Elsevier

Brain Research Bulletin

Volume 152, October 2019, Pages 285-296
Brain Research Bulletin

FGF9 alters the Wallerian degeneration process by inhibiting Schwann cell transformation and accelerating macrophage infiltration

https://doi.org/10.1016/j.brainresbull.2019.06.011Get rights and content

Highlights

  • FGF9 was expressed in both Shcwann cells (SCs) and axons, and it was downregulated in nerve injury.

  • Fgf9 knockout mildly changed motor function, impaired debris clearance and nerve regeneration after injury.

  • Fgf9 knockout promoted SCs dedifferentiation and delayed macrophages activation.

  • Exogenous FGF9 inhibited SCs dedifferentiation and promoted macrophages infiltration partially by activating ERK1/2.

Abstract

In vitro experiments have proved that Fibroblast Growth Factor 9 (FGF9) was decreased in Schwann cells (SCs) in which Wallerian degeneration (WD) occurred after nerve injury. We hypothesize that FGF9 downregulation in WD has some biological influence on Schwann cells (SCs) and macrophages – the two most important cell components involved in WD. In this study, we employed strategies to regulate FGF9 in sciatic nerve crush by generating a mouse model, wherein Fgf9 was specifically knocked-out in SCs, and an intraneural injection of human FGF9 protein administered to overexpress FGF9 independently. Furthermore, an inhibitor of extracellular-regulated kinases 1/2 (ERK1/2), PD0325901, was used to clarify the underlying downstream mechanism of ERK1/2 activated by FGF9. Analysis of WD revealed the novel features of FGF9: (i) FGF9 was widely expressed in axons and SCs, and was decreased during WD process. (ii) Fgf9 knockout in SCs impaired the debris clearance and eventually impeded the regeneration of nerve fibers after damage. (iii) Fgf9 knockout in SCs promoted the dedifferentiation of SCs and delayed the infiltration of macrophages by decreasing Mcp1, Tnfα, Il1β levels and leaky blood-nerve-barrier (BNB) in WD. (iv) FGF9 injection preserved the nerve fibers, inhibited SCs dedifferentiation and accelerated macrophages infiltration. (v) ERK1/2 phosphorylation was increased by exogenous FGF9 injection. P75, Cyclin D1, Mcp1, Tnfα, Il1β, c-Jun changes by FGF9 intraneural injection were partially reversed by the ERK1/2 inhibitor. Conclusion was that FGF9 inhibited the dedifferentiation of SCs and accelerated the accumulation of macrophages in WD, and exogenous FGF9 took effects partially by ERK1/2.

Introduction

The peripheral nerves undergo a series of complex pathological changes after nerve trauma—Wallerian degeneration (WD). The major events include: (i) axonal degeneration, (ii) transient proliferation of Schwann cells (SCs) with the reversal of molecular expression patterns reversed from that the characteristics of mature SCs to the one that resembles the immature state, known as the repair cells, which possess the ability to direct axonal regrowth, and remyelination, (iii) activation of resident macrophages and subsequent invasion of homogenous macrophages, ingestion and breakdown of the debris and blood-nerve-barrier (BNB) (Homs et al., 2011; Kanda et al., 1999). SCs constitute over 80% of the cells in the adult peripheral nerves, act as the key orchestrator of degeneration and subsequently the regeneration process in adult peripheral nervous system (PNS) after injury (Brosius Lutz and Barres, 2014).

PNS has powerful regeneration ability than the central nervous system (CNS); however, the repair abilities after nerve trauma can be incredibly variable, and only a part of axons may fully regrow to reinnervate their targets (Kanda et al., 1999). When the distal stumps of nerves are left denervated for a prolonged period, they become largely unsupportive to regeneration (Dahlin, 2008; Zhang et al., 2008). Thus, understanding the pathology and regulators of WD, then further target these regulators to increase the repair potential of peripheral nerves post trauma might be valuable.

FGF9 belongs to FGF9/16/20 subfamily and plays a role in the autocrine or paracrine pathway (Goetz and Mohammadi, 2013; Jungnickel et al., 2004). The extracellular-regulated kinases (ERK1/2, p44/p42 MAPKs) are classical downstream signaling pathways of FGFs (Lin et al., 2016). FGF9 is widely expressed in the nervous system and affects the nerve tissues. In CNS, FGF9 stimulates the mitogen-activated protein kinase (MAPK) phosphorylation and downregulates the myelin related proteins expression in oligodendrocytes (Cohen and Chandross, 2000). The in vitro experiment in PNS showed that FGF9 is downregulated in WD; silencing the molecule inhibits the proliferation and migration of SCs (Wang et al., 2016a). An investigation of FGF2, another FGF family member, demonstrated that Fgf2 knockout mice had regenerated myelinated axons with increased myelin and axonal diameter, as well as, rapid WD (Jessen and Mirsky, 2008).

Whether FGF9 influences the WD process and the underlying mechanisms are yet to be characterized. Identifying the functions of FGF9 to WD would aid in identifying the research and theraputic targets for nerve trauma. To address these issues, we used nerve crush to mimic the WD animal model. Both Fgf9 knockout and FGF9 intraneural injection strategies were used to investigate the FGF9 functions in WD. Specifically, we constructed a Fgf9fl/fl−p0 mouse line to knockout Fgf9 in SCs and administered intraneural human FGF9 protein to the crush site of wild-type (WT) nerves, then the influences of FGF9 on WD were evaluated. We found that FGF9 alters the WD process by regulating SCs and macrophages. ERK1/2 phosphorylation was upregulated by FGF9 intraneural injection. ERK1/2 inhibitor was used to further identify the molecules downstream of ERK1/2 activated by FGF9. Our results identified FGF9 as a regulator of the WD process.

Section snippets

Mouse line preparation

B6N.FVB-Tg(Mpz-cre)26Mes/J (RRID:IMSR_JAX:017927) was represented by P0-cre mice (Jackson Mice, Stock No:017927). Fgf9fl/fl mice were designed and generated by Biocytogen, with floxp sites inserted across exon 1 of Fgf9 gene. The WT phenotype mice were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were C57/B6 background. B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (RRID:IMSR_JAX:007676) was presented by (ROSA)26Tomato-EGFP(Jackson Mice, Stock

Distribution and expression area of FGF9 were widespread on SCs, neuronal components and macrophages

Normal peripheral nerves primarily consist of axons and myelin sheath formed by SCs. To investigate the distribution of FGF9 in peripheral nerves, we selected S100 to mark the SCs, myelin basic protein (MBP) to mark the myelin shealth, and 160 kDa neurofilament (NF160) to mark the axons. The immunolabeling of sciatic nerves showed that there were some FGF9 distribution in SCs soma (Fig. 1a). FGF9 was distributed external to the MBP distribution area in a typical node of Ranvier morphology (Fig.

Discussion

In this study, we confirmed that FGF9 was expressed in axons, SCs, and macrophages, and was located on both axons and SCs in uncrushed nerves. FGF9 expression was downregulated in nerve injury. By combining the analysis of a mouse model with a specific Fgf9 knockout in SCs and FGF9 intraneural injection, we found that FGF9 inhibited the transformation of SCs into repair cells by retarding the upregulation of repair cells markers and proliferation; and promoted the accumulation of macrophages by

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors would like to thank the financial support and experimental design provided by Prof. Chunyan Li. Contract grant sponsor the Natural Science Foundation of China; Contract grant number: 30371089, 81171210.

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