Periodic Reporting for period 1 - NeoPur (New treatments and novel diagnostic tests for neonatal seizures based on purinergic signaling.)
Reporting period: 2019-10-14 to 2021-10-13
New-borns are predisposed to an increased risk of developing seizures following brain injury higher than in any other moment in a person´s life. Seizures in neonates, most commonly caused as a consequence of hypoxic-ischemic events occurring during birth, are recognized as a medical emergency and require rapid treatment. Mounting evidence from experimental models and clinical studies suggest seizures to be detrimental to the developing brain. Seizures in neonates may also cause increased mortality and a higher risk of adverse neurological outcomes. Consequently, the long-term health costs associated with seizures and neonatal brain damage are very high. To date, both diagnosis and treatment of neonatal seizures remain a clinical challenge with seizure misdiagnosis estimated to be as high as 50% and only every second patient responding to current therapies. The adenosine-triphosphate (ATP)-gated P2X7 receptor, a gatekeeper of inflammation has recently emerged as a promising target for seizure control, showing anticonvulsant and disease-modifying properties in animals. Moreover, recent data suggest a role for P2X7 receptor (P2X7R) during seizures in neonates. Purines are important extracellular signaling molecules, and act as neurotransmitters or neuromodulators. Released during pathological processes in the brain (e.g. seizures, inflammation) purines are also present in body fluids of humans making them very attractive as theranostic biomarkers of disorders of the central nervous system. The overall aim of NeoPur project is to establish P2X7R-targeting as novel treatment strategy for seizures in neonates and identify novel diagnostics for neonate seizures based on purinergic signalling. However, to bring P2X7R further towards a clinical application, we need evidence of seizure-induced release of ATP in the brain, we must determine what are the clinical outcomes of a genetic and pharmacological targeting of P2X7 during neonatal seizures and we must identify biomarkers to better identify patients suffering from seizures. NeoPur project has achieved most of its objectives and milestones for the period, with relatively minor deviations
The aim of the NeoPur project was to identify novel therapeutic strategies in pre-clinical models of neonatal seizures focussing on the P2X7R and to validate novel diagnostic devices in clinical testing in new-borns suffering from hypoxia-induced encephalopathy. To achieve the objectives and goals of this project, six specific work packages were planned.
WP1-Determine the dynamics of ATP release during neonatal seizures. To carry out this and the following research WPs, we used the mouse model of neonatal hypoxia-induced seizures developed previously. To establish where and when ATP is release during neonatal seizures, the expressional changes of one of the proteins involved in ATP release, VNUT, were analyzed, showing an increased expression of VNUT 24 hours post-hypoxia. Moreover, mice treated with a specific VNUT antagonist before hypoxia showed a lower seizure onset. These results suggest the involvement of VNUT in neonatal seizures and a protective role of VNUT blocking during neonatal seizures.
WP2- Evaluate the therapeutic potential of P2X7R-targeting to treat neonatal seizures. Firstly, we obtained a genetic proof of the involvement of P2X7R during the generation of neonatal seizures using the following transgenic mouse models (i) P2X7R knock-out transgenic mouse, in which the P2X7R expression is globally knocking-down and (ii) P2X7R overexpression mouse which allows the study of the impact of elevated P2X7R on neonatal pathology. Both mouse models were characterized to ensure that levels of neurotransmitter systems involved in seizure generation, inflammation markers and other purinergic receptors were unaffected due to the deficiency or overexpression of P2X7R. After the hypoxia-induction of neonatal seizures, the seizure phenotype in both transgenic models was characterized through the quantification of clinical seizure manifestations and EEG recordings. Our results show that while mice deficient in P2X7R experience less seizures, mice overexpressing P2X7R show an exacerbated seizure phenotype during and post-hypoxia. Secondly, we determined the therapeutic potential of P2X7R-targeting during seizures in neonates using highly specific brain stable P2X7R antagonists. We evaluated targeting P2X7R may prevent epilepsy development following hypoxia-induced neonatal seizures. The seizure phenotype was analyzed showing the treatment of mice with a P2X7R antagonist post-hypoxia alleviated the development of an epilepsy-like phenotype. These results suggest P2X7R-based treatments as a novel therapeutic strategy for neonatal seizures and resulting development of epilepsy.
Based on the achieved results, a scientific paper is in preparation.
WP3- Determine the potential of purine concentrations in blood as novel diagnostics for seizures in neonates. To accomplish this objective, we used purine measuring biosensors for seizures in the mouse model of neonatal hypoxia and during clinical testing in newborns. As a result of this study, a research article was published in Frontiers in molecular neuroscience journal.
WP4- Project management. The project management includes financial management, progress monitoring, risk management and the writing of Final Progress report. The partnership agreement was signed between the universities/hospitals/industry involved in NeoPur project.
