Super-Resolved Spatial Transcriptomics Reveals Early Changes in RNA Localization in the 5xFAD Hippocampus

Cell-type-specific changes in gene expression and RNA localization are hallmarks of Alzheimer's disease (AD) and other neurodegenerative disorders, yet early spatial dysregulation remains poorly defined. Here, we applied Expansion Sequencing (ExSeq) to map the spatial distribution of 101 genes at super-resolution in the hippocampus of 4-week-old 5xFAD and wild-type (WT) mice, prior to overt pathology. We uncovered early differences in RNA spatial organization that occurred independently of changes in overall expression, identifying 23 genes with altered localization but unaltered abundance in the 5xFAD hippocampus. Leveraging ExSeq nanoscale resolution, we further performed spatial RNA velocity analysis and detected cell-type- and region-specific state differences between 5xFAD and wild type mice, which were linked to local cell-cell interactions. Together, these findings suggest that disrupted RNA positioning is an early molecular feature of AD and provide a spatially resolved view of transcriptomic alterations during disease onset in a mouse model of AD.