The Effect of P2X7 Antagonism on Subcortical Spread of Optogenetically-Triggered Cortical Spreading Depression and Neuroinflammation

Abstract

Migraine is a neurological disorder characterized by episodes of severe headache. Cortical spreading depression (CSD), the electrophysiological equivalent of migraine aura, results in the opening of pannexin-1 megachannels that release ATP and triggers parenchymal neuroinflammatory signaling cascade in the cortex. Migraine symptoms suggesting subcortical dysfunction bring subcortical spread of CSD under the light. Here, we investigated the role of purinergic P2X7 receptors on the subcortical spread of CSD and its consequent neuroinflammation using a potent and selective P2X7 antagonist, JNJ-47965567. P2X7 antagonism had no effect on the CSD threshold and characteristics but increased the latency to hypothalamic voltage deflection following CSD showing that ATP acts as a mediator in the subcortical spread. P2X7 antagonism also prevented hypothalamic neuronal activation following CSD, revealed by bilateral decrease in hypothalamic c-fos positive neuron count. P2X7 antagonism further stopped the CSD-induced neuroinflammation revealed by decreased nuclear translocation of NF-kappa B-p65 in astrocytes and decreased HMGB1 release. Following CSD we observed an increase in neuronal cytoplasmic P2X7R signal in the cortex and subcortical structures (thalamus, hypothalamus, striatum, hippocampus) concordant with the neuroinflammation which is also prevented by P2X7R antagonism. In conclusion, our data suggest that P2X7R plays an imperative role in CSD-induced neuroinflammation, the subcortical spread of CSD, and CSD-induced hypothalamic neuronal activation hence can be a potential target in migraine treatment.

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

“…Interestingly, a recent finding shows that NF-κB p65 coexpresses with P2X7 receptor in mice cortical and subcortical structures. In this study, the authors report that optogenetically-triggered CSD induces NF-κB p65 nuclear translocation, which can be reversed by a P2X7 receptor antagonist [49]. Consistently, Fyn kinase activity also enhances NF-κB p65 nuclear translocation, which correlates with IL-1β gene expression in a Parkinson's disease model [50].…”

“…These data imply that Fyn-dependent NMDA receptor activity is required for mediating CSD susceptibility. Unlike disrupting the NMDA receptor-Fyn interaction, disrupting the NMDA receptor-Src interaction by TAT-Src (40)(41)(42)(43)(44)(45)(46)(47)(48)(49) does not affect CSD (Fig. 5c, d).…”

“…BzATP reversed the reduced cortical susceptibility to CSD by TAT-Fyn (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)…”

“…Two peptides that specifically block the binding of Fyn or Src from NMDA receptor complex and the Fyn-or Src-dependent NMDA receptor activity [42][43][44][45] were applied: TAT-Fyn (39-57) (YPSFGVTSIPNYNNFHAAGYGRKKRRQRRR) or TAT-Src (40-49) (KPASADGHRGYGRKKRRQRRR) and their scrambled controls, TAT-Fyn (39-57) SC (PSAYGNPG SAYFNFTNVHIYGRKKRRQRRR) or TAT-Src (40-49) SC (GAAKRPSDGHYGRKKRRQRRR). TAT-Fyn (39-57) and TAT-Src (40)(41)(42)(43)(44)(45)(46)(47)(48)(49) correspond to the amino acids 39-57 and 40-49 of the unique domain of Fyn and Src respectively [42,43]. Experiments were designed as follows in this part: (i) Kreb's (vehicle) (n = 7); (ii)1 μM TAT-Fyn (39-57) or 1 μM TAT-Src (40-49) (n = 7 for each); (iii) 1 μM TAT-Fyn (39-57) SC or 1 μM TAT-Src (40-49) SC (n = 7 for each).…”

“…We further studied whether activation of P2X7 receptor could restore the reduced cortical susceptibility to CSD by TAT-Fyn (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57) in mouse brain slices. 300 μM BzATP restored the prolonged CSD latency and the reduced CSD propagation rate by 1 μM TAT-Fyn (39-57) from 18.2 ± 1.6 seconds and 3.7 ± 0.5 mm/minute to…”