PROJECT SUMMARY Aging is the largest risk factor for a majority of neurodegenerative disorders, including Alzheimer’s disease (AD) and related dementias. Despite growing incidence for such disorders, zero therapeutics are capable of reversing progression of aging-related cognitive decline seen in disease and healthy aging. Through the use of heterochronic parabiosis however, or the surgical joining of circulatory systems between young and old mice, our group demonstrated that blood-borne factors from young animals are capable of reversing many deleterious phenotypes seen in the aged brain. Understanding the molecular mechanisms underlying cognitive improvement post-parabiosis may uncover novel therapeutic avenues for aging-related neurodegeneration. One hallmark of aging linked to the onset of AD is the collapse of protein homeostasis (proteostasis) networks that regulate the folding of newly synthesized proteins via molecular chaperones as well as the degradation of pathologic misfolded proteins. During aging, cellular proteostatic capacity declines, leading to an increase in protein aggregates with physiological consequences driven by activation of stress response pathways in downstream cell types. Recently, by profiling transcriptional changes occurring in the aging mouse brain, our group identified chaperones that decrease in brain endothelial cells (BECs) with age, while simultaneously, in the same cells, levels of stress-inducible genes known to increase upon sensing misfolded proteins (e.g. Hspa1a, Hsp90aa1) were elevated – signatures which reversed post-parabiosis. Although BECs are some of the most vulnerable cells in AD, with many patients exhibiting altered blood-brain barrier (BBB) integrity, proteostasis machinery of these critical barrier cells has not been investigated. Taken together, in aging BECs, (1) molecular chaperones decrease, while (2) stress-inducible heat shock proteins (HSPs) increase, potentially reflecting the presence of misfolded proteins throughout the aged brain. To this end, we first seek to identify proteins that aggregate in the brain during aging and upon parabiosis via mass spectrometry. In doing so, we will profile aging-associated proteins which aggregate in a deleterious, yet reversible manner. Will then assess the ability of identified aggregation-prone proteins to activate stress response pathways in BECs. While our first aim asks, what is responsible for activation of stress-inducible HSP expression in aged BECs, our second asks what impact does increased HSP expression have on BEC function? This will be assessed by modulating Hspa1a (the most differentially expressed HSP in aging) levels using primary murine and human BECs alongside AD mutation-containing human induced pluripotent stem cell (iPSC)-derived BECs via lentiviral constructs. In parallel, we will modulate levels of Hspa1a in vivo specifically within BECs of mice via adeno-associated viral vectors. Expression of BEC markers and function wil...