ß-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Integrating network pharmacology and transcriptomic omics reveals that akebia saponin D attenuates neutrophil extracellular traps-induced neuroinflammation via NTSR1/PKAc/PAD4 pathway after intracerebral hemorrhage.

Neutrophils and their production of neutrophil extracellular traps (NETs) significantly contribute to neuroinflammation and brain damage after intracerebral hemorrhage (ICH). Although Akebia saponin D (ASD) demonstrates strong anti-inflammatory activities and blood-brain barrier permeability, its role in regulating NETs formation and neuroinflammation following ICH is uncharted. Our research focused on unraveling the influence of ASD on neuroinflammation mediated by NETs and the mechanisms involved. We found that increased levels of peripheral blood neutrophils post-ICH are correlated with worse prognostic outcomes. Through network pharmacology, we identified ASD as a promising therapeutic target for ICH. ASD administration significantly improved neurobehavioral performance and decreased NETs production in neutrophils. Furthermore, ASD was shown to upregulate the membrane protein NTSR1 and activate the cAMP signaling pathway, confirmed through transcriptome sequencing, western blot, and immunofluorescence. Interestingly, the NTSR1 inhibitor SR48692 significantly nullified ASD's anti-NETs effects and dampened cAMP pathway activation. Mechanistically, suppression of PKAc via H89 negated ASD's anti-NETs effects but did not affect NTSR1. Our study suggests that ASD may reduce NETs formation and neuroinflammation, potentially involving the NTSR1/PKAc/PAD4 pathway post-ICH, underlining the potential of ASD in mitigating neuroinflammation through its anti-NETs properties.

Chelator boosted tumor-retention and pharmacokinetic properties: development of 64Cu labeled radiopharmaceuticals targeting neurotensin receptor.

PURPOSE: Accumulating evidence suggests that neurotensin (NTS) and neurotensin receptors (NTSRs) play key roles in lung cancer progression by triggering multiple oncogenic signaling pathways. This study aims to develop Cu-labeled neurotensin receptor 1 (NTSR1)-targeting agents with the potential for both imaging and therapeutic applications. METHOD: A series of neurotensin receptor antagonists (NRAs) with variable propylamine (PA) linker length and different chelators were synthesized, including [64Cu]Cu-CB-TE2A-iPA-NRA ([64Cu]Cu-4a-c, i = 1, 2, 3), [64Cu]Cu-NOTA-2PA-NRA ([64Cu]Cu-4d), [64Cu]Cu-DOTA-2PA-NRA ([64Cu]Cu-4e, also known as [64Cu]Cu-3BP-227), and [64Cu]Cu-DOTA-VS-2PA-NRA ([64Cu]Cu-4f). The series of small animal PET/CT were conducted in H1299 lung cancer model. The expression profile of NTSR1 was also confirmed by IHC using patient tissue samples. RESULTS: For most of the compounds studied, PET/CT showed prominent tumor uptake and high tumor-to-background contrast, but the tumor retention was strongly influenced by the chelators used. For previously reported 4e, [64Cu]Cu-labeled derivative showed initial high tumor uptake accompanied by rapid tumor washout at 24 h. The newly developed [64Cu]Cu-4d and [64Cu]Cu-4f demonstrated good tumor uptake and tumor-to-background contrast at early time points, but were less promising in tumor retention. In contrast, our lead compound [64Cu]Cu-4b demonstrated 9.57 ± 1.35, 9.44 ± 2.38 and 9.72 ± 4.89%ID/g tumor uptake at 4, 24, and 48 h p.i., respectively. Moderate liver uptake (11.97 ± 3.85, 9.80 ± 3.63, and 7.72 ± 4.68%ID/g at 4, 24, and 48 h p.i.) was observed with low uptake in most other organs. The PA linker was found to have a significant effect on drug distribution. Compared to [64Cu]Cu-4b, [64Cu]Cu-4a had a lower background, including a greatly reduced liver uptake, while the tumor uptake was only moderately reduced. Meanwhile, [64Cu]Cu-4c showed increased uptake in both the tumor and the liver. The clinical relevance of NTSR1 was also demonstrated by the elevated tumor expression in patient tissue samples. CONCLUSIONS: Through the side-by-side comparison, [64Cu]Cu-4b was identified as the lead agent for further evaluation based on its high and sustained tumor uptake and moderate liver uptake. It can not only be used to efficiently detect NTSR1 expression in lung cancer (for diagnosis, patient screening, and treatment monitoring), but also has the great potential to treat NTSR-positive lesions once chelating to the beta emitter 67Cu.

