Endogenous Receptor Research Articles

Pan-ErbB inhibition impairs cognition via disrupting myelination and aerobic glycolysis in oligodendrocytes

White matter (WM) abnormalities are an emerging feature of schizophrenia, yet the underlying pathophysiological mechanisms are largely unknown. Disruption of ErbB signaling, which is essential for peripheral myelination, has been genetically associated with schizophrenia and WM lesions in schizophrenic patients. However, the roles of ErbB signaling in oligodendrocytes remain elusive. Here, we used an in vivo pan-ErbB inhibition strategy and demonstrated the functions of endogenous ErbB receptors in oligodendrocytes. Through analyses of the cellular, histological, biochemical, behavioral, and electrophysiological differences in mice with manipulated ErbB activities in oligodendrocytes at different differentiation stages, we found that ErbB signaling regulates myelination and aerobic glycolysis in oligodendrocytes, and both functions are required for working memory. ErbB inhibition in oligodendrocytes at early differentiation stages induces hypomyelination by suppressing the myelinating capacity of newly formed oligodendrocytes. In contrast, ErbB inhibition in mature oligodendrocytes alters neither myelination nor oligodendrocyte numbers, but accelerates axonal conduction decline under energy stress. Mechanistically, ErbB inhibition attenuates K-Ras activities, leading to the reduced expression of lactate dehydrogenase A that promotes aerobic glycolysis in mature oligodendrocytes. Supplementation of L-lactate restores axonal conduction and working memory capacity that are suppressed by ErbB inhibition in mature oligodendrocytes. These findings emphasize the indispensable roles of ErbB signaling in WM integrity and function and provide insights into the multifaceted contributions of WM abnormalities to cognitive impairment.

Endogenous opioid receptor system mediates costly altruism in the human brain

Functional neuroimaging studies suggest that a large-scale brain network transforms others’ pain into its vicarious representation in the observer, potentially modulating helping behavior. However, the neuromolecular basis of individual differences in vicarious pain and helping is poorly understood. We investigated the role of the endogenous μ-opioid receptor (MOR) system in altruistic costly helping. MOR density was measured using [11C]carfentanil. In a separate fMRI experiment, participants could donate money to reduce a confederate’s pain from electric shocks. Participants were generally willing to help, and brain activity was observed in amygdala, anterior insula, anterior cingulate cortex (ACC), striatum, primary motor cortex, primary somatosensory cortex and thalamus when witnessing others’ pain. Haemodynamic responses were negatively associated with MOR availability in emotion circuits. However, MOR availability positively associated with the ACC and hippocampus during helping. These findings suggest that the endogenous MOR system modulates altruism in the human brain.

Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.

Kappa-opioid receptor blockade in the inferior colliculus of prey threatened by pit vipers decreases anxiety and panic-like behaviour.

The dorsal midbrain comprises dorsal columns of the periaqueductal grey matter and corpora quadrigemina. These structures are rich in beta-endorphinergic and leu-enkephalinergic neurons and receive GABAergic inputs from substantia nigra pars reticulata. Although the inferior colliculus (IC) is mainly involved in the acoustic pathways, the electrical and chemical stimulation of central and pericentral nuclei of the IC elicits a vigorous defensive behaviour. The defensive immobility and escape elicited by IC activation is commonly related to panic-like emotional states. To investigate the role of κ-opioid receptor of the IC in the antiaversive effects of endogenous opioid receptor blockade in a dangerous situation, male Wistar rats were pretreated in the IC with the κ-opioid receptor-selective antagonist nor-binaltorphimine at different concentrations and submitted to the non-enriched polygonal arena for a snake panic test in the presence of a rattlesnake and, after 24 h, prey were resubmitted to the experimental context. The snakes elicited in prey a set of antipredatory behaviours, such as the anxiety-like responses of defensive attention and risk assessment, and the panic-like reactions of defensive immobility and either escape or active avoidance during the elaboration of unconditioned and conditioned fear-related responses. Pretreatment of the IC with microinjections of nor-binaltorphimine at higher concentrations significantly decreased the frequency and duration of both anxiety- and panic-attack-like behaviours. These findings suggest that κ-opioid receptor blockade in the IC causes anxiolytic- and panicolytic-like responses in threatening conditions, and that kappa-opioid receptor-selective antagonists can be a putative coadjutant treatment for panic syndrome treatment.

