ABSTRACT Alzheimer’s disease (AD) is a burgeoning neurological disease1 that remains refractory to understanding its pathogenesis. While the accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) in the brain characterizes AD2-4, their effect on the underlying molecular mechanisms that perturb the cellular microenvironment, induce molecular changes, and lead to neurodegeneration remain elusive. Our study bridges this gap by investigating the unexplored role of alternative splicing in AD pathology and its spatial context. By developing a novel approach that leverages single-nucleus cDNA, new long-read RNA sequencing protocols, and bleeding-edge in situ sequencing (ISS) our multiomic approach will profile isoform expression at single-cell resolution in the inferior frontal gyrus (IFG), a region pertinent to AD. Through this approach, our proposal will identify cell-type specific isoform changes associated with AD and create a spatial map of AD splicing dysfunction. Additionally, immunofluorescence and H&E staining will complement ISS to elucidate the spatial distribution of splicing signatures relative to Aβ plaques and NFT pathology. The integration of these techniques will provide a unique high-resolution isoform map that will shed light on the selective vulnerability to AD pathology. This proof-of-principle study holds significant promise for identifying potential therapeutic targets and advancing prevention and treatment strategies for AD.