Investigating the Role of IGF2 mRNA-binding protein 1 (IMP1) In Regulating Reserve Intestinal Stem Cell Survival In Response to DNA Damage

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The intestinal epithelium undergoes complete turnover in 3-5 days. This is achieved through the presence of active intestinal stem cells (a-ISCs) that are self-renewing and can give rise to all differentiated epithelial cell types. Following irradiation, which ablates cycling cells in the intestine, cells that survive irradiation referred to as reserve intestinal stem cells (r-ISCs) are able to re-enter the cell cycle and contribute to tissue regeneration. Although mechanisms regulating the dormancy of these cells are beginning to be elucidated, their radioresistance and subsequent re-activation is still unclear, representing a gap in our broader knowledge of intestinal epithelial regeneration. RNA-binding proteins (RBP) are regulators of gene expression that facilitate cellular adaptation during homeostasis or pathological states. Deletion of the RBP IGF2 mRNA-binding protein 1 (IMP1) in the intestinal epithelium is associated with enhanced regenerative response following irradiation, a result that is phenocopied in mice with Imp1 deleted specifically in r-ISCs, highlighting a putative role for Imp1 in r-ISCs. Imp1 loss in the intestinal epithelium leads to an increase in autophagic vesicle content at homeostasis, suggesting that Imp1 may function as a negative regulator of the autophagy pathway. Combined deletion of Imp1 and Atg7 in the intestinal epithelium abrogates the enhanced regeneration observed with Imp1 deletion, suggesting that Imp1’s regulatory roles are dependent on the autophagy pathway. Studies show that mice lacking the autophagy gene Atg5 in intestinal epithelial cells exhibit a marked decrease in a-ISCs and impaired regeneration following irradiation. This is attributed to accumulation of dysfunctional mytochondria and a subsequent increase in reactive oxygen species levels (ROS). The role played by autophagy in radioresistant r- ISCs during homeostasis and regeneration remains unknown. I hypothesize that Imp1 modulates r-ISC survival in response to DNA-damage by enhancing autophagy mediated clearing of reactive oxygen species. In Aim 1, we will evaluate r-ISC frequency at homeostasis as well as cell death and DNA damage post- irradiation with or without Imp1 deletion. We will use a reporter mouse that labels r-ISCs and progeny with RFP following tamoxifen administration for lineage-tracing. We will use 12Gy whole-body irradiation and evaluate RFP+ cells using flow cytometry and immunohistochemistry. For Aim 2 we will use r-ISC specific Atg7 knockout as well as Atg7 and Imp1 double knockout mice to determine whether enhanced regeneration observed in the absence of Imp1 is dependent on autophagy. Regenerative microcolony frequency will be measured via immunohistochemistry. Furthermore, we will evaluate how Imp1 loss affects mitochondrial content using Mitotracker Green FM, transmembrane potential using rhodamine123, and ROS using CellROX via flow cytometry. Lastly, we will evaluate how ROS modulation affects regenerative microcolony formation with or without the antioxidant N-acetyl-L-cysteine. These studies will uncover roles played by RBPs in regulating stem cell activity and will provide a mechanistic understanding of stem cell survival following DNA damage.