SPMs exert anti-inflammatory and pro-resolving effects through positive allosteric modulation of the prostaglandin EP4 receptor

Inflammation is a protective response to pathogens and injury. To be effective it needs to be resolved by endogenous mechanisms in order to avoid prolonged and excessive inflammation, which can become chronic. Specialized pro-resolving mediators (SPMs) are a group of lipids derived from omega-3 fatty acids, which can induce the resolution of inflammation. How SPMs exert their anti-inflammatory and pro-resolving effects is, however, not clear. Here, we show that SPMs such as protectins, maresins, and D-series resolvins function as biased positive allosteric modulators (PAM) of the prostaglandin E2 (PGE2) receptor EP4 through an intracellular binding site. They increase PGE2-induced Gs-mediated formation of cAMP and thereby promote anti-inflammatory signaling of EP4. In addition, SPMs endow the endogenous EP4 receptor on macrophages with the ability to couple to Gi-type G-proteins, which converts the EP4 receptor on macrophages from an anti-phagocytotic receptor to one increasing phagocytosis, a central mechanism of the pro-resolving activity of synthetic SPMs. In the absence of the EP4 receptor, SPMs lose their anti-inflammatory and pro-resolving activity in vitro and in vivo. Our findings reveal an unusual mechanism of allosteric receptor modulation by lipids and provide a mechanism by which synthetic SPMs exert pro-resolving and anti-inflammatory effects, which may facilitate approaches to treat inflammation.

Using Reporter Gene Assays to Screen and Identify Chemical Compounds that Modulate Estrogen Receptor Activity.

Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor α Basic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors.

Autoantibody mediated deficiency of IL-36-receptor antagonist in a subset of patients with psoriasis and psoriatic arthritis

ObjectivePsoriatic arthritis (PsA) is known as a seronegative form of spondylarthropathy. The interleukin-36 cytokine family may have a major role in disease pathogenesis and particularly the related cutaneous manifestations. In light of our recent observations on (transient) autoantibody phenotypes neutralizing endogenous anti-inflammatory receptor antagonists (progranulin, IL-1Ra) in different inflammatory conditions, we set out to investigate the potential role of such antibodies targeting IL-36 cytokine family members in PsA and psoriasis without arthritic manifestations (Pso). MethodsIn the present study we screened for hypothetic autoantibodies against the anti-inflammatory mediators IL-36 receptor antagonist (IL-36Ra) and anti-inflammatory IL-38 in PsA, Pso and inflammatory and healthy controls. Serum samples of patients with PsA (n = 254), Pso (n = 100), systemic lupus erythematosus (SLE, n = 50), rheumatoid arthritis (RA, n = 100), ulcerative colitis (UC, n = 50), Crohn´s disease (CD, n = 50), and healthy controls (n = 237) were screened for autoantibodies against IL-36Ra and IL-38 as well as IL-36Ra levels by ELISA. Biochemical analysis for immune complexes and atypic protein isoforms as well as IL-36 signaling reporter assays were performed. ResultsAnti-IL-36Ra antibodies were detected in five out of 100 (5.0 %) patients with Pso, in 12 of 254 (4.72 %) patients with PsA and in one of 50 (2 %) patients with CD, but in none of the other investigated inflammatory or healthy controls. The IL-36Ra autoantibodies belonged to the IgG1 subclass and their titers ranged between 1:200 to 1:1600. They resulted in immune-complex formation, depletion of serum IL-36Ra levels and were functional in terms of facilitating unrestricted IL-36 signaling. ConclusionIL-36Ra autoantibodies were found in subgroups of patients with Pso and PsA and may drive respective pathology.

Plasma Concentrations of Multiple Oxysterols and Risk of Colorectal Adenomas.

