ABSTRACT
This study investigated the potential role of the mouse homolog of bombesin receptor-activated protein (BRAP) in imiquimod (IMQ) induced psoriasis - like skin inflammation. The expression of both human BRAP, encoded by C6orf89, and its mouse homolog, encoded by BC004004, has been found to be expressed abundantly in the keratinocytes. BC004004 knockout mice (BC004004-/-) were topically treated with IMQ daily for 7 days to test whether they were more vulnerable to psoriasis - like inflammation. We found that those mice exhibited an altered pattern of inflammation process compared to isogenic wild type control mice (BC004004+/+). BC004004-/- mice developed skin lesions with earlier and more acute onset, as well as a quicker remission. The cytokines related to pathogenesis of psoriasis also exhibited different expression patterns in IMQ treated BC004004-/- mice. On day 4 of IMQ treatment, BC004004-/- mice exhibited a higher expression level of IL-17A compared to BC004004+/+ mice, suggesting a more robust activation of Th17 cells in the knockout mice. The serum level of thymic stromal lymphopoietin (TSLP), one of the keratinocyte derived cytokines, was also increased in BC004004-/- mice and reached its peak on day 4. Knockdown of BRAP in cultured human keratinocyte-derived HaCaT cells by siRNA silencing led to increased release of TSLP. Our data suggest that the elevated of level of TSLP released from keratinocytes due to BRAP deficiency might mediate the crosstalk between the epidermal cells and immune cells and thereby contributing to the altered pathological changes observed in psoriasis - like skin lesion in knockout mice.
Subject(s)
Psoriasis , Receptors, Bombesin , Mice , Humans , Animals , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Keratinocytes/metabolism , Imiquimod/metabolism , Inflammation/pathology , Cytokines/metabolism , Mice, Knockout , Disease Models, Animal , Skin/pathology , Mice, Inbred BALB CABSTRACT
A11 dopaminergic neurons regulate somatosensory transduction by projecting from the diencephalon to the spinal cord, but the function of this descending projection in itch remained elusive. Here, we report that dopaminergic projection neurons from the A11 nucleus to the spinal dorsal horn (dopaminergicA11-SDH ) are activated by pruritogens. Inhibition of these neurons alleviates itch-induced scratching behaviors. Furthermore, chemogenetic inhibition of spinal dopamine receptor D1-expressing (DRD1+ ) neurons decreases acute or chronic itch-induced scratching. Mechanistically, spinal DRD1+ neurons are excitatory and mostly co-localize with gastrin-releasing peptide (GRP), an endogenous neuropeptide for itch. In addition, DRD1+ neurons form synapses with GRP receptor-expressing (GRPR+ ) neurons and activate these neurons via AMPA receptor (AMPAR). Finally, spontaneous itch and enhanced acute itch induced by activating spinal DRD1+ neurons are relieved by antagonists against AMPAR and GRPR. Thus, the descending dopaminergic pathway facilitates spinal itch transmission via activating DRD1+ neurons and releasing glutamate and GRP, which directly augments GRPR signaling. Interruption of this descending pathway may be used to treat chronic itch.
Subject(s)
Receptors, Bombesin , Spinal Cord , Humans , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Gastrin-Releasing Peptide/genetics , Gastrin-Releasing Peptide/metabolism , Spinal Cord/metabolism , Glutamic Acid/metabolism , Dopamine/metabolism , Pruritus/genetics , Pruritus/metabolism , Dopaminergic Neurons/metabolism , Receptors, AMPA/genetics , Receptors, AMPA/metabolismABSTRACT
Bombesin receptor-activated protein (BRAP) and its homologous protein in mice, which is encoded by bc004004 gene, were expressed abundantly in brain tissues with unknown functions. We treated bc004004-/- mice with chronic unpredictable mild stress (CUMS) to test whether those mice were more vulnerable to stress-related disorders. The results of forced swimming test, sucrose preference test, and open field test showed that after being treated with CUMS for 28 days or 35 days both bc004004-/- and bc004004+/+ mice exhibited behavioural changes and there was no significant difference between bc004004+/+ and bc004004-/-. However, behavioural changes were observed only in bc004004-/- mice after being exposed to CUMS for 21 days, but not in bc004004+/+ after 21-day CUMS exposure, indicating that lack of BRAP homologous protein may cause vulnerability to stress-related disorders in mice. In addition, bc004004-/- mice showed a reduction in recognition memory as revealed by novel object recognition test. Since memory changes and stress related behavioural changes are all closely related to the hippocampus function we further analyzed the changes of dendrites and synapses of hippocampal neurons as well as expression levels of some proteins closely related to synaptic function. bc004004-/- mice exhibited decreased dendritic lengths and increased amount of immature spines, as well as altered expression pattern of synaptic related proteins including GluN2A, synaptophysin and BDNF in the hippocampus. Those findings suggest that BRAP homologous protein may have a protective effect on the behavioural response to stress via regulating dendritic spine formation and synaptic plasticity in the hippocampus.
