetic program promotes C. elegans longevity at cold temperatures via a thermosensitive TRP channel.

Both poikilotherms and homeotherms live longer at lower body temperatures, highlighting a general role of temperature reduction in lifespan extension. However, the underlying mechanisms remain unclear. One prominent model is that cold temperatures reduce the rate of chemical reactions, thereby slowing the rate of aging. This view suggests that cold-dependent lifespan extension is simply a passive thermodynamic process. Here, we challenge this view in C. elegans by showing that genetic programs actively promote longevity at cold temperatures. We find that TRPA-1, a cold-sensitive TRP channel, detects temperature drop in the environment to extend lifespan. This effect requires cold-induced, TRPA-1-mediated calcium influx and a calcium-sensitive PKC that signals to the transcription factor DAF-16/FOXO. Human TRPA1 can functionally substitute for worm TRPA-1 in promoting longevity. Our results reveal a previously unrecognized function for TRP channels, link calcium signaling to longevity, and, importantly, demonstrate that genetic programs contribute to lifespan extension at cold temperatures.

Slow-wave sleep drives sleep-dependent renormalization of synaptic AMPA receptor levels in the hypothalamus.

According to the synaptic homeostasis hypothesis (SHY), sleep serves to renormalize synaptic connections that have been potentiated during the prior wake phase due to ongoing encoding of information. SHY focuses on glutamatergic synaptic strength and has been supported by numerous studies examining synaptic structure and function in neocortical and hippocampal networks. However, it is unknown whether synaptic down-regulation during sleep occurs in the hypothalamus, i.e., a pivotal center of homeostatic regulation of bodily functions including sleep itself. We show that sleep, in parallel with the synaptic down-regulation in neocortical networks, down-regulates the levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in the hypothalamus of rats. Most robust decreases after sleep were observed at both sites for AMPARs containing the GluA1 subunit. Comparing the effects of selective rapid eye movement (REM) sleep and total sleep deprivation, we moreover provide experimental evidence that slow-wave sleep (SWS) is the driving force of the down-regulation of AMPARs in hypothalamus and neocortex, with no additional contributions of REM sleep or the circadian rhythm. SWS-dependent synaptic down-regulation was not linked to EEG slow-wave activity. However, spindle density during SWS predicted relatively increased GluA1 subunit levels in hypothalamic synapses, which is consistent with the role of spindles in the consolidation of memory. Our findings identify SWS as the main driver of the renormalization of synaptic strength during sleep and suggest that SWS-dependent synaptic renormalization is also implicated in homeostatic control processes in the hypothalamus.

Structural basis of GABAB receptor-Gi protein coupling.

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Structures of human mGlu2 and mGlu7 homo- and heterodimers.

The metabotropic glutamate receptors (mGlus) are involved in the modulation of synaptic transmission and neuronal excitability in the central nervous system1. These receptors probably exist as both homo- and heterodimers that have unique pharmacological and functional properties2-4. Here we report four cryo-electron microscopy structures of the human mGlu subtypes mGlu2 and mGlu7, including inactive mGlu2 and mGlu7 homodimers; mGlu2 homodimer bound to an agonist and a positive allosteric modulator; and inactive mGlu2-mGlu7 heterodimer. We observed a subtype-dependent dimerization mode for these mGlus, as a unique dimer interface that is mediated by helix IV (and that is important for limiting receptor activity) exists only in the inactive mGlu2 structure. The structures provide molecular details of the inter- and intra-subunit conformational changes that are required for receptor activation, which distinguish class C G-protein-coupled receptors from those in classes A and B. Furthermore, our structure and functional studies of the mGlu2-mGlu7 heterodimer suggest that the mGlu7 subunit has a dominant role in controlling dimeric association and G-protein activation in the heterodimer. These insights into mGlu homo- and heterodimers highlight the complex landscape of mGlu dimerization and activation.

Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4.

