Ovarian Follicle Development in Ascidians.

Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A (Ciona rubusta) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona, and comparative views of the follicle development processes of mammals and teleosts.

Long-term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co-administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus.

This study evaluates the sustained antidepressant-like effects and neurogenic potential of a 3-day intranasal co-administration regimen of galanin receptor 2 (GALR2) agonist M1145 and neuropeptide Y Y1 receptor (NPY1R) agonist [Leu31, Pro34]NPY in the ventral hippocampus of adult rats, with outcomes analyzed 3 weeks post-treatment. Utilizing the forced swimming test (FST), we found that this co-administration significantly enhances antidepressant-like behaviors, an effect neutralized by the GALR2 antagonist M871, highlighting the synergistic potential of these neuropeptides in modulating mood-related behaviors. In situ proximity ligation assay (PLA) indicated a significant increase in GALR2/NPYY1R heteroreceptor complexes in the ventral hippocampal dentate gyrus, suggesting a molecular basis for the behavioral outcomes observed. Moreover, proliferating cell nuclear antigen (PCNA) immunolabeling revealed increased cell proliferation in the subgranular zone of the dentate gyrus, specifically in neuroblasts as evidenced by co-labeling with doublecortin (DCX), without affecting quiescent neural progenitors or astrocytes. The study also noted a significant uptick in the number of DCX-positive cells and alterations in dendritic morphology in the ventral hippocampus, indicative of enhanced neuronal differentiation and maturation. These morphological changes highlight the potential of these agonists to facilitate the functional integration of new neurons into existing neural circuits. By demonstrating the long-lasting effects of a brief, 3-day intranasal administration of GALR2 and NPY1R agonists, our findings contribute significantly to the understanding of neuropeptide-mediated neuroplasticity and herald novel therapeutic strategies for the treatment of depression and related mood disorders, emphasizing the therapeutic promise of targeting neurogenesis and neuronal maturation processes.

Sex differences and sex-specific regulation of motivated behavior by Melanin-concentrating hormone: a short review.

Recent preclinical research exploring how neuropeptide transmitter systems regulate motivated behavior reveal the increasing importance of sex as a critical biological variable. Neuropeptide systems and their central circuits both contribute to sex differences in a range of motivated behaviors and regulate sex-specific behaviors. In this short review, we explore the current research of how sex as a biological variable influences several distinct motivated behaviors that are modulated by the melanin-concentrating hormone (MCH) neuropeptide system. First, we review how MCH regulates feeding behavior within the context of energy homeostasis differently between male and female rodents. Then, we focus on MCH's role in lactation as a sex-specific process within the context of energy homeostasis. Next, we discuss the sex-specific effects of MCH on maternal behavior. Finally, we summarize the role of MCH in drug-motivated behaviors. While these topics are traditionally investigated from different scientific perspectives, in this short review we discuss how these behaviors share commonalities within the larger context of motivated behaviors, and that sex differences discovered in one area of research may impact our understanding in another. Overall, our review highlights the need for further research into how sex differences in energy regulation associated with reproduction and parental care contribute to regulating motivated behaviors.

Local age-dependent neuromodulation in Rhodnius prolixus antennae.

Kissing bugs do not respond to host cues when recently molted and only exhibit robust host-seeking several days after ecdysis. Behavioral plasticity has peripheral correlates in antennal gene expression changes through the week after ecdysis. The mechanisms regulating these peripheral changes are still unknown, but neuropeptide, G-protein coupled receptor, nuclear receptor, and takeout genes likely modulate peripheral sensory physiology. We evaluated their expression in antennal transcriptomes along the first week postecdysis of Rhodnius prolixus 5th instar larvae. Besides, we performed clustering and co-expression analyses to reveal relationships between neuromodulatory (NM) and sensory genes. Significant changes in transcript abundance were detected for 50 NM genes. We identified 73 sensory-related and NM genes that were assigned to nine clusters. According to their expression patterns, clusters were classified into four groups: two including genes up or downregulated immediately after ecdysis; and two with genes with expression altered at day 2. Several NM genes together with sensory genes belong to the first group, suggesting functional interactions. Co-expression network analysis revealed a set of genes that seem to connect with sensory system maturation. Significant expression changes in NM components were described in the antennae of R. prolixus after ecdysis, suggesting that a local NM system acts on antennal physiology. These changes may modify the sensitivity of kissing bugs to host cues during this maturation interval.