Subject(s)
Bombesin , Dendritic Spines , Hippocampus , Neuronal Plasticity , Receptors, Bombesin , Stress, Psychological , Animals , Mice , Bombesin/genetics , Bombesin/metabolism , Chronic Disease , Dendritic Spines/genetics , Dendritic Spines/metabolism , Dendritic Spines/pathology , Depression/genetics , Depression/metabolism , Depression/pathology , Disease Models, Animal , Hippocampus/metabolism , Hippocampus/pathology , Neuronal Plasticity/genetics , Neuronal Plasticity/physiology , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Stress, Psychological/genetics , Stress, Psychological/metabolism , Stress, Psychological/pathologyABSTRACT
ABSTRACT: Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are believed to transmit nociceptive information. In this study, we have used a GRPR CreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for â¼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus, and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain-related and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.
Subject(s)
Posterior Horn Cells , Receptors, Bombesin , Mice , Animals , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Gastrin-Releasing Peptide/genetics , Gastrin-Releasing Peptide/metabolism , Posterior Horn Cells/metabolism , Spinal Cord Dorsal Horn/metabolism , Spinal Cord/metabolism , Interneurons/metabolism , Pruritus/metabolism , Pain/metabolismABSTRACT
Abdominal obesity coupled with polygenic hereditary defects is considered the initial event in the development of metabolic syndrome (MS). The purpose of this study was to analyse the frequency with which polymorphic loci of adiponectin (ADIPOQ) and leptin (LEP) genes occur in patients with MS and the association between the symptoms of MS and these polymorphisms. DNA was isolated from the whole blood of 207 patients with MS and 100 healthy individuals (control group) using the phenol-chloroform method. Gene polymorphisms were determined using real-time polymerase chain reaction (PCR). The most common variant of the ADIPOQ (rs2241766) gene among MS patients was the GT genotype. The A allele of the LEP (rs7799039) gene was found to be the most frequent in MS patients. The highest systolic blood pressure was found in carriers of the GG genotype of the LEP (rs7799039) gene. The carriers of the ADIPOQ (rs2241766) GT genotype were associated with the highest systolic blood pressure and body mass index (BMI); carriers of the ADIPOQ (rs2241766) GG genotype were associated with the highest diastolic blood pressure, hyperglycaemia, and elevated glycated haemoglobin (HbA1c). The results of this study allowed us to establish the unique gene variants associated with the risk of developing MS in the Crimean population.
Subject(s)
Adiponectin , Metabolic Syndrome , Receptors, Leptin , Adiponectin/genetics , Chloroform , Glycated Hemoglobin/genetics , Humans , Leptin/genetics , Metabolic Syndrome/genetics , Phenols , Polymorphism, Single Nucleotide , Receptors, Bombesin/genetics , Receptors, Colony-Stimulating Factor/genetics , Receptors, Formyl Peptide/genetics , Receptors, Leptin/genetics , Ryanodine Receptor Calcium Release ChannelABSTRACT
Bombesin receptor-activated protein (BRAP) was found to express in the interstitial cells of human fibrotic lungs with unknown function. Its homologous protein, encoded by BC004004 gene, was also present in mouse lung tissues. We used BC004004 -/- mice which lack BRAP homologous protein expression to establish a bleomycin-induced lung fibrotic model. After bleomycin treatment, BC004004 -/- mice exhibited attenuation of pulmonary injury and less pulmonary fibrosis. Fibroblasts from BC004004 -/- mice proliferated at a lower rate and produced less collagen. Autophagy-related gene 5 (ATG5) was identified as a partner interacting with human BRAP. Lacking BRAP homologous protein led to enhanced autophagy activity in mouse lung tissues as well as in isolated lung fibroblasts, indicating a negative regulatory role of this protein in autophagy via interaction with ATG5. Enhanced autophagy process in fibroblasts due to lack of BRAP homologous protein might contribute to the resistance of BC004004 -/- mice to pulmonary fibrosis.
