Neuromedin U induces an invasive phenotype in CRC cells expressing the NMUR2 receptor.

BACKGROUND: Successful colorectal cancer (CRC) therapy often depends on the accurate identification of primary tumours with invasive potential. There is still a lack of identified pathological factors associated with disease recurrence that could help in making treatment decisions. Neuromedin U (NMU) is a secretory neuropeptide that was first isolated from the porcine spinal cord, and it has emerged as a novel factor involved in the tumorigenesis and/or metastasis of many types of cancers. Previously associated with processes leading to CRC cell invasiveness, NMU has the potential to be a marker of poor outcome, but it has not been extensively studied in CRC. METHODS: Data from The Cancer Genome Atlas (TCGA) were used to analyse NMU and NMU receptor (NMUR1 and NMUR2) expression in CRC tissues vs. normal tissues, and real-time PCR was used for NMU and NMU receptor expression analysis. NMU protein detection was performed by immunoblotting. Secreted NMU was immunoprecipitated from cell culture-conditioned media and analysed by immunoblotting and protein sequencing. DNA demethylation by 5-aza-CdR was used to analyse the regulation of NMUR1 and NMUR2 expression. NMU receptor activity was monitored by detecting calcium mobilisation in cells loaded with fluo-4, and ERK1/2 kinase activation was detected after treatment with NMU or receptor agonist. Cell migration and invasion were investigated using membrane filters. Integrin expression was evaluated by flow cytometry. RESULTS: The obtained data revealed elevated expression of NMU and NMUR2 in CRC tissue samples and variable expression in the analysed CRC cell lines. We have shown, for the first time, that NMUR2 activation induces signalling in CRC cells and that NMU increases the motility and invasiveness of NMUR2-positive CRC cells and increases prometastatic integrin receptor subunit expression. CONCLUSIONS: Our results show the ability of CRC cells to respond to NMU via activation of the NMUR2 receptor, which ultimately leads to a shift in the CRC phenotype towards a more invasive phenotype.

Effects of neuromedin U-8 on stress responsiveness and hypothalamus-pituitary-adrenal axis activity in male C57BL/6J mice.

Neuromedin U (NMU) is a highly conserved neuropeptide that has been implicated in the stress response. To better understand how it influences various aspects of the stress response, we studied the effects of intracerebroventricular NMU-8 administration on stress-related behavior and activity of the hypothalamus-pituitary-adrenal (HPA) axis in male C57BL/6J mice. We investigated these NMU-8 effects when mice remained in their home cage and when they were challenged by exposure to forced swim stress. NMU-8 administration resulted in increased grooming behavior in mice that remained in their home cage and in a significant increase in c-Fos immunoreactivity in the paraventricular hypothalamus (PVH) and arcuate nucleus (ARC). Surprisingly, NMU-8 administration significantly decreased plasma corticosterone concentrations. Furthermore, NMU-8 administration increased immobility in the forced swim test in both naïve mice and mice that were previously exposed to swim stress. The effect of NMU-8 on c-Fos immunoreactivity in the PVH was dependent on previous exposure to swim stress given that we observed no significant changes in mice exposed for the first time to swim stress. In contrast, in the ARC we observed a significant increase in c-Fos immunoreactivity regardless of previous stress exposure. Interestingly, NMU-8 administration also significantly decreased plasma corticosterone concentrations in mice that were exposed to single forced swim stress, while this effect was no longer observed when mice were exposed to forced swim stress for a second time. Taken together, our data indicate that NMU-8 regulates stress responsiveness and suggests that its effects depend on previous stress exposure.

Neuromedin U and Structural Analogs: An Overview of their Structure, Function and Selectivity.

The neuromedin U peptide sequence is highly conserved between various species. Neuromedin U is involved in a variety of physiological processes. It exerts its effects via two neuromedin U receptors, NMUR1 and NMUR2. These receptors are characterized by a distinct, yet complementary, tissue distribution with NMUR1 mostly found in the periphery, while NMUR2 is most abundant in the central nervous system. The capability of the neuropeptide to reduce food intake in rodents triggered the design and synthesis of a broad range of modified peptide ligands. The purpose of these ligands is to develop novel therapeutics which could be beneficial in the treatment of obesity and diabetes. Most compounds are derived either from the full-length neuromedin U sequence or are based on the truncated orthologs of this neuropeptide. Only a few non-peptidic ligands were developed. This review provides an overview on various neuromedin U analogs and mimetics that have been reported to date.

Synthesis and in Vitro Evaluation of Stabilized and Selective Neuromedin U-1 Receptor Agonists.

Neuromedin U (NMU) is a multifunctional neuropeptide which is characterized by a high conservation through all species. Herein, we describe the synthesis of a novel set of NMU-analogs based on the truncated NMU-8. Through combination of previously reported modifications, an elaborate structure-activity relationship study was performed aiming for the development of peptides with an increased selectivity toward NMU receptor 1 (NMUR1). Compound 7 possessed the highest NMUR1 selectivity (IC50 = 0.54 nM, selectivity ratio = 5313) together with an increased potency (EC50 = 3.7 nM), an 18% increase of the maximal effect at NMUR1, and a higher resistance against enzymatic degradation as compared to the native NMU-8. The development of a potent NMUR1 agonist with extended half-life could represent an attractive tool to further unveil the role of NMUR1 in NMU signaling.

