A class of secreted mammalian peptides with potential to expand cell-cell communication.

Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Kobayashi Award 2021: Neuropeptides, receptors, and follicle development in the ascidian, Ciona intestinalis Type A: New clues to the evolution of chordate neuropeptidergic systems from biological niches.

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.

Female Infertility Is Associated with an Altered Expression Profile of Different Members of the Tachykinin Family in Human Granulosa Cells.

Neurokinin B (NKB) and its cognate receptor, NK3R, play a key role in the regulation of reproduction. NKB belongs to the family of tachykinins, which also includes substance P and neurokinin A, both encoded by the by the gene TAC1, and hemokinin-1, encoded by the TAC4 gene. In addition to NK3R, tachykinin effects are mediated by NK1R and NK2R, encoded by the genes TACR1 and TACR2, respectively. The role of these other tachykinins and receptors in the regulation of women infertility is mainly unknown. We have analyzed the expression profile of TAC1, TAC4, TACR1, and TACR2 in mural granulosa and cumulus cells from women presenting different infertility etiologies, including polycystic ovarian syndrome, advanced maternal age, low ovarian response, and endometriosis. We also studied the expression of MME, the gene encoding neprilysin, the most important enzyme involved in tachykinin degradation. Our data show that TAC1, TAC4, TACR1, TACR2, and MME expression is dysregulated in a different manner depending on the etiology of women infertility. The abnormal expression of these tachykinins and their receptors might be involved in the decreased fertility of these patients, offering a new insight regarding the diagnosis and treatment of women infertility.

BmGr4 responds to sucrose and glucose and expresses in tachykinin-related peptide-secreting enteroendocrine cells.

The regulatory hormones known as tachykinin-related peptides (TRPs) are identified as brain-gut peptides in insects. Dietary components from mulberry leaves, including glucose, induce secretion of TRPs from Bombyx mori midgut. However, the sensory molecules that recognize these compounds are still unknown. Here, we identified the gustatory receptor, BmGr4, as a sucrose and glucose receptor using Ca2+ imaging. Immunostaining revealed BmGr4 expression not only in the midgut, but also in the brain. In addition, BmGr4 expression was found to co-localize with TRP-expressing cells in both midgut enteroendocrine cells (EECs) and brain neurosecretory cells (NSCs). Furthermore, dietary nutrients after food intake result in an increase of TRP-level in hemolymph of silkworm larvae. These results provide significant circumstantial evidence for the involvement of the sucrose and glucose receptor, BmGr4, in the elicitation of TRP secretion in midgut EECs and brain NSCs.

Multiple tachykinins and their receptors characterized in the gastropod mollusk Pacific abalone: Expression, signaling cascades, and potential role in regulating lipid metabolism.

Tachykinin (TK) families, including the first neuropeptide substance P, have been intensively explored in bilaterians. Knowledge of signaling of TK receptors (TKRs) has enabled the comprehension of diverse physiological processes. However, TK signaling systems are largely unknown in Lophotrochozoa. This study identified two TK precursors and two TKR isoforms in the Pacific abalone Haliotis discus hannai (Hdh), and characterized Hdh-TK signaling. Hdh-TK peptides harbored protostomian TK-specific FXGXRamide or unique YXGXRamide motifs at the C-termini. A phylogenetic analysis showed that lophotrochozoan TKRs, including Hdh-TKRs, form a monophyletic group distinct from arthropod TKRs and natalisin receptor groups. Although reporter assays demonstrated that all examined Hdh-TK peptides activate intracellular cAMP accumulation and Ca2+ mobilization in Hdh-TKR-expressing mammalian cells, Hdh-TK peptides with N-terminal aromatic residues and C-terminal FXGXRamide motifs were more active than shorter or less aromatic Hdh-TK peptides with a C-terminal YXGXRamide. In addition, we showed that ligand-stimulated Hdh-TKRs mediate ERK1/2 phosphorylation in HEK293 cells and that ERK1/2 phosphorylation is inhibited by PKA and PKC inhibitors. In three-dimensional in silico Hdh-TKR binding modeling, higher docking scores of Hdh-TK peptides were consistent with the lower EC50 values in the reporter assays. The transcripts for Hdh-TK precursors and Hdh-TKR were highly expressed in the neural ganglia, with lower expression levels in peripheral tissues. When abalone were starved for 3 weeks, Hdh-TK1 transcript levels, but not Hdh-TK2, were increased in the cerebral ganglia (CG), intestine, and hepatopancreas, contrasting with the decreased lipid content and transcript levels of sterol regulatory element-binding protein (SREBP). At 24 h post-injection in vivo, the lower dose of Hdh-TK1 mixture increased SREBP transcript levels in the CG and hepatopancreas and accumulative food consumption of abalone. Higher doses of Hdh-TK1 and Hdh-TK2 mixtures decreased the SREBP levels in the CG. When Hdh-TK2-specific siRNA was injected into abalone, intestinal SREBP levels were significantly increased, whereas administration of both Hdh-TK1 and Hdh-TK2 siRNA led to decreased SREBP expression in the CG. Collectively, our results demonstrate the first TK signaling system in gastropod mollusks and suggest a possible role for TK peptides in regulating lipid metabolism in the neural and peripheral tissues of abalone.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