Probiotics modulation of the endotoxemic effect on the gut and liver of the lipopolysaccharide challenged mice.

The multistrain probiotics' efficacy in ameliorating the endotoxemic effect in Lipopolysaccharide (LPS) challenged mice was evaluated with the agonist of anti-inflammatory peptide, neurotensin (NTS), especially targeting the inflammation of the gut and liver. Swiss Albino Mice (Female, 8 weeks old) were maintained in eight groups: Group I as Control, Group II-Group V were exposed to intraperitoneal (i.p.) LPS (1 mg/kg bw) for 5 days. After that, Group III and Group VI were administered probiotics orally (0.6 gm/kg bw/day), Group IV and Group VII with NTS receptor 1 (NTSR1) agonist PD149163 (50 µg/kg bw/day i.p.), and Group V and Group VIII co-administered with probiotics and PD149163 for 28 days. Group II (LPS-exposed) was maintained without any further treatment; mice of all the groups were sacrificed at day 34. In the LPS-exposed mice, endotoxemia was distinct from a significant (P < 0.001) increase of plasma pro-inflammatory cytokines (TNF-α; IL-6), a decrease of anti-inflammatory cytokine (IL-10), oxidative stress, and inflammation of the gut and liver. Increased serum transaminases indicated hepatic inflammation. A decreased population of the bifidobacteria and increased clostridia indicated microbiota dysbiosis. Probiotics when used as an adjunct along with PD149163 have shown better efficacy in inflammation modulation as reflected in the significantly decreased (P < 0.001) inflammatory mediators, oxidative stress, restoration of the beneficial bacterial population, along with a significant reduction in histopathological scores of the gut and the liver than when used alone. This study suggests probiotics could be used as an adjunct in clinical practice along with anti-inflammatory drugs for better therapeutic efficacy.

Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration.

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Neurotensin receptor 1 is a new therapeutic target for human undifferentiated pleomorphic sarcoma growth.

Undifferentiated pleomorphic sarcoma (UPS) is the second most common soft tissue sarcoma. For patients with unresectable or metastatic disease, chemotherapies are considered, but in many cases they are not curative. There is a need to identify specific molecular dysregulations that can be therapeutic targets. We focused on neurotensin receptor 1 (NTSR1), which belongs to the G-protein-coupled receptor. NTSR1 expression was upregulated in specimens from patients with UPS. Real-time polymerase chain reaction showed that expression of NTSR1 messenger RNA was 5- to 7-fold increased in UPS cells compared with myoblasts. Western blot showed a high expression of NTSR1 protein in UPS cell lines. Knockdown of NTSR1 prevented UPS cell proliferation and invasion. We confirmed that SR48692, an inhibitor of NTSR1, exhibited antitumor activities in UPS cells. The combination index showed that SR48692 and standard chemotherapeutic drugs prevented UPS cell proliferation synergistically. Mouse xenograft models showed that SR48692 inhibited extracellular signal-regulated kinase phosphorylation and enhanced the response to standard chemotherapeutic drugs. Inhibition of NTSR1 improved the effect of standard chemotherapeutic drugs for UPS. SR48692 may be a new drug for targeted UPS therapy.

Differential effect on myelination through abolition of activity-dependent synaptic vesicle release or reduction of overall electrical activity of selected cortical projections in the mouse.

