Hematopoietic EphA4 deficiency alters microglial heterogeneity and improves chronic spatial memory after brain injury.

Traumatic brain injury (TBI) elicits a sustained innate immune response involving both resident microglia and infiltrating peripheral immune cells. However, the influence of peripheral immune-derived signals on microglial dynamics and functional recovery remains poorly understood. We previously identified the receptor tyrosine kinase EphA4 as a regulator of acute neuroinflammation following TBI. In this study, we employed bone marrow chimeric mice with hematopoietic-specific EphA4 knockout and subjected them to controlled cortical impact (CCI) to investigate the role of EphA4 in modulating acute and chronic microglial responses. Mice lacking EphA4 in hematopoietic cells exhibited reduced microglial apoptosis and proliferation at 3 days post-injury (dpi), accompanied by increased microglial sphericity at 3- and 60-days post-injury (dpi) within the injured cortex. These morphological changes coincided with enhanced spatial proximity to peripheral-derived macrophages. Behaviorally, EphA4-deficient chimeric mice demonstrated improved performance in the novel object recognition test at 3 dpi, and in the T-maze at both 60 and 90 dpi. Single-cell RNA sequencing of hippocampal tissue at 90 dpi revealed microglial subpopulations uniquely enriched in EphA4-deficient chimeric mice, with transcriptional profiles associated with leukocyte differentiation, immune regulation, hematopoiesis, and endothelial development. Together, these findings demonstrate that peripheral immune-derived EphA4 signaling influences microglial heterogeneity and negatively regulates long-term neuroimmune remodeling and functional recovery following TBI.