Bioactive Neuropeptides Research Articles

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.

Collaborative Expression: Transcriptomics of Conus virgo Suggests Contribution of Multiple Secretory Glands to Venom Production

Venomous marine gastropods of the family Conidae are among the most diversified predators in marine realm—in large due to their complex venoms. Besides being a valuable source of bioactive neuropeptides conotoxins, cone-snails venoms are an excellent model for molecular evolution studies, addressing origin of key innovations. However, these studies are handicapped by scarce current knowledge on the tissues involved in venom production, as it is generally assumed the sole prerogative of the venom gland (VG). The role of other secretory glands that are present in all Conus species (salivary gland, SG) or only in some species (accessory salivary gland, ASG) remains poorly understood. Here, for the first time, we carry out a detailed analysis of the VG, SG, and ASG transcriptomes in the vermivorous Conus virgo. We detect multiple transcripts clusters in both the SG and ASG, whose annotations imply venom-related functions. Despite the subsets of transcripts highly-expressed in the VG, SG, and ASG being very distinct, SG expresses an L-, and ASG—Cerm08-, and MEFRR- superfamily conotoxins, all previously considered specific for VG. We corroborate our results with the analysis of published SG and VG transcriptomes from unrelated fish-hunting C. geographus, and C. striatus, possibly fish-hunting C. rolani, and worm-hunting Conus quercinus. In spite of low expression levels of conotoxins, some other specific clusters of putative venom-related peptides are present and may be highly expressed in the SG of these species. Further functional studies are necessary to determine the role that these peptides play in envenomation. In the meantime, our results show importance of routine multi-tissue sampling both for accurate interpretation of tissue-specific venom composition in cone-snails, and for better understanding origin and evolution of venom peptides genes.

Application of the neuropeptide NPVF to enhance angiogenesis and osteogenesis in bone regeneration

The brain-bone regulatory system regulates skeletal homeostasis via bioactive neuropeptides, yet the underlying mechanism remains elusive. Here, we report the role of the neuropeptide VF (NPVF, VPNLPQRF-NH2) in enhancing both angiogenesis and osteogenesis in a rat skeletal system and the potential pathways involved. An in vitro study revealed that NPVF not only promotes migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) by activating NPFFR1, which leads to upregulation of miR-181c-3p and downregulation of Argonaute1 (AGO1), but also mediates osteogenic differentiation of bone mesenchymal stem cells (BMSCs) via the Wnt/β-catenin signaling pathway. To improve the stability and bioavailability and thus efficacy of NPVF as a promoter of in vivo bone regeneration, we genetically engineered amyloid-NPVF-fusion proteins and utilized them as self-assembling nanofiber coatings to treat bone defects in a rat calvarial defect model. We found that a porous hydroxyapatite scaffold loaded with the NPVF peptide-fused amyloid coating substantially enhanced angiogenesis and site-specific fresh bone in-growth when implanted in calvarial defects. Taken together, our work uncovered a previously undefined crosstalk between the brain and bone by unveiling the role of NPVF in bone tissue and demonstrated a viable method for promoting bone tissue repairs based upon self-assembling NPVF-containing protein coatings.

Immunohistochemical localisation of vasopressin/oxytocin-type, corazonin-type and luqin-type neuropeptide expression in the starfish Asterias rubens using antibodies to the C-terminal region of precursor proteins