Myelination of axons by oligodendrocytes in the central nervous system is crucial for fast, saltatory conduction of action potentials. As myelination is central for brain development and plasticity, and deficits are implicated in several neural disorders such as multiple sclerosis, major depressive disorder, bipolar disorder and schizophrenia, it is important to elucidate the underlying mechanisms regulating myelination. Numerous mechanisms have been proposed by which the communication between oligodendrocytes and active axons may regulate the onset and maintenance of activity-dependent myelination. We compared two models of 'silencing' layer V and/or VI cortical projection neurons from early stages by either decreasing their excitability through Kir2.1 expression, an inward rectifying potassium channel, introduced through in utero electroporation at embryonic day (E)13.5, or inhibiting regulated vesicular release through Cre-dependent knock-out of synaptosomal associated protein 25 kDA (SNAP25). SNAP25 is a component of the soluble N-ethylmaleimide fusion protein attachment protein receptor (SNARE) complex, which, among others, is needed for calcium-dependent regulated vesicle release from synapses. In layer VI cortical projection neurons in the Ntsr1-Cre;Ai14;Snap25 fl/fl mouse, we found that inhibiting regulated vesicular release significantly decreased the amount of myelin basic protein (MBP, used as marker for myelination) and the amount of myelinated projections at postnatal day (P)14 without affecting the initial timing of onset of myelination in the brain (at P7/P8). Additionally, overall oligodendrocyte maturation appears to be affected. A strong trend towards reduced node of Ranvier (NoR) length was also observed in Ntsr1-Cre;Ai14;Snap25 fl/fl corpus callosum. An equally strong trend towards reduced NoR length was observed in Rbp4-Cre;Ai14;Snap25 fl/fl corpus callosum at P14, and the g-ratio in the spinal cord dorsal column was reduced at P18. However, no measurable differences in levels of MBP were detected in the striatum when comparing Rbp4-Cre;Ai14;Snap25 fl/fl and control brains. Conversely, Kir2.1 in utero electroporation at E13.5 did not significantly affect the amount of MBP or number of myelinated callosal axons at P14 but did significantly decrease the NoR length measured in the corpus callosum. It therefore seems likely that the excitability of the neuron can potentially perform a modulating function of myelin characteristics, whereas regulated vesicular release has the potential to have a more pronounced effect on overall myelination, but in a cell-type specific manner.

Interference with NTSR1 Expression Exerts an Anti-Invasion Effect via the Jun/miR-494/SOCS6 Axis of Glioblastoma Cells.

BACKGROUND/AIMS: Glioblastoma is the most common and aggressive brain tumor and carries a poor prognosis. Previously, we found that neurotensin receptor 1 (NTSR1) contributes to glioma progression, but the underlying mechanisms of NTSR1 in glioblastoma invasion remain to be clarified. The aim of this study was to investigate the molecular mechanisms of NTSR1 in glioblastoma invasion. METHODS: Cell migration and invasion were evaluated using wound-healing and transwell assays. Cell proliferation was detected using CCK-8. The expression of NTSR1, Jun, and suppressor of cytokine signaling 6 (SOCS6) was detected using western blotting. The expression of miR-494 was detected by Quantitative real-time PCR. Chromatin immunoprecipitation assay was performed to examine the interaction between Jun and miR-494 promoter. Dual-luciferase reporter assay and western blotting were performed to identify the direct regulation of SOCS6 by miR-494. An orthotopic xenograft mouse model was conducted to assess tumor growth and invasion. RESULTS: NTSR1 knockdown attenuated the invasion of glioblastoma cells. Jun was positively regulated by NTSR1, which promoted miR-494 expression through binding to miR-494 promoter. SOCS6 was confirmed as a direct target of miR-494, thus, NTSR1-induced miR-494 upregulation resulted in SOCS6 downregulation. Both miR-494 and SOCS6 were involved in the NTSR1-induced invasion of glioblastoma cells. In vivo, tumor invasion and growth were inhibited by NTSR1 knockdown, but were restored with miR-494 overexpression. CONCLUSION: NTSR1 knockdown inhibited glioblastoma invasion via the Jun/miR-494/SOCS6 axis.

