A14 INVESTIGATING THE IMPACT OF PARKINSON’S DISEASE-ASSOCIATED GENE <i>PINK1</i> ON INTESTINAL HOMEOSTASIS AND RESPONSE TO INFECTION

Abstract Background Intestinal epithelial cells (IECs) provide an essential physical barrier between luminal contents and host tissue. Dysregulation of IECs leads to barrier dysfunction, causing pathologies in both intestinal and extra-intestinal diseases. While Parkinson’s Disease (PD) is primarily a neurodegenerative disorder, increasing evidence links PD progression and gastrointestinal dysfunction. Our group developed a model to investigate the role of the gut in PD, demonstrating that mice with genetic ablation of the PD-associated gene Pink1 exhibited motor phenotypes only when previously infected with gram-negative Citrobacter rodentium intestinal bacteria. As Pink1 is expressed in IECs and the colonic lamina propria, we hypothesize that PD-associated gene mutations directly affect the epithelium and impact early PD pathophysiology. Aims 1. To characterize the transcriptional profiles of Pink1 WT and KO IECs at baseline and following in vivo C. rodentium infection. 2. Use in vitro cell line and colonic organoid systems to study the effect of Pink1 on epithelial activity in an isolated system. Methods Single-cell RNA sequencing was performed on colonic IECs isolated from Pink1 WT and KO mice, at steady state and following in vivo C. rodentium infection. Mice were sacrificed at D6 post infection to elucidate transcriptional differences between IEC lineages of each genotype. To validate scRNAseq findings, we derived ex vivo colonoids from Pink1 WT and KO mouse crypts and used a Pink1 KO CMT-93 epithelial cell line with WT clone as control. The cultures were treated with lipopolysaccharide (LPS), or infected with gram-negative pathogens to determine how PINK1 loss-of-function affects the inflammatory response of the epithelium. Results At baseline, our data revealed upregulation of interferon (IFN)-signaling genes (ISGs) in Pink1 KO enterocytes (ECs) compared to WT; whereby more than 70% of upregulated differentially expressed genes were linked to type I or II IFN signaling. In vitro stimulation of Pink1 KO epithelial cell line with IFN resulted in stark upregulation of ISGs compared to WT. During infection, Pink1 KO ECs demonstrated significant alterations in actin-cytoskeleton-related GO term pathways compared to WT. Infection of Pink1 KO CMT-93 epithelial cells revealed morphological changes in F-actin structures. Conclusions Using in vivo and in vitro models encompassing PD-genetic susceptibility and environmental stimuli, we identified dysregulation in Pink1 KO epithelium, suggesting altered inflammatory responses at baseline and infection. By investigating PD-associated genes in IECs, we will contribute to better understanding the role of the intestine in PD initiation, progression, and pathogenesis. Funding Agencies CAG, CIHR