Neuropeptides derived from larger precursor proteins are secreted as signalling molecules by neurons and regulate diverse physiological and behavioural processes in animals. Recently, we reported the discovery of ArCRZ (HNTFTMGGQNRWKAG-NH2) and ArLQ (EEKTRFPKFMRW-NH2)—novel neuropeptides in the starfish Asterias rubens that are orthologs of arthropod corazonins and molluscan luqins, respectively. However, our efforts to generate antibodies to ArCRZ and ArLQ have been unsuccessful, precluding immunohistochemical analysis of their expression. Here, we investigated an alternative experimental approach for neuropeptide immunohistochemistry by generating antibodies to peptides corresponding to the C-terminal region of the precursor proteins. As proof of principle, we generated antibodies to the C-terminal region of the precursor of the vasopressin/oxytocin-type neuropeptide asterotocin and show that these reveal immunostaining in A. rubens that is very similar to that observed with asterotocin antibodies. Furthermore, antibodies to the C-terminal region of the ArCRZ precursor (ArCRZP) and the ArLQ precursor (ArLQP) produced patterns of immunostaining consistent, respectively, with the distribution of ArCRZP and ArLQP transcripts revealed by mRNA in situ hybridisation. Detailed immunohistochemical analysis revealed widespread expression of ArCRZP and ArLQP in A. rubens, including the central nervous system, digestive system and the body wall and its associated appendages (e.g. tube feet), providing a neuroanatomical framework for investigation and interpretation of the pharmacological actions of ArCRZ and ArLQ in A. rubens. Furthermore, our findings provide a basis for use of antibodies to the C-terminal region of neuropeptide precursor proteins in other species where the production of antibodies to the bioactive neuropeptides is unsuccessful.

Changes in Neuropeptide Prohormone Genes among Cetartiodactyla Livestock and Wild Species Associated with Evolution and Domestication

The impact of evolution and domestication processes on the sequences of neuropeptide prohormone genes that participate in cell–cell signaling influences multiple biological process that involve neuropeptide signaling. This information is important to understand the physiological differences between Cetartiodactyla domesticated species such as cow, pig, and llama and wild species such as hippopotamus, giraffes, and whales. Systematic analysis of changes associated with evolutionary and domestication forces in neuropeptide prohormone protein sequences that are processed into neuropeptides was undertaken. The genomes from 118 Cetartiodactyla genomes representing 22 families were mined for 98 neuropeptide prohormone genes. Compared to other Cetartiodactyla suborders, Ruminantia preserved PYY2 and lost RLN1. Changes in GNRH2, IAPP, INSL6, POMC, PRLH, and TAC4 protein sequences could result in the loss of some bioactive neuropeptides in some families. An evolutionary model suggested that most neuropeptide prohormone genes disfavor sequence changes that incorporate large and hydrophobic amino acids. A compelling finding was that differences between domestic and wild species are associated with the molecular system underlying ‘fight or flight’ responses. Overall, the results demonstrate the importance of simultaneously comparing the neuropeptide prohormone gene complement from close and distant-related species. These findings broaden the foundation for empirical studies about the function of the neuropeptidome associated with health, behavior, and food production.

Ion Mobility Coupled to a Time-of-Flight Mass Analyzer Combined With Fragment Intensity Predictions Improves Identification of Classical Bioactive Peptides and Small Open Reading Frame-Encoded Peptides.

Bioactive peptides exhibit key roles in a wide variety of complex processes, such as regulation of body weight, learning, aging, and innate immune response. Next to the classical bioactive peptides, emerging from larger precursor proteins by specific proteolytic processing, a new class of peptides originating from small open reading frames (sORFs) have been recognized as important biological regulators. But their intrinsic properties, specific expression pattern and location on presumed non-coding regions have hindered the full characterization of the repertoire of bioactive peptides, despite their predominant role in various pathways. Although the development of peptidomics has offered the opportunity to study these peptides in vivo, it remains challenging to identify the full peptidome as the lack of cleavage enzyme specification and large search space complicates conventional database search approaches. In this study, we introduce a proteogenomics methodology using a new type of mass spectrometry instrument and the implementation of machine learning tools toward improved identification of potential bioactive peptides in the mouse brain. The application of trapped ion mobility spectrometry (tims) coupled to a time-of-flight mass analyzer (TOF) offers improved sensitivity, an enhanced peptide coverage, reduction in chemical noise and the reduced occurrence of chimeric spectra. Subsequent machine learning tools MS2PIP, predicting fragment ion intensities and DeepLC, predicting retention times, improve the database searching based on a large and comprehensive custom database containing both sORFs and alternative ORFs. Finally, the identification of peptides is further enhanced by applying the post-processing semi-supervised learning tool Percolator. Applying this workflow, the first peptidomics workflow combined with spectral intensity and retention time predictions, we identified a total of 167 predicted sORF-encoded peptides, of which 48 originating from presumed non-coding locations, next to 401 peptides from known neuropeptide precursors, linked to 66 annotated bioactive neuropeptides from within 22 different families. Additional PEAKS analysis expanded the pool of SEPs on presumed non-coding locations to 84, while an additional 204 peptides completed the list of peptides from neuropeptide precursors. Altogether, this study provides insights into a new robust pipeline that fuses technological advancements from different fields ensuring an improved coverage of the neuropeptidome in the mouse brain.

