Pharmacological intervention of mitochondrial mechanisms in hemin toxicity and ferroptosis

The growing prevalence of neurological disorders, such as Parkinson's disease, Alzheimer's disease, and strokes, represents a growing challenge for our aging society and is one of the leading causes of morbidity and mortality. These diseases are closely linked to oxidative stress, and inte...

Full description

Saved in:
Bibliographic Details
Main Author: Merkel, Melanie
Contributors: Culmsee, Carsten (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Summary:The growing prevalence of neurological disorders, such as Parkinson's disease, Alzheimer's disease, and strokes, represents a growing challenge for our aging society and is one of the leading causes of morbidity and mortality. These diseases are closely linked to oxidative stress, and intensive research over the last decades has established a link to various cell death mechanisms such as apoptosis, oxytosis, ferroptosis, necroptosis, and hemin toxicity. This is based on evidence of different pathophysiological features, including lipid peroxidation, loss of iron homeostasis, and mitochondrial impairment. In order to gain a more comprehensive understanding of the previously unexplained factors and molecular mechanisms that contribute to neurodegenerative diseases and whose interrelationships have not yet been fully explored, this study investigated different aspects of ferroptosis. In the first section of this study, mitochondrial involvement in ACSL4/LPCAT2-driven ferroptosis was investigated in more detail. For this purpose, stably transfected HEK293T cell lines with overexpression of the enzymes ACSL4 and LPCAT2 or a control line with an empty vector were used to uncover the missing insights. The overexpression of lipid synthesis-related enzymes resulted in increased sensitivity to the ferroptosis inducer RSL3 compared to the control cell line. Elevated mitochondrial damage was observed through increased mitochondrial ROS production, loss of mitochondrial membrane potential, and reduced mitochondrial respiration. These effects were prevented by ferroptosis inhibitors such as deferoxamine, ferrostatin-1, and 5- and 12/15-LOX inhibitors. Inhibition of the overexpressed enzyme ACSL4 by thiazolidinediones also protected against ferroptosis. VDAC1 inhibitor Akos-22 and the mitochondrial ROS scavenger MitoQ were used to investigate mitochondrial involvement in more detail. Both showed high efficacy in the overexpressing cells and provided protective effects at the cytosolic and mitochondrial levels. MitoQ prevented ferroptosis through its antioxidant effect and influenced metabolism. To determine whether metabolic intervention was sufficient, inhibitors of mitochondrial complex I of the respiratory chain and glutamine deprivation were examined. However, these interventions failed to protect HEK293T cells from ferroptosis. The results suggest that mitochondrial ROS production determines ACSL4/LPCAT2-driven ferroptosis while mitochondria-targeted antioxidants protect against it. In the second section of this research, the link between erastin- and hemin-induced ferroptosis in neuronal HT22 cells was characterized in more detail, focusing on mitochondrial involvement. Differences in the response to FCS deprivation were observed. Furthermore, the pharmacological 12/15-LOX inhibitor PD146176 appeared to protect against erastin-induced ferroptosis but was ineffective against hemin toxicity. In contrast, the 5-LOX inhibitors were effective against both forms of ferroptosis. The protection was also able to prevent mitochondrial ROS production and the loss of mitochondrial membrane potential induced by hemin. MitoQ was utilized for a more detailed investigation of the role of mitochondrial ROS formation, showing protective effects against erastin but not against hemin-mediated ferroptosis. Metabolic intervention by the complex I inhibitor metformin also failed to protect the HT22 cells from hemin toxicity. As a result, hemin was found to induce a form of ferroptosis that is 5-LOX-dependent and associated with mitochondrial damage, but the latter was not causal for oxidative death. Erastin, on the other hand, induces a 5- and 12/15-LOX-dependent form of ferroptosis with major mitochondrial involvement, as metformin and MitoQ application were effective. Thus, the hemin toxicity differs from the classical ferroptosis induced by erastin. Finally, in the last section of the study, novel selenium compounds were investigated for their efficacy against ferroptosis in HT22 cells. These compounds are modifications of the already-known GPx4-mimicking compound ebselen. The selenium-containing compounds showed a remarkably potent effect against ferroptosis which was 15-20 fold more protective compared to ebselen. These compounds acted at cytosolic and mitochondrial levels by affecting lipid peroxidation and ROS production. Additionally, they exhibited strong antioxidant properties, as glutathione levels were maintained and one of the compounds even increased GPx4 protein expression. The diselenides were also found to be more potent in comparison to the benzisoselenazoles and were able to withstand an increasing ferroptosis stimulus. Furthermore, differences in efficacy against erastin- or RSL3-mediated ferroptosis were identified with lower concentrations being sufficient for erastin-induced damage. Application up to 8 hours after the onset of the cell death stimulus was still sufficient to preserve cell integrity. These promising in vitro results characterize the novel selenium compounds as potent agents against ferroptosis, warranting further studies to gain new insight into the efficacy of the selenium compounds in vivo.