The functional assay identified authentic interactions between CAPA peptides and the CAPA receptor isoforms in Bemisia tabaci (Gennadius).

CAPA neuropeptides regulate the diuresis/ antidiuresis process in insects by activating specific cognate receptor, CAPAr. In this study, we characterized the CAPAr gene (BtabCAPAr) in the whitefly, Bemisia tabaci Asia II 1. The two alternatively spliced isoforms of BtabCAPAr gene, BtabCAPAr-1 and BtabCAPAr-2, having six and five exons, respectively, were identified. The BtabCAPAr gene expression was highest in adult whitefly as compared to gene expression in egg, nymphal and pupal stages. Among the three putative CAPA peptides, CAPA-PVK1 and CAPA-PVK2 strongly activated the BtabCAPAr-1 with very low EC50 values of 0.067 nM and 0.053 nM, respectively, in heterologous calcium mobilization assays. None of the peptide activated the alternatively spliced isoform BtabCAPAr-2 that has lost the transmembrane segments 3 and 4. Significant levels of mortality were observed when whiteflies were fed with CAPA-PVK1 at 1.0 µM (50.0%), CAPA-PVK2 at 100.0 nM (43.8%) and CAPA-tryptoPK 1.0 µM (40.0%) at the 96 h after the treatment. This study provides valuable information to design biostable peptides to develop a class of insecticides.

Imaging and quantification of neuropeptides in mouse pituitary tissue by atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry has enabled the untargeted analysis and imaging of neuropeptides and proteins in biological tissues under ambient conditions. Sensitivity in AP-MALDI can be improved by using sample-specific preparation methods. METHODS: A comprehensive and detailed optimization strategy including instrument parameters, matrix spraying and sample tissue washing pretreatment was implemented to enhance the sensitivity and coverage of neuropeptides in mouse pituitary tissues by commercial AP-MALDI mass spectrometry imaging (MSI). RESULTS: The sensitivity of a commercial AP-MALDI system for endogenous neuropeptides in mouse pituitary was enhanced by up to 15.2-fold by shortening the transmission gap from the sample plate to the inlet, attaching copper adhesive tape to an indium tin oxide-coated glass slide, optimizing the matrix spray solvent and using sample tissue washing pretreatment. Following careful optimization, the distributions of nine endogenous neuropeptides were successfully visualized in the pituitary. Furthermore, the quantitative capability of AP-MALDI for neuropeptides was evaluated and the concentrations of neuropeptides oxytocin and vasopressin in the pituitary posterior lobe were increased approximately twofold under hypertonic saline stress. CONCLUSION: Mouse pituitary neuropeptides have emerged as important signaling molecules due to their role in stress response. This work indicates the potential of modified AP-MALDI as a promising AP MSI method for in situ visualization and quantification of neuropeptides in complex biological tissues.

The Formation and Function of the VTA Dopamine System.

The midbrain dopamine system is a sophisticated hub that integrates diverse inputs to control multiple physiological functions, including locomotion, motivation, cognition, reward, as well as maternal and reproductive behaviors. Dopamine is a neurotransmitter that binds to G-protein-coupled receptors. Dopamine also works together with other neurotransmitters and various neuropeptides to maintain the balance of synaptic functions. The dysfunction of the dopamine system leads to several conditions, including Parkinson's disease, Huntington's disease, major depression, schizophrenia, and drug addiction. The ventral tegmental area (VTA) has been identified as an important relay nucleus that modulates homeostatic plasticity in the midbrain dopamine system. Due to the complexity of synaptic transmissions and input-output connections in the VTA, the structure and function of this crucial brain region are still not fully understood. In this review article, we mainly focus on the cell types, neurotransmitters, neuropeptides, ion channels, receptors, and neural circuits of the VTA dopamine system, with the hope of obtaining new insight into the formation and function of this vital brain region.

From pain to tumor immunity: influence of peripheral sensory neurons in cancer.

