The analgesic effect of neuropeptide S (NPS) in alcohol-dependent male and female rats.

Insects possess a well-developed innate immune system, which encompasses both cellular and humoral mechanisms. On the basis of the similarities in neuropeptide actions between insects and vertebrates, we assume that neuropeptides such as tachykinin-related peptides (TRPs) regulate insect immune responses and are themselves modulated following infection. In this study, we examined how immune activation affects the expression of genes encoding TRP precursors and receptors (TRP and TRPR) and whether TRPs directly modulate selected immune mechanisms in the pest species Tenebrio molitor. In our experiment, we combined cytokine stimulation, genetic knockdown, pharmacological inhibition, immune profiling, and survival analysis to dissect neuropeptide function. Our results revealed two important insights. First, after activation of the immune system, TRP and TRPR genes were significantly downregulated in the nervous system and immune-related cells. These changes are closely correlated with the changes of the expression level of immune genes. We then show, using Spantide II, a potent antagonist of TRPR, and RNAi knock down of TRP and TRPR, the modulation of key processes of the T. molitor humoral response. This includes the over expression of genes encoding antimicrobial peptides and the important arthropod immune effector phenoloxidase activity. Our findings highlight a compelling association between the TRP and immune regulation in Tenebrio and provide insights into the hormonal regulation of physiological processes in insects. Our research also provides novel insights that can contribute to the development of sustainable pest control strategies amid increasing insecticide resistance.

Singapore blue arboreal tarantula peptide Lv1a preferentially targets NaV1.6 with new world-like pharmacological mechanism.

Macrophage-derived NPS drives acute lung injury by inducing CaMKII/NFATc3-Mediated M1 polarization.

This study is the first to comprehensively explore both intracellular and computational mechanisms through which Neuropeptide S (NPS) protects against paraquat-induced dopaminergic toxicity in a Parkinson’s disease (PD)-like SH-SY5Y cell model. Paraquat induces oxidative stress, mitochondrial dysfunction, and dopaminergic neuron loss, mimicking key PD features. Bioinformatic analyses, including Reactome pathway mapping and molecular docking, confirmed a high-affinity interaction between NPS and its receptor NPSR1, activating GPCR-associated signaling. NPS treatment restored intracellular dopamine and ATP levels and increased tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT) expression. Cell viability was assessed using the MTT assay, while dopamine levels were measured via LC–MS/MS. p-ERK1/2, total ERK1/2, and Nrf2 were quantified by ELISA and western blot. Oxidative stress markers, including TBARS, MAO-A, MAO-B, and COMT, were analyzed by ELISA. Gene expression of Bax, Bcl-2, Caspase-3, Caspase-8, DAT, and VMAT was evaluated by qRT-PCR. TH, c-Fos, and NPSR1 were visualized using immunofluorescence. NPS significantly improved cell viability and restored ATP levels compromised by paraquat exposure. It also reduced TBARS, MAO-B, and COMT levels, reversed paraquat-induced ERK1/2 phosphorylation, and restored Nrf2 and MAO-A expression. Additionally, NPS upregulated the anti-apoptotic marker Bcl-2. Most of these protective effects were abolished in the presence of the NPSR antagonist ML154, indicating a receptor-mediated mechanism of action. In conclusion, NPS was found to attenuate oxidative stress, support mitochondrial and dopaminergic function, and influence apoptosis-related signaling in our cellular model. These findings suggest that targeting the NPS/NPSR1 system may hold therapeutic potential in neurodegenerative diseases such as PD, warranting further in vivo validation.Graphical

Design of a peripherally biased NPSR1 antagonist for neuropeptide S induced inflammation.

Marine cone snails produce a diverse array of bioactive peptides, known as conotoxins, in their venom. Given their high target potency and specificity, conotoxins are attractive compounds for the development of precision research tools and pharmacological agents. Here, we provide the first experimental characterization of a conotoxin from the MKAVA superfamily, conkazal-M1, from Conus magus. Using NMR spectroscopy, we show that conkazal-M1 adopts a fold characteristic of the Kazal-type protease inhibitor family, featuring a Glu residue at the inhibitory P1 position. Recombinantly expressed conkazal-M1 inhibits the proteolytic activity of Subtilisin A with an apparent Ki of 1.1 μM. In addition, conkazal-M1 partially inhibits calcium transients in mouse sensory neurons, suggesting a potential role in modulating ion-channel activity, as seen for many other toxins. The dual function of conkazal-M1 in protease inhibition and neuroactivity is analogous to the dual function of several toxins harboring a Kunitz-type fold. The well-conserved sequence of the MKAVAs indicates an evolutionary trajectory in which these proteins face an adaptive conflict, where mutations that enhance one activity compromise the other. Collectively, this work provides new structural and functional insights into a previously uncharacterized toxin superfamily in cone snails, illustrates how structural scaffolds can be repurposed for functions that diverge from the original while retaining their overall structure, and expands our understanding of the toxin arsenal available to venomous animals.