A Novel Positive Feedback Loop Between NTSR1 and Wnt/ß-Catenin Contributes to Tumor Growth of Glioblastoma.

BACKGROUND/AIMS: Neurotensin (NTS), an intestinal hormone, is profoundly implicated in cancer progression through binding its primary receptor NTSR1. The conserved Wnt/ß-Catenin pathway regulates cell proliferation and differentiation via activation of the ß-catenin/T-cell factor (TCF) complex and subsequent modulation of a set of target genes. In this study, we aimed to uncover the potential connection between NTS/NTSR1 signaling and Wnt/ß-Catenin pathway. METHODS: Genetic silencing, pharmacological inhibition and gain-of-function studies as well as bioinformatic analysis were performed to uncover the link between NTS/ NTSR1 signaling and Wnt/ß-Catenin pathway. Two inhibitors were used in vivo to evaluate the efficiency of targeting NTS/NTSR1 signaling or Wnt/ß-Catenin pathway. RESULTS: We found that NTS/NTSR1 induced the activation of mitogen-activated protein kinase (MAPK) and the NF-κB pathway, which further promoted the expression of Wnt proteins, including Wnt1, Wnt3a and Wnt5a. Meanwhile, the mRNA and protein expression levels of NTSR1 were increased by the Wnt pathway activator Wnt3a and decreased by the Wnt inhibitor iCRT3 in glioblastoma cells. Furthermore, pharmacological inhibition of NTS/NTSR1 or Wnt/ß-Catenin signaling suppressed tumor growth in vitro and in vivo. CONCLUSION: These results reveal a positive feedback loop between NTS/NTSR1 and Wnt/ß-Catenin signaling in glioblastoma cells that might be important for tumor development and provide potential therapeutic targets for glioblastoma.

Cortical control of adaptation and sensory relay mode in the thalamus.

A major synaptic input to the thalamus originates from neurons in cortical layer 6 (L6); however, the function of this cortico-thalamic pathway during sensory processing is not well understood. In the mouse whisker system, we found that optogenetic stimulation of L6 in vivo results in a mixture of hyperpolarization and depolarization in the thalamic target neurons. The hyperpolarization was transient, and for longer L6 activation (>200 ms), thalamic neurons reached a depolarized resting membrane potential which affected key features of thalamic sensory processing. Most importantly, L6 stimulation reduced the adaptation of thalamic responses to repetitive whisker stimulation, thereby allowing thalamic neurons to relay higher frequencies of sensory input. Furthermore, L6 controlled the thalamic response mode by shifting thalamo-cortical transmission from bursting to single spiking. Analysis of intracellular sensory responses suggests that L6 impacts these thalamic properties by controlling the resting membrane potential and the availability of the transient calcium current IT, a hallmark of thalamic excitability. In summary, L6 input to the thalamus can shape both the overall gain and the temporal dynamics of sensory responses that reach the cortex.

Complex Effects on In Vivo Visual Responses by Specific Projections from Mouse Cortical Layer 6 to Dorsal Lateral Geniculate Nucleus.

Understanding the role of corticothalamic projections in shaping visual response properties in the thalamus has been a longstanding challenge in visual neuroscience. Here, we take advantage of the cell-type specificity of a transgenic mouse line, the GN220-Ntsr1 Cre line, to manipulate selectively the activity of a layer 6 (L6) corticogeniculate population while recording visual responses in the dorsal lateral geniculate nucleus (dLGN). Although driving Ntsr1 projection input resulted in reliable reduction in evoked spike count of dLGN neurons, removing these same projections resulted in both increases and decreases in visually evoked spike count. Both increases and decreases are contrast dependent and the sign is consistent over the full range of contrasts. Tuning properties suggest wide convergence of Ntsr1 cells with similar spatial and temporal frequency tuning onto single dLGN cells and we did not find evidence that Ntsr1 cells sharpen spatiotemporal filtering. These nonspecific changes occur independently of changes in burst frequency, indicating that Ntsr1 corticogeniculate activity can result in both net excitation and net inhibition.