Oxysterols are metabolites of cholesterol that regulate the homeostasis of cholesterol, fatty acids, and glucose. These metabolites are generated throughout the body, either enzymatically or from oxidative stress, and are detectable in peripheral circulation. We previously reported that circulating 27-hydroxycholesterol (27-OHC), an endogenous selective estrogen receptor modulator, may be a risk factor for colorectal adenomas. Here, in addition to 27-OHC, we report on four other circulating oxysterols: 25-hydroxycholesterol, 24(S)-hydroxycholesterol, 7ɑ-hydroxycholesterol, and 4β-hydroxycholesterol. Oxysterol concentrations were measured using liquid chromatography/mass spectrometry from fasting plasma collected at baseline from 1,246 participants of the Vitamin D/Calcium Polyp Prevention Study, a multicenter adenoma chemoprevention trial. To evaluate multiple oxysterols simultaneously, we used both log-linear regression and Bayesian kernel machine regression models developed for analyses of complex mixtures adjusted for potential confounding factors. Higher circulating 7ɑ-hydroxycholesterol was associated with higher adenoma risk (Bayesian kernel machine regression-based multivariable-adjusted risk ratios (RR; for the 75th vs. 25th percentile, 1.22; 95% credible interval, CI, 1.04-1.42). In contrast, higher circulating 4β-hydroxycholesterol was associated with lower risk of these polyps (RR, 0.84; 95% CI, 0.71-0.99). The positive association with advanced adenoma risk that we previously reported for circulating 27-OHC persisted when controlling for other oxysterols (RR, 1.26; 95% CI, 0.98-1.62), including among those with advanced adenomas at baseline (RR, 1.75; 95% CI, 1.01-3.06). Prevention Relevance: Circulating concentrations of multiple oxysterols measured at the time of an initial colorectal adenoma diagnosis may be risk factors for subsequent incidence of these lesions. Novel colorectal cancer prevention strategies may target oxysterol formation.

Endogenous cell membrane interactome mapping for the GLP-1 receptor in different cell types.

The GLP-1 receptor, one of the most successful drug targets for the treatment of type 2 diabetes and obesity, is known to engage multiple intracellular signaling proteins. However, it remains less explored how the receptor interacts with proteins on the cell membrane. Here, we present a ligand-based proximity labeling approach to interrogate the native cell membrane interactome for the GLP-1 receptor after agonist simulation. Our study identified several unreported putative cell membrane interactors for the endogenous receptor in either a pancreatic β cell line or a neuronal cell line. We further uncovered new regulators of GLP-1 receptor-mediated signaling and insulinotropic responses in β cells. Additionally, we obtained a time-resolved cell membrane interactome map for the receptor in β cells. Therefore, our study provides a new approach that is generalizable to map endogenous cell membrane interactomes for G-protein-coupled receptors to decipher the molecular basis of their cell-type-specific functional regulation.

Mu opioid receptors gate the locus coeruleus pain generator.

The locus coeruleus (LC) plays a paradoxical role in chronic pain. Although largely known as a potent source of endogenous analgesia, increasing evidence suggests injury can transform the LC into a chronic pain generator. We sought to clarify the role of this system in pain. Here, we show optogenetic inhibition of LC activity is acutely antinociceptive. Following long-term spared nerve injury, the same LC inhibition is analgesic - further supporting its pain generator function. To identify inhibitory substrates that may naturally serve this function, we turned to endogenous LC mu opioid receptors (LC-MOR). These receptors provide powerful LC inhibition and exogenous activation of LC-MOR is antinociceptive. We therefore hypothesized that endogenous LC-MOR-mediated inhibition is critical to how the LC modulates pain. Using cell type-selective conditional knockout and rescue of LC-MOR receptor signaling, we show these receptors bidirectionally regulate thermal and mechanical hyperalgesia - providing a functional gate on the LC pain generator.

