Cellular and molecular mechanisms governing bladder aging.

PROJECT SUMMARY / ABSTRACT Bladder conditions such as incontinence and urinary tract infections (UTI) are increasingly common with aging and driven by poorly understood changes that occur at the cellular and molecular levels. The objective of this project is to define the molecular pathways that underpin how aging makes the bladder epithelium (urothelium)

prone to UTIs and develop new therapeutics to target those pathways. Data indicate that IFRD1 plays a key role in ribosomal quality control, translation, ER trafficking, and ER stress; and loss of IFRD1 produces aging phenotypes. The central hypothesis is that aging disrupts the cellular function of proteostasis, particularly

ribosome dynamics during protein translation, modification, and trafficking between the ER and Golgi. Aim 1 will determine the role of ribosomal and translational quality control machinery in the maintenance of bladder homeostasis in young, aged, or Ifrd1−/− urothelium of mice. Cycloheximide treatment will be used to inhibit

translation in young mice, and translation efficiency will be quantified with a puromycinylation assay to test if treatment is sufficient to induce phenotypes of aging or Ifrd1−/− bladders. UTI will be induced using uropathogenic E. coli., and bladder tissue will be collected to assess molecular-cellular and inflammatory responses using

histological analysis, western blots, and qRT-PCR to measure expression of ribosome-associated quality control markers and IFRD1 expression. To determine urothelial-intrinsic role, we will use a newly generated state of the art 3D- murine urothelial organoid system, with the potential to extend investigations to a human urothelial

organoid system. Aim 2 will determine whether chronic ER stress is sufficient for promoting aging phenotypes, and if alleviating ER stress will ameliorate the phenotypes. In aged, young, and Ifrd1−/− mice bladders and organoids, the expression of ER stress proteins and uroplakins will be compared. Western blots, qRT-PCR, and

immunostaining will be used to determine if uroplakin trafficking is altered in aged bladders, as in the Ifrd1−/− phenotype. Inflammation, DNA damage, lysosomes, mitochondria, urothelial cell shedding, and expression of ER stress response proteins will be assessed in response to the ER-stressor tunicamycin and ER stress

alleviator TUDCA to determine if this stress drives aging in the urothelium using organoid models. Aim 3 will determine the effects of pharmacological interventions on aging bladder and identify novel proteins governing bladder aging. Understanding the effects and mechanism of action in these drugs will establish a foundation on

which translational research can work to develop novel interventions for the treatment of urinary tract conditions that are known to progress with age.