The metabotropic glutamate receptors (mGlus) have key roles in modulating cell excitability and synaptic transmission in response to glutamate (the main excitatory neurotransmitter in the central nervous system)1. It has previously been suggested that only one receptor subunit within an mGlu homodimer is responsible for coupling to G protein during receptor activation2. However, the molecular mechanism that underlies the asymmetric signalling of mGlus remains unknown. Here we report two cryo-electron microscopy structures of human mGlu2 and mGlu4 bound to heterotrimeric Gi protein. The structures reveal a G-protein-binding site formed by three intracellular loops and helices III and IV that is distinct from the corresponding binding site in all of the other G-protein-coupled receptor (GPCR) structures. Furthermore, we observed an asymmetric dimer interface of the transmembrane domain of the receptor in the two mGlu-Gi structures. We confirmed that the asymmetric dimerization is crucial for receptor activation, which was supported by functional data; this dimerization may provide a molecular basis for the asymmetric signal transduction of mGlus. These findings offer insights into receptor signalling of class C GPCRs.

Pharmacological inhibition of Kir4.1 evokes rapid-onset antidepressant responses.

Major depressive disorder, a prevalent and severe psychiatric condition, necessitates development of new and fast-acting antidepressants. Genetic suppression of astrocytic inwardly rectifying potassium channel 4.1 (Kir4.1) in the lateral habenula ameliorates depression-like phenotypes in mice. However, Kir4.1 remains an elusive drug target for depression. Here, we discovered a series of Kir4.1 inhibitors through high-throughput screening. Lys05, the most potent one thus far, effectively suppressed native Kir4.1 channels while displaying high selectivity against established targets for rapid-onset antidepressants. Cryogenic-electron microscopy structures combined with electrophysiological characterizations revealed Lys05 directly binds in the central cavity of Kir4.1. Notably, a single dose of Lys05 reversed the Kir4.1-driven depression-like phenotype and exerted rapid-onset (as early as 1 hour) antidepressant actions in multiple canonical depression rodent models with efficacy comparable to that of (S)-ketamine. Overall, we provided a proof of concept that Kir4.1 is a promising target for rapid-onset antidepressant effects.

Brain-gut communications via distinct neuroendocrine signals bidirectionally regulate longevity in C. elegans.

Tissue-tissue communications are integral to organismal aging, orchestrating a body-wide aging process. The brain plays a key role in this process by detecting and processing signals from the environment and then communicating them to distal tissues such as the gut to regulate longevity. How this is achieved, however, is poorly understood. Here, using Caenorhabditis elegans as a model, we identified two distinct neuroendocrine signaling circuits by which the worm nervous system senses cool and warm environmental temperatures through cool- and warm-sensitive neurons and then signals the gut to extend and shorten life span, respectively. The prolongevity "cool" circuit uses the small neurotransmitters glutamate and serotonin, whereas the anti-longevity "warm" circuit is mediated by insulin-like neuropeptides. Both types of neuroendocrine signals converge on the gut through their cognate receptors to differentially regulate the transcription factor DAF-16/FOXO, leading to opposing outcomes in longevity. Our study illustrates how the brain detects and processes environmental signals to bidirectionally regulate longevity by signaling the gut.

Cross-Species Comparative DNA Methylation Reveals Novel Insights into Complex Trait Genetics among Cattle, Sheep, and Goats.

The cross-species characterization of evolutionary changes in the functional genome can facilitate the translation of genetic findings across species and the interpretation of the evolutionary basis underlying complex phenotypes. Yet, this has not been fully explored between cattle, sheep, goats, and other mammals. Here, we systematically characterized the evolutionary dynamics of DNA methylation and gene expression in 3 somatic tissues (i.e. brain, liver, and skeletal muscle) and sperm across 7 mammalian species, including 3 ruminant livestock species (cattle, sheep, and goats), humans, pigs, mice, and dogs, by generating and integrating 160 DNA methylation and transcriptomic data sets. We demonstrate dynamic changes of DNA hypomethylated regions and hypermethylated regions in tissue-type manner across cattle, sheep, and goats. Specifically, based on the phylo-epigenetic model of DNA methylome, we identified a total of 25,074 hypomethylated region extension events specific to cattle, which participated in rewiring tissue-specific regulatory network. Furthermore, by integrating genome-wide association studies of 50 cattle traits, we provided novel insights into the genetic and evolutionary basis of complex phenotypes in cattle. Overall, our study provides a valuable resource for exploring the evolutionary dynamics of the functional genome and highlights the importance of cross-species characterization of multiomics data sets for the evolutionary interpretation of complex phenotypes in cattle livestock.

