Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

BACKGROUND: Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and neuroinflammation after CSD. METHODS: CSD was recorded using electrophysiology in rats and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA levels were detected using qPCR. Glutamate release from mouse brain slices was detected using glutamate assay. RESULTS: The data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1β and TNF-α gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, IL-1β gene expression and glutamate release after CSD induction were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. CONCLUSION: This study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway and promote neuroinflammation, which is of particular relevance to migraine

Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway

The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14-22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization

Src Family Kinases as a Molecular Switch in Migraine Pathogenesis

Background: Migraine has complicated pathological processes, including cortical spreading depression (CSD), trigeminovascular sensitisation, neuroinflammation, and abnormal brain perfusion. However, the underlying molecular mechanism is still elusive. Src family kinases (SFKs), the non-receptor tyrosine kinases, have shown an emerging role in migraine pathogenesis. Direct brain delivery of a SFKs inhibitor PP2 reduces CSD susceptibility, mechanical allodynia, and cerebral arterial contraction in animal models of migraine. However, whether and how systemic modulation of SFKs activity mediates these pathological events are unclear. Aims: This project aims to understand the effects of (1) systemic deactivation of SFKs on CSD and CSD-induced cortical neuroinflammation reflected by inflammatory cytokine gene expression in rats and (2) modulating SFKs activity on trigeminal ganglion (TG) activation and sensitisation reflected by the known target for migraine therapy calcitonin gene-related peptide (CGRP) release and inflammatory cytokine release and gene expression in mouse TG. Furthermore, (3) how SFKs mediate the above phenomena is investigated by examining the hypotheses that SFKs transmit P2X7 receptor pathway to facilitate CSD and CSD-induced neuroinflammation and that SFKs transmit a stress-sensing channel transient receptor potential ankyrin 1 (TRPA1) pathway to facilitate TG activation and sensitisation. Methods: Multi-disciplinary methods were used in this project - high potassium-induced CSD induction and recording in rats via electrophysiology and in mouse brain slices via intrinsic optical imaging; mouse TG culture; quantitative polymerase chain reaction for gene expression analysis; glutamate assay, enzyme-linked immunosorbent assay, multiplex immunoassay, western blot for protein level analysis, and immunohistochemistry. Results: Systemic deactivation of SFKs by their inhibitor PP2 reduced CSD susceptibility and CSD-induced interleukin 1 beta (IL-1β) and tumour necrosis factor alpha gene expression in rat cerebral cortices ipsilateral to CSD. Disrupting the interaction between SFKs and P2X7 receptor by an inhibitory peptide TAT-P2X7 reduced CSD susceptibility and CSD-induced IL-1β gene expression and glutamate release in mouse brain slices. The reduced CSD susceptibility was restored by activation of N-methyl-Daspartate (NMDA) receptor using its agonist NMDA. Besides, deactivation of SFKs by another SFKs inhibitor saracatinib dose-dependently reduced CGRP release and the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release and IL-1β, CCL2 gene expression in mouse TG, which alleviated TG activation and sensitisation. Furthermore, SFKs activity was increased by activation of TRPA1 using its agonist umbellulone via protein kinase A to facilitate TRPA1-dependent CGRP release and IL-1β gene expression in mouse TG. Conclusion: These findings, for the first time, reveal that: 1. SFKs contribute to CSD susceptibility and CSD-induced cortical neuroinflammation by transmitting P2X7 receptor signalling involving glutamatergic pathway activation; 2. SFKs contribute to TG activation and sensitisation by transmitting TRPA1 signalling. It is concluded that SFKs facilitate central sensitisation and trigeminovascular sensitisation in migraine pathogenesis by being a molecular switch for different pathways, which highlights the therapeutic potential of SFKs-targeted migraine therapy

TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization

Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1β gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1β mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14–22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization

Additional file 1: of Autologous cytokine-induced killer cell transfusion increases overall survival in advanced pancreatic cancer

Demographic and clinical characteristics of individual patient in chemotherapy group. (DOC 85 kb