The nervous and immune systems are the primary sensory interfaces of the body, allowing it to recognize, process, and respond to various stimuli from both the external and internal environment. These systems work in concert through various mechanisms of neuro-immune crosstalk to detect threats, provide defense against pathogens, and maintain or restore homeostasis, but can also contribute to the development of diseases. Among peripheral sensory neurons (PSNs), nociceptive PSNs are of particular interest. They possess a remarkable capability to detect noxious stimuli in the periphery and transmit this information to the brain, resulting in the perception of pain and the activation of adaptive responses. Pain is an early symptom of cancer, often leading to its diagnosis, but it is also a major source of distress for patients as the disease progresses. In this review, we aim to provide an overview of the mechanisms within tumors that are likely to induce cancer pain, exploring a range of factors from etiological elements to cellular and molecular mediators. In addition to transmitting sensory information to the central nervous system, PSNs are also capable, when activated, to produce and release neuropeptides (e.g., CGRP and SP) from their peripheral terminals. These neuropeptides have been shown to modulate immunity in cases of inflammation, infection, and cancer. PSNs, often found within solid tumors, are likely to play a significant role in the tumor microenvironment, potentially influencing both tumor growth and anti-tumor immune responses. In this review, we discuss the current state of knowledge about the degree of sensory innervation in tumors. We also seek to understand whether and how PSNs may influence the tumor growth and associated anti-tumor immunity in different mouse models of cancer. Finally, we discuss the extent to which the tumor is able to influence the development and functions of the PSNs that innervate it.

The Adipokinetic Hormone (AKH) and the Adipokinetic Hormone/Corazonin-Related Peptide (ACP) Signalling Systems of the Yellow Fever Mosquito Aedes aegypti: Chemical Models of Binding.

Neuropeptides are the main regulators of physiological, developmental, and behavioural processes in insects. Three insect neuropeptide systems, the adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone/corazonin-related peptide (ACP), and their cognate receptors, are related to the vertebrate gonadotropin (GnRH) system and form the GnRH superfamily of peptides. In the current study, the two signalling systems, AKH and ACP, of the yellow fever mosquito, Aedes aegypti, were comparatively investigated with respect to ligand binding to their respective receptors. To achieve this, the solution structure of the hormones was determined by nuclear magnetic resonance distance restraint methodology. Atomic-scale models of the two G protein-coupled receptors were constructed with the help of homology modelling. Thereafter, the binding sites of the receptors were identified by blind docking of the ligands to the receptors, and models were derived for each hormone system showing how the ligands are bound to their receptors. Lastly, the two models were validated by comparing the computational results with experimentally derived data available from the literature. This mostly resulted in an acceptable agreement, proving the models to be largely correct and usable. The identification of an antagonist versus a true agonist may, however, require additional testing. The computational data also explains the exclusivity of the two systems that bind only the cognate ligand. This study forms the basis for further drug discovery studies.

Effects of Banhabaekchulcheonma-Tang on Brain Injury and Cognitive Function Impairment Caused by Bilateral Common Carotid Artery Stenosis in a Mouse Model.

Vascular dementia (VD) is the second most prevalent dementia type, with no drugs approved for its treatment. Here, the effects of Banhabaekchulcheonma-Tang (BBCT) on ischemic brain injury and cognitive function impairment were investigated in a bilateral carotid artery stenosis (BCAS) mouse model. Mice were divided into sham-operated, BCAS control, L-BBCT (40 ml/kg), and H-BBCT (80 ml/kg) groups. BBCT's effects were characterized using the Y-maze test, novel object recognition test (NORT), immunofluorescence staining, RNA sequencing, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The NORT revealed cognitive function improvement in the H-BBCT group, while the Y-maze test revealed no significant difference among the four groups. The CD68+ microglia and GFAP+ astrocyte numbers were reduced in the H-BBCT group. Furthermore, H-BBCT treatment restored the dysregulation of gene expression caused by BCAS. The major BBCT targets were predicted to be cell division cycle protein 20 (CDC20), Epidermal growth factor (EGF), and tumor necrosis factor receptor-associated factor 1 (TRAF1). BBCT regulates the neuroactive ligand-receptor interaction and neuropeptide signaling pathways, as predicted by KEGG and GO analyses, respectively. BBCT significantly improved cognitive impairment in a BCAS mouse model by inhibiting microglial and astrocyte activation and regulating the expression of CDC20, EGF, TRAF1, and key proteins in the neuroactive ligand-receptor interaction and neuropeptide signaling pathways.

Bioinformatics for Prohormone and Neuropeptide Discovery.

