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Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research.

Backgrounds: Elucidation of peptide-receptor pairs is a prerequisite for many studies in the neuroendocrine, endocrine, and neuroscience fields. Recent omics analyses have provided vast amounts of peptide and G protein-coupled receptor (GPCR) sequence data. GPCRs for homologous peptides are easily characterized based on homology searching, and the relevant peptide-GPCR interactions are also detected by typical signaling assays. In contrast, conventional evaluation or prediction methods, including high-throughput reverse-pharmacological assays and tertiary structure-based computational analyses, are not useful for identifying interactions between novel and omics-derived peptides and GPCRs. Summary: Recently, an approach combining machine learning-based prediction of novel peptide-GPCR pairs and experimental validation of the predicted pairs have been shown to breakthrough this bottleneck. A machine learning method, logistic regression for human class A GPCRs and the multiple subsequent signaling assays led to the deorphanization of human class A orphan GPCRs, namely, the identification of 18 peptide-GPCR pairs. Furthermore, using another machine learning algorithm, the support vector machine (SVM), the peptide descriptor-incorporated SVM was originally developed and employed to predict GPCRs for novel peptides characterized from the closest relative of vertebrates, Ciona intestinalis Type A (Ciona robusta). Experimental validation of the predicted pairs eventually led to the identification of 11 novel peptide-GPCR pairs. Of particular interest is that these newly identified GPCRs displayed neither significant sequence similarity nor molecular phylogenetic relatedness to known GPCRs for peptides. Key Messages: These recent studies highlight the usefulness and versatility of machine learning for enabling the efficient, reliable, and systematic identification of novel peptide-GPCR interactions. [ABSTRACT FROM AUTHOR]

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuropeptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona. In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona , and their evolutionary aspects. • Deuterostomes possess a variety of endocrine, neuroendocrine, and nervous systems. • Ciona , a deuterostome invertebrate, is the closest relative of vertebrates. • Metamorphosis is regulated by neurotransmitters and neuropeptides in Ciona larva. • Ovarian follicle development in Ciona adults is regulated by neuropeptides. • Recent studies highlight common and unique neuroendocrine and nervous systems of Ciona in deuterostomes. [ABSTRACT FROM AUTHOR]

• Several neuropeptide signaling systems have been characterized in Ciona robusta. • Representatives of bilaterian familes and Ciona -specific neuropeptides identified. • Cognate receptors for Ciona neuropeptides have also been elucidated. • Neuropeptide function in follicle development and ovulation has been determined. Ciona intestinalis Type A (Ciona robusta) is a cosmopolitan species belonging to the phylum Urochordata, invertebrate chordates that are phylogenetically the most closely related to the vertebrates. Therefore, this species is of interest for investigation of the evolution and comparative physiology of endocrine, neuroendocrine, and nervous systems in chordates. Our group has identified>30 Ciona neuropeptides (80% of all identified ascidian neuropeptides) primarily using peptidomic approaches combined with reference to genome sequences. These neuropeptides are classified into two groups: homologs or prototypes of vertebrate neuropeptides and novel (Ciona -specific) neuropeptides. We have also identified the cognate receptors for these peptides. In particular, we elucidated multiple receptors for Ciona -specific neuropeptides by a combination of a novel machine learning system and experimental validation of the specific interaction of the predicted neuropeptide-receptor pairs, and verified unprecedented phylogenies of receptors for neuropeptides. Moreover, several neuropeptides were found to play major roles in the regulation of ovarian follicle development. Ciona tachykinin facilitates the growth of vitellogenic follicles via up-regulation of the enzymatic activities of proteases. Ciona vasopressin stimulates oocyte maturation and ovulation via up-regulation of maturation-promoting factor- and matrix metalloproteinase-directed collagen degradation, respectively. Ciona cholecystokinin also triggers ovulation via up-regulation of receptor tyrosine kinase signaling and the subsequent activation of matrix metalloproteinase. These studies revealed that the neuropeptidergic system plays major roles in ovarian follicle growth, maturation, and ovulation in Ciona , thus paving the way for investigation of the biological roles for neuropeptides in the endocrine, neuroendocrine, nervous systems of Ciona , and studies of the evolutionary processes of various neuropeptidergic systems in chordates. [ABSTRACT FROM AUTHOR]

Abstract: The midgut plays a major role in digestion and absorption of nutrients in insects, and contains endocrine cells throughout the epithelial layer that express neuropeptides, including crustacean cardioactive peptide (CCAP). In the present study, we demonstrate regulation of digestive enzyme activities by CCAP in response to nutrient ingestion in the cockroach, Periplaneta americana. The midgut of the cockroach exhibits maximal α-amylase and protease activities 3h after intake of either starch or casein, but not of non-nutrients. Similar time-dependent responses of CCAP expression in midgut endocrine cells were observed after feeding starch and casein, but not after non-nutrients. We also show that incubation of the dissected midgut with CCAP leads to an increase in α-amylase and protease activity in a time-dependent manner, with the maximal activity at 2h. Taken together, our data indicate the existence of an inducible mechanism where endocrine cells in the midgut are stimulated to synthesize and secrete CCAP by nutrients, and CCAP then up-regulates the activity of digestive enzymes. [Copyright &y& Elsevier]

