Neuroanatomical characterization of the Nmu-Cre knock-in mice reveals an interconnected network of unique neuropeptidergic cells.

Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the Nmu promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions, in situ hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.

Prognostic value of left ventricular global constructive work in patients with cardiac amyloidosis.

PURPOSE: The aim of the present study was to evaluate the role of ejection fraction (EF), left ventricular (LV) global longitudinal strain (LVGLS) and global constructive work (GCW) as prognostic variables in patients with cardiac amyloidosis (CA). METHODS: CA patients were retrospectively identified between 2015 and 2021 at a tertiary care hospital. Comprehensive clinical, biochemical, and imaging evaluation including two-dimensional (2D) echocardiography with myocardial work (MW) analysis was performed. A clinical combined endpoint was defined as all-cause mortality and heart failure readmission. RESULTS: 70 patients were followed for 16 (7-37) months and 37 (52.9%) reached the combined endpoint. Patient with versus without clinical events had a significantly lower LVEF (40.71% vs. 48.01%, p = 0.039), LVGLS (-9.26 vs. -11.32, p = 0.034) and GCW (1034.47mmHg% vs. 1424.86mmHg%, p = 0.011). Multivariable analysis showed that LVEF ( odds ratio (OR): 0.904; 95% confidence interval (CI): 0.839-0.973, p = 0.007), LVGLS ( OR: 0.620; 95% CI: 0.415-0.926, p = 0.020) and GCW ( OR: 0.995; 95% CI: 0.990-0.999, p = 0.016) were significant predictors of outcome, but the model including GCW had the best discriminative ability to predict the combined endpoint (C-index = 0.888). A GCW less than 1443mmHg% was able to predict the clinical endpoint with a sensitivity of 94% and a specificity of 64% (Area under the curve (AUC): 0.771 (95% CI: 0.581-0.961; p = 0.005)). CONCLUSION: In CA patients, GCW may be of additional prognostic value to LVEF and GLS in predicting heart failure hospitalization and all-cause mortality.

Feasibility study on exhaled-breath analysis by untargeted Selected-Ion Flow-Tube Mass Spectrometry in children with cystic fibrosis, asthma, and healthy controls: Comparison of data pretreatment and classification techniques.

Selected-Ion Flow-Tube Mass Spectrometry (SIFT-MS) has been applied in a clinical context as diagnostic tool for breath samples using target biomarkers. Exhaled breath sampling is non-invasive and therefore much more patient friendly compared to bronchoscopy, which is the golden standard for evaluating airway inflammation. In the actual pilot study, 55 exhaled breath samples of children with asthma, cystic-fibrosis and healthy individuals were included. Rather than focusing on the analysis of target biomarkers or on the identification of biomarkers, different data analysis strategies, including a variety of pretreatment, classification and discrimination techniques, are evaluated regarding their capacity to distinguish the three classes based on subtle differences in their full scan SIFT-MS spectra. Proper data-analysis strategies are required because these full scan spectra contain much external, i.e. unwanted, variation. Each SIFT-MS analysis generates three spectra resulting from ion-molecule reactions of analyte molecules with H3O+, NO+ and O2+. Models were built with Linear Discriminant Analysis, Quadratic Discriminant Analysis, Soft Independent Modelling by Class Analogy, Partial Least Squares - Discriminant Analysis, K-Nearest Neighbours, and Classification and Regression Trees. Perfect models, concerning overall sensitivity and specificity (100% for both) were found using Direct Orthogonal Signal Correction (DOSC) pretreatment. Given the uncertainty related to the classification models associated with DOSC pretreatments (i.e. good classification found also for random classes), other models are built applying other preprocessing approaches. A Partial Least Squares - Discriminant Analysis model with a combined pre-processing method considering single value imputation results in 100% sensitivity and specificity for calibration, but was less good predictive. Pareto scaling prior to Quadratic Discriminant Analysis resulted in 41/55 correctly classified samples for calibration and 34/55 for cross-validation. In future, the uncertainty with DOSC and the applicability of the promising preprocessing methods and models must be further studied applying a larger representative data set with a more extensive number of samples for each class. Nevertheless, this pilot study showed already some potential for the untargeted SIFT-MS application as a rapid pattern-recognition technique, useful in the diagnosis of clinical breath samples.

Aged xCT-Deficient Mice Are Less Susceptible for Lactacystin-, but Not 1-Methyl-4-Phenyl-1,2,3,6- Tetrahydropyridine-, Induced Degeneration of the Nigrostriatal Pathway.