Beyond Neuronal Heat Sensing: Diversity of TRPV1 Heat-Capsaicin Receptor-Channel Functions.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel best known for its ability to be gated by the pungent constituent of red chili pepper, capsaicin, and related chemicals from the group of vanilloids as well as by noxious heat. As such, it is mostly expressed in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Its activation is also sensitized by the numerous endogenous inflammatory mediators and second messengers, making it an important determinant of nociceptive signaling. Except for such signaling, though, neuronal TRPV1 activation may influence various organ functions by promoting the release of bioactive neuropeptides from sensory fiber innervation organs. However, TRPV1 is also found outside the sensory nervous system in which its activation and function is not that straightforward. Thus, TRPV1 expression is detected in skeletal muscle; in some types of smooth muscle; in epithelial and immune cells; and in adipocytes, where it can be activated by the combination of dietary vanilloids, endovanilloids, and pro-inflammatory factors while the intracellular calcium signaling that this initiates can regulate processes as diverse as muscle constriction, cell differentiation, and carcinogenesis. The purpose of the present review is to provide a clear-cut distinction between neurogenic TRPV1 effects in various tissues consequent to its activation in sensory nerve endings and non-neurogenic TRPV1 effects due to its expression in cell types other than sensory neurons.

Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production.

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient–derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.

Cocaine has some effect on neuromedin U expressing neurons related to the brain reward system

Neuromedin U (NMU) is a bioactive neuropeptide, highly distributed in the gastrointestinal tract and the central nervous system. NMU has various physiological functions related to feeding behavior, energy metabolism, stress responses, circadian rhythmicity and inflammation. Recently, several reports indicate that the central NMU system plays an important role in the reward systems in the brain. However, the underlying molecular mechanisms are not yet fully defined. In this study, we found that some of cocaine-induced c-Fos immunoreactive cells were co-localized with NMU in the nucleus accumbens (NAc), caudate putamen (CPu), and basolateral amygdala (BLA), which are key brain regions associated with the brain reward system, in wild type mice. Whereas, a treatment with cocaine did not influence the kinetics of NMU or NMU receptors mRNA expression in these brain regions, and NMU-knockout mice did not show any higher preference for cocaine compared with their control mice. These results indicate that cocaine has some effect on NMU expressing neurons related to the brain reward system, and this suggests NMU system may have a role on the brain reward systems activated by cocaine.

Identification of neuropeptides in gastropod mollusks. - Classical and brand-new approaches –

