Peptidomics profiling identifies endogenous peptides associated with meningeal afferent activation in a chronic migraine mouse model

Abstract

Migraine is a highly disabling disorder with a high prevalence, significantly impairing quality of life. Emerging evidence suggests that circulating endogenous peptides play critical roles in neurovascular regulation and nociceptive signaling. However, the contribution of blood-derived endogenous peptides to the activation of meningeal afferents and the pathophysiology of migraine remains poorly understood. Therefore, investigating the role of these peptides in the meninges is crucial for elucidating the mechanisms underlying migraine. To elucidate the role of endogenous peptides in activating meningeal afferents during migraine, we established a chronic migraine mouse model using nitroglycerin (NTG). Serum peptidomic analysis was performed to identify differentially expressed peptides between the Negative Group (NEG) and NTG group. Functional enrichment analysis revealed significant upregulation of pathways associated with prolactin signaling and hypoxia-inducible factor-1 (HIF-1) signaling. Subsequently, peptidomic profiling of the meninges was conducted in both groups. Integration of meningeal and serum peptidomic datasets, together with a curated set of endogenous secreted proteins, enabled cross-comparative analysis to identify circulating peptides with potential effects on the meninges. This analysis revealed a marked increase in both pituitary adenylate cyclase-activating polypeptide (PACAP) and calcitonin gene-related peptide (CGRP), two key mediators critically implicated in migraine pathophysiology.In addition, several PACAP-related short peptide fragments were identified, some of which exhibited sequence homology to known bioactive domains. Collectively, these findings suggest that PACAP and CGRP, along with their derived peptide fragments, may contribute to the activation of meningeal nociceptive fibers and thereby participate in the initiation and maintenance of migraine. Peptidomics sequencing of the TNC region was performed, and functional enrichment analysis of the differentially expressed proteins indicated the dysregulation of diverse biological pathways. These findings reveal that CGRP and PACAP may play a key role in the activation of meningeal afferents in a chronic migraine mouse model. Several protein modification sites are thought to be crucial mechanisms in endogenous peptides-mediated meningeal activation.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s10194-026-02426-8.

Introduction

Migraine is a highly disabling disorder with a high prevalence, significantly impacting individuals’ quality of life [1–4]. Emerging evidence suggests that circulating endogenous peptides play important roles in neurovascular regulation and nociceptive signaling [5]. In particular, blood-derived secreted peptides may access or interact with meningeal tissues, thereby influencing the activity of meningeal afferents [6]. The meninges are increasingly recognized as a critical interface in migraine pathophysiology, where peripheral signals can initiate and modulate nociceptive transmission [7]. However, the specific contribution of circulating endogenous secreted peptides to meningeal activation and migraine development remains poorly understood [8]. Therefore, investigating the role of blood-derived endogenous peptides in the meninges is essential for elucidating the underlying mechanisms of migraine [9]. A deeper understanding of this process may provide novel insights into potential therapeutic targets for migraine prevention and treatment.

Identifying the neurochemical mediators involved in meningeal nociception is essential for delineating the neurobiological framework underlying headache disorders and for guiding the development of targeted therapeutic strategies [10–13]. One promising mediator is PACAP-38, a multifunctional peptide implicated in the regulation of vascular tone, nociceptive transmission, and immune responses [14, 15]. The potential role of PACAP-38 in activating meningeal afferent nerves may be closely associated with both the initiation and chronification of migraine. In the meninges, the PACAP receptor VPAC1-R has been identified on sensory nerve fibers, vascular smooth muscle cells (VSMCs), and mast cells, suggesting that PACAP signaling may integrate neuronal, vascular, and immune components within the meningeal environment to promote migraine pathophysiology [3, 16, 17]. Peptidomics has been employed as a specialized approach to investigate the structure, function, and variability of endogenous peptides and low–molecular weight proteins [18, 19]. Peptides, composed of amino acids naturally present within biological organisms, represent a diverse class of bioactive molecules with a wide range of physiological activities. Understanding migraine initiation and chronification from the perspective of peptidomics provides a novel framework through which the complex molecular mechanisms underlying this disorder can be elucidated.