How neuropeptides act within the neural circuits that control social behavior is not well understood. While the prevailing view is that neuropeptides act through synaptic release and then activation of their canonical receptors on postsynaptic membranes, we investigated the role of a very different form of neuropeptide action in a neural circuit regulating social communication. Specifically, we tested the hypothesis that non-synaptically released oxytocin (OT) can act via the non-canonical receptors vasopressin V1a receptors (V1aR) to regulate social communication in Syrian hamsters. Scent marking, a key form of hamster social communication, can be enhanced by the α-melanocortin stimulating hormone (α-MSH), which stimulates OT but not arginine-vasopressin (AVP) release. Here, we employed hypothalamic injections of α-MSH and the α-MSH MC4R receptor antagonist MCL-0020 to determine the role of α-MSH in the expression of flank marking. To determine if these effects were intracellular calcium (iCa2+) dependent, hamsters were injected with AVP to induce flank marking and with the iCa2+ antagonist TMB-8 to test whether it was possible to block this behavioral effect. Further, a highly selective AVP V1a receptor (V1aR) antagonist and an OT receptor (OTR) antagonist were injected into the hypothalamus to investigate the receptor responsible for activating flank marking. Finally, we employed an in vitro hypothalamic slice preparation using "Sniffer cells" biosensors to confirm that α-MSH induced the release of OT but not AVP. First, we found that the in vivo hypothalamic injection of α-MSH increased odor-stimulated scent marking, whereas blockade of its receptor with MCL-0020 reduced this behavior. Hypothalamic infusion of the iCa2+ antagonist TMB-8 significantly reduced both AVP-induced and α-MSH-induced flank marking. Moreover, only the V1aR antagonist, and not the OTR antagonist, significantly decreased scent marking in response to hypothalamic infusion of α-MSH. Finally, biosensor recordings from hypothalamic slices confirmed that α-MSH stimulates OT, but not AVP, release. Together, these results demonstrate that α-MSH triggers non-synaptic OT release that regulates scent marking via V1aR activation, revealing a novel mechanism by which neuropeptides modulate social behavior.

Alcohol Use Disorder (AUD) is a global health concern, with stress playing a crucial role in its development and persistence. Currently, no pharmacotherapies specifically targeting stress are approved for AUD treatment. Neuropeptide S (NPS) plays a dual role in stress regulation, exhibiting both anxiolytic and stress-enhancing effects. While NPS reduces alcohol self-administration (ASA) in alcohol preferring rats, its role in AUD-related stress and anxiety remains unclear. This study investigated the behavioral effects of NPS in male and female Marchigian Sardinian alcohol-preferring (msP) rats. To assess its impact on locomotion, anxiety, and fear memory, we conducted an open-field, an elevated plus maze (EPM), and a fear conditioning (FC) paradigm following intracerebroventricular administration of NPS. Furthermore, we examined the effects of NPS on ASA and yohimbine-induced reinstatement of alcohol-seeking in msP rats. Our results indicate that NPS administration increased locomotor activity in both sexes and selectively alleviated generalized anxiety levels in male rats in the EPM test. In the FC task, administration of NPS immediately after FC test facilitated the extinction of fear memories in females but not in males. Notably, NPS reduced ASA in both female and male rats but did not alter yohimbine-induced reinstatement of alcohol-seeking. In conclusion, NPS modulates anxiety in a sex-dependent manner. Since both alcohol and NPS alleviate anxiety and fear conditioning in msP rats, NPS may reduce alcohol intake by replacing the anxiolytic properties of alcohol. These effects appear to be sex-dependent, with NPS primarily alleviating generalized anxiety in males and facilitating fear extinction in females.

