Dynamic Atlas and Functional Regulatory Network of the Whole-Brain Proteome Across 24-Hour Temporal States in Vespertilio sinensis

Abstract As the only mammals capable of powered flight, bats exhibit extreme metabolic fluctuations adapted to flight and a nocturnal lifestyle, making them unique models for studying diurnal rhythms and energy homeostasis. We performed directDIA-based quantitative proteomics on whole-brain tissues of Vespertilio sinensis across four distinct 24-hour physiological states: Rest, Sleep, Wake, and Activity. Among the 7652 identified proteins, a total of 643 differentially expressed proteins (DEPs) were screened via pairwise comparisons across timepoints. Time-series clustering further resolved two statistically significant temporal expression modules (Module 3 and Module 9). Combined with functional enrichment of DEPs and phase set enrichment analysis (PSEA) of 574 rhythmic proteins, our multi-layered omics results collectively uncovered stage-specific molecular adaptive patterns. The Active state upregulated oxidative phosphorylation and thermogenesis for high energy demands, the Rest state activated immune clearance and autophagy to eliminate flight-induced metabolic damage, the Sleep state suppressed global transcription, calcium signaling and DNA repair to reduce neural energy consumption, and the Wake state (pre-dusk) pre-activated sulfur biosynthesis, antioxidant defense, and energy metabolic pathways to prepare for upcoming nocturnal activity. Parallel transcriptomic and proteomic rhythmic analysis further identified 19 conserved oscillatory molecules at both molecular layers, revealing partial transcript-protein rhythmic decoupling in the bat brain and refining the diurnal regulatory landscape. As the first systematic atlas of the bat whole-brain proteome across a 24-hour cycle, this study uncovers molecular strategies maintaining brain homeostasis, providing a foundation for understanding diurnal physiological adaptation, flight energy regulation, and circadian output pathways.