In this study, targeted neuropeptidomic analysis was performed on meningeal tissues obtained from NTG-treated and VEH mice. Multiple peptides related to both PACAP and CGRP were identified, along with several post-translational modification (PTM) sites that may influence their structural and functional properties. Further Peptidomics profiling of serum samples from VEH and NTG groups revealed significant alterations in enriched biological pathways, which may be associated with the elevated levels of PACAP and CGRP observed in the meninges. Finally, Peptidomics analysis of the trigeminal nucleus caudalis (TNC) was conducted to investigate the molecular mechanisms by which activation of meningeal nociceptors impacts central pain-processing regions. Collectively, these findings suggest that PACAP and CGRP, together with their modified peptide forms, play critical roles in driving trigeminovascular activation and central sensitization, thereby contributing to the initiation and persistence of migraine.

Methods

Animals

All experimental procedures involving animals were conducted in strict compliance with institutional guidelines and were approved by the Animal Ethics Committee of The First Affiliated Hospital of Henan Medical University (Approval No: EC-025-615). Animals were housed individually in standard rodent cages under a controlled 12:12 h light–dark cycle, with an ambient temperature maintained at approximately 25 °C. Food (standard rodent chow provided ad libitum) and water were available without restriction.

Chronic migraine mouse model

NTG, was administered for establishing chronic migraine mouse models, as previously described [20]. NTG (Yimin Pharmaceutical Co., Ltd., Beijing, China) was diluted to 0.9% saline. For the chronic migraine model, mice received 5 intraperitoneal (i.p.) injections of NTG (10 mg/kg) every other day over a 9-day period (Fig. 1A). Behavioral experiments were conducted 30 min prior to each NTG injection and 2 h after each injection.

Fig. 1.

NTG group exhibited decreased pain thresholds and increased neuronal excitability in the TNC. A. The schematic diagram of an experiment. B. Schematic diagram of periorbital pain threshold vehavioral experiment. C. Basal mechanical threshold of periorbital mechanical threshold (g).(n = 6). D. Post - Treatment mechanical threshold of periorbital mechanical threshold (g).(n = 6). E. Schematic diagram of paw pain threshold vehavioral experiment. F. Basal mechanical threshold of paw mechanical threshold (g).(n = 6). G. Post - Treatment mechanical threshold of paw mechanical threshold (g).(n = 6). H. Immunofluorescence staining of the TNC brain region Scale bar: 200 μm. I. Quantification of CGRP fluorescence intensity. Data were averaged from 3 fields of view per mouse (n = 3 mice per group). Repeated measures two-way ANOVA Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 6/group. Data is presented as mean ± SEM

Mechanical withdrawal threshold tests

Adult mice were first acclimated to the behavioral testing environment for 2 consecutive days, with each habituation session lasting at least 30 min [21]. Following acclimation, mechanical withdrawal thresholds were evaluated using Von Frey filaments ranging from 0.008 to 2 g, and the up-down paradigm was applied to determine sensitivity. For the assessment of periorbital mechanical thresholds, mice were gently restrained in plastic cups, and filaments were applied perpendicularly to the forehead. Facial stroking, head withdrawal, or head shaking was considered a positive nocifensive response. For the evaluation of hind paw plantar mechanical thresholds, mice were individually placed on wire-mesh platforms within transparent acrylic chambers and allowed to habituate for 10 min prior to testing. Filaments were then applied to the plantar surface of each hind paw, and paw withdrawal, shaking, or licking was recorded as a positive response. The 50% withdrawal threshold was calculated using the online up-down analysis tool (https://bioapps.shinyapps.io/von_frey_app/) in accordance with established criteria.

