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.