GPR39-1b, the 5-transmembrane isoform of GPR39 interacts with neurotensin receptor NTSR1 and modifies its function.

G-protein-coupled receptor 39 (GPR39), a member of the ghrelin receptor family, has a full-length isoform GPR39-1a and a truncated isoform GPR39-1b. While GPR39-1a was clarified as a receptor of Zn(2+), the characteristic property of GPR39-1b remains unknown. Therefore, in this study, the molecular functions of GPR39-1b were explored in cell culture. In contrast to GPR39-1a, GPR39-1b showed no response to Zn(2+) stimulation in calcium mobilization assays, suggesting that GPR39-1b is not a functional receptor of Zn(2+). To understand the signaling interaction of GPR39-1b, we investigated the dimerization between the isoforms, and conducted bioluminescence resonance energy transfer (BRET(2)) assays. The results indicated that GPR39-1b homodimerized, but did not heterodimerize with GPR39-1a. We subsequently attempted to search the heterodimeric counterparts of GPR39-1b. Neurotensin receptor 1 (NTSR1) was also targeted as a GPR39-1b interacting partner because of its highly conserved amino acid sequence and mRNA localization, which was similar to GPR39-1b. BRET(2) assays demonstrated that GPR39-1b heterodimerized with NTSR1. To examine the effect of GPR39-1b on NTRS1-mediated cAMP/PKA signaling, we used the cAMP responsive element-luciferase assays and observed that GPR39-1b attenuated neurotensin-induced NTSR1 signaling. Taken together, our results provided a novel regulatory mechanism for GPR39-1b in NTRS1 signaling.

NTSR1 promotes epithelial-mesenchymal transition and metastasis in lung adenocarcinoma through the Wnt/ß-catenin pathway.

BACKGROUND: Lung adenocarcinoma (LUAD) patients are implicated in poor prognoses and increased mortality rates. Metastasis, as a leading cause of LUAD-related deaths, requires further investigation. Highly metastatic cancer cells often exhibit extensive characteristics of epithelial-mesenchymal transition (EMT). This study attempted to identify novel targets associated with LUAD metastasis and validate their specific molecular mechanisms. METHODS: Bioinformatics was conducted to determine NTSR1 expression in LUAD and the enriched pathways. Immunohistochemical analysis was used to assess NTSR1 expression in LUAD tissue. qRT-PCR examined expressions of NTSR1 and Wnt/ß-Catenin pathway-related genes in LUAD cells. Transwell assayed cell migration and invasion. Cell adhesion experiments were conducted to evaluate cell adhesion capacity. Western blot analysis was employed to examine expression of EMT, Wnt/ß-Catenin pathway, and cell adhesion markers. RESULTS: NTSR1 was upregulated in LUAD tissues and cells, and enriched in EMT pathway. Knockdown of NTSR1 reduced migration, invasion, and adhesion abilities in LUAD cells, and inhibited EMT progression and Wnt/ß-Catenin pathway. Rescue experiments demonstrated that ß-Catenin activator SKL2001 reversed repressive influence of NTSR1 knockdown on LUAD cell malignant phenotypes and EMT progression. CONCLUSION: The data obtained in this study suggested that NTSR1 stimulated EMT and metastasis in LUAD via Wnt/ß-Catenin pathway. This finding may provide options for overcoming LUAD metastasis.

Ntsr1 contributes to pulmonary hypertension by enhancing endoplasmic reticulum stress via JAK2-STAT3-Thbs1 signaling.

