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

Abstract

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 in vitro and in vivo. 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. In vivo, spi1 mutation in zebrafish and neuron-specific SPI1 knockdown in rats facilitated axonal regeneration and functional recovery after SCI, as evidenced by significant increases in GAP43, NF200, and 5-HT expression and improved motor function compared with the control animals. The depletion of SPI1 or Rassf10 increased p-p38 mitogen-activated protein kinase levels and promoted axonal growth, whereas p38 inhibition abrogated these effects. 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.

Keywords

SPI1 / Rassf10 / Axonal outgrowth / p38 MAPK / Spinal cord injury

References

[1] Hilton BJ, Griffin JM, Fawcett JW, Bradke F. Neuronal maturation and axon regeneration: unfixing circuitry to enable repair. Nat Rev Neurosci 2024; 25:649-67. https://doi.org/10.1038/s41583-024-00849-3.

[2] Mahar M, Cavalli V. Intrinsic mechanisms of neuronal axon regeneration. Nat Rev Neurosci 2018; 19:323-37. https://doi.org/10.1038/s41583-018-0001-8.

[3] Oksuz O, Henninger JE, Warneford-Thomson R, Zheng MM, Erb H, Vancura A, et al. Transcription factors interact with RNA to regulate genes. Mol Cell 2023; 83:e13. https://doi.org/10.1016/j.molcel.2023.06.012.

[4] Stadhouders R, Vidal E, Serra F, Di Stefano B, Le Dily F, Quilez J, et al. Transcription factors orchestrate dynamic interplay between genome topology and gene regulation during cell reprogramming. Nat Genet 2018; 50:238-49. https://doi.org/10.1038/s41588-017-0030-7.

[5] Lerch JK, Martinez-Ondaro YR, Bixby JL, Lemmon VP. cJun promotes CNS axon growth. Mol Cell Neurosci 2014; 59:97-105. https://doi.org/10.1016/j.mcn.2014.02.002.

[6] Lloberas J, Soler C, Celada A. The key role of PU.1/SPI-1 in B cells, myeloid cells and macrophages. Immunol Today 1999; 20:184-9. https://doi.org/10.1016/s0167-5699(99)01442-5.

[7] Zhao X, Bartholdy B, Yamamoto Y, Evans EK, Alberich-Jorda M, Staber PB, et al. PU.1-c-Jun interaction is crucial for PU.1 function in myeloid development. Commun Biol 2022; 5:961. https://doi.org/10.1038/s42003-022-03888-7.

[8] Shi Y, Zheng M, Luo Y, Li J, Ouyang F, Zhao Y, et al. Targeting transcription factor pu.1 for improving neurologic outcomes after spinal cord injury. Front Neurosci 2024; 18:1418615. https://doi.org/10.3389/fnins.2024.1418615.

[9] Yu M, Ou Y, Wang H, Gu W. PU.1 interaction with p50 promotes microglial-mediated inflammation in secondary spinal cord injury in SCI rats. Int J Neurosci 2023; 133:389-402. https://doi.org/10.1080/00207454.2021.1923017.

[10] Zhang Z, Jin P, Guo Z, Tu Z, Yang H, Hu M, et al. Integrated analysis of chromatin and transcriptomic profiling identifies PU.1 as a core regulatory factor in microglial activation induced by chronic cerebral hypoperfusion. Mol Neurobiol 2024; 61:2569-89. https://doi.org/10.1007/s12035-023-03734-9.

[11] Li C, Wu Z, Zhou L, Shao J, Hu X, Xu W, et al. Temporal and spatial cellular and molecular pathological alterations with single-cell resolution in the adult spinal cord after injury. Signal Transduct Target Ther 2022; 7:65. https://doi.org/10.1038/s41392-022-00885-4.

[12] Han X, Dong Q, Wu J, Luo Y, Rong X, Han Q, et al. Rassf10 suppresses lung cancer proliferation and invasion by decreasing the level of phosphorylated LRP6. Mol Carcinog 2019; 58:1168-80. https://doi.org/10.1002/mc.23000.

[13] Wang Y, Ma T, Bi J, Song B, Zhou Y, Zhang C, et al. Rassf10 is epigenetically inactivated and induces apoptosis in lung cancer cell lines. Biomed Pharmacother 2014; 68:321-6. https://doi.org/10.1016/j.biopha.2013.12.005.

[14] Kase Y, Sato T, Okano Y, Okano H. The GADD45G/p38 MAPK/CDC25B signaling pathway enhances neurite outgrowth by promoting microtubule polymerization. iScience 2022; 25:104089. https://doi.org/10.1016/j.isci.2022.104089.

[15] Zhang T, Wang K, Luo J, Guo L. MiR-152 inhibits neuronal axon growth after spinal cord injury through regulating p38 MAPK signaling pathway. Minerva Med 2021; 112:300-2. https://doi.org/10.23736/S0026-4806.19.06224-4.