Progress monitoring was carried out during weekly supervision meetings with the supervisor and regular meetings with our clinical, academic and industry partners for data discussion and troubleshooting. Financial management of the project was carried out through the Finance Department of the host institution. Finally, an initial risk impact assessment was performed including the partnerships.
WP5- Communication, Dissemination and Exploitation. Some of the planned activities could not to be carried out due to COVID-19. However, it was promoted the public engagement and communication of findings through the participation in several outreach activities (TY programme, Dublin Science week, Rose week). The results of NeoPur were disseminated through scientific publications (Beamer et al, Front Mol Neurosci (2021) PMID: 34566578; Menéndez et al, Int J Mol Sci (2020) PMID: 33105750), organized events and seminars (FENS 2020 Virtual Forum).
WP6- Training/Transfer of Knowledge. Finally, NeoPur project has allowed to Individual to increase a number of critical transferable skills highly relevant for becoming and independent researcher.
WP1-Determine the dynamics of ATP release during neonatal seizures. To carry out this and the following research WPs, we used the mouse model of neonatal hypoxia-induced seizures developed previously. To establish where and when ATP is release during neonatal seizures, the expressional changes of one of the proteins involved in ATP release, VNUT, were analyzed, showing an increased expression of VNUT 24 hours post-hypoxia. Moreover, mice treated with a specific VNUT antagonist before hypoxia showed a lower seizure onset. These results suggest the involvement of VNUT in neonatal seizures and a protective role of VNUT blocking during neonatal seizures.
WP2- Evaluate the therapeutic potential of P2X7R-targeting to treat neonatal seizures. Firstly, we obtained a genetic proof of the involvement of P2X7R during the generation of neonatal seizures using the following transgenic mouse models (i) P2X7R knock-out transgenic mouse, in which the P2X7R expression is globally knocking-down and (ii) P2X7R overexpression mouse which allows the study of the impact of elevated P2X7R on neonatal pathology. Both mouse models were characterized to ensure that levels of neurotransmitter systems involved in seizure generation, inflammation markers and other purinergic receptors were unaffected due to the deficiency or overexpression of P2X7R. After the hypoxia-induction of neonatal seizures, the seizure phenotype in both transgenic models was characterized through the quantification of clinical seizure manifestations and EEG recordings. Our results show that while mice deficient in P2X7R experience less seizures, mice overexpressing P2X7R show an exacerbated seizure phenotype during and post-hypoxia. Secondly, we determined the therapeutic potential of P2X7R-targeting during seizures in neonates using highly specific brain stable P2X7R antagonists. We evaluated targeting P2X7R may prevent epilepsy development following hypoxia-induced neonatal seizures. The seizure phenotype was analyzed showing the treatment of mice with a P2X7R antagonist post-hypoxia alleviated the development of an epilepsy-like phenotype. These results suggest P2X7R-based treatments as a novel therapeutic strategy for neonatal seizures and resulting development of epilepsy.
Based on the achieved results, a scientific paper is in preparation.
WP3- Determine the potential of purine concentrations in blood as novel diagnostics for seizures in neonates. To accomplish this objective, we used purine measuring biosensors for seizures in the mouse model of neonatal hypoxia and during clinical testing in newborns. As a result of this study, a research article was published in Frontiers in molecular neuroscience journal.
WP4- Project management. The project management includes financial management, progress monitoring, risk management and the writing of Final Progress report. The partnership agreement was signed between the universities/hospitals/industry involved in NeoPur project.
Progress monitoring was carried out during weekly supervision meetings with the supervisor and regular meetings with our clinical, academic and industry partners for data discussion and troubleshooting. Financial management of the project was carried out through the Finance Department of the host institution. Finally, an initial risk impact assessment was performed including the partnerships.
WP5- Communication, Dissemination and Exploitation. Some of the planned activities could not to be carried out due to COVID-19. However, it was promoted the public engagement and communication of findings through the participation in several outreach activities (TY programme, Dublin Science week, Rose week). The results of NeoPur were disseminated through scientific publications (Beamer et al, Front Mol Neurosci (2021) PMID: 34566578; Menéndez et al, Int J Mol Sci (2020) PMID: 33105750), organized events and seminars (FENS 2020 Virtual Forum).
WP6- Training/Transfer of Knowledge. Finally, NeoPur project has allowed to Individual to increase a number of critical transferable skills highly relevant for becoming and independent researcher.
NeoPur brings together a team of experts in purinergic signaling, industrial partners developing novel P2X7R antagonists and diagnostic devices and clinicians specialised in neonates. This highly interdisciplinary and intersectoral approach will add a significant contribution to the understanding of purinergic signaling during neonatal seizures providing novel diagnostic and therapeutic approaches. Finally, NeoPur has contributed to improve the European contribution to research in neonate research through the development of a novel research tool which can be available to researchers and clinicians (e.g. blood purine biosensors).