Poster #038, Charles R. Drew University of Medicine and Science
Trisomy 21 is Associated with Impaired Lysosomal Function in Human Pulmonary Fibroblasts
Mentors: Denise Al Alam, PhD; Soula Danopoulos, PhD
Trisomy 21 (T21), resulting in Down syndrome, is a chromosomal abnormality caused by chromosome 21 triplication. Cardiopulmonary defects are one of the main causes of morbidity and mortality in individuals with T21. Our lab recently demonstrated that T21 lungs present developmental anomalies. These abnormalities were associated with excess senescence (SEN), defined by cell cycle arrest, within prenatal fibroblast and persists postnatally. Lysosome dysfunction has been strongly associated with cellular SEN. Therefore, we hypothesize that T21 lung cells exhibit compromised lysosomal activity. Three pairs of T21 and non-T21 postnatal human lung fibroblasts (HLF) were cultured for 48 hours. Cells were subsequently collected for protein extraction and western blot (WB) analysis or fixed for immunofluorescent (IF) staining to determine protein localization. Our proteins of interest include LAMP1 as a global lysosome marker, and Galectin-3, Cathepsin D, and P62/Ubiquitin as function state markers. Cultured cells were also stained with LysoTracker, a live cell dye, to assess lysosomal morphology, function and activity in T21-postnatal HLF compared to non-T21 controls. WB analysis demonstrated decreased LAMP1 and increased p62 protein levels in T21 fibroblasts compared to non-T21 controls. Galectin-3 levels varied between samples, thus failing to show a consistent trend. Additionally, LysoTracker staining was lower in T21 cells, suggesting lysosomal alkalinization and impaired lysosomal function relative to non-T21 HLF. Further characterization of lysosomal architecture and protein localization in T21 and non-T21 HLF will be examined by IF. Based on current results, we are expecting increased p62 in T21 lysosomes. These preliminary data suggest altered lysosomal function in T21 lung fibroblasts, supported by decreased LAMP1 and LysoTracker staining and increased p62 levels. Although further analyses are needed to confirm these findings, the results implicate lysosomal dysfunction as a potential contributor to the elevated senescence observed in T21 lungs.