Immunofluorescent

Mice were anesthetized with 2% sodium pentobarbital and transcardially perfused with 20 mL of phosphate-buffered saline (PBS) followed by 20 mL of 4% paraformaldehyde (PFA). The brains were post-fixed in 4% PFA overnight at 4 °C and subsequently cryoprotected in 30% sucrose in PBS for 48 h. Coronal Sect.  (25 μm thick) were obtained using a Leica VT1000S vibratome. After thorough washing with PBS, free-floating sections were incubated overnight at 4 °C with primary antibodies (anti-CGRP, 1:200, Santa Cruz Biotechnology, sc-57053) diluted in 1% bovine serum albumin (BSA) in PBS. Then, triple 5 min washes with PBS, sections were incubated for 2 h at room temperature with Alexa Fluor-conjugated secondary antibodies (goat anti-mouse Alexa Fluor 552, 1:5000, Yeasen 33213ES60). Nuclei were counterstained with DAPI (1:1000, Yeasen), and sections were washed again to remove excess dye. All antibody incubations were followed by triple 5 min washes with PBS to ensure minimal background staining.

Immunofluorescent imaging data analysis

Confocal imaging was carried out using a Zeiss LSM980 microscope (Carl Zeiss AG, Germany) under randomized acquisition and analysis protocols. Fluorescence images were captured using consistent exposure and gain settings to maintain signal uniformity. Three brain sections containing the TNC were analysed for each animal (n = 3 per group) at magnifications of 10× and 20×. Quantitative analyses were conducted in ImageJ by an investigator blinded to experimental grouping.

Sample preparation

For serum analysis, a total of 200 µL of serum was collected from each mouse and subjected to peptidomic sequencing [22]. For meningeal tissue, the skull of each mouse was first carefully removed, and the dura mater was gently separated from the inner surface of the skull. The meninges obtained from five VEH group mice were pooled as one biological sample, and those from five NTG group mice were combined as another. The two samples (VEH vs. NTG) were subsequently subjected to peptidomic sequencing analysis to identify and compare endogenous peptide profiles between the two experimental conditions.

For TNC tissue, approximately 50 mg of tissue sample was weighed and placed in a grinding tube. A total of 200 µL of 8 M urea and 2 µL of PMSF were added, and the samples were homogenized thoroughly using a cryogenic grinding instrument. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was transferred to a pre-cooled 1.5 mL microcentrifuge tube for further processing. For ultrafiltration, the supernatant was transferred to a 10 kDa ultrafiltration tube and centrifuged at 12,000 rpm for 10 min at 4 °C. The filtrate containing peptides smaller than 10 kDa was collected and stored in a 1.5 mL centrifuge tube. Protein concentration was measured using a NanoDrop spectrophotometer at 205 nm, and all readings were blank-corrected.

For meningeal tissue, samples from 5 mice in the VEH group and 5 mice in the NTG group were pooled respectively to generate one sample per group for peptidomics sequencing. The remaining steps were performed exactly as described for the TNC tissue samples.

To detect protein modifications, all samples were adjusted to a final volume of 300 µL. Then, 3 µL of 1 M DTT was added to achieve a final concentration of 10 mmol/L, and the samples were incubated at 56 °C for 1 h. Next, 6 µL of 1 M iodoacetamide (IAM) was added to reach a final concentration of 20 mmol/L, followed by incubation at room temperature in the dark for 40 min. Finally, an additional 3 µL of 1 M DTT was added to neutralize the remaining IAM. For desalting, C18 desalting columns were activated twice with 300 µL of 100% acetonitrile (ACN), equilibrated twice with 200 µL of 0.1% formic acid (FA), and then loaded with the peptide samples. The columns were washed twice with 200 µL of 0.1% FA and eluted once with 300 µL of 80% ACN containing 0.1% FA. The eluates were vacuum-dried at 45 °C and stored for subsequent analysis.