Neuropeptide S system mediates nicotine-induced reward-facilitatory behavior.

Background: Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder with limited treatment options. Emerging evidence reveals bidirectional crosstalk between gut and brain through inflammatory signaling, leading us to hypothesize that anti-neuroinflammatory agents may concurrently ameliorate intestinal inflammation. The scorpion venom-derived heat-resistant synthetic peptide (SVHRSP), a bioactive peptide initially identified in scorpion venom and subsequently synthesized by our laboratory, possesses neuroprotective, anti-inflammatory, and antioxidative activities. Its properties make SVHRSP a promising candidate for investigating the therapeutic potential of anti-neuroinflammatory strategies in mitigating intestinal inflammation. Methods: Using a chronic dextran sodium sulfate (DSS)-induced colitis model in wild-type and α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice, along with lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, we assessed SVHRSP’s effects on inflammation, histopathology, gut permeability, oxidative stress markers, and α7nAChR-Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling. Results: SVHRSP treatment significantly ameliorated colitis symptoms in wild-type mice by reducing inflammation, repairing histological damage, restoring gut barrier function, and attenuating oxidative stress, with these effects abolished in α7nAChR knockout mice. Mechanistically, SVHRSP activated JAK2/STAT3 signaling through α7nAChR engagement, suppressing proinflammatory cytokine production in macrophages. Conclusion: These results demonstrated that SVHRSP alleviated intestinal inflammation via α7nAChR-dependent JAK2/STAT3 activation. Combined with its known neuroprotective properties, our findings support the repurposing of this neuroactive peptide, SVHRSP, for treating intestinal inflammatory disorders.

Neuroendocrine neoplasms (NEN) are rare but increasingly recognized malignancies. Fatigue is common among individuals with NEN, but factors associated with fatigue in this population are not well described. Understanding these factors is essential for optimizing supportive care and quality of life. This study aimed to investigate factors associated with fatigue in patients with NEN using a biopsychosocial model. Participants enrolled on a rare tumor natural history study (NCT03739827) were included in this analysis if they had NEN, were ≥ 18years old, and completed at least one evaluation of patient-reported outcome measures. Fatigue was assessed using the Patient-Reported Outcome Measurement Information System (PROMIS). Sixty-four adult participants with NEN (mean age = 59years; SD = 12.0) completed PROMIS measures. Over half (53%) had PROMIS Fatigue T-scores > 55, indicating fatigue, including 31% in the moderate-to-severe range. Fatigue was highly correlated with pain, functional status, and psychosocial factors including depression, anxiety, and emotional support. There were no significant differences in fatigue based on identified sociodemographic or disease-related factors, although a significant association between fatigue and serotonin, a neuroactive peptide secreted by some NENs, was found in a subset of patients. Fatigue is common in patients with NEN, with one-third having moderate to severe fatigue impacting daily functioning. Pain, anxiety, and depression were significantly associated with fatigue, suggesting that interventions targeting these symptoms also may improve fatigue in these patients. Further work is needed to investigate additional NEN-specific factors to optimize management of fatigue in individuals living with this long-term disease.

NeuroCL: A deep learning approach for identifying neuropeptides based on contrastive learning.

Neuropeptides (NPs) are small molecular messengers synthesized in large dense core vesicles (LDCVs) and secreted to the extracellular space. In the central nervous system (CNS), NPs are secreted to the synaptic space, playing crucial roles in modulating neurons, astrocytes, microglia, oligodendrocytes, and other glial cells, through G-protein-coupled receptors, thereby influencing complex multicellular responses. During neuroinflammation, NPs regulate glial and neuronal reactions to inflammatory signals, promoting resolution and preventing chronic, non-resolving inflammation. For example, NPs inhibit apoptosis in neurons and oligodendrocytes while inducing anti-inflammatory effects in microglia and astrocytes, modulating cytokine secretion. Here, we present the notion that neuropeptides could participate in neuroinflammatory progression, altering glial responses, leading to excessive, non-resolutive inflammation when dysregulated. NP signaling—whether excessive or deficient—can disrupt specific cellular processes, leading to pathological inflammation, gliosis, and functional loss—hallmarks of neurodegenerative diseases. Despite their significance, the precise mechanisms underlying NP-mediated effects remain incompletely understood. This review synthesizes experimental and translational evidence highlighting the pivotal role of NPs in resolving neuroinflammation and explores how targeting NPs or their receptors could offer novel therapeutic strategies for neurodegenerative disorders. Further research is needed to elucidate the specific signaling pathways and receptor dynamics involved, which could pave the way for innovative treatments that address the root causes of these debilitating conditions.

