ELUCIDATION OF THE ROLE OF AUTOPHAGY AND MITOPHAGY IN RADIATION RESPONSE

Abstract

Autophagy is well preserved cellular recycling machinery which elicits upon numerous cellular insults including hypoxia, starvation, pathogenic infection etc. for salvaging damaged cellular cargos, thus helping in maintaining cellular homeostasis. Autophagic pathway starts with formation of a double membrane organelle i.e. autophagosome, which engulfs cellular cargos destined to recycle. Major categories of autophagy includes macroautophagy, microautophagy and chaperon mediated autophagy. Among the other forms of autophagy, Macroautophagy is most widely studied. Autophagy has become an emerging scientific filed of research in the context of cancer resistance/ sensitivity, hypoxic signalling, nutrient starvation and in numerous harmful pathogenic infections including TB etc. or other harmful stress conditions. Due to advancements in technologies, incidents and accidents of radiation exposure (due to natural calamities) leading to leakage of radioactive material in the environment imposes a great threat to mankind. Accidental/ incidental radioactivity leakage demands trained personnel (first responders) to go to the accident site for the help of individuals‟ stuck in these sites, who are in need of immediate medical help. The first responders going to those sites impose themselves to radioactivity exposure. Under these conditions, radioprotection strategy may be helpful. In order to attain better radioprotective response, a deep knowledge of cell survival responses and their modulation under radiation exposure in order to attain improved protection strategies is required. Since radiation-induced macromolecular damage is associated with ROS generation and UPR induction, we hypothesised that autophagy may get induced to recycle damaged macromolecules (cargos), thereby protecting the cell against the radiation stress. The present study was started with the specific aim to understand the link between autophagy, ER stress and ROS generation, and the impact of this link on cell survival during radiation induced stress conditions. Macrophages serve as an important line of defense under most of the stress conditions in our body. Therefore, in this study, we investigated the induction of autophagy following irradiation in murine macrophage cells both in-vitro and ex vivo. We found that radiation induced autophagy is ROS-dependent and proceeded by UPR, specifically through the activation of EIF2AK3/Eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) and ERN1/Endoplasmic reticulum to nucleus signaling 1 ix (IRE1) UPR pathways. Further we confirmed our in-vitro findings at systemic levels as well. In next part of our study, we extended our work in murine model system using pharmacological modulators of autophagy; Rapamycin and Chloroquine. We found enhancement in mice survival and better intestinal recovery in autophagy inducer treated mice following radiation exposure. Nucleus is a vital cell organelle, containing genetic information in the form of DNA. Exposure to ionizing radiation can lead to direct as well as indirect DNA damage depending on the dose of exposure. Ionizing radiation generates excessive ROS/RNS leading to an indirect DNA damage and genomic instability. Therefore, there are indirect evidences available which suggests the possible role of radiation induced autophagy in genome integrity maintenance. We further performed study to comprehend the differential response of tumorigenic colon carcinoma and normal intestinal cells towards radiation exposure induced autophagy and its association with radiation induced DNA damage response. We found that cells (normal/tumorigenic) were showing differential response in autophagy levels as well as final cell fate. Furthermore, distinct DNA damage repair pathway induction was found in different cells in a cell type specific manner. After investigating the differential role of autophagy, we also investigated the specific induction of mitochondrial recycling through autophagy (mitophagy) following radiation exposure and its role in cellular providence if any. Under conditions of extensive mitochondrial damage, the cell adapts mitophagy in order to exterminate the damaged and dysfunctional mitochondria. In this way, mitophagy results in cell survival after harmful stress condition injury. Most of the radiation exposure induced ROS/RNS is largely produced in the mitochondria. Mitochondria are known to play an important role in radiation-induced cellular response, but the underlying mechanisms are largely unknown. Therefore, after establishing the role of radiation induced autophagy in deciding cellular fate decision under radiation exposed conditions, We next elucidated the role of mitophagy during radiation exposure (if at all its induction occurs specifically or not after radiation exposure). We found cell survival improvement following mitophagy induction in radiation exposed cells further suggesting prosurvival role of mitophagy post radiation exposure by recycling of damaged mitochondria.