Peptide sequencing

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was performed for peptide detection and sequencing. The chromatographic separation was carried out on a PEPMAP NEO C18 pre-column (300 μm × 5 mm) and an analytical column (150 μm i.d. × 170 mm, Reprosil-Pur 120 C18-AQ, 1.9 μm particle size). The mobile phase consisted of solvent A (0.1% FA in water) and solvent B (0.1% FA in 80% ACN), with a flow rate maintained at 600 nL/min. Each fraction was analyzed for a total run time of 66 min.

Mass spectrometric analysis was conducted in data-dependent acquisition (DDA) mode. The MS1 spectra were acquired at a resolution of 120,000 with an AGC target set to “standard” a maximum injection time of 200 ms, and a scan range of 200–4000 m/z. MS2 spectra were acquired at a resolution of 15,000 with an AGC target set to “standard,” a maximum injection time of 200 ms, a cycle time of 2 s, and a collision energy of 30. The raw MS data obtained were used for subsequent peptide identification and quantification.

Data processing and statistical analysis

Peptidomics data processing

Peptidomic data were normalized to the total ion current (TIC) and processed using the Q Module before further analysis. Distinct peptides with similar m/z values and retention times were occasionally assigned to a single peak in the absence of MS/MS spectra. Data preprocessing was performed in R (version 4.2.1). Differential peptide abundance was evaluated using both Student’s t-test and Wilcoxon rank-sum test, followed by Benjamini–Hochberg correction for multiple comparisons. Peptides with adjusted p < 0.05 in both tests were considered significantly altered. Fold change (FC) values were calculated to estimate the magnitude of abundance differences, and principal component analysis (PCA) was conducted using the pcaMethods package to visualize sample clustering.

Protein abundance calculation

The first step of MS data processing involved converting the data files to mzML format using ProteoWizard. Initially, peptides identified in the sequencing results with an estimation value of p < 0.05 were included for peptidomic data analysis. The peak area of each detected peptide in the mass spectrometry profile was used to represent its relative abundance for subsequent statistical analysis. Each peptide sequence was matched to the corresponding parent protein by Homo_sapiens_prosit_generated_library, dlib with a peptide mass tolerance of 20 ppm and a fragment mass tolerance of 10.0 ppm. The molecular weight of each peptide was then calculated, and the ratio of peptide molecular weight to that of the full-length protein was determined. Finally, the expression level of each protein was obtained by calculating the weighted sum of all its corresponding peptide abundances, with weighting based on peptide molecular mass, to provide a quantitative estimation of protein expression.

Peptidomics informatic analysis

Statistical analyses were performed using Perseus, R programming language, and GraphPad Prism software. Comparisons between two independent groups were conducted using an unpaired two-tailed Student’s t-test, whereas multiple-group comparisons were analyzed by one-way analysis of variance (ANOVA). Statistical significance was defined as a false discovery rate (FDR) less than 0.05. Pathway enrichment and functional annotation analyses were carried out using Cytoscape (version 3.7.1), Reactome, and DAVID software, with a significance threshold set at p < 0.05. The composition of Protein segment–specific Peptidomics profiles was visualized using Venny software.

Statistical analysis

All experimental data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). For two-group comparisons, data were analyzed using unpaired t tests, paired t tests, Mann-Whitney tests or Wilcoxon tests. One-way or two-way ANOVA or two-way repeated measures followed by one-way ANOVA was applied for comparison of multiple groups with Dunnett’s post hoc test, Dunn’s post hoc test or Bonferroni correction. Complete methodological details for specific analyses are provided in corresponding figure legends. Statistical significance threshold was set at p < 0.05.

Results

NTG group exhibited decreased pain thresholds and increased level of CGRP in the TNC

Behavioral experiments were conducted 30 min prior to each NTG injection and 2 h after each injection in both the VEH and NTG groups. The results showed that, compared with VEH mice, NTG mice displayed a significant reduction in periorbital mechanical thresholds, indicating the development of periorbital allodynia (Fig. 1B–D). Similarly, in the hind paw mechanical sensitivity test, NTG mice exhibited a significant decrease in withdrawal thresholds relative to controls (Fig. 1E–G), further confirming the establishment of a chronic migraine-like pain state.