Subject(s)
Bleomycin , Pulmonary Fibrosis , Animals , Bleomycin/adverse effects , Bleomycin/metabolism , Bombesin/adverse effects , Bombesin/metabolism , Humans , Lung/metabolism , Mice , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolismABSTRACT
Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.
Subject(s)
Gastrin-Releasing Peptide , Macaca fuscata , Sense Organs , Animals , DNA, Complementary , Gastrin-Releasing Peptide/physiology , Humans , Ligands , Pruritus/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Sense Organs/physiologyABSTRACT
Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.
Subject(s)
Bombesin , Receptors, Bombesin , Animals , Anura/metabolism , Bombesin/metabolism , Bombesin/pharmacology , Mammals/metabolism , Molecular Docking Simulation , Peptides/pharmacology , Rats , Rats, Wistar , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Stomach , Tandem Mass SpectrometryABSTRACT
Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.
Subject(s)
Hyperalgesia , Pruritus , Animals , Gastrin-Releasing Peptide/genetics , Gastrin-Releasing Peptide/metabolism , Gastrin-Releasing Peptide/pharmacology , Hyperalgesia/metabolism , Neurokinin B/analogs & derivatives , Pain/metabolism , Primates/metabolism , Pruritus/chemically induced , Pruritus/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Spinal Cord , Spinal Cord Dorsal Horn/metabolismABSTRACT
Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole body energy homeostasis, however, the presence of NB-NBR signaling and its effects in SM are unknown. Here, we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase and Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females' gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. In addition, in L6 myocytes, NB treatment (5 µg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.NEW & NOTEWORTHY This study describes neuromedin B (NB) and NB receptor as new regulators of skeletal muscle mitochondrial function. The white skeletal muscle mitochondrial oxidative phosphorylation capacity was increased by NB receptor genetic disruption in female mice. These findings may contribute to the resistance to diet-induced obesity, previously found in these mice, which requires future studies. Thus, investigations are necessary to clarify if blockade of NB receptor may be an approach to develop drugs to combat obesity.
Subject(s)
Oxidative Phosphorylation , Receptors, Bombesin , Adenosine Triphosphate/metabolism , Animals , Female , Male , Mice , Mice, Knockout , Mitochondria/metabolism , Mitochondria, Muscle/metabolism , Muscle, Skeletal/metabolism , Obesity/metabolism , RNA, Messenger/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolismABSTRACT
Vascular calcification is the heterotopic accumulation of calcium phosphate salts in the vascular tissue and is highly correlated with increased cardiovascular morbidity and mortality. In this study, we found that the expression of neuromedin B (NMB) and NMB receptor is upregulated in phosphate-induced calcification of vascular smooth muscle cells (VSMCs). Silencing of NMB or treatment with NMB receptor antagonist, PD168368, inhibited the phosphate-induced osteogenic differentiation of VSMCs by inhibiting Wnt/ß-catenin signaling and VSMC apoptosis. PD168368 also attenuated the arterial calcification in cultured aortic rings and in a rat model of chronic kidney disease. The results of this study suggest that NMB-NMB receptor axis may have potential therapeutic value in the diagnosis and treatment of vascular calcification. [BMB Reports 2021; 54(11): 569-574].
Subject(s)
Calcium/metabolism , Neurokinin B/analogs & derivatives , Osteogenesis , Phosphates/toxicity , Receptors, Bombesin/metabolism , Renal Insufficiency, Chronic/complications , Vascular Calcification/pathology , Animals , Cell Differentiation , Cells, Cultured , Male , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Neurokinin B/genetics , Neurokinin B/metabolism , Rats , Rats, Wistar , Receptors, Bombesin/genetics , Vascular Calcification/etiology , Vascular Calcification/metabolism , Wnt Signaling PathwayABSTRACT
Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.
