Neuronal SPI1 suppression enhances axonal regeneration after spinal cord injury through Rassf10 downregulation.

Background: Axonal regeneration following spinal cord injury (SCI) is hindered by the intrinsic inhibitory properties of spinal neurons. While the role of the transcription factor SPI1 (PU.1) has been well established in myeloid cells, its function in neurons, particularly in regulating axonogenesis, remains poorly defined. The data from recent single-cell sequencing studies have indicated that SPI1 is clearly expressed in spinal neurons. Thus, we aimed to explore the effect of SPI1 on axonal growth and its underlying molecular mechanisms. Methods: Primary cultured rat spinal neurons, CRISPR/Cas9-mediated SPI1 mutation in zebrafish, and neuron-specific AAV9-shRNA delivery after rat spinal cord hemisection were utilized to manipulate SPI1 expression. RNA-seq, luciferase reporter assays, ChIP-qPCR, and Cut&Tag assays were employed to identify and validate Rassf10 as a candidate target gene of SPI1. Axonal outgrowth and functional recovery postinjury were assessed both Results: Neuronal SPI1 expression declined progressively during spinal cord development but was markedly upregulated after SCI. SPI1 knockdown promoted axonal outgrowth, whereas SPI1 overexpression significantly reduced the axonal length. The transcriptomic analysis revealed that Rassf10 is a target gene of SPI1, which was subsequently validated by the results of luciferase reporter, ChIP-qPCR, and Cut&Tag assays. Conclusions: SPI1 impairs axonal growth and regrowth by transcriptionally activating Rassf10 expression, with this inhibitory effect mediated by the suppression of p38 MAPK signaling. These findings suggest that targeting the SPI1-Rassf10 pathway represents a promising therapeutic strategy for improving neural repair after SCI.