Neurokinin-1 receptors in the nucleus accumbens shell influence sensitivity to social defeat stress and stress-induced alcohol consumption in male mice

Chronic social defeat stress (SDS) is a widely employed preclinical model of depression involving repeated exposure to physical defeats using a resident-intruder model in male mice. Exposure to SDS induces depressive-like phenotypes including anhedonia, social withdrawal, and increased drug and alcohol consumption. Previously, we found that expression of the neurokinin-1 receptor (NK1R) is increased in the nucleus accumbens (NAC) of mice that are sensitive to this stressor and increase their alcohol intake. The NK1R is the endogenous receptor for the neuropeptide substance P (SP) and plays a prominent role in stress, anxiety, and addiction. In the present study, we assessed changes in NK1R protein levels in the NAC shell and implemented viral vector strategies to demonstrate a functional role of the NK1R in sensitivity to SDS. Specifically, we found that NK1R protein levels were increased in the NAC shell following SDS exposure. Next, we found that NK1R overexpression in the NAC shell increased the sensitivity to SDS and stress-induced alcohol consumption. Together, these experiments provide evidence for a role of the NK1R in the NAC shell in the sensitivity to SDS and the subsequent escalation in alcohol intake.

Association of Adiposity Phenotypes with 27-Hydroxycholesterol and Sex Hormones: The Multiethnic Cohort Study.

The distribution of body fat has been linked to circulating levels of lipids and sex-steroid hormones. The cholesterol metabolite and endogenous selective estrogen receptor modulator, 27-hydroxychlolesterol (27HC), may be influenced by adiposity phenotypes, particularly among females. No study has examined the relationships of 27HC and steroid hormones with adiposity phenotypes. To investigate the associations of 27HC and steroid hormones with detailed adiposity phenotypes among a multiethnic population of postmenopausal females. A cross-sectional study was conducted among 912 postmenopausal females from the Multiethnic Cohort- Adiposity Phenotype study. Multivariable linear regression examined the associations of circulating levels of 27HC, steroid hormones, and sex hormone-binding globulin (SHBG) with detailed adiposity phenotypes, adjusting for demographics, lifestyle factors, diabetes status, and use of lipid lowering drugs. Subgroup analyses were conducted across race and ethnicity. Total fat mass (P-trend=0.003), subcutaneous adipose tissue (SAT) (P-trend=0.006), and superficial subcutaneous adipose tissue (sSAT) (P-trend=4.41x10-4) were inversely associated with circulating 27HC levels. In contrast, visceral adipose tissue (VAT) (P-trend=0.003) and liver fat (P-trend=0.005) were positively associated with 27HC levels. All adiposity phenotypes were associated with higher levels of free estradiol, testosterone and lower levels of SHBG. Generally, similar patterns of associations were observed across race and ethnicity. Adiposity phenotypes, such as SAT, VAT, and liver fat, were differentially associated with circulating 27HC, while consistent directions of associations were seen for circulating hormones among postmenopausal females. Future studies are warranted to further understand the biology and relationships of 27HC and adiposity-related diseases.

Photoproximity labeling of endogenous receptors in the live mouse brain in minutes.

Understanding how protein-protein interaction networks in the brain give rise to cognitive functions necessitates their characterization in live animals. However, tools available for this purpose require potentially disruptive genetic modifications and lack the temporal resolution necessary to track rapid changes in vivo. Here we leverage affinity-based targeting and photocatalyzed singlet oxygen generation to identify neurotransmitter receptor-proximal proteins in the live mouse brain using only small-molecule reagents and minutes of photoirradiation. Our photooxidation-driven proximity labeling for proteome identification (named PhoxID) method not only recapitulated the known interactomes of three endogenous neurotransmitter receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), inhibitory γ-aminobutyric acid type A receptor and ionotropic glutamate receptor delta-2) but also uncovered age-dependent shifts, identifying NECTIN3 and IGSF3 as developmentally regulated AMPAR-proximal proteins in the cerebellum. Overall, this work establishes a flexible and generalizable platform to study receptor microenvironments in genetically intact specimens with an unprecedented temporal resolution.

