Analysis of receptor-mediated transcytosis pathways implicated in Alzheimer's disease

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PROJECT SUMMARY The blood-brain barrier (BBB) is a highly selective barrier comprised of brain microvascular endothelial cells (BMECs) that presents a major bottleneck for drug delivery to treat disorders such as Alzheimer's disease (AD). In addition to preventing therapeutics from entering the brain, BBB dysregulation is observed in the earliest stages of AD, leading to a decreased ability to clear amyloid beta (Aβ) that largely contributes to the accumulation of Aβ in the brain. Studying receptor-mediated transcytosis in the BBB is central to both discovering novel routes for delivery of therapeutics into the brain for treatment of AD and developing a mechanistic understanding of early pathological changes to the BBB that lead to eventual Aβ accumulation. The parent grant is focused on developing novel high-throughput screening technologies to identify the full set of transcytosis receptors in the intestinal epithelium for applications in oral drug delivery. The objective of the proposed supplemental work is to apply these technologies to a stem cell-derived model of the BBB. The use of human induced pluripotent stem cell-derived BMECs will enable studies of transcytosis in the AD BBB using cell lines with varying copy numbers of the APOE-ε4 allele, which is the greatest genetic risk factor for late-onset AD. While the precise connections between the APOE-ε4 allele and BBB dysfunction are not well characterized, it has been shown that expression of apoE4, encoded by APOE-ε4, alters Aβ transcytosis across the BBB, inhibiting its proper clearance. The proposed research consists of two aims: (1) identification of novel receptors for transcytosis-mediated drug delivery to the brain for AD treatment and (2) identification of Aβ receptors in BMECs. In the first aim, BMECs will be differentiated from hiPSC lines containing zero, one or two copies of the APOE-ε4 allele, and a novel transcytosis-based directed evolution method, as detailed in the parent grant, will be used to discover new protein ligands that are able to transcytose the BMEC monolayer. The receptors mediating transcytosis of these ligands will then be identified as potential targets for delivery of therapeutics to the AD BBB. In the second aim, crosslinking mass spectrometry (CLMS) will be utilized to identify the full repertoire of Aβ receptors in BMECs differentiated from iPSC lines with varying APOE-ε4 status. These studies will enable analysis of any genotype- specific differences in expression of known Aβ receptors, such as LRP1 and RAGE, as well as discovery of new Aβ receptors among the different BMECs. These pilot studies will be incorporated into a new R01 proposal to elucidate new mechanisms of transcytosis related to Aβ clearance as well as delivery of therapeutic payloads to the brains of AD patients.