MAGE: metafounders-assisted genomic estimation of breeding value, a novel additive-dominance single-step model in crossbreeding systems.

MOTIVATION: Utilizing both purebred and crossbred data in animal genetics is widely recognized as an optimal strategy for enhancing the predictive accuracy of breeding values. Practically, the different genetic background among several purebred populations and their crossbred offspring populations limits the application of traditional prediction methods. Several studies endeavor to predict the crossbred performance via the partial relationship, which divides the data into distinct sub-populations based on the common genetic background, such as one single purebred population and its corresponding crossbred descendant. However, this strategy makes prediction inaccurate due to ignoring half of the parental information of crossbreed animals. Furthermore, dominance effects, although playing a significant role in crossbreeding systems, cannot be modeled under such a prediction model. RESULTS: To overcome this weakness, we developed a novel multi-breed single-step model using metafounders to assess ancestral relationships across diverse breeds under a unified framework. We proposed to use multi-breed dominance combined relationship matrices to model additive and dominance effects simultaneously. Our method provides a straightforward way to evaluate the heterosis of crossbreeds and the breeding values of purebred parents efficiently and accurately. We performed simulation and real data analyses to verify the potential of our proposed method. Our proposed model improved prediction accuracy under all scenarios considered compared to commonly used methods. AVAILABILITY AND IMPLEMENTATION: The software for implementing our method is available at https://github.com/CAU-TeamLiuJF/MAGE.

Glucose Sensing by Skeletal Myocytes Couples Nutrient Signaling to Systemic Homeostasis.

Skeletal muscle is a major site of postprandial glucose disposal. Inadequate insulin action in skeletal myocytes contributes to hyperglycemia in diabetes. Although glucose is known to stimulate insulin secretion by ß cells, whether it directly engages nutrient signaling pathways in skeletal muscle to maintain systemic glucose homeostasis remains largely unexplored. Here we identified the Baf60c-Deptor-AKT pathway as a target of muscle glucose sensing that augments insulin action in skeletal myocytes. Genetic activation of this pathway improved postprandial glucose disposal in mice, whereas its muscle-specific ablation impaired insulin action and led to postprandial glucose intolerance. Mechanistically, glucose triggers KATP channel-dependent calcium signaling, which promotes HDAC5 phosphorylation and nuclear exclusion, leading to Baf60c induction and insulin-independent AKT activation. This pathway is engaged by the anti-diabetic sulfonylurea drugs to exert their full glucose-lowering effects. These findings uncover an unexpected mechanism of glucose sensing in skeletal myocytes that contributes to homeostasis and therapeutic action.

Assembly of novel sequences for Chinese domestic pigs reveals new genes and regulatory variants providing new insights into their diversity.

There is an increasing understanding that a reference genome representing an individual cannot capture all the gene repertoire of a species. Here, we conduct a population-scale missing sequences detection of Chinese domestic pigs using whole-genome sequencing data from 534 individuals. We identify 132.41 Mb of sequences absent in the reference assembly, including eight novel genes. In particular, the breeds spread in Chinese high-altitude regions perform significantly different frequencies of new sequences in promoters than other breeds. Furthermore, we dissect the role of non-coding variants and identify a novel sequence inserted in the 3'UTR of the FMO3 gene, which may be associated with the intramuscular fat phenotype. This novel sequence could be a candidate marker for meat quality. Our study provides a comprehensive overview of the missing sequences in Chinese domestic pigs and indicates that this dataset is a valuable resource for understanding the diversity and biology of pigs.

Parvalbumin-Positive Interneurons in the Medial Prefrontal Cortex Regulate Stress-Induced Fear Extinction Impairments in Male and Female Rats.

