TBI is the leading cause of injury, disability, and death across the world, with age, sex, and population being associated with risk for injury (Cheng et al., 2017). TBI is a disruption in the normal function of the brain caused by a bump, blow, or jolt to the head, or penetrating injury (Traumatic Brain Injury and Concussion, 2024). Individuals frequently suffer from long-term physical and neurocognitive disabilities that are often dictated by early pathological events (Azouvi et al., 2017; Haarbauer-Krupa et al., 2021). TBI elicits a series of sequelae, including neuroinflammation, blood-brain barrier disruption, excitotoxicity, oxidative stress, and neuronal cell death in the acute phase post-injury (Rola et al., 2006; Evanson et al., 2018; McKee and Daneshvar, 2015; Wu et al., 2021; Cash et al., 2023). These events often proceed through interconnected cellular and molecular pathways that contribute to both primary damage (initial injury) and secondary damage (delayed injury processes that exacerbate the initial damage) (Ng and Lee, 2019). Understanding the cellular and molecular drivers of these events, particularly in specific cell types, may open new avenues for treatment. One promising molecular target for addressing TBI pathology is the EphA4 receptor, a member of the erythropoietin-producing hepatocellular carcinoma (Eph) receptor family of tyrosine kinases. Eph receptors, along with their ephrin ligands, play essential roles in axonal guidance, neuronal development, and synaptic plasticity during development and are increasingly recognized for their role in postnatal neural injury responses (Lai and Ip, 2009). EphA4, in particular, is expressed in various central nervous system (CNS) cells, including neurons, astrocytes, and endothelial cells, and regulates cellular behavior by binding to membrane-bound ephrin ligands on adjacent cells (Kania and Klein, 2016). These interactions induce bidirectional signaling that influences cell adhesion, migration, and cytoskeletal dynamics, thereby shaping tissue architecture and cellular interactions (Coulthard et al., 2012). In the context of CNS injuries, such as spinal cord injury, stroke, and TBI, EphA4 has been shown to exacerbate injury by promoting axonal retraction, neuroinflammation, and glial scarring (Frugier et al., 2012; Herrmann et al., 2010).

Our recent findings show that endothelial-derived EphA4 plays a novel role in mediating tissue damage following TBI, in part through regulation of the blood-brain barrier (BBB) (Cash et al., 2023). Endothelial cells and astrocytes in the brain form the BBB, a critical interface that regulates the entry of cells and molecules into the CNS and maintains homeostasis (Kadry et al., 2020). Following TBI, BBB disruption is commonly observed, is a key determinant in patient outcome, and is associated with increased infiltration of immune cells, elevated neuroinflammation, and neuronal injury (Hay et al., 2015; Xiong et al., 2018). Given EphA4's role in modulating endothelial cell responses and BBB integrity, it is plausible that endothelial-derived EphA4 may have additional contributions to the cellular milieu that may be identified through single-cell RNA sequencing (scRNA-seq). However, the precise mechanisms by which endothelial-derived EphA4 influences other CNS cells, such as astrocytes, within the damaged cortex are poorly understood. Astrocytes are essential for neuronal support, BBB maintenance, and neuroinflammation regulation (Manu et al., 2023). Following injury, astrocytes undergo reactive changes that can either exacerbate or mitigate tissue damage, depending on the specific signaling context (Anderson et al., 2016; Cieri and Ramos, 2025). Understanding how endothelial-derived EphA4 influences astrocyte behavior at the transcriptional level could, therefore, yield valuable insights into the cellular networks underlying TBI pathology.

To address these questions, we utilize scRNA-seq to examine cell-specific transcriptional changes in the cortex following TBI. Single-cell transcriptomics provides the resolution needed to investigate the molecular responses of individual cell populations, allowing us to identify distinct signaling pathways and gene expression patterns that may mediate TBI pathology in specific cell types. We identified that endothelial cell-specific EphA4 deletion (EC-KO) had the greatest effect on the differential gene expression of cortical endothelial cells and astrocytes; revealing a potential role for endothelial-astrocyte communication following TBI. In addition, we identified through scRNA-seq a subset of Sox9-postive cortical astrocytes that express the MerTK receptor, a known regulator of efferocytosis (Bae et al., 2022; Vandivier et al., 2006)and mediator of neuroinflammation (Cai et al., 2016).Moreover, the loss of endothelial EphA4 altered key genes involved in metabolic pathways, such as cholesterol, spermine, putrescine, and ceramide biosynthesis, in astrocytes and endothelial cells, suggesting that EphA4 signaling is critical for maintaining cellular homeostasis and metabolic balance in the neurovascular unit. These metabolic disruptions may contribute to impaired BBB integrity and increased vulnerability to neuroinflammatory processes. This study highlights the role of endothelial-derived EphA4 in driving transcriptional alterations in the cortical cellular milieu, advancing our understanding of TBI's cellular pathology and identifying potential metabolic and signaling targets for therapeutic intervention aimed at preserving BBB integrity and reducing neuroinflammation.

Our transcriptional analysis indicates that astrocytes and endothelial cells are most profoundly impacted by endothelial deletion of EphA4. Largely, this study supports a role for endothelial EphA4 in mediating cortical tissue damage and cell-specific transcriptomic changes following TBI. These findings provide greater insight into the molecular underpinnings and cellular mechanisms of endothelial EphA4 following TBI.