Periodic Reporting for period 1 - EpiPur (The ATP-P2X7R axis: a target for drug-refractory epilepsy)
Periodo di rendicontazione: 2018-01-01 al 2019-12-31
"Epilepsy is a debilitating neurological disorder, with multiple causes. Currently, treatment for epilepsy includes only drugs which control seizures, rather than tackling the underlying causes. Further, approximately 30% of epilepsy patients are resistant to all available drugs. Epilepsy which develops following an acute insult to the brain, such as a seizure or head injury are particularly commonly associated with resistance to drugs. Understanding the processes which lead to the brain becoming chronically epileptic and the causes of drug resistance is important for developing solutions.
When the brain is mechanically injured, deprived of oxygen, deprived of blood or hyperexcited, ATP, a molecule involved in providing energy to cells, is released into the extracellular space. Here, it acts as a trigger for inflammation which is important for limiting damage to local sites, killing pathogens, removing debris from damage, etc. Inflammation, however, when chronic, can contribute to pathology. It has previously been demonstrated that inflammation contributes to the development of epilepsy and is involved in resistance to antiepileptic treatments.
The purpose of this project was to investigate the contribution of ATP to this process via the ATP receptor, P2X7, which has been widely reported as a ""gatekeeper"" of inflammation. This has involved characterizing ATP release in the brain during and following seizures and investigating the contribution of P2X7 to epilepsy and resistance to anti-convulsants, using an array of transgenic mice and P2X7-targeting drugs. The overall objective of the project was to characterise the role of the ATP-P2X7 axis in drug-resistance and development of epilepsy and test methods for potentially intervening."
When the brain is mechanically injured, deprived of oxygen, deprived of blood or hyperexcited, ATP, a molecule involved in providing energy to cells, is released into the extracellular space. Here, it acts as a trigger for inflammation which is important for limiting damage to local sites, killing pathogens, removing debris from damage, etc. Inflammation, however, when chronic, can contribute to pathology. It has previously been demonstrated that inflammation contributes to the development of epilepsy and is involved in resistance to antiepileptic treatments.
The purpose of this project was to investigate the contribution of ATP to this process via the ATP receptor, P2X7, which has been widely reported as a ""gatekeeper"" of inflammation. This has involved characterizing ATP release in the brain during and following seizures and investigating the contribution of P2X7 to epilepsy and resistance to anti-convulsants, using an array of transgenic mice and P2X7-targeting drugs. The overall objective of the project was to characterise the role of the ATP-P2X7 axis in drug-resistance and development of epilepsy and test methods for potentially intervening."
Microelectrode biosensors have been used to measure extracellular concentrations of ATP during and following induced seizures in different sub regions of the hippocampus and dentate gyrus. This has not previously been achieved. Further, the method applied offered a high level of temporal resolution for investigating the relationship between seizures and ATP release into the extracellular space.
Downstream of ATP release, the contribution of the P2X7 receptor to seizures and resistance to anticonvulsants was assessed using a wide array of transgenic mouse lines and pharmacological agents. Our principle finding from this work is that, while P2X7 has no impact on the severity of seizures, its activation confers resistance to anticonvulsant drugs. This finding is consistent across two transgenic mouse lines (overexpressing and knockout) and with four different anticonvulsants (lorazepam, midazolam, phenytoin and carbamazepine) and may be the principle cause of changes in epileptogenesis and the severity of chronic epilepsy in this model.
We have found that, when pretreated with the anti-inflammatory, minocycline, mice overexpressing P2X7 show no greater resistance to lorazepam than wildtypes, suggesting that overexpression of P2X7 mediates responses to anticonvulsants via downstream immune effectors, such as interleukin-1beta. Experiments using P2X7 antagonists have demonstrated that this approach may be efficacious as an adjuct treatment alongside an anticonvulsant drug, during persistent seizures, when resistance to the drug is demonstrated.
Downstream of ATP release, the contribution of the P2X7 receptor to seizures and resistance to anticonvulsants was assessed using a wide array of transgenic mouse lines and pharmacological agents. Our principle finding from this work is that, while P2X7 has no impact on the severity of seizures, its activation confers resistance to anticonvulsant drugs. This finding is consistent across two transgenic mouse lines (overexpressing and knockout) and with four different anticonvulsants (lorazepam, midazolam, phenytoin and carbamazepine) and may be the principle cause of changes in epileptogenesis and the severity of chronic epilepsy in this model.
We have found that, when pretreated with the anti-inflammatory, minocycline, mice overexpressing P2X7 show no greater resistance to lorazepam than wildtypes, suggesting that overexpression of P2X7 mediates responses to anticonvulsants via downstream immune effectors, such as interleukin-1beta. Experiments using P2X7 antagonists have demonstrated that this approach may be efficacious as an adjuct treatment alongside an anticonvulsant drug, during persistent seizures, when resistance to the drug is demonstrated.
We have demonstrated the potential for in vivo measurements of ATP with a high level of temporal resolution. Further, we have demonstrated a mechanism whereby ATP is released following insult and contributes to the development and persistence of epilepsy and resistance to drug treatments. Further, we have demonstrated that targeting P2X7 increases the efficacy of anti-seizure drugs. This may be of clinical importance.