APOE 4/4 promotes dysfunctional and inflammatory phenotypes concomitant with impaired maturation of hiPSC-derived astrocytes

Alzheimer’s disease (AD) is the leading cause of dementia in the aging population, with the ε4 allele of apolipoprotein E ( APOE ) being the strongest genetic risk factor. Although astrocytes are a major source of APOE, how APOE alleles affect astrocyte maturation and function remains unclear. We generated human induced pluripotent stem cell (hiPSC)-derived astrocytes from AD patients carrying ε3/ε3 and ε4/ε4 alleles and from healthy controls (HC). We also used isogenic gene-edited hiPSC lines homozygous for each APOE allele and an APOE knock-out line to identify allele-specific phenotypes and distinguish gain- from loss-of-function mechanisms. APOE 4/4 astrocyte cultures showed significant reductions in GFAP- and S100β-positive cell percentages compared to APOE 2/2 and APOE 3/3, with no changes in GLT-1- and AQP4-positive cells. Astrocytes of all genotypes responded to IL-1β + TNFα by increasing proinflammatory cytokine expression and release, and to both IL-1β + TNFα and Aβ _1-42 by changing morphology, with APOE 4/4 astrocytes showing increased IL6 mRNA and morphological branching upon IL-1β + TNFα stimulation. Notably, under basal conditions, APOE 4/4 astrocytes showed significant reductions in glutamate uptake capacity and cell size alongside increased IL-6 release and CXCL3 mRNA expression. In Aβ _1-42 uptake experiments, the proportion of Aβ ⁺ astrocytes was higher in APOE 4/4 than in APOE KO cultures. Most phenotypes were absent in APOE KO astrocytes, suggesting that the effects of APOE 4/4 were predominantly mediated through gain-of-function mechanisms. Our results indicate that APOE 4/4 alters astrocyte morphological and molecular maturation while promoting inflammation, disturbing glutamate and Aβ handling under basal conditions. It suggests that APOE ε4/ε4 genotype disrupts astrocyte development and key processes of cellular homeostasis early in Alzheimer’s disease etiopathology.