The Role of the Wnt3a and the Beta-Catenin Signaling Pathway at the Motor Endplate following Traumatic Nerve Injury

Introduction: Major peripheral nerve injuries lead to significant functional deficits. Although the general belief is that the regenerative capacity of the PNS when compared to the CNS is much more robust after nerve insult, this does not translate in the clinical setting. A reason for this phenomenon may be explained by end-organ atrophy. We recently showed that preservation of the motor endplate after traumatic nerve injury improves functional recovery after surgical repair1. Wnt signaling proteins, particularly the canonical Wnt/beta-catenin pathway, play an important role in the development and the maintenance of motor endplates. Previous studies have shown that multiple signaling pathways and proteins including agrin, MuSK, and the Wnt pathway influence the neuromuscular junction (NMJ). In particular, Wnt3a has been shown to inhibit agrin-induced acetylcholine receptor (AChR) clustering by suppressing rapsyn expression via beta-catenin dependent but TCF/LEF independent signaling. The current study focuses on assessing the NMJ after long-term denervation injury. Levels of Wnt3a and activated beta-catenin are quantified throughout various timepoints to determine if destabilization of the NMJ corresponds to an increase in the concentration of these proteins within the motor endplate. As such, we explored the role of Wnt/beta-catenin at the NMJ after traumatic nerve injury. Methods: Animal Model. All procedures involving living animals were approved by the Institutional animal care and Use committee of the University of California, Irvine. Homozygous pairs of the 129 SV/EV wildtype mice were a generous gift from Dr. Wee Yong at the University of Calgary. Genotyping was performed by Transnetyx (Cordova, TN). Surgery. For denervation studies, 6-week old male mice were anesthetized with ketamine/xylazine. A 10 mm segment of the right sciatic nerve was excised. Immunohistochemistry and Immunoblotting. Whole mounts of plantaris muscles were extracted from wildtype mice ipsilateral and contralateral to the side of denervation at 1 month and 2 months. IHC analysis was performed to define the integrity of the nerve terminal and motor endplates after denervation and for Wnt3a. Gastroc-soleus muscle complexes were harvested for Wnt3a and beta-catenin levels and quantified using standard western blot techniques. A t test was performed with p value < 0.05 constituting significance. Additionally, Wnt3a staining in uninjured muscles was not present. However, after denervation injury, Wnt3a was upregulated and recruited into the post-synaptic muscle, specifically to the degrading AChRs and motor endplate band at increasing levels until 2 months. Discussion: The NMJ undergoes a gradual degradation with loss of its different constitutents after …