INVESTIGATION OF SMOOTH MUSCLE CELL DEATH AND GENOME INSTABILITY IN HUTCHINSON-GILFORD PROGERIA SYNDROME

Hutchinson–Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) from large arteries in HGPS patient and mouse models, suggesting a causal connection between SMC loss and cardiovascular malfunction. The primary aim of this dissertation is to elucidate the molecular mechanisms underlying the massive SMC loss in HGPS. To study this, I develop an in vitro differentiation protocol to generate HGPS SMCs from induced pluripotent stem cells (iPSCs). My results indicate that HGPS SMCs exhibit a profound cell death phenotype, potentially recapitulating the in vivo SMC loss. Mechanistically, I find that HGPS SMCs bear deficient homologous recombination (HR). In addition, progerin accumulation strongly suppresses PARP1 and consequently triggers an activation of the error-prone non-homologous end joining (NHEJ) response during S/G2 phase. As a result, HGPS SMCs exhibit prolonged mitosis and mitotic catastrophe. Mis-regulated DNA damage response (DDR) is proposed to induce genome instability and various cellular phenotypes in HGPS, including HGPS SMC cell death. To better understand HGPS DDR misregulation, I examine HR and NHEJ in HGPS fibroblasts at different cell cycle phases. My analysis indicates that HR is deficient in S/G2 phase, whereas NHEJ, the dominant G0/G1 phase DDR pathway, is impaired in G0/G1 phase but active in S/G2 phase HGPS fibroblasts. The mis-regulation of HR and NHEJ may jeopardize genome integrity in both G0/G1 and S/G2 phase HGPS cells. Mechanistic study reveals that H2AX, a crucial upstream DDR signal, is reduced in G0/G1 but normal in S/G2 phase HGPS cells, implicating a potential cause of the cell cycle-dependent NHEJ mis-regulation. Furthermore, this reduction is correlated with impaired ATM activation and loss of H3K9me3 in HGPS. Restoration of H3K9me3 by methylene blue treatment can stimulate ATM activity, improve H2AX signaling and rescue NHEJ in G0/G1 phase HGPS cells. This dissertation not only is the first mechanistic study on HGPS SMC loss but also provides a molecular basis and therapeutic approach for the HGPS DDR deficiencies