Development of potent and proteolytically stable human neuromedin U receptor agonists.

Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the development of a set of novel NMU-analogs based on NMU-8, by introducing unnatural amino acids into the native sequence. This approach shows that it is possible to generate molecules with increased potency and improved plasma stability without major changes of the peptidic nature or the introduction of large conjugates. When compared to the native NMU-8 peptide, compounds 16, 18 and 20 have potent agonist activity and affinity for both NMU receptors. Selectivity towards NMUR1 was observed when the Phe residue in position 4 was modified, whereas higher potencies at NMUR2 were found when substitutions of the Pro residue in position 6 were executed. To study the effect of the modifications on the proteolytic stability of the molecules, an in vitro stability assay in human plasma at 37 °C was performed. All analyzed analogs possessed an increased resistance against enzymatic degradation in human plasma resulting in half-lifes from 4 min for NMU-8, up to more than 23 h for compound 42.

Challenges for the in vivo quantification of brain neuropeptides using microdialysis sampling and LC-MS.

In recent years, neuropeptides and their receptors have received an increased interest in neuropharmacological research. Although these molecules are considered relatively small compared with proteins, their in vivo quantification using microdialysis is more challenging than for small molecules. Low microdialysis recoveries, aspecific adsorption and the presence of various multiply charged precursor ions during ESI-MS/MS detection hampers the in vivo quantification of these low abundant biomolecules. Every step in the workflow, from sampling until analysis, has to be optimized to enable the sensitive analysis of these compounds in microdialysates.

In-depth behavioral characterization of the corticosterone mouse model and the critical involvement of housing conditions.

Depression and anxiety are disabling and highly prevalent psychiatric disorders. To better understand the neurobiological basis of mood and anxiety disorders, relevant animal models are needed. The corticosterone mouse model is frequently used to study depression. Chronic stress and accompanying glucocorticoid elevation causes pathological changes in the central nervous system, which are related to psychiatric symptoms. Exogenous administration of corticosterone is therefore often used to induce depressive-like behavior in mice and in some cases also features of anxiety-like behavior are shown. However, a thorough characterization of this model has never been conducted and housing conditions of the used subjects often differ between the implemented protocols. We chronically administered a subcutaneous corticosterone bolus injection to single- and group-housed mice, and we subsequently evaluated the face validity of this model by performing a battery of behavioral tests (forced swim test, mouse-tail suspension test, saccharin intake test, novelty-suppressed feeding test, elevated plus maze, light/dark paradigm and open field test). Our results show that corticosterone treatment has a substantial overall effect on depressive-like behavior. Increases in anxiety-like behavior on the other hand are mainly seen in single housed animals, independent of treatment. The current study therefore does not only show a detailed behavioral characterization of the corticosterone mouse model, but furthermore also elucidates the critical influence of housing conditions on the behavioral outcome in this model.

Neuropeptide FF receptors as novel targets for limbic seizure attenuation.

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepileptic neuropeptide. In this study, the controversial role of NPY1 receptors in epilepsy was reassessed by testing two highly selective NPY1 receptor ligands and a mixed NPY1/NPFF receptor antagonist BIBP3226 in a rat model for limbic seizures. While BIBP3226 significantly attenuated the pilocarpine-induced seizures, neither of the highly selective NPY1 receptor ligands altered the seizure severity. Administration of the NPFF1/NPFF2 receptor antagonist RF9 also significantly attenuated limbic seizure activity. To further prove the involvement of NPFF receptors in these seizure-modulating effects, low and high affinity antagonists for the NPFF receptors were tested. We observed that the low affinity ligand failed to exhibit anticonvulsant properties while the two high affinity ligands significantly attenuated the seizures. Continuous NPFF1 receptor agonist administration also inhibited limbic seizures whereas bolus administration of the NPFF1 receptor agonist was without effect. This suggests that continuous agonist perfusion could result in NPFF1 receptor desensitization and mimic NPFF1 receptor antagonist administration. Our data unveil for the first time the involvement of the NPFF system in the management of limbic seizures.

Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish.

Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically diverse antiepileptic drugs (AEDs) were used. Three out of the five AEDs (levetiracetam, phenytoin, and topiramate) showed only a limited protective effect (mainly mortality delay) at doses close to the TD50 (dose inducing motor impairment in 50% of animals) in mice. The two remaining AEDs (diazepam and sodium valproate) displayed protective activity against AG-induced seizures. Experiments performed in zebrafish larvae revealed behavioral AED activity profiles highly analogous to those obtained in mice. Having demonstrated cross-species similarities and limited efficacy of tested AEDs, we propose the use of AG in zebrafish as a convenient and high-throughput model of treatment-resistant seizures.