Pulmonary hypertension (PH) is a severe clinical syndrome with pulmonary vascular remodeling and poor long-term prognosis. Neurotensin receptor 1 (Ntsr1), serve as one of the G protein-coupled receptors (GPCRs), implicates in various biological processes, but the potential effects of Ntsr1 in PH development are unclear. The Sugen/Hypoxia (SuHx) or monocrotaline (MCT) induced rat PH model was used in our study and the PH rats showed aggravated pulmonary artery remodeling and increased right ventricular systolic pressure (RVSP). Our results revealed that Ntsr1 induced endoplasmic reticulum (ER) stress response via ATF6 activation contributed to the development of PH. Moreover, RNA-sequencing (RNA-seq) and phosphoproteomics were performed and the Ntsr1-JAK2-STAT3-thrombospondin 1 (Thbs1)-ATF6 signaling was distinguished as the key pathway. In vitro, pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition showed enhanced proliferation and migration properties, which could be inhibited by Ntsr1 knockdown, JAK2 inhibitor (Fedratinib) treatment, STAT3 inhibitior (Stattic) treatment, Thbs1 knockdown or ATF6 knockdown. In addition, adeno-associated virus 1 (AAV1) were used to knockdown the expression of Ntsr1, Thbs1 or ATF6 in rats and reversed the phenotype of PH. In summary, our results reveal that Ntsr1-JAK2-STAT3-Thbs1 pathway can induce enhanced ER stress via ATF6 activation and increased PASMC proliferation and migration capacities, which can be mechanism of the pulmonary artery remodeling and PH. Targeting Ntsr1 might be a novel therapeutic strategy to ameliorate PH.

Neurotensin receptor-1 antagonist SR48692 modulation of high-fat diet induced reproductive impairment in male mice.

Neurotensin (NTS), a tridecapeptide of the gastrointestinal tract, has been implicated in the facilitation of lipid absorption on ingestion of a high-fat diet (HFD) especially via NTS receptors, NTSR1, NTSR2, and NTSR3, to cause lipid metabolic dysregulation and imbalance of the oxidant-antioxidant system. Oxidative stress induced a negative impact on reproductive function, affecting the reproductive organ and related reproductive hormones. The present study elucidated the efficacy of NTSR1 antagonist SR48692 in the modulation of HFD-induced reproductive impairment in male mice. Swiss albino mice (male, 23 ± 2 g) were maintained (6/group) for eight weeks; Group-I chow diet (CD), Group-II HFD, Group-III (HFD+SR48692L), Group-IV (HFD+SR48692H), Group-V (CD+SR48692L) and Group-VI (CD+SR48692H). SR48692 low (100 µg/kg b.w./SR48692L) and high-dose (400 µg/kg b.w./SR48692H) were given intraperitoneally for the last four weeks. Treatment with low-dose (SR48692L) to HFD-fed mice showed some efficacy in mitigating lipid dysregulation, oxidative stress, and reproductive impairment as evidenced by decreased triglycerides, total cholesterol, low-density lipoprotein cholesterol, leptin, and increased high-density lipoprotein cholesterol, increased antioxidant defense enzymes, reduction of histopathological scores in testis and increase in plasma level of LH, FSH and testosterone compared to that of HFD, but not up to CD. With the high-dose of antagonist (SR48692H) showed more adverse effects even from that of HFD. Treatment of both doses of SR48692 to CD-fed mice these effects become more extended. Less effectiveness of NTSR1 antagonist with the doses tried (low and high) in normalizing the lipid dysregulation and reproductive impairments might be due to the persistence of NTSR2/NTSR3-mediated lipid absorption.

Time to drink: Activating lateral hypothalamic area neurotensin neurons promotes intake of fluid over food in a time-dependent manner.

