The Role of the Wnt3a and the Beta-Catenin Signaling Pathway at the Motor Endplate Following Traumatic Nerve Injury
James Jung1, Derek Frump2, Marian Waterman1, Tahseen Mozaffar2 and Ranjan Gupta3,
1University of California, Irvine, Irvine, CA, 2Orthopaedic Surgery, University of California, Irvine, Orange, CA, 3Orthopaedic Surgery, University of California Irvine, Orange, CA
Hypothesis: 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 repair. 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 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: 6-week old male 129SV/EV mice were denervated by trasnection of the sciatic nerve. Whole mounts of plantaris muscles were extracted 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. Results: Wnt3a protein levels were elevated at 1 month (0.633±0.0540 vs 0.937±0.128) and 2 months post-injury (0.488±0.0170 vs. 0.970±0.232; p<0.002) relative to control. Moreover, activated beta-catenin showed a similar increase (0.532±0.0250 vs. 1.050±0.204; p<0.026). In contrast to uninjured muscles, Wnt3a staining remained localized at the motor endplate after denervation where it was upregulated near degrading AChRs. Summary: Long-term denervation leads to extensive atrophy of the motor endplate, which translates to deficits in functional recovery. Post-synaptic AChRs at the NMJ appear to destabilize after denervation by a process that involves the Wnt/beta-catenin pathway. Our data implicates this pathway as a potential source of this motor endplate instability after injury. Our data presents a potential novel target to optimize functional outcomes following surgical management of traumatic nerve injuries.
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