Identification of a tachykinin receptor and its implication in carbohydrate and lipid homeostasis in Rhodnius prolixus, a chagas disease vector.

Neuropeptides and their receptors are fundamentally important in regulating many physiological and behavioural processes in insects. In this work, we have identified, cloned, and sequenced the tachykinin receptor (Rhopr-TKR) from Rhodnius prolixus, a vector of Chagas disease. The receptor is a G protein-coupled receptor belonging to the Rhodopsin Family A. The total length of the open reading frame of the Rhopr-TKR transcript is 1110 bp, which translates into a receptor of 338 amino acids. Fluorescent in-situ RNA-hybridization (FISH) for the Rhopr-TKR transcript shows a signal in a group of six bilaterally paired neurons in the protocerebrum of the brain, localized in a similar region as the insulin producing cells. To examine the role of tachykinin signaling in lipid and carbohydrate homeostasis we used RNA interference. Downregulation of the Rhopr-TKR transcript led to a decrease in the size of blood meal consumed and a significant increase in circulating carbohydrate and lipid levels. Further investigation revealed a close relationship between tachykinin and insulin signaling since the downregulation of the Rhopr-TKR transcript negatively affected the transcript expression for insulin-like peptide 1 (Rhopr-ILP1), insulin-like growth factor (Rhopr-IGF) and insulin receptor 1 (Rhopr-InR1) in both the central nervous system and fat body. Taken together, these findings suggest that tachykinin signaling regulates lipid and carbohydrate homeostasis via the insulin signaling pathway.

Tachykinins, new players in the control of reproduction and food intake: A comparative review in mammals and teleosts.

In vertebrates, the tachykinin system includes tachykinin genes, which encode one or two peptides each, and tachykinin receptors. The complexity of this system is reinforced by the massive conservation of gene duplicates after the whole-genome duplication events that occurred in vertebrates and furthermore in teleosts. Added to this, the expression of the tachykinin system is more widespread than first thought, being found beyond the brain and gut. The discovery of the co-expression of neurokinin B, encoded by the tachykinin 3 gene, and kisspeptin/dynorphin in neurons involved in the generation of GnRH pulse, in mammals, put a spotlight on the tachykinin system in vertebrate reproductive physiology. As food intake and reproduction are linked processes, and considering that hypothalamic hormones classically involved in the control of reproduction are reported to regulate also appetite and energy homeostasis, it is of interest to look at the potential involvement of tachykinins in these two major physiological functions. The purpose of this review is thus to provide first a general overview of the tachykinin system in mammals and teleosts, before giving a state of the art on the different levels of action of tachykinins in the control of reproduction and food intake. This work has been conducted with a comparative point of view, highlighting the major similarities and differences of tachykinin systems and actions between mammals and teleosts.

Tachykinins amplify the action of capsaicin on central histaminergic neurons.

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

Regulatory Peptides in Asthma.

Numerous regulatory peptides play a critical role in the pathogenesis of airway inflammation, airflow obstruction and hyperresponsiveness, which are hallmarks of asthma. Some of them exacerbate asthma symptoms, such as neuropeptide Y and tachykinins, while others have ameliorating properties, such as nociception, neurotensin or ß-defensin 2. Interacting with peptide receptors located in the lungs or on immune cells opens up new therapeutic possibilities for the treatment of asthma, especially when it is resistant to available therapies. This article provides a concise review of the most important and current findings regarding the involvement of regulatory peptides in asthma pathology.