The astrocytic cystine/glutamate antiporter system x c - (with xCT as the specific subunit) imports cystine in exchange for glutamate and has been shown to interact with multiple pathways in the brain that are dysregulated in age-related neurological disorders, including glutamate homeostasis, redox balance, and neuroinflammation. In the current study, we investigated the effect of genetic xCT deletion on lactacystin (LAC)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal pathway, as models for Parkinson's disease (PD). Dopaminergic neurons of adult xCT knock-out mice (xCT-/-) demonstrated an equal susceptibility to intranigral injection of the proteasome inhibitor LAC, as their wild-type (xCT+/+) littermates. Contrary to adult mice, aged xCT-/- mice showed a significant decrease in LAC-induced degeneration of nigral dopaminergic neurons, depletion of striatal dopamine (DA) and neuroinflammatory reaction, compared to age-matched xCT+/+ littermates. Given this age-related protection, we further investigated the sensitivity of aged xCT-/- mice to chronic and progressive MPTP treatment. However, in accordance with our previous observations in adult mice (Bentea et al., 2015a), xCT deletion did not confer protection against MPTP-induced nigrostriatal degeneration in aged mice. We observed an increased loss of nigral dopaminergic neurons, but equal striatal DA denervation, in MPTP-treated aged xCT-/- mice when compared to age-matched xCT+/+ littermates. To conclude, we reveal age-related protection against proteasome inhibition-induced nigrostriatal degeneration in xCT-/- mice, while xCT deletion failed to protect nigral dopaminergic neurons of aged mice against MPTP-induced toxicity. Our findings thereby provide new insights into the role of system x c - in mechanisms of dopaminergic cell loss and its interaction with aging.

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.

5-HTR2A and 5-HTR3A but not 5-HTR1A antagonism impairs the cross-modal reactivation of deprived visual cortex in adulthood.

Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation (ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR1A and 5-HTR3A were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR1A, 5-HTR2A and 5-HTR3A subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR1A and 5-HTR3A protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR1A in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR2A and 5-HTR3A but not 5-HTR1A during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.

Inhibition of astroglial connexin43 hemichannels with TAT-Gap19 exerts anticonvulsant effects in rodents.

Accumulating evidence shows a key function for astrocytic connexin43 (Cx43) signaling in epilepsy. However, the lack of experimental distinction between Cx43 gap junction channels (GJCs) and hemichannels (HCs) has impeded the identification of the exact contribution of either channel configurations to epilepsy. We therefore investigated whether TAT-Gap19, a Cx mimetic peptide that inhibits Cx43 HCs but not the corresponding Cx43 GJCs, influences experimentally induced seizures in rodents. Dye uptake experiments in acute hippocampal slices of mice demonstrated that astroglial Cx43 HCs open in response to the chemoconvulsant pilocarpine and this was inhibited by TAT-Gap19. In vivo, pilocarpine-induced seizures as well as the accompanying increase in D-serine microdialysate levels were suppressed by Cx43 HC inhibition. Moreover, the anticonvulsant action of TAT-Gap19 was reversed by exogenous D-serine administration, suggesting that Cx43 HC inhibition protects against seizures by lowering extracellular D-serine levels. The anticonvulsive properties of Cx43 HC inhibition were further confirmed in electrical seizure mouse models, i.e. an acute 6 Hertz (Hz) model of refractory seizures and a chronic 6 Hz corneal kindling model. Collectively, these results indicate that Cx43 HCs play a role in seizures and underscore their potential as a novel and druggable target in epilepsy treatment.

Glutamate released in the preoptic area during sexual behavior controls local estrogen synthesis in male quail.

Estrogens are known to act rapidly, probably via membrane estrogen receptors, to induce fast effects on physiological and behavioral processes. Engaging in some of these behaviors, such as sexual behavior, results in an acute modulation of the production of estrogens in the brain by regulating the efficiency of the estrogen synthase enzyme, aromatase. We recently demonstrated that aromatase activity (AA) in the male quail brain is rapidly inhibited in discrete brain regions including the medial preoptic nucleus (POM) following exposure to a female. Evidence from in vitro studies point to glutamate release as one of the mechanisms controlling these rapid regulations of the aromatase enzyme. Here, we show that (a) the acute injection of the glutamatergic agonist kainate into the POM of anesthetized male quail inhibits AA and (b) glutamate is released in the POM during copulation. These results provide the first set of in vivo data demonstrating a role for glutamate release in the rapid control of AA in the context of sexual behavior.

Caloric Restriction Protects against Lactacystin-Induced Degeneration of Dopamine Neurons Independent of the Ghrelin Receptor.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.

LC-method development for the quantification of neuromedin-like peptides. Emphasis on column choice and mobile phase composition.

In this study, the separation of four neuromedin-like peptides is investigated on four different core-shell stationary phases. Moreover, the effect of the mobile phase composition, i.e. organic modifier (acetonitrile and methanol) and additive (trifluoroacetic acid, formic acid, acetic acid, ammonium formate and ammonium acetate) on the chromatographic performance is studied. An improvement in chromatographic performance is observed when using the ammonium salt instead of its corresponding acid as additive, except for the column containing a positively charged surface (C18+). In general, the RP-Amide column provided the highest separation power with different mobile phases. However, for the neuromedin-like peptides of interest, the C18+ column in combination with a mobile phase containing methanol as organic modifier and acetic acid as additive provided narrower and higher peaks. A three-factor, three-level design is applied to further optimize the method in terms of increased peak height and reduced solvent consumption, without loss in resolution. The optimized method was subsequently used to assess the in vitro microdialysis recovery of the peptides of interest. Recovery values between 4 and 8% were obtained using a perfusion flow rate of 2µL/min.

