Purinergic signaling-induced neuroinflammation and status epilepticus

KEYWORDS:

• Status epilepticus
• inflammation
• ATP signaling
• P2 receptors
• P2X7 receptor

Current state of the art

Status epilepticus (SE, prolonged seizures) is a common and serious clinical neurological emergency defined as ‘a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms, which lead to abnormally, prolonged seizures’. Operationally, SE is defined as continuous seizure activity over 5 minutes or multiple seizures with incomplete return to baseline electroencephalogram activity between seizures. The annual incidence of SE is up to 41/100 000 with an overall mortality of about 20%.[1] Common causes of SE include non-compliance with anti-epileptic drugs (AED), stroke, metabolic abnormalities, or traumatic brain injuries.[1] First-line treatment usually includes the gamma-aminobutyric acid (GABA)-potentiating benzodiazepines such as lorazepam or midazolam. If SE persists after first-line treatment, patients receive AEDs such as phenytoin, valproic acid, or levetiracetam. Failure of second-line treatment indicates patients have entered drug-refractory SE.[2] Data from experimental models and patients have shown that SE, in particular drug-refractory SE, can lead to widespread brain damage, gliosis, cognitive deficits, and an increased risk of developing epilepsy. Pharmacoresistance in SE patients remains as high as 43%.[2] This and the fact that prolonged seizure activity may lead to a decrease in available functional GABA receptors[2] emphasizes the need to develop new drugs with new mechanisms of action independent of GABA signaling.

Research to identify new drug targets for the treatment of SE has shifted toward neuroinflammation and gliosis.[3] Astrocytosis may interfere with the clearance of extracellular neurotransmitters or with the extracellular levels of the anti-convulsive adenosine.[4,5] Indeed, a recent study showed that astrocytosis on its own is sufficient to trigger seizures in mice.[6] Activated microglia might also contribute to seizure generation and the resulting brain damage. In this context, particular interest has been on the pro-inflammatory cytokine Interleukin-1β (IL-1β). IL-1β has been shown to increase glutamatergic signaling and reduced GABA_A currents[3,7] and targeting of IL-1β signaling has been reported to reduce acute seizures in animal models.[8] The involvement of glial cells as well as neurons in network hyperexcitability and the mediation of inflammatory processes modulating/increasing the release of neurotransmitters and pro-inflammatory cytokines implies that a fruitful approach to identify novel drug targets to treat SE could be to target genes which are expressed and function on both neurons and glia to influence communication and damage responses between these cells.

Adenosine triphosphate (ATP), the main universal cellular energy currency, is also a key upstream trigger of neuroinflammation and a potential link between inflammation and hyperexcitability.[9,10] Usually present at very low levels in the extracellular medium, ATP levels rapidly increase during pathological conditions occurring during SE due to increased neuronal activity, inflammation, or cell death to act as either a sole transmitter or co-transmitter.[11] ATP can be released from neurons and glial cells in an uncontrolled fashion (e.g. during necrosis or from damaged cells) or via vesicular release. After its release, ATP is rapidly degraded by ectonucleotidases into breakdown products including adenosine.[9]

Once released into the extracellular compartment, ATP activates metabotropic P2Y and ionotropic P2X receptors.[11] Both P2X and P2Y receptor subtypes are distributed throughout the central nervous system and are expressed and functional on glial cells as well as on neurons; however, controversy about the expression and function on different cell types for some receptors still remains.[11,12] P2X receptors have been associated with mainly facilitatory roles in fast synaptic transmission becoming most prominent during increased neuronal activity states by increasing Ca²⁺ influx and potentially influencing the release of neurotransmitters. On the other side, P2Y receptors have been suggested to play a mainly inhibitory role on synaptic transmission.[11] Most SE studies to date have focused on the P2X receptor subtype; however, there is now also convincing evidence showing disease-contributing functions of the metabotropic P2Y receptor family in SE.[13] Whereas data on expressional changes have been reported for almost all P2 receptors after experimental SE, functional studies have been restricted to a rather limited number of receptor subtypes. This might be partly due to the lack of specific receptor agonists/antagonists or due to the lack of genetic models with a modulated receptor activity.[13]

