A Two-Hit Model of Traumatic Brain Injury and Stress-Enhanced Fear Learning Reveals Altered RNF25–GKAP Regulation in the Medial Prefrontal Cortex

<title>Abstract</title> <p> <bold>Background</bold> Traumatic brain injury (TBI) is associated with an increased risk of post-traumatic stress disorder (PTSD), but the molecular alterations through which prior brain injury enhances vulnerability to subsequent trauma-related fear remain incompletely understood. This study investigated the behavioral and molecular responses to combined physical brain injury and psychological trauma, with a focus on protein homeostasis in the medial prefrontal cortex (mPFC). <bold>Methods</bold> A two-hit mouse model was established by combining controlled cortical impact-induced TBI with stress-enhanced fear learning (SEFL). Fear-related behavior, anxiety-like behavior, and locomotor activity were assessed. Exploratory transcriptomic and proteomic profiling of the mPFC was performed using three biological replicates per group. Candidate molecular features were prioritized through differential-expression analysis, least absolute shrinkage and selection operator regression, and random forest analysis. The relationship between RNF25 and guanylate kinase-associated protein (GKAP; encoded by <italic>Dlgap1</italic> ) was investigated using structural modeling, reciprocal co-immunoprecipitation, and an <italic>in vitro</italic> ubiquitination assay. Structure-based virtual screening was performed to prioritize compounds predicted to bind RNF25. <bold>Results</bold> Prior TBI enhanced SEFL-associated contextual freezing and anxiety-like behavior without detectable changes in locomotor activity or initial fear acquisition. Exploratory multi-omics profiling revealed distinct but partially convergent molecular responses to TBI and SEFL. TBI was associated predominantly with enrichment of inflammatory, extracellular matrix, and metabolic pathways, whereas SEFL was associated mainly with synaptic and cytoskeletal processes. Combined TBI and SEFL induced relatively limited transcriptomic changes but broader proteomic alterations involving intracellular trafficking, protein quality control, cellular stress responses, and synaptic regulation. GKAP was prioritized as a candidate molecular feature of the combined condition and showed increased protein abundance despite reduced <italic>Dlgap1</italic> transcript levels. Reciprocal co-immunoprecipitation of endogenous proteins supported an association between RNF25 and GKAP, and RNF25 promoted GKAP ubiquitination in an <italic>in vitro</italic> reconstituted system. Total RNF25 abundance in the mPFC was not detectably altered following combined TBI and SEFL, whereas GKAP was enriched in the postsynaptic density-enriched fraction. Virtual screening further prioritized dobutamine and several AG-205-related scaffolds as putative RNF25-binding compounds. <bold>Conclusions</bold> Altered GKAP abundance and postsynaptic distribution were identified as candidate molecular features of TBI-exacerbated fear sensitization. Together with the observed RNF25–GKAP association and RNF25-mediated GKAP ubiquitination <italic>in vitro</italic> , these findings raise the hypothesis that altered RNF25-associated GKAP proteostasis may be relevant to postsynaptic remodeling in the mPFC after combined physical and psychological trauma. The causal contribution of this pathway to the behavioral phenotype, as well as the biochemical and pharmacological activities of the computationally prioritized compounds, requires further experimental validation. </p>