The lateral hypothalamic area (LHA) is essential for ingestive behavior but has primarily been studied in modulating feeding, with comparatively scant attention on drinking. This is partly because most LHA neurons simultaneously promote feeding and drinking, suggesting that ingestive behaviors track together. A notable exception are LHA neurons expressing neurotensin (LHANts neurons): activating these neurons promotes water intake but modestly restrains feeding. Here we investigated the connectivity of LHANts neurons, their necessity and sufficiency for drinking and feeding, and how timing and resource availability influence their modulation of these behaviors. LHANts neurons project broadly throughout the brain, including to the lateral preoptic area (LPO), a brain region implicated in modulating drinking behavior. LHANts neurons also receive inputs from brain regions implicated in sensing hydration and energy status. While activation of LHANts neurons is not required to maintain homeostatic water or food intake, it selectively promotes drinking during the light cycle, when ingestive drive is low. Activating LHANts neurons during this period also increases willingness to work for water or palatable fluids, regardless of their caloric content. By contrast, LHANts neuronal activation during the dark cycle does not promote drinking, but suppresses feeding during this time. Finally, we demonstrate that the activation of the LHANts â†’ LPO projection is sufficient to mediate drinking behavior, but does not suppress feeding as observed after generally activating all LHANts neurons. Overall, our work suggests how and when LHANts neurons oppositely modulate ingestive behaviors.

Effect of NTSR1 on the Proliferation and Invasion of Gastric Adenocarcinoma Cells, the Underlying Mechanism, and Clinical Role.

OBJECTIVE: This work aimed to explore the effect of NTSR1 on oncogenesis and the potential clinical role of gastric adenocarcinoma (GAC). METHODS: Immunohistochemistry was used to assay NTSR1 and EGFR/HER2 expression. NTSR1 and MET were disrupted using shRNA. The role of 19 genes related to cancer phenotype signaling pathways was explored. The expression of genes was verified by Western blotting or quantitative real-time polymerase chain reaction. The interactions among genes were analyzed by STRING. RESULTS: There was a significant positive correlation between the expression of NTSR1 and EGFR/HER2. The proliferation and invasion rate of MKN-45 cells was significantly reduced by the NTSR1 shRNA. The expression of MET and EGFR/HER2 was downregulated by the NTSR1 shRNA. NTSR1 modulated the invasion ability of gastric cancer cells via MET. NTSR1 interacted with MET via PIK3CA. Combined knockout of NTSR1 and MET further reduced the PIK3CA mRNA level and the invasion ability of MKN-45 cells. CONCLUSIONS: NTSR1 plays an important role in the occurrence, invasion, and metastasis of GAC in a manner involving several other genes, such as MET and EGFR/HER2. Therefore, NTSR1 constitutes a potential therapeutic target for GAC via synthetic lethality, and assessment of NTSR1 signaling may be necessary when performing tyrosine kinase inhibitor therapy.

Neurotensin receptor 1 signaling promotes pancreatic cancer progression.

Pancreatic cancer is one of the cancers with the poorest prognosis, with a 5-year survival rate of approximately 5-10%. Thus, it is urgent to identify molecular targets for the treatment of pancreatic cancer. Using serial transplantations in a mouse pancreatic orthotopic inoculation model, we previously produced highly malignant pancreatic cancer sublines with increased tumor-forming abilities in vivo. Here, we used these sublines to screen molecular targets for the treatment of pancreatic cancer. Among the genes with increased expression levels in the sublines, we focused on those encoding cell surface receptors that may be involved in the interactions between cancer cells and the tumor microenvironment. Based on our previous RNA-sequence analysis, we found increased expression levels of neurotensin (NTS) receptor 1 (NTSR1) in highly malignant pancreatic cancer sublines. Furthermore, re-analysis of clinical databases revealed that the expression level of NTSR1 was increased in advanced pancreatic cancer and that high NTSR1 levels were correlated with a poor prognosis. Overexpression of NTSR1 in human pancreatic cancer cells Panc-1 and SUIT-2 accelerated their tumorigenic and metastatic abilities in vivo. In addition, RNA-sequence analysis showed that MAPK and NF-κB signaling pathways were activated upon NTS stimulation in highly malignant cancer sublines and also revealed many new target genes for NTS in pancreatic cancer cells. NTS stimulation increased the expression of MMP-9 and other pro-inflammatory cytokines and chemokines in pancreatic cancer cells. Moreover, the treatment with SR48692, a selective NTSR1 antagonist, suppressed the activation of the MAPK and NF-κB signaling pathways and induction of target genes in pancreatic cancer cells in vitro, while the administration of SR48692 attenuated the tumorigenicity of pancreatic cancer cells in vivo. These findings suggest that NTSR1 may be a prognostic marker and a molecular target for pancreatic cancer treatment.