For the understanding of the neural regulation of homeostasis, reproduction and behaviors, elucidation of structure and function of neuropeptides is inevitable. A classical technique for peptide purification is the separation of peptidic extract from nervous tissue by the high-performance liquid chromatography, which was followed by screening of fractions with biological or immunological assays. Although this approach requires relatively large amount of tissues for extraction and the screening is time- consuming, we have identified more than 15 kinds of bioactive neuropeptides in the nervous tissue of a marine snail, Thais clavigera. The purified peptides include TEP (Thais excitatory peptide)-1/-2, FRFamide, WWamide and others. The molecular cloning of precursor for each neuropeptide demonstrated that structurally related peptides are aligned in tandem on most of the precursor proteins. To identify the peptides on the precursors, we conducted the de novo sequencing of peptides with the nanoLC-Orbitrap-MS/MS analysis. It identified most of the neuropeptides found on the precursor proteins in the extract from 50 of Thais ganglia. Thus, this technique is suitable for the comprehensive identification of peptides from relatively small amount of tissues. However, several peptides that had been identified by the classical technique, such as APGWamide and leucokinin, were not identified, suggesting the limitation of this brand-new technique.

Strategies for the Identification of Bioactive Neuropeptides in Vertebrates.

Neuropeptides exert essential functions in animal physiology by controlling e.g., reproduction, development, growth, energy homeostasis, cardiovascular activity and stress response. Thus, identification of neuropeptides has been a very active field of research over the last decades. This review article presents the various methods used to discover novel bioactive peptides in vertebrates. Initially identified on the basis of their biological activity, some neuropeptides have also been discovered for their ability to bind/activate a specific receptor or based on their biochemical characteristics such as C-terminal amidation which concerns half of the known neuropeptides. More recently, sequencing of the genome of many representative species has facilitated peptidomic approaches using mass spectrometry and in silico screening of genomic libraries. Through these different approaches, more than a hundred of bioactive neuropeptides have already been identified in vertebrates. Nevertheless, researchers continue to find new neuropeptides or to identify novel functions of neuropeptides that had not been detected previously, as it was recently the case for nociceptin.

SAT-156 Sex-Specific, Diet-Induced Metabolic Dysfunction in F1 Progeny as a Consequence of Maternal Adolescent Preconception Opioid Exposure

Adverse metabolic conditions (obesity and type-2 diabetes) continue to be prevalent in developed nations. While the etiology of these diseases is multifactorial (i.e., genetic, environmental), parental exposures/experiences can also impart unfavorable metabolic phenotypes in future non-exposed progeny. We are currently in the midst of an opioid epidemic, with adolescents representing a growing population now exposed to this class of drugs. Endogenous opioids regulate reward- and motivation- related behaviors and chronic exposure to opioids significantly alter the brain’s reward system, an effect that can be transmitted across multiple generations. Indeed, data from animal models demonstrate that adolescent opioid exposure in females can impact their future progeny, even when that exposure occurs prior to conception. Effects observed in the offspring of morphine exposed females (MORF1) when compared to the offspring of saline exposed females (SALF1) include increased transcription of proopiomelanocortin (POMC) within the hypothalamic arcuate nucleus (ARC) of MORF1 offspring. POMC is differentially processed into several bioactive neuropeptides that play key roles in homeostatic and adaptive mechanisms. For example, β- endorphin (β-EP) and α- melanocyte stimulating hormone (α-MSH) regulate both reward/motivation and metabolic pathways, thus influencing reward liability as well as energy balance. Utilizing a preclinical model of preconception female adolescent opioid exposure, the current study demonstrates that when fed an obesogenic diet for 6-weeks, MORF1 offspring sex-specifically develop several deleterious metabolic effects; they consume more food, gain more weight, and develop fasting-induced hyperglycemia, hyperinsulinemia, and hypercorticosteronemia. MORF1 effects are associated with diet-specific alterations in ARC expression of neuropeptides known to regulate energy homeostasis. Further, while analysis across the POMC locus revealed no differences related to DNA methylation status in F1 male offspring at baseline, RNA sequencing of ARC tissue revealed an overall down-regulation of genes associated with synaptic plasticity in MORF1 males maintained on an obesogenic diet. Moreover, this transcriptional effect is coupled with an upregulation of inflammatory transcriptional networks. Thus, females adolescently exposed to morphine, conferred innate changes to the transcriptional response of the ARC to an obesogenic diet in the next generation. Such an effect induces the development of features suggestive of the emergence of metabolic syndrome in F1 offspring. Collectively, these findings suggest a potential confluence of two major global health issues (the opioid epidemic and metabolic syndrome) and provides evidence that maternal indices of opioid exposure can adversely influence metabolic predisposition in future progeny.