To date, diverse neurotransmitters, steroid hormones, neuropeptides and peptide hormones, and their receptors have been characterized, and some of them have been shown to participate in regulation of a variety of biological processes in invertebrate deuterostomes. Using the starfish I Asterias rubens i as an experimental model system, Tinoco et al. report the molecular characterization of a receptor for the neuropeptide NGFFYamide, which is orthologous to vertebrate neuropeptide-S (NPS) and the arthropod neuropeptide crustacean cardioactive peptide (CCAP). In a second review article, Sekiguchi discusses the evolution of calcitonin/calcitonin gene-related peptide (CT/CGRP)-type signaling in deuterostomes and the characterization of CT/CGRP-type neuropeptides and their receptors in invertebrate chordates. [Extracted from the article]

Ascidians are the closest phylogenetic neighbors to vertebrates and are believed to conserve the evolutionary origin in chordates of the endocrine, neuroendocrine, and nervous systems involving neuropeptides and peptide hormones. Ciona intestinalis harbors various homologs or prototypes of vertebrate neuropeptides and peptide hormones including gonadotropin-releasing hormones (GnRHs), tachykinins (TKs), and calcitonin, as well as Ciona -specific neuropeptides such as Ciona vasopressin, LF, and YFV/L peptides. Moreover, molecular and functional studies on Ciona tachykinin (Ci-TK) have revealed the novel molecular mechanism of inducing oocyte growth via up-regulation of vitellogenesis-associated protease activity, which is expected to be conserved in vertebrates. Furthermore, a series of studies on Ciona GnRH receptor paralogs have verified the species-specific regulation of GnRHergic signaling including unique signaling control via heterodimerization among multiple GnRH receptors. These findings confirm the remarkable significance of ascidians in investigations of the evolutionary processes of the peptidergic systems in chordates, leading to the promising advance in the research on Ciona peptides in the next stage based on the recent development of emerging technologies including genome-editing techniques, peptidomics-based multi-color staining, machine-learning prediction, and next-generation sequencing. These technologies and bioinformatic integration of the resultant “multi-omics” data will provide unprecedented insights into the comprehensive understanding of molecular and functional regulatory mechanisms of the Ciona peptides, and will eventually enable the exploration of both conserved and diversified endocrine, neuroendocrine, and nervous systems in the evolutionary lineage of chordates. [ABSTRACT FROM AUTHOR]

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH 2 ). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH in neuronal fibers elongating to peripheral organs suggests that it is multi-functional. Time-resolved fluorescent immunoassays (TR-FIA) enabled the quantification of each py-GnRH form in the single CPG or VG tissue obtained from one individual. In addition, we observed greater abundance of mature py-GnRH in VG compared with its level in CPG, suggesting that VG is the main producing organ of mature py-GnRH peptide and that py-GnRH may play a central regulatory role in neurons of scallops. Our study provides evidence, for the first time, for the presence of precursor and mature forms of invGnRH peptides in the nerve ganglia of an invertebrate. [ABSTRACT FROM AUTHOR]

Abstract: The gonadotropin-releasing hormone (GnRH) family peptides are most widely distributed neuropeptides and/or neurophysial hormones. GnRH is involved in diverse neuroendocrine, paracrine, autocrine, and neurotransmitter/neuromodulatory functions in the central and peripheral nervous system as well as peripheral tissues. In the present study, we show the identification of a novel GnRH-related peptide, Ci-GnRH-X, in the ascidian, Ciona intestinalis. Intriguingly, Ci-GnRH-X possesses a unique primary sequence consisting of 16 amino acids, although typical GnRH family peptides are composed of 10 amino acids. On the other hand, Ci-GnRH-X shares the GnRH consensus motifs, including the N-terminal pQHWS (‘pQ’ indicates a pyro-glutamic acid) and C-terminal Gly-amide. Reverse transcription (RT)-PCR analysis shows that the Ci-GnRH-X gene is expressed exclusively in the central nervous system. Moreover, in situ hybridization demonstrated that the Ciona GnRH-1 gene encoding Ciona GnRHs (t-GnRH-3, -5 and -6) was co-expressed with the Ci-GnRH-X gene in neurons of the cerebral ganglion. Of particular interest is that Ci-GnRH-X exhibited moderate (10–50%) inhibitory activity against t-GnRHs at their cognate receptors. Ci-GnRH-X repressed the elevation of the intracellular calcium and cAMP production by t-GnRH-6 at Ci-GnRHR-1, and cAMP production by t-GnRH-3, and t-GnRH-5 via Ci-GnRHR-3 was also inhibited by Ci-GnRH-X. In contrast, no inhibitory effect of Ci-GnRH-X at Ci-GnRHR-2 was observed. The localization and biochemical assays revealed that Ci-GnRH-X acts as an endogenous antagonist for the Ciona GnRHergic system. This is the first molecular and functional characterization of an endogenous inhibitor of GnRHs in an animal species. [Copyright &y& Elsevier]