Presentation description

Stroke is a leading cause of death and disability worldwide, with aging as the greatest risk factor. The cellular mechanisms responsible for worse stroke outcomes in older individuals remain unclear. In published studies, we reported steady-state autophagy and autophagic flux are repressed by aging in non-cerebral endothelial cells (ECs). In pilot studies, we find steady-state autophagy is blunted in cerebral ECs in response to AIS in older vs. adult mice. The purpose of my summer project is to optimize procedures to assess autophagic flux in cerebral ECs. Autophagy is a process whereby damaged or dysregulated intracellular organelles are targeted for removal by tethering proteins (i.e., SQSTM1/p62), escorted into and engulfed by mature autophagosomes, and delivered to the lysosome for degradation and recycling. Steady-state autophagy can be quantified via immunoblotting to assess accrual of proteins that represent mature autophagosome formation (i.e., LC3-II) and degradation of tethering proteins (i.e., SQSTM1/p62). In this regard, we find AIS-induced LC3-II accrual is greater in cerebral ECs from adult vs. older mice. However, it is unclear whether increased LC3-II protein expression reflects heightened autophagosome formation or impaired lysosomal degradation. To differentiate increased trafficking of the autophagosome to the lysosome (i.e., autophagic flux) from impaired lysosomal degradation of the autophagosome, selective lysosomal inhibitors can be used (i.e., chloroquine, bafilomycin A1). Currently, I am using autophagy stimulants (e.g., nutrient deprivation in cells, fasting in mice) combined with lysosomal inhibitors (i.e., chloroquine, bafilomycin), and evaluating LC3-II and p62 accrual in primary mouse brain microvascular ECs and immortalized human brain microvascular ECs, to test the hypothesis that autophagic flux is impaired by aging in cerebral endothelial cells. If intrinsic AIS-induced cytoprotection afforded by EC autophagy is blunted by aging, this conserved intracellular mechanism designed to maintain proteostasis might be targeted therapeutically in stroke.