Hemokinin-1 induces transcriptomic alterations in pain-related signaling processes in rat primary sensory neurons independent of NK1 tachykinin receptor activation.

The tachykinin hemokinin-1 (HK-1) is involved in immunological processes, inflammation, and pain. Although the neurokinin 1 receptor (NK1R) is described as its main target, several effects are mediated by currently unidentified receptor(s). The role of HK-1 in pain is controversial, depending on the involvement of peripheral and central sensitization mechanisms in different models. We earlier showed the ability of HK-1 to activate the trigeminovascular system, but the mechanisms need to be clarified. Therefore, in this study, we investigated HK-1-induced transcriptomic alterations in cultured rat trigeminal ganglion (TRG) primary sensory neurons. HK-1 was applied for 6 or 24 h in 1 µM causing calcium-influx in these neurons, 500 nM not inducing calcium-entry was used for comparison. Next-generation sequencing was performed on the isolated RNA, and transcriptomic changes were analyzed to identify differentially expressed (DE) genes. Functional analysis was performed for gene annotation using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. NK1R and Neurokinin receptor 2 (NK2R) were not detected. Neurokinin receptor 3 (NK3R) was around the detection limit, which suggests the involvement of other NKR isoforms or other receptors in HK-1-induced sensory neuronal activation. We found protease-activated receptor 1 (PAR1) and epidermal growth factor receptor (EGFR) as DE genes in calcium signaling. The transmembrane protein anthrax toxin receptor 2 (ANTXR2), a potential novel pain-related target, was upregulated. Acid-sensing ion channel 1; 3 (Asic1,3), N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors decreased, myelin production and maintenance related genes (Mbp, Pmp2, Myef2, Mpz) and GNDF changed by HK-1 treatment. Our data showed time and dose-dependent effects of HK-1 in TRG cell culture. Result showed calcium signaling as altered event, however, we did not detect any of NK receptors. Presumably, the activation of TRG neurons is independent of NK receptors. ANTXR2 is a potential new target, PAR-1 has also important role in pain, however their connection to HK-1 is unknown. These findings might highlight new targets or key mediators to solve how HK-1 acts on TRG.

Novel peptide calcitonin gene-related peptide antagonists for migraine therapy.

OBJECTIVES: It has previously been shown that the peptide (34Pro,35Phe)CGRP27-37 is a potent calcitonin gene-related peptide, CGRP receptor antagonist, and in this project we aimed to improve the antagonist potency through the structural modification of truncated C-terminal CGRP peptides. METHODS: Six peptide analogues were synthesized and the anti-CGRP activity confirmed using both in vitro and in vivo studies. KEY FINDINGS: A 10 amino acid-containing peptide VPTDVGPFAF-NH2 (P006) was identified as a key candidate to take forward for in vivo evaluation, where it was shown to be an effective antagonist after intraperitoneal injection into mice. P006 was formulated as a preparation suitable for nasal administration by spray drying with chitosan to form mucoadhesive microcarriers (9.55 ± 0.91 mm diameter) and a loading of 0.2 mg peptide per 20 mg dose. CONCLUSIONS: The project has demonstrated the potential of these novel small peptide CGRP antagonists, to undergo future preclinical evaluation as anti-migraine therapeutics.

Disease- and headache-specific microRNA signatures and their predicted mRNA targets in peripheral blood mononuclear cells in migraineurs: role of inflammatory signalling and oxidative stress.

BACKGROUND: Migraine is a primary headache with genetic susceptibility, but the pathophysiological mechanisms are poorly understood, and it remains an unmet medical need. Earlier we demonstrated significant differences in the transcriptome of migraineurs' PBMCs (peripheral blood mononuclear cells), suggesting the role of neuroinflammation and mitochondrial dysfunctions. Post-transcriptional gene expression is regulated by miRNA (microRNA), a group of short non-coding RNAs that are emerging biomarkers, drug targets, or drugs. MiRNAs are emerging biomarkers and therapeutics; however, little is known about the miRNA transcriptome in migraine, and a systematic comparative analysis has not been performed so far in migraine patients. METHODS: We determined miRNA expression of migraineurs' PBMC during (ictal) and between (interictal) headaches compared to age- and sex-matched healthy volunteers. Small RNA sequencing was performed from the PBMC, and mRNA targets of miRNAs were predicted using a network theoretical approach by miRNAtarget.com™. Predicted miRNA targets were investigated by Gene Ontology enrichment analysis and validated by comparing network metrics to differentially expressed mRNA data. RESULTS: In the interictal PBMC samples 31 miRNAs were differentially expressed (DE) in comparison to healthy controls, including hsa-miR-5189-3p, hsa-miR-96-5p, hsa-miR-3613-5p, hsa-miR-99a-3p, hsa-miR-542-3p. During headache attacks, the top DE miRNAs as compared to the self-control samples in the interictal phase were hsa-miR-3202, hsa-miR-7855-5p, hsa-miR-6770-3p, hsa-miR-1538, and hsa-miR-409-5p. MiRNA-mRNA target prediction and pathway analysis indicated several mRNAs related to immune and inflammatory responses (toll-like receptor and cytokine receptor signalling), neuroinflammation and oxidative stress, also confirmed by mRNA transcriptomics. CONCLUSIONS: We provide here the first evidence for disease- and headache-specific miRNA signatures in the PBMC of migraineurs, which might help to identify novel targets for both prophylaxis and attack therapy.