Background/Objectives: Receptor-mediated transcytosis utilizing the native transporters at the blood–brain barrier (BBB) is a growing strategy for the delivery of therapeutics to the brain. One of the major challenges in identifying appropriate human transcytosis targets is that there is a species-specific transporter expression profile at the BBB, complicating translation of successful preclinical candidates into humans. In an effort to overcome this obstacle and identify proteins capable of binding human-relevant BBB ligands, we generated and screened a BBB-targeting library against human-induced pluripotent stem cell-derived brain microvascular endothelial-like cells (iPSC-derived BMEC-like cells). As targeting molecules, we used lamprey antibodies, known as variable lymphocyte receptors (VLRs), and generated a VLR library by immunizing lamprey with iPSC-derived BMEC-like cells, and inserting the resultant VLR repertoire into the yeast surface display system. Methods: The yeast displayed VLR library was then panned against human iPSC-derived BMEC-like cells and lead VLRs were validated using human in vitro models and mouse and human ex vivo brain tissue sections. Results: Finally, brain uptake for a set of VLRs was validated in mice. Of the 15 lead VLR candidates, 14 bound to human BBB antigens, and 10 bound to the murine BBB. Pharmacodynamic testing using the neuroactive peptide neurotensin indicated that the lead candidate, VLR2G, could cross the mouse BBB after intravenous injection and deliver sufficient neurotensin payload to generate a pharmacological response and lower systemic body temperature. Conclusions: Together, these results demonstrate the application of a novel screening technique capable of identifying a VLR with human relevance that can cross the BBB and deliver a payload.

Neuropeptides (NPs) and their receptors (NPRs) play critical roles in modulating physiological processes and behaviors across species. While the transcriptional regulation of NP genes has been extensively studied, how NPRs contribute to context‐dependent behavioral plasticity remains poorly understood. Here, we investigate the genomic features and expression patterns of NPRs in Drosophila melanogaster, leveraging comparative genomics, single‐cell RNA sequencing (scRNA‐seq), transcription factor (TF) network analysis, and empirical validation to uncover the regulatory mechanisms that involve NPRs and play roles in context‐dependent adaptation. We demonstrate that NPR genes exhibit more complex cis‐regulatory landscapes, with greater numbers of enhancers compared to NP genes. Also, NPRs are regulated via a broader network of TFs, particularly in response to environmental and physiological cues such as temperature shifts. Through analysis of scRNA‐seq data and qRT‐PCR, we show that the expression level of NPRs is dynamically modulated in a context‐dependent manner, while NP levels remain relatively stable. This “NPR‐biased” gene regulation is evident across diverse combinations of NP‐NPR pairs, with a distinct pattern of TF control in the head and body of D. melanogaster. Furthermore, the expression level of NPR genes increases during aging of the fly, suggesting a key role in aging and developmental processes. Our findings highlight the importance of NPR transcriptional control in shaping neuropeptidergic signaling and adaptive behaviors.

Animals respond to changes in their environment and internal states via neuromodulation. Neuropeptides modulate neural circuits with flexibility because 1 gene can produce either multiple copies of the same neuropeptide or different neuropeptides. However, with this architectural complexity, the function of discrete and active neuropeptides is muddled. Here, we design a genetic tool that facilitates functional analysis of individual peptides. We engineered Escherichia coli bacteria to express active peptides, fed loss-of-function Caenorhabditis elegans, and rescued the activity of genes with varying lengths and functions: pdf-1, flp-3, ins-6, and ins-22. Some peptides were functionally redundant, while others exhibit unique and previously uncharacterized functions. We postulate our rescue-by-feeding approach can elucidate the functional landscape of neuropeptides, identifying the circuits and complex peptidergic pathways that regulate different behavioral and physiological processes.