In silico prediction reveals the existence of potential bioactive neuropeptides produced by the human gut microbiota

This work reports on a large-scale potential neuropeptide activity screening in human gut microbiomes deposited in public databases. In our experimental approach, the sequences of the bioactive peptides collected in the MAHMI database, mainly predicted as immunomodulatory or antitumoral, were crossed with those of the neuroactive/digestive peptides. From 91,325,790 potential bioactive peptides, only 581 returned a match when crossed against the 5949 neuroactive peptides from the NeuroPep database and the 15 digestive hormones. Relevant bacterial taxa, such as Ruminococcus sp., Clostridium sp. were found among the main producers of the matching sequences, and many of the matches corresponded to adiponectin and the hormone produced by adipocites, which is involved in glucose homeostasis. These results show, for the first time, the presence of potentially bioactive peptides produced by gut microbiota members over the nervous cells, most notably, peptides with already predicted immunomodulatory or anti-inflammatory activity. Classical (Lactobacillus sp.) and next-generation (Faecalibacterium sp.) probiotics are shown to produce these peptides, which are proposed as a potential mechanism of action of psychobiotics. Our previous experimental results showed that many of these peptides were active when incubated with immune cells, such as dendritic cells, so their effect over the nervous system innervating the gut mucosa holds significant potential and should be explored.

Evidence for antimicrobial and anticancer activity of pituitary adenylate cyclase-activating polypeptide (PACAP) from North African catfish (Clarias gariepinus): Its potential use as novel therapeutic agent in fish and humans

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide that belongs to the secretin/glucagon superfamily, of which some members have shown antimicrobial activities. Contrasting to mammals, published studies on the action of PACAP in non-mammalian vertebrate immune system remain scarce. Some of our recent studies added this peptide to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in teleost fish. Regulation of PACAP and expression of its receptor genes has been demonstrated during an immune response mounted against acute bacterial infection in fish, though the direct effect of PACAP against fish pathogenic bacteria has never been addressed. Current work provides evidence of antimicrobial activity of Clarias gariepinus PACAP against a wide spectrum of Gram-negative and Gram-positive bacteria and fungi of interest for human medicine and aquaculture, in which computational prediction studies supported the putative PACAP therapeutic activity. Results also indicated that catfish PACAP not only exhibits inhibitory effects on pathogen growth, but also affects the proliferation of human non-small cell lung cancer cell line H460 in a dose-dependent manner. The observed cytotoxic activity of catfish PACAP against human tumor cells and pathogenic microorganisms, but not healthy fish and mammalian erythrocytes support a potential physiological role of this neuropeptide in selective microbial and cancer cell killing. All together, our findings extend the mechanisms by which PACAP could contribute to immune responses, and open up new avenues for future therapeutic application of this bioactive neuropeptide.

EGL-3 and EGL-21 are required to trigger nocifensive response of Caenorhabditis elegans to noxious heat