Novel Pituitary Actions of TAC4 Gene Products in Teleost.

Tachykinin 4 (TAC4) is the latest member of the tachykinin family involved in several physiological functions in mammals. However, little information is available about TAC4 in teleost. In the present study, we firstly isolated TAC4 and six neurokinin receptors (NKRs) from grass carp brain and pituitary. Sequence analysis showed that grass carp TAC4 could encode two mature peptides (namely hemokinin 1 (HK1) and hemokinin 2 (HK2)), in which HK2 retained the typical FXGLM motif in C-terminal of tachyinin, while HK1 contained a mutant VFGLM motif. The ligand-receptor selectivity showed that HK2 could activate all 6 NKRs but with the highest activity for the neurokinin receptor 2 (NK2R). Interestingly, HK1 displayed a very weak activation for each NKR isoform. In grass carp pituitary cells, HK2 could induce prolactin (PRL), somatolactin α (SLα), urotensin 1 (UTS1), neuromedin-B 1 (NMB1), cocaine- and amphetamine-regulated transcript 2 (CART2) mRNA expression mediated by NK2R and neurokinin receptor 3 (NK3R) via activation cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC) and calcium2+ (Ca2+)/calmodulin (CaM)/calmodulin kinase-II (CaMK II) cascades. However, the corresponding stimulatory effects triggered by HK1 were found to be notably weaker. Furthermore, based on the structural base for HK1, our data suggested that a phenylalanine (F) to valine (V) substitution in the signature motif of HK1 might have contributed to its weak agonistic actions on NKRs and pituitary genes regulation.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

Metabolic control of daily locomotor activity mediated by tachykinin in Drosophila.

Metabolism influences locomotor behaviors, but the understanding of neural curcuit control for that is limited. Under standard light-dark cycles, Drosophila exhibits bimodal morning (M) and evening (E) locomotor activities that are controlled by clock neurons. Here, we showed that a high-nutrient diet progressively extended M activity but not E activity. Drosophila tachykinin (DTk) and Tachykinin-like receptor at 86C (TkR86C)-mediated signaling was required for the extension of M activity. DTk neurons were anatomically and functionally connected to the posterior dorsal neuron 1s (DN1ps) in the clock neuronal network. The activation of DTk neurons reduced intracellular Ca2+ levels in DN1ps suggesting an inhibitory connection. The contacts between DN1ps and DTk neurons increased gradually over time in flies fed a high-sucrose diet, consistent with the locomotor behavior. DN1ps have been implicated in integrating environmental sensory inputs (e.g., light and temperature) to control daily locomotor behavior. This study revealed that DN1ps also coordinated nutrient information through DTk signaling to shape daily locomotor behavior.

Sex differences in fear memory consolidation via Tac2 signaling in mice.

Memory formation is key for brain functioning. Uncovering the memory mechanisms is helping us to better understand neural processes in health and disease. Moreover, more specific treatments for fear-related disorders such as posttraumatic stress disorder and phobias may help to decrease their negative impact on mental health. In this line, the Tachykinin 2 (Tac2) pathway in the central amygdala (CeA) has been shown to be sufficient and necessary for the modulation of fear memory consolidation. CeA-Tac2 antagonism and its pharmacogenetic temporal inhibition impair fear memory in male mice. Surprisingly, we demonstrate here the opposite effect of Tac2 blockade on enhancing fear memory consolidation in females. Furthermore, we show that CeA-testosterone in males, CeA-estradiol in females and Akt/GSK3ß/ß-Catenin signaling both mediate the opposite-sex differential Tac2 pathway regulation of fear memory.

Characterization of putative tachykinin peptides in Caenorhabditis elegans.

Tachykinin-like peptides, such as substance P, neurokinin A, and neurokinin B, are among the earliest discovered and best-studied neuropeptide families, and research on them has contributed greatly to our understanding of the endocrine control of many physiological processes. However, there are still many orphan tachykinin receptor homologs for which cognate ligands have not yet been identified, especially in small invertebrates, such as the nematode Caenorhabditis elegans (C. elegans). We here show that the C. elegans nlp-58 gene encodes putative ligands for the orphan G protein-coupled receptor (GPCR) TKR-1, which is a worm ortholog of tachykinin receptors. We first determine, through an unbiased biochemical screen, that a peptide derived from the NLP-58 preprotein stimulates TKR-1. Three mature peptides that are predicted to be generated from NLP-58 show potent agonist activity against TKR-1. We designate these peptides as C. elegans tachykinin (CeTK)-1, -2, and -3. The CeTK peptides contain the C-terminal sequence GLR-amide, which is shared by tachykinin-like peptides in other invertebrate species. nlp-58 exhibits a strongly restricted expression pattern in several neurons, implying that CeTKs behave as neuropeptides. The discovery of CeTKs provides important information to aid our understanding of tachykinin-like peptides and their functional interaction with GPCRs.

