Spatial learning-specific remodeling of the hippocampal palmitoylome

Protein S-palmitoylation is a reversible lipid modification that regulates protein trafficking, membrane association, and synaptic signaling, yet its role in learning-induced hippocampal plasticity remains incompletely understood. Here, we investigated how spatial learning remodels the hippocampal palmitoylome in rats trained in a Morris water maze using short-term training (1 session, 15 trials; test at 1 h, STT) or long-term training (4 sessions over 4 days; 4 trials/session; test at 24 h, LTT). Palmitoylated proteins were profiled using acyl-biotin exchange followed by tandem mass tag labeling and LC-MS/MS. In total, 5,260 proteins were identified, including 763 palmitoylated species. Spatial learning induced robust and time-dependent remodeling of protein S-palmitoylation, with pronounced differences between STT and LTT. Comparison of trained and yoked controls revealed 186 differentially palmitoylated proteins (DPPs) in STT and 62 in LTT, indicating stronger early molecular reorganization. Notably, yoked animals also displayed substantial palmitoylation changes versus cage controls, indicating that locomotor activity and mild stress independently reshape the hippocampal palmitoylome. DPPs were broadly distributed across cellular compartments, with enrichment of synaptic proteins at both stages. STT preferentially engaged functional enrichment in synaptic vesicle cycling, GTPase signaling, cytoskeletal remodeling, mitochondrial metabolism, and secretory pathways, whereas LTT was associated with protein translation, synaptic membrane organization, and structural plasticity, consistent with consolidation processes. Protein-protein interaction and KEGG analyses supported a transition from widespread early network remodeling toward more selective regulation of synaptic and translational machinery. Site-specific analysis further identified previously unreported palmitoylation sites in rat hippocampal proteins. Together, these data demonstrate that spatial learning dynamically reshapes the hippocampal palmitoylome in a temporally structured manner, suggesting a key role for S-palmitoylation in coordinating metabolic and synaptic adaptations underlying memory formation.