PROJECT SUMMARY We will test the hypothesis that choroid plexus epithelial cells (CPECs) are affected in late onset Alzheimer's Disease (AD) associated with the type 4 isoform of apolipoprotein E (APOE4). At least one copy of APOE4 is present in 56-65% of people with AD, and two copies are highly predictive of AD. APOE4 has been implicated in faulty clearance of the toxic Aβ peptide associated with AD, as well as in numerous other AD-related functions in various cell types. Rodent CPECs are known to remove toxins from the cerebrospinal fluid (CSF), express high levels of APOE, and take up and transport Aβ peptides. However, human CPECs have not been well studied, in part due to the difficulty in acquiring healthy human cells. Our laboratory has developed a protocol to derive human CPECs from pluripotent stem cells, which provides the opportunity to study basic human CPEC functions and their roles in AD and other diseases. We propose in Aim 1 to derive CPECs using existing induced pluripotent stem cells (iPSCs) from APOE4/4 AD patients and from their isogenic APOE3/3 counterparts that have been edited using CRISP/Cas9. We will monitor the CPEC derivations for changes in differentiation efficiency, then test the specific hypothesis that Aβ uptake is impaired in APOE4/4 CPECs. In Aim 2, we will characterize the transcriptomes of individual human CPECs using single cell RNA sequencing (scRNAseq) to look for gene expression changes in CPEC subpopulations that correspond to the Aβ uptake findings from Aim 1. Based on a prior study that examined neurons, astrocytes, and microglia derived from APOE isogenic iPSCs, we anticipate a broad spectrum of gene expression differences that are unique to CPECs and predictive of altered functions. We will then validate scRNAseq findings by RT-qPCR and immunostaining, then cross-validate in vivo by immunostaining postmortem choroid plexus specimens from patients with known APOE genotypes. We envision this R21 proposal leading to subsequent R01 submissions that extend this work to CPECs derived from additional isogenic pairs involving APOE and other AD risk genes, to test additional emergent hypotheses regarding altered CPEC functions in AD, and to explore potential therapeutic interventions to correct impaired CPEC functions.