Subject(s)
Circadian Clocks/genetics , Circadian Rhythm/genetics , Gastrointestinal Hormones/genetics , Neuropeptides/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Suprachiasmatic Nucleus/metabolism , Transcriptome , Animals , Gastrin-Releasing Peptide/genetics , Gastrin-Releasing Peptide/metabolism , Gastrointestinal Hormones/metabolism , Gene Expression Regulation , Gene Regulatory Networks , Mice , Neurons/cytology , Neurons/metabolism , Neuropeptides/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Receptors, Vasopressin/genetics , Receptors, Vasopressin/metabolism , Signal Transduction , Single-Cell Analysis , Suprachiasmatic Nucleus/cytology , Vasoactive Intestinal Peptide/genetics , Vasoactive Intestinal Peptide/metabolism , Vasopressins/genetics , Vasopressins/metabolismABSTRACT
G-protein-coupled receptors (GPCRs) are increasingly being considered as possible therapeutic targets in cancers. Activation of GPCR on tumors can have prominent growth effects, and GPCRs are frequently over-/ectopically expressed on tumors and thus can be used for targeted therapy. CNS/neural tumors are receiving increasing attention using this approach. Gliomas are the most frequent primary malignant brain/CNS tumor with glioblastoma having a 10-year survival <1%; neuroblastomas are the most common extracranial solid tumor in children with long-term survival<40%, and medulloblastomas are less common, but one subgroup has a 5-year survival <60%. Thus, there is an increased need for more effective treatments of these tumors. The Bombesin-receptor family (BnRs) is one of the GPCRs that are most frequently over/ectopically expressed by common tumors and is receiving particular attention as a possible therapeutic target in several tumors, particularly in prostate, breast, and lung cancer. We review in this paper evidence suggesting why a similar approach in some CNS/neural tumors (gliomas, neuroblastomas, medulloblastomas) should also be considered.
Subject(s)
Central Nervous System Neoplasms/drug therapy , Molecular Targeted Therapy/trends , Receptors, Bombesin/agonists , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/metabolism , Child , Female , Glioma/drug therapy , Glioma/genetics , Glioma/metabolism , Humans , Male , Molecular Targeted Therapy/methods , Multigene Family , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Neuroblastoma/metabolism , Receptors, Bombesin/genetics , Therapies, Investigational/methods , Therapies, Investigational/trendsABSTRACT
The preoptic area (POA) is a key brain region for regulation of body temperature (Tb), dictating thermogenic, cardiovascular, and behavioral responses that control Tb. Previously characterized POA neuronal populations all reduced Tb when activated. Using mice, we now identify POA neurons expressing bombesin-like receptor 3 (POABRS3) as a population whose activation increased Tb; inversely, acute inhibition of these neurons reduced Tb. POABRS3 neurons that project to either the paraventricular nucleus of the hypothalamus or the dorsomedial hypothalamus increased Tb, heart rate, and blood pressure via the sympathetic nervous system. Long-term inactivation of POABRS3 neurons caused increased Tb variability, overshooting both increases and decreases in Tb set point, with RNA expression profiles suggesting multiple types of POABRS3 neurons. Thus, POABRS3 neuronal populations regulate Tb and heart rate, contribute to cold defense, and fine-tune feedback control of Tb. These findings advance understanding of homeothermy, a defining feature of mammalian biology.
Subject(s)
Body Temperature Regulation , Heart Rate , Neurons/physiology , Preoptic Area/metabolism , Receptors, Bombesin/metabolism , Animals , Body Temperature/genetics , Body Temperature Regulation/genetics , Heart Rate/genetics , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Neurons/cytology , Neurons/metabolism , Preoptic Area/cytology , Receptors, Bombesin/genetics , Signal Transduction/genetics , Sympathetic Nervous System/physiology , Thermogenesis/geneticsABSTRACT
Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.
Subject(s)
Molecular Imaging/methods , Nanostructures/chemistry , Prostatic Neoplasms/diagnostic imaging , Saline Solution, Hypertonic/chemistry , Acoustics , Animals , Cell Line, Tumor , Contrast Media/chemistry , Drug Stability , Humans , Hydrocarbons, Fluorinated/chemistry , Male , Mice, Inbred NOD , Molecular Imaging/instrumentation , Phantoms, Imaging , Prostatic Neoplasms/metabolism , Radio Waves , Receptors, Bombesin/genetics , Receptors, Bombesin/immunology , Receptors, Bombesin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Intractable or recurrent chronic itch greatly reduces the patients' QOL and impairs their daily activities. In this study, we investigated whether there are certain key signaling molecules downstream of the recently identified peptides mediating itch in the spinal cord. RNA sequencing analysis of mouse spinal cord in chronic itch models induced by squaric acid dibutylester and imiquimod showed that extracellular signal-regulated kinase (ERK) 1/2 cascade is the most significantly upregulated gene cluster in both models. In four different mouse models of chronic itch, sustained ERK phosphorylation was detected mainly in spinal neurons, and MAPK/ERK kinase inhibitors significantly inhibited chronic itch in these models. Phosphorylated ERK was observed in the interneurons expressing the receptors of different neuropeptides for itch, including gastrin-releasing peptide receptor, natriuretic peptide receptor A, neuromedin B receptor, and sst2A. Blocking gastrin-releasing peptide receptor and natriuretic peptide receptor A by genetic approaches or toxins in mice significantly attenuated or ablated spinal phosphorylated ERK. When human embryonic kidney 293T cells transfected with these receptors were exposed to their respective agonists, ERK was the most significantly activated intracellular signaling molecule. Together, our work showed that phosphorylated ERK is a unique marker for itch signal transmission in the spinal cord and an attractive target for the treatment of chronic itch.