Following completion of the chronic migraine induction protocol on day 9, brain tissues were collected for immunofluorescence analysis of the TNC region. The results demonstrated that NTG mice exhibited markedly elevated levels of CGRP compared with VEH mice (Fig. 1H–I). These findings indicated that repeated administration of NTG induced both mechanical hypersensitivity and an increased level of CGRP in the TNC, thereby validating the successful establishment of the chronic migraine-like mouse model.

Exploring the hemodynamic mechanisms underlying increased PACAP in the meninges

Peptidomics analysis of serum samples from the VEH and NTG groups was performed. PCA of the Peptidomics data revealed a clear clustering distinction between the two groups, indicating substantial differences in serum protein profiles (Fig. 2A). Quantitative analysis of the proteins identified by mass spectrometry demonstrated a significant reduction in the total serum protein abundance in NTG mice compared with VEH mice (Fig. 2B). Representative differentially expressed proteins were visualized in a heatmap to illustrate the expression patterns across groups (Fig. 2C), and a volcano plot was used to display the distribution of upregulated and downregulated proteins between the two conditions (Fig. 2D). Functional enrichment analysis of the differential expressed proteins was subsequently conducted using KEGG pathway mapping (Fig. 4E) and Gene Ontology (GO) annotation (Fig. 4F). The results showed a significant increase in pathways related to prolactin signaling pathway, HIF-1 signaling pathway and ferroptosis signaling pathway. This suggests that these biological processes may contribute to the systemic mechanisms that lead to NTG-induced chronic migraine-like phenotypes in mice.

Fig. 2.

Multiple signaling pathways are altered in serum. A. PCA metabolic pattern analysis. B. The total serum protein abundance in NTG mice and VEH mice. C. Heatmap illustrates the expression of different representative proteins. D. Volcano plot was used to display the distribution of upregulated and downregulated proteins. E. KEGG pathway mapping. Repeated measures two-way ANOVA Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 6/group. Data is presented as mean ± SEM

Fig. 4.

Peptidomics revealed a significant increase in PACAP-related peptides in the meninges. A-C. Amino acid sequences of PACAP31–38, PACAP38, and PACAP27 peptides identified in the peptidomic analysis. D. PTM analysis of PACAP-38. E. Structural modeling using the AlphaFold2 online tool

Overlap between serum secretome and meningeal proteome indicates potential peripheral regulation of the meninges

After the establishment of a chronic migraine-like mouse model induced by NTG, the meninges were carefully dissected following skull removal. The meninges obtained from five VEH group mice were pooled as one biological sample, and those from five NTG group mice were combined as another. The two pooled samples (VEH vs. NTG) were subsequently subjected to peptidomic sequencing analysis to identify and compare endogenous peptide profiles between the two experimental conditions. Peptides were extracted, separated, and analyzed by LC–MS/MS. We first screened endogenous secreted proteins from the serum proteome and performed a cross-comparative analysis with proteins identified in the meninges.(Fig. 3A) This approach enabled us to identify a subset of serum-derived endogenous secreted proteins that were also present in meningeal tissues.(Fig. 3B). Among these, a proportion corresponded to neuropeptide-related proteins, yielding a total of 37 endogenous secreted neuropeptides. These peptides may have the potential to modulate the function of meningeal nerve endings.(Fig. 3D) Therefore, subsequent analyses will focus on elucidating the functional roles of these 37 neuropeptides within the meninges. Among these, the expression levels of 10 neuropeptides were significantly decreased, whereas 6 neuropeptides were significantly increased in the meninges of NTG group mice compared with NTG group (Fig. 3E). Further evaluation of neuropeptide diversity revealed a notable increase in the number of PACAP-related peptides in the NTG group, accompanied by moderate increases in peptides related to Secretin, Motilin, VIP, GLP-1, and IGF-1 (Fig. 3F).

Fig. 3.