[16] Nix P, Hisamoto N, Matsumoto K, Bastiani M. Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways. Proc Natl Acad Sci USA 2011; 108:10738-43. https://doi.org/10.1073/pnas.1104830108.

[17] Wu R, Lin H, Zhang W, Sun Y, Qian X, Lin G, et al. Cooperation of long noncoding RNA LOC100909675 and transcriptional regulator CTCF modulates Cdk1 transcript to control astrocyte proliferation. J Biol Chem 2023; 299:105153. https://doi.org/10.1016/j.jbc.2023.105153.

[18] Qian X, Lin G, Wang J, Zhang S, Ma J, Yu B, et al. CircRNA_01477 influences axonal growth via regulating miR-3075/FosB/Stat3 axis. Exp Neurol 2022; 347:113905. https://doi.org/10.1016/j.expneurol.2021.113905.

[19] Feng M, Li J, Zhang J, Zhang H, Li W, Liu Y, et al. Targeting the Jun-Irf8-CD36 axis attenuates fibrotic scar formation and promotes functional recovery after spinal cord injury. Burns Trauma 2026; 14:tkag020. https://doi.org/10.1093/burnst/tkag020.

[20] Ishino T, Hashimoto M, Amagasa M, Saito N, Dochi O, Kirisawa R, et al. Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation. Biomed Res 2018; 39:95-104. https://doi.org/10.2220/biomedres.39.95.

[21] Ma C, Teng L, Lin G, Guo B, Zhuo R, Qian X, et al. L-leucine promotes axonal outgrowth and regeneration via mTOR activation. FASEB J 2021; 35:e21526. https://doi.org/10.1096/fj.202001798RR.

[22] Wu R, Sun Y, Zhou Z, Dong Z, Liu Y, Liu M, et al. MEF2C contributes to axonal branching by regulating Kif2c transcription. Eur J Neurosci 2024; 59:3389-402. https://doi.org/10.1111/ejn.16344.

[23] Hossainian D, Shao E, Jiao B, Ilin VA, Parris RS, Zhou Y, et al. Quantification of functional recovery in a larval zebrafish model of spinal cord injury. J Neurosci Res 2022; 100:2044-54. https://doi.org/10.1002/jnr.25118.

[24] Xu M, Zhao Y, Gong M, He Z, Wang W, Li Y, et al. Dehydroevodiamine ameliorates neurological dysfunction after traumatic brain injury in mice via regulating the SIRT1/FOXO3a/Bim pathway. Phytomedicine 2024; 125:155321. https://doi.org/10.1016/j.phymed.2023.155321.

[25] Wu R, Yan Y, Ma C, Chen H, Dong Z, Wang Y, et al. HMGB1 contributes to SASH1 methylation to attenuate astrocyte adhesion. Cell Death Dis 2019; 10:417. https://doi.org/10.1038/s41419-019-1645-7.

[26] Shen P, Zhang T, Chen G, Zhang B, Huang A, Duan L, et al. Recombinant P40 protein of Schistosoma japonicum inhibits TREM-1 expression in RAW264.7 cells via FOXO3a. Biomed Pharmacother 2022; 149:112826. https://doi.org/10.1016/j.biopha.2022.112826.

[27] Xu T, Ye L, Li W, Liu F, Wei T, Gu W, et al. Targeting HDAC3 suppresses ferroptosis and demyelination in white matter injury by restoring PDK4-mediated iron homeostasis. CNS Neurosci Ther 2025; 31:e70471. https://doi.org/10.1111/cns.70471.

[28] Hong H, Xu G, Bao G, Zhang J, Chen C, Chen J, et al. VIRMA promotes neuron apoptosis via inducing m6A methylation of STK10 in spinal cord injury animal models. CNS Neurosci Ther 2024; 30:e14453. https://doi.org/10.1111/cns.14453.

[29] Guo B, Qi M, Luo X, Guo L, Xu M, Zhang Y, et al. GIP attenuates neuronal oxidative stress by regulating glucose uptake in spinal cord injury of rat. CNS Neurosci Ther 2024; 30:e14806. https://doi.org/10.1111/cns.14806.

[30] Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995; 12:1-21. https://doi.org/10.1089/neu.1995.12.1.

[31] Liu S, Lin G, Yang Q, Wang P, Ma C, Qian X, et al. Depletion of SASH1, an astrocyte differentiation-related gene, contributes to functional recovery in spinal cord injury. CNS Neurosci Ther 2023; 29:228-38. https://doi.org/10.1111/cns.13998.

