Crow, A.

Abstract Background The rare APOE3 ‐Christchurch ( APOE3Ch ) variant is associated with remarkable protective effects, delaying the age at onset of PSEN1 ‐E280A driven autosomal dominant Alzheimer's disease (AD). While much of the research on APOE3C h has focused on its role in AD, little is known about how this variant impacts APOE biology under non‐disease conditions. Using cortical organoids as a model system, we aim to investigate the fundamental mechanisms by which APOE3Ch alters APOE biology, including its effects on metabolism and cellular interactions, to better understand its unique properties and potential implications for broader applications. Method Cortical organoids were derived from the isogenic neurodegeneration iPSC line series, with either the APOE3 or APOE3Ch genotype. Single‐cell RNA sequencing (scRNA‐seq) was performed on three independent batches of 6‐month‐old cerebral organoids to identify pathways differentially regulated by APOE3Ch. To further investigate APOE lipidation and its effects on lipid metabolism, lipidomics analysis was conducted on 8‐month‐old cerebral organoids, offering additional insights into APOE3Ch ‐specific biological mechanisms. Validation was also carried out using iPSC‐derived astrocytes and neurons to confirm key findings. Result ScRNA‐seq revealed differences in cell populations and astrocyte subtypes between APOE3 and APOE3Ch organoids, with mitochondrial‐related pathways differentially regulated in APOE3Ch cerebral organoids. These findings were validated in iPSC‐derived astrocytes. APOE3Ch iPSC‐derived astrocytes exhibited increased APOE secretion compared to APOE3 . They also demonstrated enhanced mitochondrial function, as evidenced by increased spare respiratory capacity measured by Seahorse stress assays. Furthermore, APOE3Ch astrocytes showed reduced lipid droplet formation following oleic acid treatment. Lipidomics analysis of APOE3Ch cerebral organoids revealed decreased cholesterol ester levels, suggesting alterations in lipid metabolism. Conclusion Our findings provide novel insights into how the APOE3Ch variant alters APOE biology, revealing differences in mitochondrial function and lipid metabolism. The differential regulation of mitochondrial pathways in APOE3Ch cerebral organoids, along with the enhanced mitochondrial capacity and altered lipid metabolism observed in APOE3Ch astrocytes, highlights the unique properties of this variant. These results offer valuable mechanistic understanding that may inform future therapeutic strategies targeting APOE biology, particularly in the context of AD.

AbstractBackgroundHomozygosity for the rare APOE3‐Christchurch (APOE3Ch) variant, encoding for apoE3‐R136S (apoE3‐Ch), was linked to resistance against an aggressive form of familial Alzheimer’s disease (AD). Carrying two copies of APOE3Ch was sufficient to delay autosomal AD onset by 30 years. This remarkable protective effect makes it a strong candidate for uncovering new therapies against AD. Thus, we aim to explore the protective mechanisms of APOE3Ch against AD onset, to inform therapy.MethodsWe used both amyloid mouse models and human induced‐pluripotent stem cells (iPSC) models to address this aim. We examined whether astrocytic expression of apoE3‐Ch through an AAV‐mediated approach can mitigate AD‐related pathology and toxicity in early (3.5 months old) and late (8 months old) amyloid pathology in 5xFAD mice. We also used isogenic iPSC lines carrying the APOE3Ch variant that were generated from APOE3 parental lines using CRISPR/Cas9 technology. Biochemical and biophysical properties of both recombinant and native apoE3‐Ch lipoprotein particles secreted by iPSC‐derived astrocytes have been investigated using different techniques including heparin affinity chromatography and size‐exclusion chromatography.ResultsWe investigated the impact of astrocytic apoE3‐Ch on amyloid pathology and toxicity in 3.5‐month‐old and 8‐month‐old 5xFAD mice. We confirmed successful AAV‐mediated overexpression of apoE3 and apoE3‐Ch in astrocytes. We found apoE3‐Ch specific changes in protein levels and solubility. Astrocytic expression of apoE3‐Ch reduced soluble Aβ oligomer levels and ameliorated ER stress response compared to apoE3 in the 8‐month‐old 5xFAD mice. Furthermore, our findings showed altered biochemical properties of HEK cell‐derived apoE3‐Ch protein, including decreased heparin binding, altered receptor binding, and enhanced lipid accepting capacity. We also characterized APOE3Ch iPSC‐derived cell models, including iPSC‐derived astrocytes, neurons, and cerebral organoids, and found differences between APOE3 and APOE3Ch at the functional level in these iPSC models.ConclusionsThe proposed work and the developed methods to study apoE3‐Ch promises to provide new insights into the possible roles of this rare, mutated protein in protecting against AD, offering new therapeutic avenues for AD treatment.