A cell type-specific cortico-subcortical brain circuit for investigatory and novelty-seeking behavior.

Exploring the physical and social environment is essential for understanding the surrounding world. We do not know how novelty-seeking motivation initiates the complex sequence of actions that make up investigatory behavior. We found in mice that inhibitory neurons in the medial zona incerta (ZIm), a subthalamic brain region, are essential for the decision to investigate an object or a conspecific. These neurons receive excitatory input from the prelimbic cortex to signal the initiation of exploration. This signal is modulated in the ZIm by the level of investigatory motivation. Increased activity in the ZIm instigates deep investigative action by inhibiting the periaqueductal gray region. A subpopulation of inhibitory ZIm neurons expressing tachykinin 1 (TAC1) modulates the investigatory behavior.

A Second Wave for the Neurokinin Tac2 Pathway in Brain Research.

Despite promising advances in basic research of the neurokinin B/Tac2 pathway in both animals and humans, clinical applications are yet to be implemented. This is likely because of our limited understanding of the action of the pathway in the brain. While this system controls neuronal activity in multiple regions, the precise impact of Tac2-induced cellular responses on behavior remains unclear. Recently, elegant studies revealed a key contribution to stress-related behaviors and memory. Here, we discuss the crucial importance of bridging the gap between the Tac2 pathway's involvement in cell physiology and cognition to comprehend its role in health and disease. We propose that a better understanding of the Tac2 pathway in the brain could provide an essential perspective for basic investigations, which in turn will feed clinical research.

Characterization of the Action of Tachykinin Signaling on Pulsatile LH Secretion in Male Mice.

The alternation of the stimulatory action of the tachykinin neurokinin B (NKB) and the inhibitory action of dynorphin within arcuate (ARH) Kiss1 neurons has been proposed as the mechanism behind the generation of gonadotropin-releasing hormone (GnRH) pulses through the pulsatile release of kisspeptin. However, we have recently documented that GnRH pulses still exist in gonadectomized mice in the absence of tachykinin signaling. Here, we document an increase in basal frequency and amplitude of luteinizing hormone (LH) pulses in intact male mice deficient in substance P, neurokinin A (NKA) signaling (Tac1KO), and NKB signaling (Tac2KO and Tacr3KO). Moreover, we offer evidence that a single bolus of the NKB receptor agonist senktide to gonad-intact wild-type males increases the basal release of LH without changing its frequency. Altogether, these data support the dispensable role of the individual tachykinin systems in the generation of LH pulses. Moreover, the increased activity of the GnRH pulse generator in intact KO male mice suggests the existence of compensation by additional mechanisms in the generation of kisspeptin/GnRH pulses.

Tachykinin signaling inhibits task-specific behavioral responsiveness in honeybee workers.

Behavioral specialization is key to the success of social insects and leads to division of labor among colony members. Response thresholds to task-specific stimuli are thought to proximally regulate behavioral specialization, but their neurobiological regulation is complex and not well understood. Here, we show that response thresholds to task-relevant stimuli correspond to the specialization of three behavioral phenotypes of honeybee workers in the well-studied and important Apis mellifera and Apis cerana. Quantitative neuropeptidome comparisons suggest two tachykinin-related peptides (TRP2 and TRP3) as candidates for the modification of these response thresholds. Based on our characterization of their receptor binding and downstream signaling, we confirm a functional role of tachykinin signaling in regulating specific responsiveness of honeybee workers: TRP2 injection and RNAi-mediated downregulation cause consistent, opposite effects on responsiveness to task-specific stimuli of each behaviorally specialized phenotype but not to stimuli that are unrelated to their tasks. Thus, our study demonstrates that TRP signaling regulates the degree of task-specific responsiveness of specialized honeybee workers and may control the context specificity of behavior in animals more generally.