Subject(s)
MAP Kinase Signaling System/immunology , Pruritus/immunology , Receptors, Atrial Natriuretic Factor/metabolism , Receptors, Bombesin/metabolism , Spinal Cord/metabolism , Animals , Chronic Disease/drug therapy , Cyclobutanes/immunology , Disease Models, Animal , HEK293 Cells , Humans , Interneurons/metabolism , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/drug effects , Phosphorylation/immunology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pruritus/drug therapy , Pruritus/pathology , RNA-Seq , Receptors, Atrial Natriuretic Factor/genetics , Receptors, Bombesin/genetics , Skin/immunology , Skin/innervation , Skin/pathology , Spinal Cord/cytology , Spinal Cord/pathologyABSTRACT
Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.
Subject(s)
Antineoplastic Agents/pharmacology , Positron Emission Tomography Computed Tomography , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/drug therapy , Receptors, Bombesin/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Cobalt Radioisotopes , Copper Radioisotopes , Humans , Male , Mice , Neoplasms, Experimental/diagnostic imaging , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , PC-3 Cells , Prostatic Neoplasms/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolismABSTRACT
INTRODUCTION: Radiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. METHODS: DOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. RESULTS: Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70-80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). CONCLUSIONS: Comparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.
Subject(s)
Bombesin/antagonists & inhibitors , Breast Neoplasms/pathology , Gallium Radioisotopes , Positron-Emission Tomography/methods , Prostatic Neoplasms/pathology , Radioisotopes , Scandium , Animals , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Isotope Labeling , MCF-7 Cells , Male , PC-3 Cells , RNA, Messenger/genetics , Receptors, Bombesin/geneticsABSTRACT
The neurokinin-1 receptor (NK1R; encoded by Tacr1) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch in rodents. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. To address this limitation, we leveraged a newly developed Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization (FISH) to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn (DDH), as well as the lateral spinal nucleus (LSN), of mouse and human spinal cord. Retrograde labeling studies in mice from the parabrachial nucleus (PBN) show that less than 20% of superficial Tacr1CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn (SDH) neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry.SIGNIFICANCE STATEMENT The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons process itch input and how itch signals are encoded within the spinal cord is not fully understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These NK1R neurons comprise a subset of gastrin-releasing peptide receptor (GRPR) interneurons and are thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in human spinal cord, underscoring the translational relevance of our findings in mice. This work is the first to suggest a role for NK1R interneurons in itch and extends our understanding of the complexities of spinal itch circuitry.
Subject(s)
Interneurons , Nerve Net/physiopathology , Pruritus/physiopathology , Receptors, Bombesin/biosynthesis , Receptors, Bombesin/genetics , Receptors, Neurokinin-1/biosynthesis , Receptors, Neurokinin-1/genetics , Spinal Cord/metabolism , Spinal Cord/physiopathology , Adult , Animals , Behavior, Animal , Brachial Plexus/physiopathology , Female , Humans , Male , Mice, Inbred C57BL , Middle Aged , Pain/psychology , Posterior Horn Cells/metabolism , Pruritus/genetics , Pruritus/psychologyABSTRACT
Itch, in particular chronic forms, has been widely recognized as an important clinical problem, but much less is known about the mechanisms of itch in comparison with other sensory modalities such as pain. Recently, considerable progress has been made in dissecting the circuit mechanisms of itch at both the spinal and supraspinal levels. Major components of the spinal neural circuit underlying both chemical and mechanical itch have now been identified, along with the circuits relaying ascending transmission and the descending modulation of itch. In this review, we summarize the progress in elucidating the neural circuit mechanism of itch at spinal and supraspinal levels.