A key role for BID-mediated mitochondrial damage in oxidative cell death

Age-related neuropathologies, such as Alzheimer’s and Parkinson’s disease as well as acute brain injury commonly involve oxidative stress-induced disruption of the intracellular calcium homeostasis, disturbed redox balance and impaired energy metabolism attributed to mitochondrial damage which event...

Full description

Saved in:
Bibliographic Details
Main Author: Jelinek, Anja Maria
Contributors: Culmsee, Carsten (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2018
Subjects:
BID
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Age-related neuropathologies, such as Alzheimer’s and Parkinson’s disease as well as acute brain injury commonly involve oxidative stress-induced disruption of the intracellular calcium homeostasis, disturbed redox balance and impaired energy metabolism attributed to mitochondrial damage which eventually drives neuronal cells to death. Thus, identifying biochemical features underlying the detrimental impairment of mitochondrial integrity and function is key to develop therapeutic strategies preventing neuronal loss. To date, a great variety of regulated cell death modalities has been established in neuronal death. In particular, apoptosis, excitotoxicity and regulated necrosis were shown to play a prominent role, and tight crosstalk between these paradigms of cell death exists, especially via convergence at the mitochondria. Despite increasing knowledge on cell death pathways, for many neurodegenerative diseases no curative treatment is available, so far. Over the last decades, a number of publications proposed the involvement of BCL-2-family proteins like BID and BAX in mediating mitochondrial cell death. For the protein BID, the contribution to neuronal apoptosis during ischemia has been widely established in vivo, exposing BID inhibition as a promising future therapy option. However, in non-apoptotic models of oxidative cell death, for instance ferroptosis, the role of BID and involvement of mitochondrial damage in cell death execution remained to be elucidated. In addition, the exact mechanisms how lipid ROS formation triggers BID activation and mitochondrial demise are still unknown. Due to high energy utilization for the maintenance of the membrane potential, neurotransmitter synthesis and restoring intracellular ion pools after action potentials, neurons strongly rely on oxygen and functional energy metabolism, thus being vulnerable to loss of mitochondrial function. The aim of this study was to determine hallmarks of regulated oxidative cell death pathways with respect to their time-dependent progression, involvement of BID as well as mitochondrial damage. Therefore, CRISPR/Cas9 technology was applied to generate neuronal HT22 cell lines lacking BID in order to analyze their sensitivity to oxidative stress induced by erastin and RSL3. Further, protective effects of MitoQ were investigated to evaluate the therapeutic potential of this mitochondria-targeted ROS scavenger in models of ferroptosis. Additionally, this work aimed to improve BID crystallization with novel recombinant protein constructs and optimized crystallization conditions. The first part of the thesis reports on the involvement of mitochondrial damage in oxidative death signaling in neuronal HT22 cells and mouse embryonic fibroblasts. In the model of glutamate-induced oxytosis, which is concentration- and cell density-dependent, impairment of mitochondrial respiration occurred in a time-dependent manner. In this cell death paradigm mitochondrial damage was represented the point of no return in the cell’s commitment to die as the well-established BID inhibitor BI-6c9 could rescue the cells within a time window of up to 8 hours after onset of glutamate exposure when massive mitochondrial damage was observed. The comprehensive analysis of erastin-induced oxidative death revealed a close interconnection of the previously separated cell death pathways of oxytosis and ferroptosis. In a time-dependent manner, erastin induced loss of mitochondrial membrane potential and lipid peroxidation followed by cell death 8 to 10 hours after treatment onset. Mitochondrial ROS production and loss of mitochondrial function was observed after 6 hours, and was tightly connected to severe mitochondrial fission thereby underlining the major finding of mitochondrial damage as the converging point of death signaling in neural and MEF cells in this paradigm of erastin-induced ferroptosis. In addition, siRNA-mediated AIF knockdown mitigated cell death in HT22 cells exposed to erastin, revealing a significant role for AIF release from mitochondria in ferroptosis similar to earlier findings in glutamate-induced oxytosis. The second part of the thesis focused on the involvement of BID in mitochondrial cell death pathways. SiRNA approach and CRISPR/Cas9 Bid knockout in HT22 cells revealed a significant contribution of BID in mediating mitochondrial demise upon oxidative stress. BID deprived cells were not only protected against glutamate- or erastin-induced cell death but also their mitochondrial parameters, such as membrane potential, ROS production and morphology were preserved at control levels. In contrast, tBID overexpression in Bid KO cells reversed the protective effects of BID absence and led to a significant increase in lipid ROS formation and cell death. In addition to shared BID involvement, mechanistic overlap of oxytosis and ferroptosis could be shown by the comparable cell protection against glutamate and erastin challenge by the ferroptosis inhibitor liproxstatin. Direct GPX4 inhibition by 1S, 3R-RSL3 and protection mediated by the mitochondria-targeted antioxidant MitoQ further established significant contribution of ROS formation to mitochondrial damage and again highlighted a role for BID as Bid KO cells were less sensitive to RSL3-mediated ferroptosis. Overall, these findings highlight a key role for BID in both paradigms of oxytosis and ferroptosis and expose BID transactivation to the mitochondria, mitochondrial oxidative damage and AIF-release as common mechanistic hallmarks linking these pathways. Finally, the 3D structure of BID should be elucidated by X-ray crystallography and, therefore, novel recombinant Bid constructs were established. For the first time, BID crystals could be obtained in a reproducible manner and were optimized by improving protein constructs and crystallization conditions. In order to solve the phase problem, selenomethionine crystals were grown, however, the resolution of electron density data was not sufficient to solve the molecular 3D structure of BID but provide a promising basis for further optimization.
Physical Description:223 Pages
DOI:https://doi.org/10.17192/z2018.0088