Single-cell, spatial, and machine learning analyses identify ANKIB1 as an oligodendrocyte-anchored palmitoylation-associated candidate biomarker of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cognitive decline, yet most candidate therapies targeting amyloid-β have failed to robustly modify disease progression, underscoring an incomplete mechanistic understanding. Oligodendrocytes (ODCs) have recently emerged as active contributors to AD pathology, but the cell-type-specific role of protein S-palmitoylation - a reversible lipid post-translational modification regulating membrane targeting and trafficking - in AD remains uncharacterized. Here we performed an integrated multi-omics analysis combining single-cell RNA sequencing (GSE233208; 48,714 cells), five bulk transcriptomic cohorts (769 samples), and 10× Visium spatial transcriptomic data from 16 brain sections. Palmitoylation activity was quantified at single-cell resolution using a curated 46-gene set; machine learning frameworks (LASSO, random forest, and XGBoost) were used to distil a parsimonious biomarker panel; and four complementary metrics (Jaccard index, overlap coefficient, colocalization percentage, and odds ratio) were applied to evaluate spatial gene-cell colocalization. Oligodendrocytes emerged as the cell population most strongly enriched for palmitoylation activity in the AD brain, supported by per-cell-type scoring, odds ratio enrichment, and an ODC-anchored 658-gene palmitoylation module. Intersection with AD-associated differentially expressed genes yielded 89 candidate genes, from which machine learning identified a seven-gene biomarker panel (ANKIB1, SPAG9, HBP1, SLC25A29, FAM171A1, ATXN10, and NDUFV1). ANKIB1 consistently topped every layer of analysis, with the highest AUC for AD discrimination (0.813, 95% CI 0.782-0.844) and persistent spatial colocalization with ODC-enriched regions across early AD and AD. Network pharmacology nominated AHR and TRIM11 as candidate upstream regulators and catalogued compounds with reported gene interactions. These findings identify the ODC-palmitoylation axis as a previously underappreciated dimension of AD biology and position ANKIB1 as a leading candidate biomarker of ODC dysfunction in AD.