Neuropeptides (NPs) are a diverse group of signaling molecules involved in regulating key physiological processes such as pain perception, stress response, mood, appetite, and circadian rhythms. Acting as neurotransmitters, neuromodulators, or neurohormones, they play a critical role in modulating and fine-tuning neural signaling networks. Despite their biological significance, identifying NPs through experimental techniques remains time-consuming and resource-intensive. To support this effort, computational prediction tools have emerged as a cost-effective approach for prioritizing candidate sequences for experimental validation. In this study, we propose EnsembleNPPred, an ensemble learning framework that integrates traditional machine learning (ML) models with a deep learning (DL) component. By combining the complementary strengths of these approaches, the model aims to improve generalization and predictive robustness. EnsembleNPPred employs a majority voting mechanism to aggregate the outputs from three classifiers: Support Vector Machine (SVM), Extra Trees (ET), and a CNN-based DL model. When evaluated on independent datasets, EnsembleNPPred demonstrated consistently competitive performance, achieving improvements in both accuracy and sensitivity-specificity balance compared to several existing methods. Furthermore, testing on multiple neuropeptide families from the NeuroPep database yielded an average accuracy of 91.92%, suggesting the model’s potential to generalize across diverse peptide classes. These results suggest that EnsembleNPPred may be a useful tool for early-stage neuropeptide candidate identification and for supporting downstream experimental validation.

Insulin-regulated aminopeptidase (IRAP) is a zinc-dependent metalloenzyme identified as a novel target for combating diabetes-induced diseases due to its crucial role in glucose metabolism and insulin sensitivity regulation. IRAP's catalytic domain catalyzes the N-terminal peptide bond hydrolysis of natural substrate oxytocin, a neuroactive peptide linked to improved cognition and other elemental brain functions. Angiotensin IV and similar peptides are recognized as cognitive enhancers due to their ability to competitively inhibit IRAP's proteolytic activity, thereby mitigating natural neuropeptide degradation. Despite a very similar binding complex between the substrate and the inhibitor with IRAP, particularly around the scissile bond, it is unclear why the enzyme metabolizes oxytocin but does not efficiently degrade angiotensin IV. We employed enhanced sampling quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations and higher-level QM/MM calculations to explore the reaction of these two peptides in IRAP. The calculated energy barrier for oxytocin cleavage was in very good agreement with the experimental data. A significantly higher energy barrier for the formation of the oxyanion tetrahedral intermediate (TI) and a higher overall barrier for the peptide cleavage were observed for the reaction with angiotensin IV. Comprehensive electronic structure analysis utilizing NBO and NCI methods unveiled the molecular basis for different reactivity, a stabilizing interaction between the sigma hole of the N-terminus disulfide bond and the hybridizing lone pair of the scissile peptide nitrogen in oxytocin. The interplay between a weak noncovalent spodium bond and strong bidentate coordination of the catalytic Zn2+ by angiotensin IV caused a larger deviation of the valine C-Cα-Cβ angle from ideal tetrahedral geometry, consequently destabilizing the TI. These results underscore the critical importance of analyzing the dynamics, interactions, and electronic properties of reaction intermediates and transition states in enzymatic processes. Our findings have significant implications for the rational design and development of IRAP inhibitors as potential therapeutic agents for memory disorders, neurodegenerative diseases, and diabetes.

The somal distributions of orexigenic orexin-A, anorexigenic corticotropin-releasing factor (CRF) and melanin-concentrating hormone (MCH) were immunohistochemically investigated in the whitespotted conger eel (Conger myriaster) brain, with particular focus on the preoptic nuclei and hypothalamus. Immunoreactive somata of these three neuroactive peptides were categorised into large (around 30 μm in diameter) and small (around 10 μm in diameter) types. Large-type orexin-A-, CRF- and MCH-immunoreactive (ir) somata were present in the magnocellular preoptic nucleus. Large-type MCH-ir somata were also seen in the lateral tuberal nucleus (TL). Fibres of these large-type somata may penetrate the neurointermediate lobe of the pituitary. In contrast, the small-type immunoreactive somata of these three neuroactive peptides showed differential distributions in the hypothalamus. Small-type orexin-A-ir somata were mainly seen in the paraventricular organ. CRF-ir somata were seen in the posterior parvocellular preoptic nucleus, around the dorsal and ventral areas of the anterior lateral recesses, and around the dorsal area of the posterior lateral recesses. MCH-ir somata were seen in the TL and around the medial region of the posterior lateral recesses. Based on the hypothalamic differential localisations of these peptides, of which association with feeding behaviour is suggested, the presence of intra-hypothalamic functional localisation related to feeding is likely.