Augmentative effects of leukemia inhibitory factor reveal a critical role for TYK2 signaling in vascular calcification

Medial vascular calcification in chronic kidney disease (CKD) involves pro-inflammatory pathways induced by hyperphosphatemia. Several interleukin 6 family members have been associated with pro-calcific effects in vascular smooth muscle cells (VSMCs) and are considered as therapeutic targets. Therefore, we investigated the role of leukemia inhibitory factor (LIF) during VSMC calcification. LIF expression was found to be increased following phosphate exposure of VSMCs. LIF supplementation aggravated, while silencing of endogenous LIF or LIF receptor (LIFR) ameliorated the pro-calcific effects of phosphate in VSMCs. The soluble LIFR mediated antagonistic effects towards LIF and reduced VSMC calcification. Mechanistically, LIF induced phosphorylation of the non-receptor tyrosine-protein kinase 2 (TYK2) and signal transducer and activator of transcription-3 (STAT3) in VSMCs. TYK2 inhibition by deucravacitinib, a selective, allosteric oral immunosuppressant used in psoriasis treatment, not only blunted the effects of LIF, but also interfered with the pro-calcific effects induced by phosphate. Conversely, TYK2 overexpression aggravated VSMC calcification. Ex vivo calcification of mouse aortic rings was ameliorated by Tyk2 pharmacological inhibition and genetic deficiency. Cholecalciferol-induced vascular calcification in mice was improved by Tyk2 inhibition and in the Tyk2-deficient mice. Similarly, calcification was ameliorated in Abcc6/Tyk2-deficient mice after adenine/high phosphorus-induced CKD. Thus, our observations indicate a role for LIF in CKD-associated vascular calcification. Hence, the effects of LIF identify a central pro-calcific role of TYK2 signaling, which may be a future target to reduce the burden of vascular calcification in CKD.

Glucocorticoid receptor controls atopic dermatitis inflammation via functional interactions with P63 and autocrine signaling in epidermal keratinocytes

Atopic dermatitis (AD), a prevalent chronic inflammatory disease with multifactorial etiology, features epidermal barrier defects and immune overactivation. Synthetic glucocorticoids (GCs) are widely prescribed for treating AD due to their anti-inflammatory actions; however, mechanisms are incompletely understood. Defective local GC signaling due to decreased production of endogenous ligand and/or GC receptor (GR) levels was reported in prevalent inflammatory skin disorders; whether this is a consequence or contributing factor to AD pathology is unclear. To identify the chromatin-bound cell-type-specific GR protein interactome in keratinocytes, we used rapid immunoprecipitation of endogenous proteins and mass spectrometry identifying 145 interactors that increased upon dexamethasone treatment. GR-interacting proteins were enriched in p53/p63 signaling, including epidermal transcription factors with critical roles in AD pathology. Previous analyses indicating mirrored AD-like phenotypes between P63 overexpression and GR loss in epidermis, and our data show an intricate relationship between these transcription factors in human keratinocytes, identifying TP63 as a direct GR target. Dexamethasone treatment counteracted transcriptional up-regulation of inflammatory markers by IL4/IL13, known to mimic AD, causing opposite shifts in GR and P63 genomic binding. Indeed, IL4/IL13 decreased GR and increased P63 levels in cultured keratinocytes and human epidermal equivalents (HEE), consistent with GR down-regulation and increased P63 expression in AD lesions vs normal skin. Moreover, GR knockdown (GRKD) resulted in constitutive increases in P63, phospho-P38 and S100A9, IL6, and IL33. Also, GRKD culture supernatants showed increased autocrine production of TH2-/TH1-/TH17-TH22-associated factors including IL4, CXCL10, CXCL11, and CXCL8. GRKD HEEs showed AD-like features including hyperplasia and abnormal differentiation, resembling phenotypes observed with GR antagonist or IL4/IL13 treatment. The simultaneous GR/P63 knockdown partially reversed constitutive up-regulation of inflammatory genes in GRKD. In summary, our data support a causative role for GR loss in AD pathogenesis via functional interactions with P63 and autocrine signaling in epidermal keratinocytes.