Neuropeptides and peptide hormones are signaling molecules produced via complex posttranslational modifications of precursor proteins known as prohormones. Neuropeptides activate specific receptors and are associated with the regulation of physiological systems and behaviors. The identification of prohormones-and the neuropeptides created by these prohormones-from genomic assemblies has become essential to support the annotation and use of the rapidly growing number of sequenced genomes. Here we describe a well-validated methodology for identifying the prohormone complement from genomic assemblies that employs widely available public toolsets and databases. The uncovered prohormone sequences can then be screened for putative neuropeptides to enable accurate proteomic discovery and validation.

Nuclear parcellation and numbers of orexinergic neurons in five species of larger brained birds.

The orexinergic/hypocretinergic system, while having several roles, appears to be a key link in the balance between arousal and food intake. In birds, to date, this system has only been examined anatomically in four species, all with brains smaller than 3.5 g and of limited phylogenetic range. Here, using orexin-A immunohistochemistry, we describe the distribution, morphology, and nuclear parcellation of orexinergic neurons within the hypothalami of a Congo gray and a Timneh gray parrot, a pied crow, an emu, and a common ostrich. These birds represent a broad phylogeny, with brains ranging in size from 7.85 to 26.5 g. Within the hypothalami of the species studied, the orexinergic neurons were organized in two clusters, and a densely packed paraventricular hypothalamic nucleus cluster located within the medial hypothalamus (Hyp), but not contacting the ventricle, and a more loosely packed lateral hypothalamic cluster in the lateral Hyp. Stereological analysis revealed a strong correlation, using phylogenetic generalized least squares regression analyses, between brain mass and the total number of orexinergic neurons, as well as soma parameters such as volume and area. Orexinergic axonal terminals evinced two types of boutons, larger and the smaller en passant boutons. Unlike the orexinergic system in mammals, which has several variances in cluster organization, that of the birds studied, in the present and previous studies, currently shows organizational invariance, despite the differences in brain and body mass, phylogenetic relationships, and life-histories of the species studied.

Investigating the role of phenylalanine residues for amyloid formation of the neuropeptide neurokinin B.

Neurokinin B (NKB) is a tachykinin peptide that has diverse roles in biology, including in human reproductive development. Cellular processing of this peptide is thought to involve formation of a dense core vesicle during transit through the regulated secretory pathway. The ability of NKB to rapidly form an amyloid can contribute to formation of the secretory granule but features that support amyloid formation of NKB are not well understood. NKB contains a diphenylalanine sequence well recognised as an important motif for self-assembly of other peptides including amyloid ß. Using mutations of the diphenylalanine motif we show that this motif in NKB is necessary for amyloid formation, and it is the unique combination of aromaticity and hydrophobicity of phenylalanine that is crucial for aggregation. Using disulfide cross-linking we propose that phenylalanine at sequence position 6 is important for stabilising inter-sheet interactions in the NKB amyloid fibril. Although having a highly conserved sequence, the NKB peptide from zebrafish only contains a single phenylalanine and does not fibrillise as extensively as mammalian NKB. Analysis of self-assembly of NKB-like peptides from different species may help in elucidating their biological roles. Taken together, this work shows that mammalian NKB has evolved, within only 10 residues, a sequence optimised for rapid self-assembly, whilst also containing residues for metal-binding, receptor binding and receptor discrimination.

Neuropeptides as Potential Biomarkers in Vascular Dementia.

Neuropeptides are endogenous active substances within the central and peripheral nervous systems that play important roles in a wide range of brain functions, including metabolism, food intake, social behavior, reproduction, learning, sleep, and wakefulness. This article reviews recent advances in the involvement of neuropeptides in vascular dementia. Neuropeptides are present in the brain as chemical signals and last for nearly 50 years. Peptide hormones are chemical signals of the endocrine system. Thus, neuropeptides are the most diverse class of signaling molecules in the brain, involving the genomes of many mammals, encoding neuropeptide precursors and many bioactive neuropeptides. Here the aim is to describe the recent advances in classical neuropeptides, as well as putative neuropeptides from other families, in the control of or as diagnostic tools for vascular dementia. Additionally, its molecular mechanisms are described to explore new avenues of treatment and early diagnosis, as there is increasing evidence that dysregulation of vascular processes is associated with different pathological conditions.

Differential Gene Regulatory Network Analysis between Azacitidine-Sensitive and -Resistant Cell Lines.

