Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Christopher J. Lengner

Abstract

Injury to the intestinal epithelium is a hallmark of numerous common clinical disorders, including radiation enteropathy, ischemia-reperfusion, and inflammatory bowel diseases. Disruption of the epithelial barrier in these conditions must be quickly restored to avoid dehydration and translocation of gut microbiota to the bloodstream. Short-term calorie restriction can enhance regenerative response of the intestinal epithelium following DNA damaging injury. However, the specific cell type responsible for this enhanced regenerative capacity and as a result the molecular determinants of this process remain unknown.

Regeneration of the intestinal epithelium is driven by multiple intestinal stem cell (ISC) types, including an active, radiosensitive Wnthigh�ISC that fuels turnover during homeostasis and a reserve, radioresistant Wntlow/off�ISC capable of generating active Wnthigh�ISCs. In this study, I utilize mouse genetic approaches to mark, isolate, and ablate intestinal stem cell (ISC) populations in order to address, for the first time, the functional importance of reserve ISCs for optimal regeneration following DNA damaging injury in response to caloric restriction. I demonstrate that modulation of mTORC1 signaling in reserve ISCs is a key factor in the regenerative response to radiation injury. I show that mTORC1 is both necessary and sufficient for the activation reserve ISCs. Loss of mTORC1 activity in reserve ISCs following injury impairs tissue regeneration due to failure of reserve stem cell activation. Conversely, promiscuous mTORC1 activation prior to injury sensitizes the epithelium to radiation damage as premature activation of reserve ISCs renders them susceptible to the radiation-induced apoptosis.

I demonstrate that Musashi (Msi) family of RNA-binding proteins are potent upstream regulator of mTORC1 signaling and metabolic genes in reserve ISCs and, similar to mTORC1 signaling, Msi proteins are necessary for intestinal regeneration despite their dispensability for intestinal basal homeostasis. I could modulate mTORC1 activity in vivo through caloric restriction, nutrient stimulation with branched chain amino acids, and pharmacological intervention with Rapamycin to govern activation of reserve ISCs and their sensitivity to radiation. These findings delineate a critical role for Msi-mTORC1 axis in regulating reserve ISC activation and epithelial regeneration and inform clinical strategies to protect reserve ISCs from genotoxic insults based on nutrient modulation.

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