98 - Lysosomal Permeabilization Facilitate Mitochondria-mediated Cell Death in Ferroptosis and Oxytosis

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The central role of mitochondria in energy production is associated with generation of reactive oxygen species (ROS). Energy failure and increased ROS levels induce autophagy or regulated cell death (RCD). Apoptosis is a well-studied form of RCD that is identified by the release of mitochondrial content into cytoplasm, through mitochondrial permeability transition pore (PTP) and activation of caspase cascade. One component of PTP is the voltage-dependent anion channel 1 (VDAC1) that has been shown to be upregulated and oligomerized in apoptosis. In this study we examined the response of neuronal VDAC1 in Oxytosis and ferroptosis, two caspase-independent models of RCD. In these models, glutathione is depleted from the cells leading to excessive oxidative stress and cell death. Results VDAC1 was highly upregulated in a time dependent manner in immortalized hippocampal neurons (HT22) in response to inducers of oxytosis and ferroptosis, glutamate and erastin, respectively and was associated with significant cell death. Application of N-acetylcysteine decreased cell death, indicating the involvement of oxidative stress. Further analysis of VDAC1 revealed that oxytosis and ferroptosis lead to VDAC1 oligomerization, which are known to induce mitochondrial membrane permeabilization, as confirmed by cytochrome C and A poptosis I nducing F actor (AIF) release, leading to cell death. Pharmacological inhibitor of VDAC1, DIDS or function-blocking antibody against VDAC1 showed significant protection against glutamate and erastin induced cytotoxicity. Incidentally, we observed that pretreatment of cells with cathepsin B inhibitor (CA-074me) significantly improved cell viability. These results suggest that lysosomal permeability may facilitate mitochondrial damage and VDAC1 oligomerization, and identify VDAC1 as a potential therapeutic target in neurogenerative diseases where oxidative stress plays a key role in progression of disease.