Caenorhabditis elegans (C. elegans) is a widely used model organism to examine nocifensive response to noxious stimuli, including heat avoidance. Recently, comprehensive analysis of the genome sequence revealed several pro-neuropeptide genes, encoding a series of bioactive neuropeptides. C. elegans neuropeptides are involved in the modulation of essentially all behaviors including locomotion, mechanosensation, thermosensation and chemosensation. The maturation of pro-neuropeptide to neuropeptide is performed by ortholog pro-protein convertases and carboxypeptidase E (e.g. EGL-3 and EGL-21). We hypothesized that C. elegans egl-3 or egl-21 mutants will have a significant decrease in mature neuropeptides and they will display an impaired heat avoidance behavior. Our data has shown that thermal avoidance behavior of egl-3 and egl-21 mutants was significantly hampered compared to WT(N2) C. elegans. Moreover, flp-18, flp-21 and npr-1 mutant C. elegans displayed a similar phenotype. EGL-3 pro-protein convertase and EGL-21 carboxypeptidase E are essential enzymes for the maturation of pro-neuropeptides to active neuropeptides in C. elegans. Quantitative mass spectrometry analyses with egl-3 and egl-21 mutant C. elegans homogenates demonstrated that proteolysis of ProFLP-18 and ProFLP-21 are severely impeded, leading to a lack of mature bioactive neuropeptides. Not only FLP-21 but also FLP-18 related mature neuropeptides, both are ligands of NPR-1 and are needed to trigger nocifensive response of C. elegans to noxious heat.

Deciphering the Role of EGL-3 for Neuropeptides Processing in Caenorhabditis elegans Using High-Resolution Quadrupole–Orbitrap Mass Spectrometry

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.

Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

Different Bioactive Neuropeptides are Expressed in Two Sub-Classes of GABAergic RME Nerve Ring Motorneurons in Ascaris suum

Neuropeptides can have significant effects on neurons and synapses, but among the ∼250 predicted peptides in nematodes, few have been characterized functionally. Here, we report new neuropeptides in the 4 RME nerve ring motorneurons of the nematode Ascaris suum. These GABAergic neurons are involved in three-dimensional head movement. Mass spectrometry (MS) of single dissected RMEs detected a total of 12 neuropeptides (encoded by five genes), nine of which are novel. None of these are expressed in the DI/VI inhibitory GABAergic motorneurons that synapse onto body wall muscle. Using peptide sequences obtained by tandem MS, we cloned the peptide-encoding transcripts and synthesized riboprobes for in situ hybridization (ISH). This complementary technique corroborated the results from single-cell MS, showing that the dissections were not contaminated with adhering tissue from other cells. We also synthesized a multiple antigenic peptide to raise a highly specific antibody against one of the endogenous peptides, which labeled the same cells detected by MS and ISH. Our results show that the RMEs can be divided into two subsets: RMED/V (expressing afp-2, afp-15, Asu-nlp-58, and high levels of afp-16) and RMEL/R (expressing afp-15 and low levels of afp-4 and afp-16). Almost all of these peptides are bioactive in A. suum.

Nematode neuropeptides as transgenic nematicides.

Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars.

Reproductive neuropeptides that stimulate spawning in the Sydney Rock Oyster (Saccostrea glomerata)

The Sydney Rock Oyster, Saccostrea glomerata, is a socioeconomically important species in Australia, yet little is known about the molecular mechanism that regulates its reproduction. To address this gap, we have performed a combination of high throughput transcriptomic and peptidomic analysis, to identify genes and neuropeptides that are expressed in the key regulatory tissues of S. glomerata; the visceral ganglia and gonads. Neuropeptides are known to encompass a diverse class of peptide messengers that play functional roles in many aspects of an animal’s life, including reproduction. Approximately 28 neuropeptide genes were identified, primarily within the visceral ganglia transcriptome, that encode precursor proteins containing numerous neuropeptides; some were confirmed through mass spectral peptidomics analysis of the visceral ganglia. Of those, 28 bioactive neuropeptides were synthesized, and then tested for their capacity to induce gonad development and spawning in S. glomerata. Egg laying hormone, gonadotropin-releasing hormone, APGWamide, buccalin, CCAP and LFRFamide were neuropeptides found to trigger spawning in ripe animals. Additional testing of APGWa and buccalin demonstrated their capacity to advance conditioning and gonadal maturation. In summary, our analysis of S. glomerata has identified neuropeptides that can influence the reproductive cycle of this species, specifically by accelerating gonadal maturation and triggering spawning. Other molluscan neuropeptides identified in this study will enable further research into understanding the neuroendocrinology of oysters, which may benefit their cultivation.