Transgenic APOEε4/4 overexpression induces reactivity in astrocytes with a European APOEε4/4 local ancestry, but not in astrocytes with an African APOEε4/4 local ancestry

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Recently we demonstrated that local genomic ancestry (LA) drives the difference in AD risk between European (EU) and African (AF) carriers of APOEε4/4. As a follow-up study using single-nuclei RNAseq, we found that AD APOEε4 homozygotes with EU Local Ancestry (LA) had a significantly increased APOEε4 expression compared to AD APOEε4/4 homozygotes with AF LA. In two of the EU LA patients, an astrocyte cluster with the highest APOEε4 expression was observed and expressed a panel of genes consistent with A1 reactive astrocytes (A1RA). No such cluster was seen in the AF LA. As a previous study in mice suggested that APOEε4 expression was a contributor to the development of A1RA, we sought to explore the relationship of increased APOEε4 expression and A1RA using inducible pluripotent stem cells (iPSC)-derived astrocytes.APOEε4/4 astrocytes from one European and one African LA were derived from iPSC lines. The astrocytes were brought to maturity (Day 54 in-vitro), and then each group was treated with either a cytokine cocktail (IL-1a, hTNFa, C1q) or overexpressed with APOEε4 by lentiviral transduction for 14-days (two replicates per line). mRNA was extracted and qPCR was performed to measure changes in APOEε4 and markers of A1RA (C3, GBP2, IFITM3).iPSCs were validated by immunocytochemistry (ICC) and qPCR for Nanog, SOX2 and OCT4 and subsequently differentiated into astrocytes. Astrocyte validation was performed by GFAP and βS100 ICC. Astrocytes treated with the cytokine cocktail had a 100- and 600-fold increase in C3 in EU and AF LA astrocytes respectively, as well as significant increase in GBP2 and IFITM3 compatible with A1RA. When overexpressing APOEε4, a significant increase in C3 was observed in the EU LA astrocytes, while no increase was observed in the AF LA astrocytes.Our preliminary results support the hypothesis that APOEe4 overexpression can increase the conversion of astrocytes to the toxic A1RA state in EU LA astrocytes. It is intriguing that this effect was not seen in the African LA astrocytes. By increasing the sample size, we will increase the significance of the association between APOEe4 overexpression and A1RA induction in EU LA astrocytes compared to their AF counterparts.