Background Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. Methods Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1β release after incubation with the P2X7-specific nbs. Results Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1β release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood–brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. Conclusion Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.
P2X7, an ion channel gated by extracellular ATP, is widely expressed on the plasma membrane of immune cells and plays important roles in inflammation and apoptosis. Several single nucleotide polymorphisms have been identified in the human P2RX7 gene. In contrast to other members of the P2X family, non-synonymous polymorphisms in P2X7 are common. Three of these occur at overall frequencies of more than 25% and affect residues in the extracellular “head”-domain of P2X7 (155 Y/H), its “lower body” (270 R/H), and its “tail” in the second transmembrane domain (348 T/A). Comparison of the P2X7 orthologues of human and other great apes indicates that the ancestral allele is Y—R—T (at 155–270–348). Interestingly, each single amino acid variant displays lower ATP-sensitivity than the ancestral allele. The originally published reference sequence of human P2X7, often referred to as “wildtype,” differs from the ancestral allele at all three positions, i.e. H—H—A. The 1,000 Genome Project determined the sequences of both alleles of 2,500 human individuals, including roughly 500 persons from each of the five major continental regions. This rich resource shows that the ancestral alleles Y155, R270, and T348 occur in all analyzed human populations, albeit at strikingly different frequencies in various subpopulations (e.g., 25%–59% for Y155, 59%–77% for R270, and 13%–47% for T348). BLAST analyses of ancient human genome sequences uncovered several homozygous carriers of variant P2X7 alleles, possibly reflecting a high degree of inbreeding, e.g., H—R—T for a 50.000 year old Neanderthal, H—R—A for a 24.000 year old Siberian, and Y—R—A for a 7,000 year old mesolithic European. In contrast, most present-day individuals co-express two copies of P2X7 that differ in one or more amino acids at positions 155, 270, and 348. Our results improve the understanding of how P2X7 structure affects its function and suggest the importance of considering P2X7 variants of participants when designing clinical trials targeting P2X7.
BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.
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