Discovery of novel targets in a complex regional pain syndrome mouse model by transcriptomics: TNF and JAK-STAT pathways.

Complex Regional Pain Syndrome (CRPS) represents severe chronic pain, hypersensitivity, and inflammation induced by sensory-immune-vascular interactions after a small injury. Since the therapy is unsatisfactory, there is a great need to identify novel drug targets. Unbiased transcriptomic analysis of the dorsal root ganglia (DRG) was performed in a passive transfer-trauma mouse model, and the predicted pathways were confirmed by pharmacological interventions. In the unilateral L3-5 DRGs 125 genes were differentially expressed in response to plantar incision and injecting IgG of CRPS patients. These are related to inflammatory and immune responses, cytokines, chemokines and neuropeptides. Pathway analysis revealed the involvement of Tumor Necrosis Factor (TNF) and Janus kinase (JAK-STAT) signaling. The relevance of these pathways was proven by abolished CRPS IgG-induced hyperalgesia and reduced microglia and astrocyte markers in pain-associated central nervous system regions after treatment with the soluble TNF alpha receptor etanercept or JAK inhibitor tofacitinib. These results provide the first evidence for CRPS-related neuroinflammation and abnormal cytokine signaling at the level of the primary sensory neurons in a translational mouse model and suggest that etanercept and tofacitinib might have drug repositioning potentials for CRPS-related pain.

PACAP-38 Induces Transcriptomic Changes in Rat Trigeminal Ganglion Cells Related to Neuroinflammation and Altered Mitochondrial Function Presumably via PAC1/VPAC2 Receptor-Independent Mechanism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a broadly expressed neuropeptide which has diverse effects in both the peripheral and central nervous systems. While its neuroprotective effects have been shown in a variety of disease models, both animal and human data support the role of PACAP in migraine generation. Both PACAP and its truncated derivative PACAP(6-38) increased calcium influx in rat trigeminal ganglia (TG) primary sensory neurons in most experimental settings. PACAP(6-38), however, has been described as an antagonist for PACAP type I (known as PAC1), and Vasoactive Intestinal Polypeptide Receptor 2 (also known as VPAC2) receptors. Here, we aimed to compare the signaling pathways induced by the two peptides using transcriptomic analysis. Rat trigeminal ganglion cell cultures were incubated with 1 µM PACAP-38 or PACAP(6-38). Six hours later RNA was isolated, next-generation RNA sequencing was performed and transcriptomic changes were analyzed to identify differentially expressed genes. Functional analysis was performed for gene annotation using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. We found 200 common differentially expressed (DE) genes for these two neuropeptides. Both PACAP-38 and PACAP(6-38) treatments caused significant downregulation of NADH: ubiquinone oxidoreductase subunit B6 and upregulation of transient receptor potential cation channel, subfamily M, member 8. The common signaling pathways induced by both peptides indicate that they act on the same target, suggesting that PACAP activates trigeminal primary sensory neurons via a mechanism independent of the identified and cloned PAC1/VPAC2 receptor, either via another target structure or a different splice variant of PAC1/VPAC2 receptors. Identification of the target could help to understand key mechanisms of migraine.

Cerebral and Systemic Stress Parameters in Correlation with Jugulo-Arterial CO2 Gap as a Marker of Cerebral Perfusion during Carotid Endarterectomy.

Intraoperative stress is common to patients undergoing carotid endarterectomy (CEA); thus, impaired oxygen and metabolic balance may appear. In this study, we aimed to identify new markers of intraoperative cerebral ischemia, with predictive value on postoperative complications during CEA, performed in regional anesthesia. A total of 54 patients with significant carotid stenosis were recruited and submitted to CEA. Jugular and arterial blood samples were taken four times during operation, to measure the jugulo-arterial carbon dioxide partial pressure difference (P(j-a)CO2), and cortisol, S100B, L-arginine, and lactate levels. A positive correlation was found between preoperative cortisol levels and all S100B concentrations. In addition, they are positively correlated with P(j-a)CO2 values. Conversely, postoperative cortisol inversely correlates with P(j-a)CO2 and postoperative S100B values. A negative correlation was observed between maximum systolic and pulse pressures and P(j-a)CO2 after carotid clamp and before the release of clamp. Our data suggest that preoperative cortisol, S100B, L-arginine reflect patients' frailty, while these parameters postoperatively are influenced by intraoperative stress and injury. As a novelty, P(j-a)CO2 might be an emerging indicator of cerebral blood flow during CEA.

