Minja Belic1*, Joanna Bons1, Sydney Alderfer1, Mikayla Hady1, Tyne L. M. McHugh1, Kizito-Tshitoko Tshilenge1, Maria A. Sanchez1, Kevin Schneider1, Long Wu1, Nikola Markov1, Birgit Schilling2, David Furman1 and Lisa M. Ellerby2
1Buck Institute for Research on Aging
2Buck Institute for Research on Aging; University of Southern California, Leonard Davis School of Gerontology
mbelic [at] buckinstitute.org
Abstract
Apolipoprotein E (APOE) genotype is a major determinant of late-onset Alzheimer’s disease risk and a modulator of neural aging, yet the effects of APOE isoforms on developmental trajectories in human neural tissue remain incompletely understood. Here, we combined single-cell RNA sequencing with quantitative proteomics in induced pluripotent stem cell-derived isogenic human cerebral organoids carrying APOE ε2/ε2, ε3/ε3, or ε4/ε4 alleles to characterize genotype- and stress-dependent remodeling across cellular, transcriptional, and secretory axes.
We profiled ~46,000 cells across control and irradiation-induced senescence conditions using single-cell RNA-seq. Raw counts were denoised with CellBender to remove ambient-RNA contamination, and scVI was applied to learn a latent embedding while adjusting for stress-related and technical covariates before clustering and pseudotime trajectory analysis. Cells were annotated using snapseed with marker panels adapted from the Human Neural Organoid Cell Atlas (HNOCA), and cell-type composition differences were tested with scCODA.
Cell-type composition differed significantly across APOE genotypes at baseline, with APOE4 organoids showing a higher relative abundance of glial and excitatory neuronal populations. After adjusting for these compositional differences, residual genotype-dependent differences in differentiation trajectories persisted, indicating that APOE4 affects both the cell types and their progression through development. Following irradiation, organoids exhibited genotype-dependent transcriptional responses and changes in inferred regulatory activity beyond the baseline compositional differences.
In parallel, data-independent acquisition mass spectrometry quantified >6,500 proteins across organoid lysates and secretomes under basal and senescence conditions. Single-sample gene-set enrichment analysis (ssGSEA) showed APOE isoforms differentially regulated aging-associated pathways, with APOE4 exhibiting increased senescence signatures and an inflammatory senescence-associated secretory phenotype (SASP), while APOE2 showed reduced senescence and a SASP enriched in antioxidant enzymes and chaperones.
Together, these results suggest that APOE4-associated risk biology already emerges in developmental tissue composition and stress-response programs prior to aging-related insults.
Keywords: scRNA, proteomics, cerebral_organoids, APOE, senescence
Acknowledgement: This work is supported by the NCRR shared instrumentation grant 1S10 OD028654 (PI: Birgit Schilling) for the Orbitrap Eclipse Tribrid system and by the NIH grant P01AG066591 (PI: Lisa M. Ellerby). T.L.M.M was supported by the NIA grant T32 AG052374. MS and LW were supported by T32-AG000266 (PI: Lisa M. Ellerby).