Sesaminol alters phospholipid metabolism and alleviates obesity-induced NAFLD.

The prevalence of obesity-induced non-alcoholic fatty liver disease (NAFLD) and insulin resistance is increasing worldwide. We previously demonstrated that sesaminol increases thermogenesis in adipocytes, improves insulin sensitivity, and mitigates obesity in mice. In this study, we demonstrated that sesaminol increased mitochondrial activity and reduced ROS production in hepatocytes. Therefore, we delve into the metabolic action of sesaminol in obesity-induced NAFLD or metabolic dysfunction-associated liver disease (MAFLD). Here, we report that sesaminol induces OXPHOS proteins and mitochondrial function invivo. Further, our data suggest that sesaminol administration reduces hepatic triacylglycerol accumulation and LDL-C levels. Prominently, the lipidomics analyses revealed that sesaminol administration decreased the major phospholipids such as PC, PE, PI, CL, and PS to maintain membrane lipid homeostasis in the liver upon HFD challenge. Besides, SML reduced ePC and SM molecular species and increased PA levels in the HFD-fed mice. Also, sesaminol renders anti-inflammatory properties and dampens fibrosis markers in the liver. Remarkably, SML lowers the hepatic levels of ALT and AST enzymes and alleviates NAFLD in diet-induced obese mice. The molecular docking analysis identifies peroxisome proliferator-activated receptors as potential endogenous receptors for sesaminol. Together, our study demonstrates plant lignan sesaminol as a potential small molecule that alters the molecular species of major phospholipids, including sphingomyelin and ether-linked PCs in the liver tissue, improves metabolic parameters, and alleviates obesity-induced fatty liver disease in mice.

27-Hydroxycholesterol acts on estrogen receptor α expressed by POMC neurons in the arcuate nucleus to modulate feeding behavior.

Oxysterols are metabolites of cholesterol that regulate cholesterol homeostasis. Among these, the most abundant oxysterol is 27-hydroxycholesterol (27HC), which can cross the blood-brain barrier. Because 27HC functions as an endogenous selective estrogen receptor modulator, we hypothesize that 27HC binds to the estrogen receptor α (ERα) in the brain to regulate energy balance. Supporting this view, we found that delivering 27HC to the brain reduced food intake and activated proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (POMCARH) in an ERα-dependent manner. In addition, we observed that inhibiting brain ERα, deleting ERα in POMC neurons, or chemogenetic inhibition of POMCARH neurons blocked the anorexigenic effects of 27HC. Mechanistically, we further revealed that 27HC stimulates POMCARH neurons by inhibiting the small conductance of the calcium-activated potassium (SK) channel. Together, our findings suggest that 27HC, through its interaction with ERα and modulation of the SK channel, inhibits food intake as a negative feedback mechanism against a surge in circulating cholesterol.

Multimodal Image Fusion Workflow Incorporating MALDI Imaging Mass Spectrometry and Microscopy for the Study of Small Pharmaceutical Compounds.

Multimodal imaging analyses of dosed tissue samples can provide more comprehensive insights into the effects of a therapeutically active compound on a target tissue compared to single-modal imaging. For example, simultaneous spatial mapping of pharmaceutical compounds and endogenous macromolecule receptors is difficult to achieve in a single imaging experiment. Herein, we present a multimodal workflow combining imaging mass spectrometry with immunohistochemistry (IHC) fluorescence imaging and brightfield microscopy imaging. Imaging mass spectrometry enables direct mapping of pharmaceutical compounds and metabolites, IHC fluorescence imaging can visualize large proteins, and brightfield microscopy imaging provides tissue morphology information. Single-cell resolution images are generally difficult to acquire using imaging mass spectrometry but are readily acquired with IHC fluorescence and brightfield microscopy imaging. Spatial sharpening of mass spectrometry images would thus allow for higher fidelity coregistration with other higher-resolution microscopy images. Imaging mass spectrometry spatial resolution can be predicted to a finer value via a computational image fusion workflow, which models the relationship between the intensity values in the mass spectrometry image and the features of a high-spatial resolution microscopy image. As a proof of concept, our multimodal workflow was applied to brain tissue extracted from a Sprague-Dawley rat dosed with a kratom alkaloid, corynantheidine. Four candidate mathematical models, including linear regression, partial least-squares regression, random forest regression, and two-dimensional convolutional neural network (2-D CNN), were tested. The random forest and 2-D CNN models most accurately predicted the intensity values at each pixel as well as the overall patterns of the mass spectrometry images, while also providing the best spatial resolution enhancements. Herein, image fusion enabled predicted mass spectrometry images of corynantheidine, GABA, and glutamine to approximately 2.5 μm spatial resolutions, a significant improvement compared to the original images acquired at 25 μm spatial resolution. The predicted mass spectrometry images were then coregistered with an H&E image and IHC fluorescence image of the μ-opioid receptor to assess colocalization of corynantheidine with brain cells. Our study also provides insights into the different evaluation parameters to consider when utilizing image fusion for biological applications.