Peptidomics revealed a significant increase in PACAP in the meninges. A. Schematic illustration showing the collection of meningeal tissue for peptidomic sequencing. B. The aptamers were classified into 2 categories based on the annotated subcellular localization of the targeted proteins: “intracellular” and “secreted” proteins. The numbers indicate proteins counts and corresponding percentages of all the aptamers and only the altered proteins. The percentages in the 2 subcellular categories are relative to the total number of assigned aptamers in each class. D. The 37 neuropeptides included in the targeted peptidomic analysis. E. Volcano plot depicting differentially expressed neuropeptides between the NTG and VEH groups. F. Changes in the number of neuropeptide-related peptides identified between NTG and VEH mice

Detailed analysis of PACAP-related peptides

Identified several novel PACAP-derived fragments in NTG group mice, with the confidence of peptide identification determined by –log₁₀P values obtained from mass spectrometric analysis (Fig. 4A–C). Additionally, PTM analysis of PACAP-38 revealed 6 modification sites, among which two sites exhibited significantly elevated modification levels in NTG mice (Fig. 4D). Structural modeling using the AlphaFold2 online tool indicated that these modification sites may induce conformational changes in the PACAP-38 peptide structure (Fig. 4E).

PACAP levels were significantly elevated in the meninges of NTG-treated mice

Peptidomic sequencing of meningeal tissue revealed an elevation of PACAP levels, which was subsequently validated by immunofluorescence analysis. A chronic migraine-like mouse model was successfully established through repeated intraperitoneal administration of NTG. Three hours after the final NTG injection, mice were euthanized, and transcardial perfusion was performed with PBS followed by 4% PFA. The meninges was then carefully isolated for immunofluorescent staining. The results showed that PACAP expression in the meninges was significantly increased in NTG mice compared with VEH mice (Fig. 5A-B). To further assess the localization of PACAP within meninges, endothelial cells were labeled with CD31. Elevated PACAP level was observed near the superior sagittal sinus (SSS) (Fig. 5C-D), and a marked increase in PACAP expression was detected in the vicinity of the middle cerebral artery (MCA) in NTG mice (Fig. 5E-F). These findings confirmed that PACAP upregulation occurred predominantly in the vascular structures of the meninges under chronic migraine-like conditions.

Fig. 5.

PACAP levels are significantly elevated in the meninges. A-B. Immunofluorescence staining revealed PACAP expression in the meninges was significantly increased in NTG mice compared with VEH mice. C-D. Elevated PACAP level was observed near the superior sagittal sinus. E-F. Elevated PACAP level was observed near the middle cerebral artery. Repeated measures two-way ANOVA Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 6/group. Data is presented as mean ± SEM

CGRP levels were significantly elevated in the meninges of NTG-treated mice

Peptidomic sequencing of meningeal tissue revealed an elevation of CGRP levels, which was subsequently validated by immunofluorescence analysis. The results showed that increase in CGRP was detected in the vicinity of the superior sagittal sinus (SSS) (Fig. 6A-B), and a marked increase in CGRP expression was detected in the vicinity of the middle cerebral artery (MCA) in NTG mice (Fig. 6C-D). These findings confirmed that CGRP upregulation occurred predominantly in the vascular structures of the meninges under chronic migraine-like conditions.

Fig. 6.

CGRP levels are significantly elevated in the meninges. A-B. Immunofluorescence staining revealed CGRP expression in the meninges near the superior sagittal sinus was significantly increased in NTG mice compared with VEH mice. C-D. Immunofluorescence staining revealed CGRP expression in the meninges near the middle cerebral artery was significantly increased in NTG mice compared with VEH mice Repeated measures two-way ANOVA Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 6/group. Data is presented as mean ± SEM

