Investigating the Role of Autophagy in Premature Vascular Aging

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ABRAR GHALI ALREHAILI
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Accumulation of the precursor of nuclear envelope protein lamin A/C, prelamin A, in human vascular smooth muscle cells (HVSMCs) develops in both physiological and pathological ageing and is a hallmark of number a of diseases such as atherosclerosis and Hutchinson- Gilford progeria syndrome HGPS, where mutated/uncleavable prelamin A, or progerin, accumulates. Therefore, understanding of the mechanism by which prelamin A accumulates is important to enhance patients’ health and prognosis. Studies have shown that Rapamycin has successfully decreased the accumulation of prelamin A and progerin in HGPS cells through autophagy. Autophagy is an intracellular degradation system that functions to regulate cellular homeostasis. Previous work in our laboratory using bioinformatic analysis shows the prelamin A gene LMNA contains a theoretical LC3-interacting region (LIR) motif,we propose prelamin A may bind to LC3, a critical protein in the autophagy machinery, through the putative LIR-motif , thus potentially targeting prelamin A for autophagic degradation. In this project, we investigated if there is a role of autophagy process in the degradation of accumulated prelamin A and whether occurs via LIR-motif. To explore our hypothesis, we first tried to establish a novel protocol to modulate autophagy in HVSMCs. Different treatments were used as autophagy inducers and inhibitors. Unfortunately, the optimized conditions for induction and inhibition of autophagy in HVSMCs didn’t work out by the used treatments, that led us to test these conditions in rat SMCs, which gave a promising result when autophagy was induced by amino acid starvation, although further experiments are required for confirmation. Furthermore, two adenoviruses were generated: Wild-type eGFP-prelamin A (WT) and eGFP- mutant prelamin A (MT) where two critical amino acids were replaced in the LIR-motif. The multiplicity of infection (MOI) titration test for both adenoviruses were performed to establish the optimized titre for infecting the HVSMCs for expressing eGFP-WT and eGFP-MT prelamin A. The expression level and subcellular localization of WT and MT prelamin A were examined and corresponding Lamin A/C and autophagy expression in the transduced cells using western blot. My data/results showed HVSMCs were successfully transduced with adenovirus that carry WT and MT after using the optimized MOI for transduction. This allowed us to measure WT and MT prelamin A expression level to see the difference between the two proteins in addition to the nuclear lamina proteins Lamin A/C. Also, to check if there is any change in subcellular localization of both WT and MT protein. Moreover, expression levels of autophagic related proteins LC3 and P62 were also measured although it was performed one due to time restriction, further experiments are required. In conclusion, autophagy could be induced with prolonged serum starvation. However, preliminary data suggests amino acid starvation may be a more robust method of inducing autophagy in HVSMCs. Mutant preLMNA accumulation suggests the mutated LIR motif confers resistance to autographic degradation
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