Identification of disease- and headache-specific mediators and pathways in migraine using blood transcriptomic and metabolomic analysis.

BACKGROUND: Recent data suggest that gene expression profiles of peripheral white blood cells can reflect changes in the brain. We aimed to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) and changes of plasma metabolite levels of migraineurs in a self-controlled manner during and between attacks. METHODS: Twenty-four patients with migraine were recruited and blood samples were collected in a headache-free (interictal) period and during headache (ictal) to investigate disease- and headache-specific alterations. Control samples were collected from 13 age- and sex-matched healthy volunteers. RNA was isolated from PBMCs and single-end 75 bp RNA sequencing was performed using Illumina NextSeq 550 instrument followed by gene-level differential expression analysis. Functional analysis was carried out on information related to the role of genes, such as signaling pathways and biological processes. Plasma metabolomic measurement was performed with the Biocrates MxP Quant 500 Kit. RESULTS: We identified 144 differentially-expressed genes in PBMCs between headache and headache-free samples and 163 between symptom-free patients and controls. Network analysis revealed that enriched pathways included inflammation, cytokine activity and mitochondrial dysfunction in both headache and headache-free samples compared to controls. Plasma lactate, succinate and methionine sulfoxide levels were higher in migraineurs while spermine, spermidine and aconitate were decreased during attacks. CONCLUSIONS: It is concluded that enhanced inflammatory and immune cell activity, and oxidative stress can play a role in migraine susceptibility and headache generation.

Human Somatostatin SST4 Receptor Transgenic Mice: Construction and Brain Expression Pattern Characterization.

Somatostatin receptor subtype 4 (SST4) has been shown to mediate analgesic, antidepressant and anti-inflammatory functions without endocrine actions; therefore, it is proposed to be a novel target for drug development. To overcome the species differences of SST4 receptor expression and function between humans and mice, we generated an SST4 humanized mouse line to serve as a translational animal model for preclinical research. A transposon vector containing the hSSTR4 and reporter gene construct driven by the hSSTR4 regulatory elements were created. The vector was randomly inserted in Sstr4-deficient mice. hSSTR4 expression was detected by bioluminescent in vivo imaging of the luciferase reporter predominantly in the brain. RT-qPCR confirmed the expression of the human gene in the brain and various peripheral tissues consistent with the in vivo imaging. RNAscope in situ hybridization revealed the presence of hSSTR4 transcripts in glutamatergic excitatory neurons in the CA1 and CA2 regions of the hippocampus; in the GABAergic interneurons in the granular layer of the olfactory bulb and in both types of neurons in the primary somatosensory cortex, piriform cortex, prelimbic cortex and amygdala. This novel SST4 humanized mouse line might enable us to investigate the differences of human and mouse SST4 receptor expression and function and assess the effects of SST4 receptor agonist drug candidates.

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization.

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system.

Transcriptional Alterations in the Trigeminal Ganglia, Nucleus and Peripheral Blood Mononuclear Cells in a Rat Orofacial Pain Model.

Orofacial pain and headache disorders are among the most debilitating pain conditions. While the pathophysiological basis of these disorders may be diverse, it is generally accepted that a common mechanism behind the arising pain is the sensitization of extra- and intracranial trigeminal primary afferents. In the present study we investigated gene expression changes in the trigeminal ganglia (TRG), trigeminal nucleus caudalis (TNC) and peripheral blood mononuclear cells (PBMC) evoked by Complete Freund's Adjuvant (CFA)-induced orofacial inflammation in rats, as a model of trigeminal sensitization. Microarray analysis revealed 512 differentially expressed genes between the ipsi- and contralateral TRG samples 7 days after CFA injection. Time-dependent expression changes of G-protein coupled receptor 39 (Gpr39), kisspeptin-1 receptor (Kiss1r), kisspeptin (Kiss1), as well as synaptic plasticity-associated Lkaaear1 (Lkr) and Neurod2 mRNA were described on the basis of qPCR results. The greatest alterations were observed on day 3 ipsilaterally, when orofacial mechanical allodynia reached its maximum. This corresponded well with patterns of neuronal (Fosb), microglia (Iba1), and astrocyte (Gfap) activation markers in both TRG and TNC, and interestingly also in PBMCs. This is the first description of up- and downregulated genes both in primary and secondary sensory neurones of the trigeminovascular system that might play important roles in neuroinflammatory activation mechanisms. We are the first to show transcriptomic alterations in the PBMCs that are similar to the neuronal changes. These results open new perspectives and initiate further investigations in the research of trigeminal pain disorders.