Molecular definition of the endogenous Toll-like receptor signalling pathways.

Innate immune pattern recognition receptors, such as the Toll-like receptors (TLRs), are key mediators of the immune response to infection and central to our understanding of health and disease1. After microbial detection, these receptors activate inflammatory signal transduction pathways that involve IκB kinases, mitogen-activated protein kinases, ubiquitin ligases and other adaptor proteins. The mechanisms that connect the proteins in the TLR pathways are poorly defined. To delineate TLR pathway activities, we engineered macrophages to enable microscopy and proteomic analysis of the endogenous myddosome constituent MyD88. We found that myddosomes form transient contacts with activated TLRs and that TLR-free myddosomes are dynamic in size, number and composition over the course of 24 h. Analysis using super-resolution microscopy revealed that, within most myddosomes, MyD88 forms barrel-like structures that function as scaffolds for effector protein recruitment. Proteomic analysis demonstrated that myddosomes contain proteins that act at all stages and regulate all effector responses of the TLR pathways, and genetic analysis defined the epistatic relationship between these effector modules. Myddosome assembly was evident in cells infected with Listeria monocytogenes, but these bacteria evaded myddosome assembly and TLR signalling during cell-to-cell spread. On the basis of these findings, we propose that the entire TLR signalling pathway is executed from within the myddosome.

Gene Expression Level and Immunohistochemical Localization of Cannabinoid and Cannabinoid-Related Receptors in The Small Intestine of Holstein Bulls (Bos Taurus Taurus)

Abstract The gastrointestinal tract plays a crucial role in nutrient absorption, secretion, and motility, ensuring proper digestion and overall homeostasis. Regulation of this complex system involves the coordination of various communication pathways, including neural and humoral mechanisms. One such mechanism is the endocannabinoid system (ECS), a signaling network comprising endogenous cannabinoids, receptors, and enzymes involved in the regulation of physiological processes in mammals and non-mammalian species. While extensive research has been conducted on the ECS in monogastric animals, limited information is available on its presence and distribution in cattle. This study aimed to investigate the distribution and localization patterns of cannabinoid receptors type 1 (CB1R) and type 2 (CB2R) and transient receptor potential vanilloid type 1 (TRPV1) in the bovine small intestine. The study included immunohistochemical analysis of intestinal tissue samples from Polish Holstein-Friesian breed bulls. Gene expression levels of CNR1, CNR2, and TRPV1 genes, encoding CB1R, CB2R, and TRPV1, respectively, were quantified using qPCR analysis. The results showed that all three receptors were expressed in the bovine small intestine, with TRPV1 exhibiting a significant upregulation in the jejunum compared to the duodenum and ileum. Immunoreactivity for CB1R and CB2R was predominantly observed in neurons of the enteric plexuses, while TRPV1 immunolabeling was detected in both enteric neurons and duodenal Brunner’s glands. These findings may establish an anatomical foundation for further investigations, lending support to the potential therapeutic efficacy of cannabinoid receptor agonists in alleviating gastrointestinal motility disorders associated with bovine enteropathies and optimizing milk production in dairy cattle.