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. 2015 May 21:294:101-8.
doi: 10.1016/j.neuroscience.2015.02.049. Epub 2015 Mar 3.

Activation of the Wnt/β-catenin signaling cascade after traumatic nerve injury

S Kurimoto  1 , J Jung  2 , M Tapadia  2 , J Lengfeld  3 , D Agalliu  3 , M Waterman  4 , T Mozaffar  5 , R Gupta  6
Affiliations

Activation of the Wnt/β-catenin signaling cascade after traumatic nerve injury

S Kurimoto et al. Neuroscience. .

Abstract

Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerve injury helps to improve functional recovery with surgical repair via matrix metalloproteinase-3 (MMP3) blockade. As such, we sought to explore additional pathways that may augment this response. Wnt3a has been shown to inhibit acetylcholine receptor (AChR) clustering via β-catenin-dependent signaling in the development of the NMJ. Therefore, we hypothesized that Wnt3a and β-catenin are associated with NMJ destabilization following traumatic denervation. A critical size nerve defect was created by excising a 10-mm segment of the sciatic nerve in mice. Denervated muscles were then harvested at multiple time points for immunofluorescence staining, quantitative real-time PCR, and western blot analysis for Wnt3a and β-catenin levels. Moreover, a novel Wnt/β-catenin transgenic reporter mouse line was utilized to support our hypothesis of Wnt activation after traumatic nerve injury. The expression of Wnt3a mRNA was significantly increased by 2 weeks post-injury and remained upregulated for 2 months. Additionally, β-catenin was activated at 2 months post-injury relative to controls. Correspondingly, immunohistochemical analysis of denervated transgenic mouse line TCF/Lef:H2B-GFP muscles demonstrated that the number of GFP-positive cells was increased at the motor endplate band. These collective data support that post-synaptic AChRs destabilize after denervation by a process that involves the Wnt/β-catenin pathway. As such, this pathway serves as a potential therapeutic target to prevent the motor endplate degeneration that occurs following traumatic nerve injury.

Keywords: Wnt signaling; neuromuscular junction; peripheral nerve; traumatic nerve injury.

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Figures

Fig. 1
Fig. 1
Wnt3a expression in denervated muscles. (A) qRT-PCR of Wnt3a and Wnt3 in at different timepoints after denervation injury. Wnt3a is elevated at 2 weeks to 2 months. However, Wnt3, a non-canonical Wnt, is decreased during this time. (B) Wnt3a is co-localized to the nerves in uninjured mice. After denervation injury, Wnt3a is upregulated. Wnt3a expression is localized at the post-synaptic muscle, preferentially at the sites of degenerating AChRs which are no longer innervated. Bar graphs represent mean ± S.E. (n = 7 per group). *p < 0.05. Red = α-bungarotoxin; blue = neurofilament; green = Wnt3a. Scale bar = 10 μm (100×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
β-catenin activity in plantaris muscles 2 months post-transection. (A1–A5) Uninjured wild-type muscles do not have inherent β-catenin activity. (B1–B5). However, after 2 months denervation injury is introduced, β-catenin is dramatically increased in the post-synaptic muscle. (C) Immunoblot analysis demonstrated that the ratio of active/total β-catenin protein level is nearly twofold elevated in the 2-month denervated muscle as compared to control, signifying activation of the Wnt/β-catenin signaling pathway. Blue = DAPI; green = active β-catenin; red = α-bungarotoxin; purple = synaptophysin. Scale bar = 10 μm (100×). Bar graphs represent mean ± S.E. (n = 9 per group). *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
H2B-GFP expression in plantaris muscles after 1 month denervation. (A, B) Sham-operated muscles show minimal GFP fluorescence at the motor endplate band with no fluorescence seen at other sites of the muscle. Inset shows a magnified AChR with absent GFP fluorescence, signifying minimal Wnt activity at baseline. (C, D) One month denervated muscles show significantly elevated GFP fluorescence and Wnt activity but similar signaling was not seen outside of the motor endplate band. Inset shows AChR with multiple GFP histones fluorescing. (E) Total number of GFP-positive cells counted at the motor endplate band and outside of the motor endplate in sham-operated and 1-month denervated muscle. (F) Immunoblot analysis probed for GFP showing a relative increase of nearly threefold in the 1-month denervated muscle as compared to control. Green = GFP; red = α-bungarotoxin; blue = DAPI. Scale bars = 100 μm (10×), 10 μm (100×). Bar graphs represent mean ± S.E. (n = 5 per group). *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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References

    1. Aguilera O, Fraga MF, Ballestar E, Paz MF, Herranz M, Espada J, García JM, Muñoz A, Esteller M, González-Sancho JM. Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer. Oncogene. 2006;25:4116–4121. - PubMed
    1. Bafico A, Liu G, Goldin L, Harris V, Aaronson SA. An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell. 2004;6:497–506. - PubMed
    1. Bentolila V, Nizard R, Bizot P, Sedel L. Complete traumatic brachial plexus palsy. Treatment and outcome after repair. J Bone Joint Surg Am. 1999;81:20–28. - PubMed
    1. Bezakova G, Helm JP, Francolini M, Lømo T. Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo. J Cell Biol. 2001;153:1441–1452. - PMC - PubMed
    1. Chao T, Frump D, Lin M, Caiozzo VJ, Mozaffar T, Steward O, Gupta R. Matrix metalloproteinase 3 deletion preserves denervated motor endplates after traumatic nerve injury. Ann Neurol. 2013;73:210–223. - PubMed

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