Eberts, Paulina2025-02-142025-02-142024-09https://hdl.handle.net/11299/269983University of Minnesota Ph.D. dissertation. September 2024. Major: Chemical Engineering. Advisors: Samira Azarin, Casim Sarkar. 1 computer file (PDF); xii, 157 pages.The blood-brain barrier (BBB) is a highly selective barrier that is crucial for maintaining brain homeostasis. Disruption of the BBB can have deleterious effects, and it is believed that certain types of BBB damage are central to the etiology of Alzheimer's disease (AD). Given its role in disease onset, disrupted BBB function could be an attractive target for potential early intervention in AD. However, more needs to be understood about its functions in relation to AD to best inform these approaches. This includes understanding the relationship between the BBB and the multitude of factors thought to contribute to AD development, including genetic and nongenetic risk factors. To facilitate this, improved models and strategies are required to identify mechanistic links between risk factors and early features of AD. Animal models have been indispensable in AD research, providing critical insights into disease mechanisms. However, they are limited in their ability to elucidate the specific interactions between hallmark features of AD and the BBB endothelium, partly due to critical species differences. Human in vitro models of the BBB offer a complementary approach, allowing for a highly controlled, tunable environment for the examination of complex, multifactorial interactions with greater throughput. This makes them well-suited for a systems approach, which is particularly beneficial for understanding complex diseases like AD. However, these models also have limitations, such as their lack of maturity, which decreases their relevance to a disease that primarily affects the aged adult population. Despite this, there are considerable opportunities to expand the types of insights that can be gained from them. The purpose of this dissertation is to enhance understanding of BBB functions as they relate to the earliest stages of AD through efforts to improve in vitro models, apply these models, and expand the model toolkit. In Chapter 1, the fundamentals of AD are reviewed and the role of the BBB in this context is presented. This includes a summary of models and approaches typically used to examine the BBB in relation to AD. Chapter 2 describes methods for improving models of the BBB comprised of brain microvascular endothelial-like cells (iBMECs) derived from human induced pluripotent stem cells (hiPSCs) to better represent the adult BBB. This work finds that extending the culture of iBMEC models of the BBB induces quiescence and improves structural organization in the barriers, indicative of a more mature, rested phenotype. Chapter 3 presents the application of in vitro models of the BBB to map out and characterize amyloid-beta binders expressed by the brain endothelium. This work identifies HspB1 as a key amyloid-beta binder expressed by the brain endothelium. Further examination of HspB1 links its behavior to features of genetic and non-genetic risk for AD, namely ApoE isoform and oxidative stress. Chapter 4 discusses the extension of a proximity labeling technology, TurboID, for use in identifying protein-protein interactions at the BBB surface. This includes recombinant expression of TurboID constructs for extracellular use. Design principles determined in AlphaFold for the robust design of constructs implicate linker selection in ensuring proper construct binding activity. Chapter 5 discusses key conclusions from each of these efforts, as well as future directions.enAlzheimer’s diseaseamyloid-betaapolipoproteinblood-brain barrierhuman induced pluripotent stem cellsTurboIDThesis or Dissertation