Azacitidine, a DNA methylation inhibitor, is employed for the treatment of acute myeloid leukemia (AML). However, drug resistance remains a major challenge for effective azacitidine chemotherapy, though several studies have attempted to uncover the mechanisms of azacitidine resistance. With the aim to identify the mechanisms underlying acquired azacitidine resistance in cancer cell lines, we developed a computational strategy that can identify differentially regulated gene networks between drug-sensitive and -resistant cell lines by extending the existing method, differentially coexpressed gene sets (DiffCoEx). The technique specifically focuses on cell line-specific gene network analysis. We applied our method to gene networks specific to azacitidine sensitivity and identified differentially regulated gene networks between azacitidine-sensitive and -resistant cell lines. The molecular interplay between the metallothionein gene family, C19orf33, ELF3, GRB7, IL18, NRN1, and RBM47 were identified as differentially regulated gene network in drug resistant cell lines. The biological mechanisms associated with azacitidine and AML for the markers in the identified networks were verified through the literature. Our results suggest that controlling the identified genes (e.g., the metallothionein gene family) and "cellular response"-related pathways ("cellular response to zinc ion", "cellular response to copper ion", and "cellular response to cadmium ion", where the enriched functional-related genes are MT2A, MT1F, MT1G, and MT1E) may provide crucial clues to address azacitidine resistance in patients with AML. We expect that our strategy will be a useful tool to uncover patient-specific molecular interplay that provides crucial clues for precision medicine in not only gastric cancer but also complex diseases.

Two Different Strategies for Stabilization of Brain Tissue and Extraction of Neuropeptides.

Neuropeptides are bioactive peptides that are synthesized and secreted by neurons in signaling pathways in the brain. Peptides and proteins are extremely vulnerable to proteolytic cleavage when their biological surrounding changes. This makes neuropeptidomics challenging due to the rapid alterations that occur to the peptidome after harvesting of brain tissue samples. For a successful neuropeptidomic study, the biological tissue sample analyzed should resemble the living state as much as possible. Heat stabilization has been proven to inhibit postmortem degradation by denaturing proteolytic enzymes, hence increasing identification rates of neuropeptides. Here, we describe two different stabilization protocols for rodent brain samples that increase the number of intact mature neuropeptides and minimize interference from degradation products of abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by sequentially using an aqueous and an organic extraction medium.

Neuropeptidomics of Genetically Defined Cell Types in Mouse Brain.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. When this method is used with mice lacking CPE activity in genetically defined cell types, it allows the detection of peptides specifically produced in that cell type.

Origins, Technological Advancement, and Applications of Peptidomics.

Peptidomics is the comprehensive characterization of peptides from biological sources instead of heading for a few single peptides in former peptide research. Mass spectrometry allows to detect a multitude of peptides in complex mixtures and thus enables new strategies leading to peptidomics. The term was established in the year 2001, and up to now, this new field has grown to over 3000 publications. Analytical techniques originally developed for fast and comprehensive analysis of peptides in proteomics were specifically adjusted for peptidomics. Although it is thus closely linked to proteomics, there are fundamental differences with conventional bottom-up proteomics. Fundamental technological advancements of peptidomics since have occurred in mass spectrometry and data processing, including quantification, and more slightly in separation technology. Different strategies and diverse sources of peptidomes are mentioned by numerous applications, such as discovery of neuropeptides and other bioactive peptides, including the use of biochemical assays. Furthermore, food and plant peptidomics are introduced similarly. Additionally, applications with a clinical focus are included, comprising biomarker discovery as well as immunopeptidomics. This overview extensively reviews recent methods, strategies, and applications including links to all other chapters of this book.

Identification and Targeted Quantification of Endogenous Neuropeptides in the Nematode Caenorhabditis elegans Using Mass Spectrometry.

The nematode Caenorhabditis elegans lends itself as an excellent model organism for peptidomics studies. Its ease of cultivation and quick generation time make it suitable for high-throughput studies. The nervous system, with its 302 neurons, is probably the best-known and studied endocrine tissue. Moreover, its neuropeptidergic signaling pathways display numerous similarities with those observed in other metazoans. Here, we describe two label-free approaches for neuropeptidomics in C. elegans: one for discovery purposes, and another for targeted quantification and comparisons of neuropeptide levels between different samples. Starting from a detailed peptide extraction procedure, we here outline the liquid chromatography tandem mass spectrometry (LC-MS/MS) setup and describe subsequent data analysis approaches.