The P2X7 receptor has attracted most attention in SE. The P2X7 receptor has the lowest affinity to ATP (high µM to low mM) and is likely activated only under pathological conditions.[12] P2X7R activation is an essential step in microglia activation and proliferation[14], and the P2X7R has been described as the key regulatory element of the inflammasome complex inducing the release of the pro-inflammatory cytokine IL-1β.[15] The role of the P2X7R in SE has been explored using genetic and pharmacologic tools.[13] Studies by our group using the intra-amygdala kainic acid model of SE have shown potent anticonvulsive and neuroprotective properties of P2X7R antagonists.[13] In particular, the antagonists brilliant blue G (BBG) and A438079 reduced seizure severity and damage in rats and mice.[16,17] However, contradictory results have been obtained using pilocarpine to trigger SE, where P2X7R inhibition worsened pathology during SE.[18] The reasons for this discrepancy are not fully understood and might include the use of different methods to trigger SE, the amount of seizure-induced neurodegeneration, the availability of extracellular ATP, or drug doses/administrations of the antagonists. Other P2 receptors studied during SE using intraperitoneal or intra-ventricular injected kainic acid to trigger SE include the P2X4 receptors and the metabotropic P2Y₆ and P2Y₁₂ receptors. P2X4 receptor inhibition led to a decrease in inflammation, microglia density, and seizure-induced cell death after SE without affecting seizure severity or levels of the cytokine IL-1β. P2Y₆ and P2Y₁₂ activation increased microglial currents, and the deletion of the P2yr12 gene led to an increased seizure phenotype.[13]

Outlook

Accumulating evidence indicates that ATP signaling is involved in the generation of seizures and that the modulation of ATP-gated receptors impacts on different pathological changes occurring in the brain during SE such as gliosis, inflammation, and neurodegeneration. What are the next questions? Our understanding of the role of purinergic signaling during SE in vivo comes from studies in only few animal models. Furthermore, the conflicting results obtained between different models needs to be resolved, perhaps by tests using kindling or another standard model. New pharmacologic tools with superior blood–brain barrier permeability and stability offer ways to achieve prolonged receptor blockade in vivo and these may represent the next generation of P2 receptor drugs for clinical use. One of the consequences of SE is the increased risk of developing epilepsy in later life.[1] We still do not know if modulating P2 receptor activity during or after SE will influence the resulting epileptic phenotype. This is critical as reports have shown that timing of P2 receptor targeting during pathology is crucial to avoid negative outcomes. Too early suppression of the immune system may lead to a reduction in neutrophil recruitment or the inhibition of microglia activation which is necessary to protect astrocytes from dying, thereby compromising tissue homeostasis.[19] We still do not know when and to what extent extracellular ATP is available to activate P2 receptors during and after SE. ATP release has been reported during increased neuronal activity; however, ATP is also released from dying cells and during inflammation. What is the cell- and sub-cellular-specific expression and function of different P2 receptors? This is an important question to answer as possible future treatments strongly depend on what cell type(s) are involved during pathology and what cellular functions we want to modulate. Is a combination of targeting different P2 receptors at the same time during SE more beneficial than single targeting? It is highly unlikely that newly developed drugs will be used as sole treatment during SE. Drugs will rather be used as adjunctive treatment in combination with already established drugs in the clinic such as benzodiazepines. Notably, we found that the specific P2X7 receptor inhibitor A438079 when given together with lorazepam suppressed seizures in a mouse model of drug-refractory SE where lorazepam on its own showed only weak anticonvulsive properties.[16] This demonstrates the potential clinical utility of P2 receptor antagonists/agonists[20] in the treatment of SE and should be validated in different animal models of SE and with different drugs currently used in the clinic.

Declaration of interest

This work was supported by funding from Science Foundation Ireland and the Health Research Board to T Engel ((13/SIRG/2098 and HRA-POR-2015-1243) and RCSI StAR. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Acknowledgments

The author would like to thank Prof. David Henshall for critical reading of the manuscript and apologize to those authors whose relevant work could not be cited here due to the strict restrictions on numbers of citations.