Stress has profound effects on fear extinction, a form of learning that is essential to behavioral therapies for trauma-related and stressor-related disorders. Recent work reveals that acute footshock stress reduces medial prefrontal cortex (mPFC) activity that is critical for extinction learning. Reductions in mPFC activity may be mediated by parvalbumin (PV)-containing interneurons via feedforward inhibition imposed by amygdala afferents. To test this hypothesis, footshock stress-induced Fos expression was characterized in PV+ and PV- neurons in the prelimbic (PL) and infralimbic (IL) cortices. Footshock stress increased the proportion of PV+ cells expressing Fos in both male and female rats; this effect was more pronounced in IL compared with PL. To determine whether PV+ interneurons in the mPFC mediate stress-induced extinction impairments, we chemogenetically silenced these neurons before an immediate extinction procedure in PV-Cre rats. Clozapine-N-oxide (CNO) did not affect conditioned freezing during the extinction procedure. However, CNO exacerbated extinction retrieval in both male and female rats with relatively high PL expression of designer receptors exclusively activated by designer drugs (DREADD). In contrast, in rats with relatively high IL DREADD expression, CNO produced a modest facilitation of extinction in the earliest retrieval trials, but in male rats only. Conversely, excitation of IL PV interneurons was sufficient to impair delayed extinction in both male and female rats. Finally, chemogenetic inhibition of IL-projecting amygdala neurons reduced the immediate extinction deficit in male, but not female rats. These results reveal that PV interneurons regulate extinction learning under stress in a sex-dependent manner, and this effect is mediated by amygdaloprefrontal projections.SIGNIFICANCE STATEMENT Stress significantly impairs the memory of fear extinction, a type of learning that is central to behavioral therapies for trauma-based and anxiety-based disorders (e.g., post-traumatic stress disorder). Here we show that acute footshock stress recruits parvalbumin (PV) interneurons in the medial prefrontal cortex (mPFC) of male and female rats. Silencing mPFC PV interneurons or mPFC-projecting amygdala neurons during immediate extinction influenced extinction retrieval in a sex-dependent manner. This work highlights the role for PV-containing mPFC interneurons in stress-induced impairments in extinction learning.

Proteomics and Metabolic Characteristics of Boar Seminal Plasma Extracellular Vesicles Reveal Biomarker Candidates Related to Sperm Motility.

Although seminal plasma extracellular vesicles (SPEVs) play important roles in sperm function, little is known about their metabolite compositions and roles in sperm motility. Here, we performed metabolomics and proteomics analysis of boar SPEVs with high or low sperm motility to investigate specific biomarkers affecting sperm motility. In total, 140 proteins and 32 metabolites were obtained through differentially expressed analysis and weighted gene coexpression network analysis (WGCNA). Seven differentially expressed proteins (DEPs) (ADIRF, EPS8L1, PRCP, CD81, PTPRD, CSK, LOC100736569) and six differentially expressed metabolites (DEMs) (adenosine, beclomethasone, 1,2-benzenedicarboxylic acid, urea, 1-methyl-l-histidine, and palmitic acid) were also identified in WGCNA significant modules. Joint pathway analysis revealed that three DEPs (GART, ADCY7, and NTPCR) and two DEMs (urea and adenosine) were involved in purine metabolism. Our results suggested that there was significant correlation between proteins and metabolites, such as IL4I1 and urea (r = 0.86). Furthermore, we detected the expression level of GART, ADCY7, and CDC42 in sperm of two groups, which further verified the experimental results. This study revealed that several proteins and metabolites in SPEVs play important roles in sperm motility. Our results offered new insights into the complex mechanism of sperm motility and identified potential biomarkers for male reproductive diseases.