Corticothalamic gating of population auditory thalamocortical transmission in mouse.

The mechanisms that govern thalamocortical transmission are poorly understood. Recent data have shown that sensory stimuli elicit activity in ensembles of cortical neurons that recapitulate stereotyped spontaneous activity patterns. Here, we elucidate a possible mechanism by which gating of patterned population cortical activity occurs. In this study, sensory-evoked all-or-none cortical population responses were observed in the mouse auditory cortex in vivo and similar stochastic cortical responses were observed in a colliculo-thalamocortical brain slice preparation. Cortical responses were associated with decreases in auditory thalamic synaptic inhibition and increases in thalamic synchrony. Silencing of corticothalamic neurons in layer 6 (but not layer 5) or the thalamic reticular nucleus linearized the cortical responses, suggesting that layer 6 corticothalamic feedback via the thalamic reticular nucleus was responsible for gating stochastic cortical population responses. These data implicate a corticothalamic-thalamic reticular nucleus circuit that modifies thalamic neuronal synchronization to recruit populations of cortical neurons for sensory representations.

A comparative study of peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 in preclinical prostate tumour models.

INTRODUCTION: Peptide-based imaging agents targeting prostate-specific membrane antigen (PSMA) have revolutionized the evaluation of biochemical recurrence of prostate cancer (PCa) but lacks sensitivity at very low serum prostate specific antigen (PSA) levels. Once recurrence is suspected, other positron emission tomography (PET) radiotracers could be of interest to discriminate between local and distant relapse. We studied [18F]fluorodeoxyglucose ([18F]FDG) targeting glucose metabolism, [18F]fluorocholine ([18F]FCH) targeting membrane metabolism and peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 targeting PSMA, gastrin releasing peptide receptor (GRPr), αvß3 integrin and neurotensin type 1 receptor (NTSR1) respectively, in different PCa tumour models. METHODS: Mice were xenografted with 22Rv1, an androgen-receptor (AR)-positive, PCa cell line that expresses PSMA and PC3, an AR-negative one that does not express PSMA. PET imaging using the different radiotracers was performed sequentially and the uptake characteristics compared to one other. NTSR1 and PSMA expression levels were analysed in tumours by immunohistochemistry. RESULTS: [18F]FDG displayed low but sufficient uptake to visualize PC3 and 22Rv1 derived tumours. We also observed a low efficacy of [18F]FCH PET imaging and a low [68Ga]Ga-NODAGA-RGD tumour uptake in those tumours. As expected, an elevated tumour uptake was obtained for [68Ga]Ga-PSMA-11 in 22Rv1 derived tumour although no uptake was measured in the androgen independent cell line PC3, derived from a bone metastasis of a high-grade PCa. Moreover, in PC3 cell line, we obtained good tumour uptake, high tumour-to-background contrast using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3. Immunohistochemistry analysis confirmed high NTSR1 expression in PC3 derived tumours and conversely high PSMA expression in 22Rv1 derived tumours. CONCLUSION: PET imaging using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3 demonstrates that GRPr and NTSR1 could represent viable alternative targets for diagnostic or therapeutic applications in PCa with limited PSMA expression levels. More preclinical and clinical studies will follow to explore this potential. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT: Peptide-based imaging agents targeting PSMA represent a major progress in the evaluation of biochemical recurrence of PCa but sometimes yield false negative results in some lesions. Continuing efforts have thus been made to evaluate other radiotracers. Our preclinical results suggest that [68Ga]labelled bombesin and neurotensin analogues could serve as alternative PET radiopharmaceuticals for diagnostic or therapy in cases of PSMA-negative PCa.