Background & Aims

Neuropathic pain is a debilitating chronic pain that lacks effective treatment. Neuroinflammation mediated by cytokines and chemokines has been implicated in the pathogenesis of neuropathic pain. Follistatin (FST) is a secreted protein that has been identified to antagonize the biological activity of the cytokine TGF-? superfamily. Microarray and RNA-seq studies have identified FST as one of the highly upregulated genes in the DRG following peripheral nerve injury in mice. In this study, we investigated whether the upregulated FST influences nerve injury-induced neuropathic pain and explored the involved mechanisms.

Methods

(1) The L4 spinal nerve was isolated and tightly ligated with 6-0 silk thread to produce the spinal nerve ligation (SNL) pain model. (2) The expression of FST and other related genes in the dorsal root ganglia (DRG) was tested using qRT-PCR, immunostaining, RNA scope, and western blot. (3) Animal’s pain behaviors were detected by von Frey and Hargreaves test. (4) Whole-cell patch-clamp recordings in DRG and CHO-K1 cells were used to test neuronal excitability and sodium currents. (5) The binding affinity of the isolated FST with IGF1R was assessed using Co-Immunoprecipitation (Co-IP) and Biolayer Interferometry (BLI).

Results

(1) FST was upregulated in A-fiber sensory neurons after SNL in mice. (2) Inhibition or deletion of FST alleviated neuropathic pain and reduced the neuronal hyperexcitability induced by SNL. (3) Over-expression of FST in the DRG or injection of recombinant FST induced pain hypersensitivity, and increased neuronal excitability and spontaneous firing of nociceptive neurons. (4) FST activated ERK and AKT signaling in DRG neurons. (5) Co-IP and BLI showed that FST has a strong affinity with insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibitor attenuated FST-induced neuronal hyperexcitability and neuropathic pain. (6) Western blot showed that the membrane IGF1R was decreased, and the cytoplasm IGF1R was increased after SNL. Inhibition or deletion of IGF1R alleviated neuropathic pain. (7) The N-terminal domain of FST has the highest affinity with IGF1R, and blocking the interaction by a peptide derived from FST attenuated SNL-induced neuronal hyperexcitability and neuropathic pain. (8) FST increased the current density in cells transfected with IGF1R and Nav1.7 plasmids. In addition, pretreatment with FST-derived peptide reduced the current density induced by FST. (9) FST increases neuronal excitability of human DRG neurons, which was reduced by IGF1R inhibitor.

Conclusions

FST, upregulated and released from A-fiber neurons after peripheral nerve injury, acts on IGF1R on nociceptive neurons to activate ERK/AKT, and further phosphorylates Nav1.7 to increase neuronal excitability and neuropathic pain.

References

1.T. B. Thompson, T. F. Lerch, R. W. Cook, T. K. Woodruff, T. S. Jardetzky, The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding. Developmental cell 9, 535-543 (2005).
2.G. Hu, K. Huang, Y. Hu, G. Du, Z. Xue, X. Zhu, G. Fan, Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons. Sci Rep 6, 31851 (2016).
3.A. D. Shaffer, B. Feng, J. H. La, S. C. Joyce, G. F. Gebhart, A novel role for follistatin in hypersensitivity following cystitis. Neurourology and urodynamics 36, 286-292 (2017).
4.D. Tavares-Ferreira, S. Shiers, P. R. Ray, A. Wangzhou, V. Jeevakumar, I. Sankaranarayanan, A. M. Cervantes, J. C. Reese, A. Chamessian, B. A. Copits, P. M. Dougherty, R. W. t. Gereau, M. D. Burton, G. Dussor, T. J. Price, Spatial transcriptomics of dorsal root ganglia identifies molecular signatures of human nociceptors. Sci Transl Med 14, eabj8186 (2022).
5.M. Q. Nguyen, L. J. von Buchholtz, A. N. Reker, N. J. Ryba, S. Davidson, Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons. Elife 10, (2021).
6.K. Kohno, R. Shirasaka, K. Yoshihara, S. Mikuriya, K. Tanaka, K. Takanami, K. Inoue, H. Sakamoto, Y. Ohkawa, T. Masuda, M. Tsuda, A spinal microglia population involved in remitting and relapsing neuropathic pain. Science 376, 86-90 (2022).

Presenting Author

Yong-Jing Gao

Poster Authors

Yong-Jing Gao

PhD

Institute of Pain Medicine, Nantong University

Lead Author

Yue-Juan Ling

Nantong University

Lead Author

Meng-Lin Xu

Nantong University

Lead Author

Jun Gu

Nantong University

Lead Author

Xiao-Bo Wu

Nantong University

Lead Author

Ouyang Chen

Duke University

Lead Author

Chang-Yu Jiang

Huazhong University of Science and Technology Union Shenzhen Hospital

Lead Author

Ru-Rong Ji

Duke University

Lead Author

Topics

  • Models: Chronic Pain - Neuropathic