In-vitro and in-vivo evaluation of the modulatory effects of the multitarget compound ASS234 on the monoaminergic system.

OBJECTIVES: To evaluate the in-vitro and in-vivo effects on monoaminergic neurotransmission of ASS234, a promising multitarget-directed ligand (MTDL), for Alzheimer's disease (AD) therapy. METHODS: In vitro was explored the effect of ASS234 on the monoaminergic metabolism in SH-SY5Y and PC12 cell lines, and remaining activity of both monoamine oxidase (MAO) isoforms was assessed. The corresponding dopamine (DA), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) and noradrenaline (NA) levels were determined by HPLC-ED. In-vivo experiments were carried out Wistar rats and intracerebral guide cannulas were implanted in the hippocampus and in the prefrontal cortex by sterotaxic coordinates. The day after microdialysis samples were collected and levels of 5-HT, DA and NA were determined by (UHPLC) with electrochemical detector. KEY FINDINGS: ASS234 induced a significant increase in serotonin (5-HT) levels in SH-SY5Y cells. In PC12 cells, ASS234 increased significantly the ratio of dopamine (DA)/(HVA + DOPAC), although no apparent differences in (NA) were observed. By in-vivo microdialysis, ASS234 showed a significant increase in the extracellular levels of 5-HT and NA in hippocampus whereas in the prefrontal cortex, DA and NA also increased significantly. CONCLUSIONS: This study reveals the ability of ASS234 a MTDL compound, to enhance the monoaminergic neurotransmission supporting its potential use in AD therapy.

Validation of the 6 Hz refractory seizure mouse model for intracerebroventricularly administered compounds.

The six hertz (6 Hz) refractory seizure model is considered an indispensable chain of the Anticonvulsant Screening Project. We here describe an adapted protocol using the intracerebroventricular (i.c.v.) delivery route, which will allow researchers to perform targetvalidation or proof-of-principle studies using promising compounds with unknown or limited blood-brain barrier permeability (e.g. neuropeptides and peptidomimetics) in this model. Seizures were induced by single application of a current intensity of 49 mA to i.c.v.-implanted NMRI mice using an ECT Unit 57800 Ugo Basile stimulator. By applying these key parameters, c-Fos immunohistochemistry revealed the recruitment of the dentate gyrus, ratifying this model as a valuable tool for testing i.c.v. administered compounds against therapy-resistant seizures. This finding was further strengthened, since i.c.v. administration of levetiracetam suppressed 6 Hz-evoked seizure severity but sodium phenytoin did not. We also propose to use "seizure duration" as an alternative, accurate parameter to express the results within this model.

An ultrasensitive nano UHPLC-ESI-MS/MS method for the quantification of three neuromedin-like peptides in microdialysates.

AIM: An ultrasensitive nano UHPLC-ESI-MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. RESULTS: Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. CONCLUSION: The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.

Toward greener analytical techniques for the absolute quantification of peptides in pharmaceutical and biological samples.

Peptide-based biopharmaceuticals represent one of the fastest growing classes of new drug molecules. New reaction types included in the synthesis strategies to reduce the rapid metabolism of peptides, along with the availability of new formulation and delivery technologies, resulted in an increased marketing of peptide drug products. In this regard, the development of analytical methods for quantification of peptides in pharmaceutical and biological samples is of utmost importance. From the sample preparation step to their analysis by means of chromatographic or electrophoretic methods, many difficulties should be tackled to analyze them. Recent developments in analytical techniques emphasize more and more on the use of green analytical techniques. This review will discuss the progresses in and challenges observed during green analytical method development for the quantification of peptides in pharmaceutical and biological samples.

Strategies to reduce aspecific adsorption of peptides and proteins in liquid chromatography-mass spectrometry based bioanalyses: an overview.

In the drug-discovery setting, the development of new peptide and protein-based biopharmaceuticals attracts increased attention from the pharmaceutical industry and consequently demands the development of high-throughput LC-MS methods. Regulatory guidelines require bioanalytical methods to be validated not only in terms of linearity, sensitivity, accuracy, precision, selectivity and stability, but also in terms of carryover. Carryover results from the aspecific adsorption of analyte(s) to parts of the analytical system and thus introduces bias in both identification and quantification assays. Moreover, nonspecific binding occurs at the surface of materials used during sample preparation, such as pipette tips, sample tubes and LC-vials. Hence, linearity, sensitivity and repeatability of the analyses are negatively affected. Due to the great diversity in physicochemical properties of biomolecules, there is no general approach available to minimize adsorption phenomena. Therefore, we aim to present different strategies which can be generically applied to reduce nonspecific binding of peptides and proteins. In the first part of this review, a systematic approach is proposed to guide the reader through the different solvents which can be used to dissolve the analyte of interest. Indeed, proper solubilization is one of the most important factors for a successful analysis. In addition, alternative approaches are described to improve analyte recovery from the sample vial. The second part focuses on strategies to efficiently reduce adsorption at components of the autosampler, column and mass spectrometer. Thereby carryover is reduced while maintaining a sufficiently wide dynamic range of the assay.