Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Jasmine S. Carter - Claflin University
Co-Author(s): Benedicth Ukhueduan, University of South Carolina, Columbia, SC; Rekha C. Patel, University of South Carolina, Columbia, SC
Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder that causes premature aging and affects young children. The affected children begin to display hair loss, growth retardation, osteoporosis, and other age-related diseases usually associated with aged individuals. HGPS is caused by a point mutation in the LMNA gene responsible for the production of Lamin A and Lamin C, which serve as scaffolding proteins for the nuclear lamina. The nuclear lamina is a structure inside the nucleus that is composed of intermediate filaments and membrane-associated proteins that provide nuclear support and chromatin organization for the cells. The nuclear lamina also plays an important role in the transcriptional regulation of several genes, including the anti-oxidant genes induced by oxidative stress. The point mutation in the LMNA gene results in the loss of 55-amino acids generated by aberrant splicing and the mutant protein is known as progerin. Progerin is responsible for the premature aging phenotype seen in individuals with HGPS. Proteotoxicity due to misfolded proteins is thought to be a central component of aging. The heat shock response (HSR) triggered by heat stress and misfolded proteins is one of the pathways that cells use to protect themselves against proteotoxicity. HSR is downregulated in old organisms in most previously established models of aging. The objective of this project is to study how HGPS cells respond to heat shock (HS). We hypothesized that progerin expressing cells would not be able to respond to HS and undergo more death due to the stress they were subjected to from the point mutation compared to the Wt-LMNA expressing cells. To test our hypothesis, we used hTERT-immortalized wild type human skin fibroblasts stably transfected with expression constructs for tetracycline-inducible expression of the GFP-Wt-Lamin A (LMNA) or GFP-progerin proteins to conduct an HSR time course experiment and a Trypan blue viability assay. After 48 hours in doxycycline-free medium, Wt-LMNA and progerin expressing cells were subjected to HS at 42°C for 1 hour and allowed a recovery period (3, 6, 12, and 24 hours) while the control cells were left at 37°C to compare HSR induction. For the Trypan blue viability assay, Wt-LMNA and progerin expressing cells plated with doxycycline or without doxycycline for 48 hours were subjected to HS (1, 2, and 3 hours) at 42°C and allowed a 24 hours recovery period. We found that progerin expressing cells had a higher basal level of HSP70 compared to the Wt-LMNA expressing cells and a lower cell viability and survival rate in the absence of doxycycline in comparison to the Wt-LMNA expressing cells. Therefore, we concluded that progerin might affect HGPS cells ability to respond to HS. For future studies, we plan to compare our current cell culture model to fibroblasts established from normal and HGPS patients in order to determine how the stability and degradation process of HSP70 is activated in HGPS cells.
Funder Acknowledgement(s): I would like to thank Dr. Rekha C. Patel, my research advisor, Benedicth Ukhueduan, the graduate student supervisor, the Patel lab, and the University of South Carolina Department of Biological Sciences. I also would like to thank the National Institute on Aging for providing the funding for my project through the South Carolina Advancing Diversity in Aging Research Program (SC-ADAR) (R25AG050484).
Faculty Advisor: Dr. Rekha C. Patel, email@example.com
Role: I conducted the heat shock experiments. I also performed western blot analyses to determine if the progerin expressing cells heat shock pathway was compromised in comparison to the Wt-LMNA expressing cells, as a result of the stress from heat shock.