NTG group TNC peptidomics results indicate activation of multiple pathways

The TNC peptidomic analysis of NTG group mice revealed the activation of multiple biological pathways relevant to migraine pathophysiology. Peptidomics analysis of TNC tissue samples from the VEH and NTG groups was performed. PCA of the Peptidomics data revealed a clear clustering distinction between the two groups, indicating substantial differences in TNC protein profiles (Fig. 7A). To obtain a deeper understanding of the differential Peptidomics alterations in the TNC of the chronic migraine mouse model, we identified a total of 109 proteins uniquely expressed in NTG mice. A volcano plot was used to display the distribution of upregulated and downregulated proteins between the two conditions (Fig. 7B). The 15 deficient proteins detected in NTG mice compared with VEH mice were of particular interest. Functional annotation revealed that these proteins were predominantly enriched in pathways (Fig. 7C). In addition, GO pathway analysis of 15 deficient proteins between groups highlighted their involvement in “protein − containing complex”, “intracellular organelle” and “intracellular membrane − bounded organelle” (Fig. 7D).

Fig. 7.

Multiple signaling pathways are altered in TNC. A. PCA metabolic pattern analysis. B. Volcano plot was used to display the distribution of upregulated and downregulated proteins. C. KEGG pathway mapping and D. Gene Ontology (GO) annotation

Discussion

In this study, we employed advanced peptidomic sequencing to systematically profile neuropeptides with high amino acid sequence resolution. Using a NTG-induced chronic migraine-like mouse model, we analyzed peptide alterations across the meninges and serum. Our investigation primarily focused on neuropeptide changes in the meninges, combined with cross-comparative analysis against the identified protein landscape. We identified multiple forms of CGRP and PACAP that are enriched in the meninges of chronic migraine-like mice, along with several key PTM sites of PACAP. Furthermore, serum Peptidomics analysis revealed that the upregulation of prolactin signaling, HIF-1α signaling, and ferroptosis-related pathways in peripheral circulation may contribute to enhanced CGRP and PACAP release, thereby promoting meningeal activation and migraine-like symptoms. A schematic illustration of the proposed mechanism is presented (Fig. 8).These findings provide important insights into the molecular basis of meningeal activation and highlight potential therapeutic targets.

Fig. 8.

Alterations in multiple molecular pathways in the blood may be associated with elevated PACAP levels in the meninges

Previous studies have suggested that PACAP analogs may undergo conformational changes that could potentially affect the ability of PACAP-related peptides to activate their receptors [23, 24]. Such structural alterations are of considerable interest as they may affect the binding affinity of the peptide and subsequent receptor activation, thereby modulating its physiological effects [25, 26]. Current research has confirmed the existence of several biologically active PACAP-related peptides. Although PACAP-38 is the primary form responsible for activating the VPAC1-R, other peptides, such as PACAP27, PACAP6-38, and PACAP31-38, have also been identified as active forms with distinct receptor-binding properties [27, 28]. These peptides have been studied for their roles in various biological processes [22].

Additionally, the PTMs of PACAP-38 are closely linked to its receptor activation ability [29]. These modifications, including phosphorylation and amidation, can significantly alter the peptide’s functional properties, such as its binding affinity for receptors and its ability to activate downstream signalling pathways. The impact of these modifications on VPAC1-R activation underscores the intricate role of Pacap-related peptides in regulating cellular responses [30]. Understanding the specific modifications and how they affect receptor activation could provide valuable insights into the development of targeted therapies for diseases associated with PACAP-related peptide signaling, including migraine and other neurovascular disorders [31].

The primary receptors for PACAP are VPAC1 and VPAC2, which are G protein-coupled receptors (GPCRs) [32, 33]. Upon binding to VPAC1, PACAP activates several intracellular signaling pathways, primarily through the activation of adenylate cyclase, leading to increased cyclic AMP (cAMP) levels. This, in turn, activates protein kinase A (PKA) and various downstream effectors involved in cell signaling [34]. Additionally, PACAP receptor activation can also stimulate phospholipase C (PLC), leading to the production of inositol trisphosphate (IP3) and diacylglycerol (DAG), which further mediate intracellular calcium release and activation of protein kinase C (PKC) [35]. These signaling cascades contribute to the diverse physiological effects of PACAP, including modulation of vascular tone, neuronal excitability, and immune responses [20]. The role of PACAP and its receptors in these processes underscores its potential as a therapeutic target for migraine and other neurovascular disorders [36, 37]. Understanding the specific mechanisms by which PACAP influences cellular signaling in the context of migraine pathophysiology could provide new insights for the development of targeted treatments.

