Necroptosis and mitophagy in the pathogenesis of hypoxic-ischaemic encephalopathy
File(s)
Author(s)
Wu, Lingzhi
Type
Thesis
Abstract
Hypoxic-ischaemic encephalopathy (HIE) is a devastating brain injury in newborns resulting from perinatal hypoxia event. A better understanding of the HIE pathophysiological mechanisms can help to identify targetable pathways for therapeutic development to improve HIE outcomes. In the present study, we hypothesise that necroptosis acts upstream to regulate mitophagy during HIE and ischaemia-reperfusion (I/R) brain injury.
Oxygen-glucose deprivation and reperfusion (OGD/Rep) was used to model I/R brain injury in vitro with mouse primary neurons or mouse neuroblastoma Neuro-2a cells. For murine model of HIE, right common carotid artery (CCA) coagulation plus hypoxia (8%O2) was performed on postnatal day7 mice.
OGD/Rep induced necroptosis in vitro with increased phosphorylation of MLKL• and translocation of necrosome into the mitochondrial fraction. OGD/Rep also upregulated mitophagy mediators Parkin and BNIP3 and increased mitochondrial localisation of autophagosome marker LC3A/B-II to reflect enhanced mitophagy. Blocking necroptosis with RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK’872 attenuated OGD/Rep-induced mitophagy, whereas siRNA-mediated MLKL knockdown further amplified mitophagy. Notwithstanding, either RIPK1/RIPK3 inhibition or MLKL knockdown restored mitochondrial function and morphology. In postnatal day7 mice, CCA plus hypoxia stimulated necroptosis and mitophagy at 24hrs of reperfusion, in particular in CA1 and CA3 of hippocampus. Intracerebrovascular administration of Nec-1s attenuated Parkin and BNIP3 in the hippocampus and decreased mitochondrial localisation of LC3A/B-II to indicate suppressed mitophagy. Moreover, RIPK1 inhibition by Nec-1s was associated with long-term improvement in motor functions in HIE-injured mice.
In summary, the current study suggests RIPK1 and RIPK3 as likely positive regulators of mitophagy whereas MLKL likely suppresses mitophagy during HIE pathopathogenesis. Mitochondrial protection was evident from RIPK1/RIP3 inhibition or MLKL silencing, and RIPK1 inhibition may improve long-term motor function in HIE. Our findings provide foundamental rationale for developing therapeutic strategy that targets necroptosis and associated mitophagy pathway in treating HIE or other forms of I/R brain injury per se.
Oxygen-glucose deprivation and reperfusion (OGD/Rep) was used to model I/R brain injury in vitro with mouse primary neurons or mouse neuroblastoma Neuro-2a cells. For murine model of HIE, right common carotid artery (CCA) coagulation plus hypoxia (8%O2) was performed on postnatal day7 mice.
OGD/Rep induced necroptosis in vitro with increased phosphorylation of MLKL• and translocation of necrosome into the mitochondrial fraction. OGD/Rep also upregulated mitophagy mediators Parkin and BNIP3 and increased mitochondrial localisation of autophagosome marker LC3A/B-II to reflect enhanced mitophagy. Blocking necroptosis with RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK’872 attenuated OGD/Rep-induced mitophagy, whereas siRNA-mediated MLKL knockdown further amplified mitophagy. Notwithstanding, either RIPK1/RIPK3 inhibition or MLKL knockdown restored mitochondrial function and morphology. In postnatal day7 mice, CCA plus hypoxia stimulated necroptosis and mitophagy at 24hrs of reperfusion, in particular in CA1 and CA3 of hippocampus. Intracerebrovascular administration of Nec-1s attenuated Parkin and BNIP3 in the hippocampus and decreased mitochondrial localisation of LC3A/B-II to indicate suppressed mitophagy. Moreover, RIPK1 inhibition by Nec-1s was associated with long-term improvement in motor functions in HIE-injured mice.
In summary, the current study suggests RIPK1 and RIPK3 as likely positive regulators of mitophagy whereas MLKL likely suppresses mitophagy during HIE pathopathogenesis. Mitochondrial protection was evident from RIPK1/RIP3 inhibition or MLKL silencing, and RIPK1 inhibition may improve long-term motor function in HIE. Our findings provide foundamental rationale for developing therapeutic strategy that targets necroptosis and associated mitophagy pathway in treating HIE or other forms of I/R brain injury per se.
Version
Open Access
Date Issued
2023-02-27
Date Awarded
2024-02-01
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Ma, Daqing
Sponsor
British Journal of Anaesthesia
Publisher Department
Department of Surgery & Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)