Characterization of the rat Atg16l1 gene and its role in autophagy and disease

Crohn's disease (CD) is a potentially life-threatening inflammatory condition of the gastrointestinal tract affecting approximately 1.6 million Americans. Previous studies confirm the important role of genetics in IBD. More than 160 genetic alleles have been linked to CD, one of which lies in the autophagy-related 16-like 1 (ATG16L1) gene. In humans, a threonine to alanine amino acid variant at position 300 (T300A) of the evolutionarily conserved autophagy-related 16-like 1 (ATG16L1) protein is correlated with increased predisposition to CD. Using CRISPR-Cas9 technology, our laboratory developed the first reported genetically modified rat model of CD by inserting the T300A variant into the rat genome. An additional rat model with a knock-out mutation of the Atg16l1 gene was also developed to perform loss-of-function analyses. This dissertation research characterizes the wild type (WT) and T300A susceptibility variant Atg16l1 genes in the rat as well as understanding the mechanistic function of rat Atg16l1 in autophagy. Prior to this research, the rat Atg16l1 gene had two known and one predicted splice variants; however, no further characterization of this gene had been done in wild type animals. Through collection and amplification of DNA from select rat tissues, we confirmed four splice variants and revealed that they exist in different combinations depending on the tissue. In addition, in vitro work revealed all splice variants could produce protein. Additional phenotypic characterization found that, like non-diseased intestinal tissue from human CD patients, Paneth cells exhibited abnormal granulation patterns. From this study we were able to determine that the T300A rat model faithfully recapitulates pre-disease signs seen in humans. However, in order to address the usefulness of the model, we began to explore methods to incite CD signs. Very few studies evaluating the effect of known environmental triggers of CD on specific genetic susceptibility variants have been performed. I developed two exposure studies, one acute using high-dose nonsteroidal anti-inflammatory (NSAID) and one chronic using both low-dose NSAID or ad libitum Western diet formulated rodent feed. These studies confirmed that rats heterozygous (HET) for the T300A variant are more susceptible to NSAID toxicity than WT littermates, and our model does express mild histologic changes comparable to CD lesions in human CD patients as compared to WT littermates when exposed to low-dose NSAID or Western diet. These studies support the T300A rat model as a valuable tool for both acute and chronic environmental studies of IBD. In addition to animal model studies, we also performed in vitro work to evaluate the effect of each WT rat splice variant on autophagy. By transfecting HEK293 cells with one of each of the four WT rat variants, we have begun to understand how each Atg16l1 variant effects autophagic flux. This information is crucial to understanding the underlying mechanism of autophagy and how autophagy functions in different tissues of the body. This research sets the foundation for using the Atg161 T300A rat model in CD research and will help elucidate the role of Atg16ll1 in autophagy. A better understanding of the T300A variant and the influence Atg16l1 on autophagy will facilitate the potential for future targets of therapeutics for CD and other autophagy-related diseases.Includes bibliographical references (pages 112-128)