PACAP within the meninges has been shown to modulate multiple physiological processes that may contribute to both the initiation and chronification of migraine [38, 39]. It is likely that PACAP promotes mast cell degranulation in the meningeal layer, leading to the release of vasoactive and pro-inflammatory mediators that can sensitize nearby nociceptive fibers and trigger migraine attacks [40, 41]. In addition, PACAP may induce excitation of meningeal C-fibers, resulting in enhanced neuronal activity and pain transmission along the trigeminovascular pathway [42]. Activation of meningeal vascular endothelial cells by PACAP may further stimulate the release of inflammatory cytokines, thereby amplifying neurogenic inflammation [21]. Moreover, PACAP-induced depolarization of vascular smooth muscle cells can lead to vasodilation, which in turn contributes to meningeal hyperemia and nociceptor activation. Collectively, these findings suggest that PACAP-mediated interactions among immune, vascular, and neuronal components within the meninges represent a critical mechanism underlying migraine pathophysiology [43].

Several limitations should be acknowledged in the present study. First, a direct molecular link between the alterations in serum protein expression and the increased PACAP levels observed in the meninges was not demonstrated, and only a correlative association could be established. In future experiments, additional investigations will be conducted to elucidate the hematological mechanisms that may contribute to the elevation of meningeal PACAP in the chronic migraine-like state.

In conclusion, a significant elevation of PACAP levels was observed in the meninges of migraine-like mice, accompanied by an increase in the diversity of PACAP-related peptides. 6 PTM sites of PACAP-38 were identified, among which two sites exhibited markedly higher modification levels in NTG-treated mice. These alterations may be associated with changes in multiple molecular pathways within the circulation, including prolactin signaling pathway, HIF-1 signaling pathway and ferroptosis signaling pathway. These findings reveal that CGRP and PACAP may play a key role in the activation of meningeal afferents in a chronic migraine mouse model. Several protein modification sites are thought to be crucial mechanisms in endogenous peptides-mediated meningeal activation.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

GPCRs

G protein-coupled receptors

DAG

Diacylglycerol

IP3

Inositol trisphosphate

PACAP

Pituitary adenylate cyclase-activating polypeptide

TNC

Trigeminal nucleus

PKC

Protein kinase C

PVS

Perivascular spaces

PPI

Protein–protein interaction

CGRP

Calcitonin gene–related peptide

PFA

Paraformaldehyde

NTG

Nitroglycerin

CSD

Cortical spreading depression

ANOVA

Analysis of variance

IF

Immunofluorescence

PBS

Phosphate-buffered saline

Author contributions

G.Z., C.Z., T.C.: Conceptualization, Investigation, Formal analysis, Data curation, Validation, Visualization, Writing - original draft, Writing - review & editing. G.Z., C.D., Y.G.: Investigation, Writing - original draft. H.W., C.D.: Data curation. J.Z., S.Z., Z.G.: Writing - original draft. G.Z., C.S., J.C.: Writing - review & editing. J.C., T.C., S.Z., C.Z.: Supervision, Writing - review & editing.

Funding

This study was funded by the Henan Provincial Department of Science and Technology Research Project (No. 242102310078, No. 242102310202).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All experimental procedures involving animals were conducted in strict compliance with institutional guidelines and were approved by the Animal Ethics Committee of The First Affiliated Hospital of Henan Medical University (Approval No: EC-025-615).

Consent for publication

This manuscript does not include details, images, or videos relating to an individual person; therefore, consent for publication is not needed beyond the informed consent provided by all study participants as described above.

Competing interests

The authors declare no competing interests.