Time-of-day effects on post-exercise phosphoproteomic profiling in mouse hippocampus.

Introduction: Exercise benefits cognition, and various aspects of exercise can impact physiological outcomes. Recently, there has been a growing interest in understanding the optimal timing of exercise to maximize its benefits. However, how timed exercise influences hippocampal function and signaling pathways remains largely unexplored. Therefore, our aim was to investigate the effects of exercise timing on hippocampal phosphoproteomic profiling. Methods: The phosphoproteome/phosphoproteome was conducted by affinity enrichment and liquid chromatography-tandem mass spectrometry on hippocampus of mice immediately after acute exercise or sham-exercise at the early rest phase (ZT3) or the early active phase (ZT15). To compare the differences in exercise-regulated phosphorylated sites and proteins between the two phases, bioinformatic analyses including functional enrichment analysis, motif analysis and kinase prediction were performed. Results: A bout of acute exercise induced significant changes in the phosphorylation status of 932 and 828 phosphosites during the rest and active phase, respectively. There were only 49 overlapped differentially regulated sites on 44 proteins. Functional enrichment analysis revealed that the breadth of signaling pathways modulated by exercise at ZT3 was considerably greater. The phosphorylation status of differentially phosphorylated proteins enriched in multiple pathways was vastly different after the timed exercise, so were the predicted upstream kinases. 29% differentially phosphorylated proteins were associated with synapse structure or function. Moreover, both timepoints converged on glutamate synapse-calcium signaling-LTP pathways via distinct molecular nodes. Daytime exercise increased the pCaMKII/CaMKII ratio ( Conclusion: Timed exercise elicited time-dependent phosphoproteomic features, and only a trivial number of phosphosites were regulated regardless of the time of exercise. Preliminary analyses suggest early daytime exercise may better support hippocampus-dependent learning, while early night-time exercise may be more beneficial for reducing neuroinflammation. This study advances exercise chronobiology and provides insights into circadian regulation of hippocampal exercise physiology, and involvement of epigenomic memory in this process would be interesting to be further studied.