[32] Tao Y, Cai X, Chen Z. Can therapeutic potency of a cancer nanomedicine be predicted by pain-related behavioral test in subcutaneous tumor model? Int J Pharm X 2026; 11:100450. https://doi.org/10.1016/j.ijpx.2025.100450.

[33] Wang Y, Liu F, Shan Q, Wang X, Liu W, Chen X, et al. Functional recovery induced by KCC2-enabled relay pathways in completely injured spinal cords in adult rats. Proc Natl Acad Sci USA 2025; 122:e2421823122. https://doi.org/10.1073/pnas.2421823122.

[34] Teng L, Guan T, Guo B, Ma C, Lin G, Wu R, et al. GIP-GIPR promotes neurite outgrowth of cortical neurons in Akt dependent manner. Biochem Biophys Res Commun 2021; 534:121-7. https://doi.org/10.1016/j.bbrc.2020.11.120.

[35] Yu B, Yao C, Wang Y, Mao S, Wang Y, Wu R, et al. The landscape of gene expression and molecular regulation following spinal cord hemisection in rats. Front Mol Neurosci 2019; 12:287. https://doi.org/10.3389/fnmol.2019.00287.

[36] Sim PL, Heese K. Ligand-dependent activation of the chimeric tumor necrosis factor receptor-amyloid precursor protein (APP) reveals increased APP processing and suppressed neuronal differentiation. Neurosignals 2010; 18:9-23. https://doi.org/10.1159/000242425.

[37] Hou Y, Li S, Du W, Li H, Wen R. The tumor suppressor role of the Ras association domain family 10. Anti Cancer Agents Med Chem 2020; 20:2207-15. https://doi.org/10.2174/1871520620666200714141906.

[38] Zheng B, Tuszynski MH. Regulation of axonal regeneration after mammalian spinal cord injury. Nat Rev Mol Cell Biol 2023; 24:396-413. https://doi.org/10.1038/s41580-022-00562-y.

[39] Nakamura Y, Ueno M, Niehaus JK, Lang RA, Zheng Y, Yoshida Y. Modulation of both intrinsic and extrinsic factors additively promotes rewiring of corticospinal circuits after spinal cord injury. J Neurosci 2021; 41:10247-60. https://doi.org/10.1523/JNEUROSCI.2649-20.2021.

[40] Varadarajan SG, Hunyara JL, Hamilton NR, Kolodkin AL, Huberman AD. Central nervous system regeneration. Cell 2022; 185:77-94. https://doi.org/10.1016/j.cell.2021.10.029.

[41] Saijilafu HEM, Zhou FQ. Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons. Nat Commun 2011; 2:543. https://doi.org/10.1038/ncomms1568.

[42] Zhang G, Lu J, Zheng J, Mei S, Li H, Zhang X, et al. Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage. Neural Regen Res 2024; 19:161-70. https://doi.org/10.4103/1673-5374.375343.

[43] Zhu P, Zheng P, Kong X, Wang S, Cao M, Zhao C. Rassf7a promotes spinal cord regeneration and controls spindle orientation in neural progenitor cells. EMBO Rep 2023; 24:e54984. https://doi.org/10.15252/embr.202254984.

[44] Park J, Kang SI, Lee SY, Zhang XF, Kim MS, Beers LF, et al. Tumor suppressor ras association domain family 5 (RASSF5/NORE1) mediates death receptor ligand-induced apoptosis. J Biol Chem 2010; 285:35029-38. https://doi.org/10.1074/jbc.M110.165506.

[45] Xia M, Zhang Y, Wu H, Zhang Q, Liu Q, Li G, et al. Forsythoside B attenuates neuro-inflammation and neuronal apoptosis by inhibition of NF-kappaB and p38-MAPK signaling pathways through activating Nrf2 post spinal cord injury. Int Immunopharmacol 2022; 111:109120. https://doi.org/10.1016/j.intimp.2022.109120.

[46] Fu Y, O’Connor LM, Shepherd TG, Nachtigal MW. The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor beta-induced Smad signaling in human ovarian cancer cells. Biochem Biophys Res Commun 2003; 310:391-7. https://doi.org/10.1016/j.bbrc.2003.09.021.

[47] Akram R, Anwar H, Javed MS, Rasul A, Imran A, Malik SA, et al. Axonal regeneration: underlying molecular mechanisms and potential therapeutic targets. Biomedicines 2022; 10:3186. https://doi.org/10.3390/biomedicines10123186.

[48] Enamorado M, Kulalert W, Han SJ, Rao I, Delaleu J, Link VM, et al. Immunity to the microbiota promotes sensory neuron regeneration. Cell 2023; 186:e17. https://doi.org/10.1016/j.cell.2022.12.037.

[49] Schnaar RL. Brain gangliosides in axon-myelin stability and axon regeneration. FEBS Lett 2010; 584:1741-7. https://doi.org/10.1016/j.febslet.2009.10.011.