2023 Julius Axelrod Symposium: Plant-Derived Molecules Acting on G Protein-Coupled Receptors.

Plant extracts have played a significant role in traditional medicine for centuries, contributing to improved health and the treatment of various human illnesses. G protein-coupled receptors (GPCRs) are crucial in numerous physiologic functions, and there is growing evidence suggesting their involvement in the therapeutic effects of many plant extracts. In recent years, scientists have identified an expanding number of isolated molecules responsible for the biologic activity of these extracts, with many believed to act on GPCRs. This article critically reviews the evidence supporting the modulation of GPCR function by these plant-derived molecules through direct binding. Structural information is now available for some of these molecules, allowing for a comparison of their binding mode with that of endogenous GPCR ligands. The final section explores future trends and challenges, focusing on the identification of new plant-derived molecules with both orthosteric and allosteric binding modes, as well as innovative strategies for designing GPCR ligands inspired by these plant-derived compounds. In conclusion, plant-derived molecules are anticipated to play an increasingly vital role as therapeutic drugs and serve as templates for drug design. SIGNIFICANCE STATEMENT: This minireview summarizes the most pertinent publications on isolated plant-derived molecules interacting with G protein-coupled receptors (GPCRs) and comments on available structural information on GPCR/plant-derived ligand pairs. Future challenges and trends for the isolation and characterization of plant-derived molecules and drug design are discussed.

Genomic evidence for human-mediated introgressive hybridization and selection in the developed breed.

BACKGROUND: The pig (Sus Scrofa) is one of the oldest domesticated livestock species that has undergone extensive improvement through modern breeding. European breeds have advantages in lean meat development and highly-productive body type, whereas Asian breeds possess extraordinary fat deposition and reproductive performance. Consequently, Eurasian breeds have been extensively used to develop modern commercial breeds for fast-growing and high prolificacy. However, limited by the sequencing technology, the genome architecture of some nascent developed breeds and the human-mediated impact on their genomes are still unknown. RESULTS: Through whole-genome analysis of 178 individuals from an Asian locally developed pig breed, Beijing Black pig, and its two ancestors from two different continents, we found the pervasive inconsistent gene trees and species trees across the genome of Beijing Black pig, which suggests its introgressive hybrid origin. Interestingly, we discovered that this developed breed has more genetic relationships with European pigs and an unexpected introgression from Asian pigs to this breed, which indicated that human-mediated introgression could form the porcine genome architecture in a completely different type compared to native introgression. We identified 554 genomic regions occupied 63.30 Mb with signals of introgression from the Asian ancestry to Beijing Black pig, and the genes in these regions enriched in pathways associated with meat quality, fertility, and disease-resistant. Additionally, a proportion of 7.77% of genomic regions were recognized as regions that have been under selection. Moreover, combined with the results of a genome-wide association study for meat quality traits in the 1537 Beijing Black pig population, two important candidate genes related to meat quality traits were identified. DNAJC6 is related to intramuscular fat content and fat deposition, and RUFY4 is related to meat pH and tenderness. CONCLUSIONS: Our research provides insight for analyzing the origins of nascent developed breeds and genome-wide selection remaining in the developed breeds mediated by humans during modern breeding.

Desorption Separation Ionization Mass Spectrometry (DSI-MS) for Rapid Analysis of COVID-19.

During the coronavirus disease 2019 (COVID-19) pandemic, which has witnessed over 772 million confirmed cases and over 6 million deaths globally, the outbreak of COVID-19 has emerged as a significant medical challenge affecting both affluent and impoverished nations. Therefore, there is an urgent need to explore the disease mechanism and to implement rapid detection methods. To address this, we employed the desorption separation ionization (DSI) device in conjunction with a mass spectrometer for the efficient detection and screening of COVID-19 urine samples. The study encompassed patients with COVID-19, healthy controls (HC), and patients with other types of pneumonia (OP) to evaluate their urine metabolomic profiles. Subsequently, we identified the differentially expressed metabolites in the COVID-19 patients and recognized amino acid metabolism as the predominant metabolic pathway involved. Furthermore, multiple established machine learning algorithms validated the exceptional performance of the metabolites in discriminating the COVID-19 group from healthy subjects, with an area under the curve of 0.932 in the blind test set. This study collectively suggests that the small-molecule metabolites detected from urine using the DSI device allow for rapid screening of COVID-19, taking just three minutes per sample. This approach has the potential to expand our understanding of the pathophysiological mechanisms of COVID-19 and offers a way to rapidly screen patients with COVID-19 through the utilization of machine learning algorithms.