VCOM Louisiana Research Day Program

Biomedical Research

Olivia B. Bee, OMS-II; Rebekah L. Morrow, PhD; Stephen DiGuiseppe, PhD; Sarah B. Voth, PhD; K. Adam Morrow, PhD Edward Via College of Osteopathic Medicine-Louisiana, Monroe, Louisiana 01 ALTERED NUCLEAR LOCALIZATION OF PULMONARY ENDOTHELIAL EXPRESSED TAR DNA-BINDING PROTEIN 43 IN RESPONSE TO PSEUDOMONAS AERUGINOSA INFECTION

Context: TAR DNA Binding Protein 43 (TDP 43) is a major component of pathologic inclusions in Amyotrophic lateral sclerosis (ALS), Frontotemporal Dementia (FTD) and is implicated in Alzheimer’s Disease (AD). TDP-43 proteinopathies in neurodegenerative diseases are characterized by loss of nuclear TDP-43 and accumulation of the ubiquitinated protein aggregates in the cytosol, with proteolytic cleavage of TDP-43 into abnormal C-terminal fragments. Recent evidence suggests roles for TDP-43 in immunity and inflammation, specifically related to the pathogenesis of such proteinopathies. While studied extensively in neuronal diseases, there is limited data on the role of TDP-43 in response to pulmonary endothelial injury. Previous work has established that Pseudomonas aeruginosa infection causes damage to pulmonary endothelial cell barriers, and that the strain of P. aeruginosa PA103 utilizes the T3SS effectors ExoU and ExoT, which has an established role in cellular injury and gap formation, in a manner characteristic of an endothelial proteinopathy. Objective: Here, we aim to establish cellular localization of TDP-43, and to determine the effect of P. aeruginosa , as a model of promoting cellular stress, on quantifiable levels, as well as location, of host expressed TDP-43.

Methods: Rat pulmonary microvascular endothelial cells were grown to confluence and infected with P. aeruginosa . Images of the cell monolayer were captured, and lysates were collected hourly during infection. TDP 43 levels were assessed via western blot and densitometry conducted using ImageJ. Separate infections were conducted across a range of multiplicities of infection (1:1 to 20:1), and immunofluorescent staining for TDP-43 was subsequently performed. Results: P. aeruginosa induces visible and time-dependent gap formation in endothelial monolayers, confirming previously published work establishing cellular injury in response to such infections. Western blot analysis shows that TDP-43 is decreased intracellularly in response to infection. Immunofluorescence of uninfected cells indicates nuclear localization of TDP-43. While still partially nuclear, there appears to be greater cytoplasmic signal, and less nuclear signal in response to infection, suggesting importance of further exploration of underlying pathophysiological cellular redistribution of TDP-43 in response to cellular stress. Conclusions: We conclude that Pseudomonas aeruginosa infection of pulmonary endothelial

cells results in diminished levels of TDP 43, and loss of nuclear TDP-43, consistent with pathologic alterations of the protein in neurodegenerative diseases.

13 2023 Via Research Recognition Day