Inhibitors of p53 and HIF-prolyl-4-hydroxylases provide mitochondrial protection in a model of oxytosis

Mitochondrial dysfunction and demise are hallmarks of many neurological disorders and neurodegenerative diseases. Since mitochondria are the key organelles providing energy, they are regarded as the power house of the cell. Under conditions of stress, mitochondria regulate the ‘point of no return’...

Full description

Saved in:
Bibliographic Details
Main Author: Neitemeier, Sandra
Contributors: Culmsee, Carsten (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2015
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Summary:Mitochondrial dysfunction and demise are hallmarks of many neurological disorders and neurodegenerative diseases. Since mitochondria are the key organelles providing energy, they are regarded as the power house of the cell. Under conditions of stress, mitochondria regulate the ‘point of no return’ in intrinsic cell death pathways, which marks the decision point between life and death. Once the mitochondria are irretrievably damaged, they drive the cell into death. Mitochondrial protection displays a promising strategy to protect neurons from death. Previously, the two proteins p53 and PHD1 have been associated with neurodegeneration. Hence, the aim of this study was to investigate the role of those two proteins in neuronal cell death and to evaluate the neuroprotective potential of their particular inhibitors PFTα and DFO, DHB, CPO and oxyquinoline, respectively. Additionally, underlying mechanisms should be elucidated by which these inhibitors mediate their neuroprotective effects. In this study, immortalised mouse hippocampal HT-22 cells were used since they represent an established model of caspase-independent cell death induced by glutamate, termed oxytosis. Moreover, erastin was applied in the same cell line to induce a mode of cell death called ferroptosis. The first part of this study revealed that siRNA-mediated knockdown of p53 delayed glutamate-induced cell death in HT-22 cells for about 2 h, but failed to prevent lipid peroxidation or mitochondrial damage depicted as enhanced mitochondrial fragmentation, depolarisation of the mitochondrial membrane, increased mitochondrial ROS formation and a loss of ATP levels. Both p53 and phospho-p53 did not translocate to the mitochondria upon glutamate challenge indicating that oxytosis was not attributed to a direct action of p53 at the level of mitochondria. The inhibition of p53 transcriptional activity by knockdown of p53, which was determined by a reporter assay established in this work, could serve as a possible explanation for the observed delay of cell death. In contrast, the pharmacological p53-inhibitor PFTα prevented glutamate-induced cell death of HT-22 cells more efficiently and was still able to rescue these cells when applied up to 4 h after the onset of glutamate treatment. Furthermore, PFTα abolished lipid peroxidation and subsequently preserved mitochondrial integrity which was indicated by reduced mitochondrial fission, attenuated formation of mitochondrial ROS and restored mitochondrial membrane potential and ATP levels. Notably, PFTα rescued HT-22 cells depleted of p53 from glutamate-induced cell death. These results exposed a pronounced neuroprotective potential of PFTα which occurred at the level of mitochondria and independently of p53. The second part of this thesis demonstrated the neuroprotective potential of PHD inhibition by the use of structural diverse PHD-inhibitors and siRNAs selectively targeting PHD1. Both concepts of PHD inhibition reduced generation of lipid peroxides and preserved mitochondrial morphology and function indicated by restored mitochondrial respiration and membrane potential and abolished mitochondrial ROS formation, revealing that PHD inhibition acts upstream of mitochondrial demise. Remarkably, the effects by siRNA-mediated PHD1 silencing were less pronounced than those achieved by pharmacological inhibitors. These differences in efficacy were likely attributed to the insufficient knockdown by the siRNA approach. Nevertheless, these findings exposed the selective inhibition of PHD1 and the broad pharmacological inhibition of the PHD enzyme family as promising strategies to achieve mitochondrial rescue and subsequent neuroprotection. Previously, PHDs have been shown to interact with the transcription factor ATF4. The present study revealed that oxyquinoline was able to prevent the glutamate-induced down regulation of ATF4. However, oxyquinoline was still able to prevent oxytosis in cells depleted of ATF4. Therefore, the observed regulation of ATF4 protein levels after oxyquinoline application emerged as dispensable for oxyquinoline mediated protection in oxytosis, although previous studies in vivo suggested a modification of ATF4 transcriptional activity as mode of action for oxyquinoline. Overall, the exact mechanism by which PHD inhibition and, in particular oxyquinoline induced neuroprotection in the paradigm of oxytosis remains elusive so far. Finally, PHD inhibition was also shown to protect HT-22 cells against erastin-induced ferroptosis further supporting the pivotal role of PHDs in neuronal demise and the potential of PHD inhibition as a promising therapeutic strategy in the treatment of neurodegenerative diseases, where oxidative stress contributes to progressive mitochondrial dysfunction and neuronal death.