Neuromuscular Junction Morphology and Gene Dysregulation in the Wobbler Model of Spinal Neurodegeneration

Abstract

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disease for which there is currently no effective treatment. The progression of ALS includes loss of motor neurons controlling the voluntary muscles, with much of this loss occurring at the neuromuscular junction. In an effort to better understand changes at the neuromuscular junction, we utilized the wobbler mouse model of motor neuron loss. We examined biceps and end plate morphologies and monitored selected factors involved in end plate function. Structural volumes were determined from 3D reconstructions that were generated for the end plates. Wobbler mice exhibited size reductions of both the muscle fibers and the end plates within the biceps, and we found that the end plate volumes were the most sensitive indicator of the degeneration. Concurrently, we found increases in calcitonin gene-related peptide (CGRP) and its receptor in wobbler biceps and spinal cord. We also found increases in gene expression of two acetylcholine receptors within the wobbler biceps, which may be a result of altered CGRP/CALCRL (calcitonin receptor-like receptor) expression.

Keywords: Calcitonin gene-related peptide (CGRP); End plate; Motor neuron disease; Neuromuscular junction; Wobbler mice.

Fig. 1

Bicep muscle fibers were stained for nuclei (Hoechst 33342, blue channel), F-actin (phalloidin Alexa488, green channel), and acetylcholine receptors (α-bungarotoxin Alexa647, red channel). It can be seen in the maximum z projections of the confocal slices that, compared with the wildtype (a) and the wobbler (b) fiber diameters, indicate atrophy. The end plate structures in the wild-type (c)muscle display normal neuromuscular junctions while the wobbler end plates (d) are significantly reduced and less organized. End plate volumes were determined by isolating the bungarotoxin positive objects in 3D. An example volume is noted by the arrow in c, and examples of the corresponding computer generated end plates are shown below c and d. Supplementary videos S1A, S1B, S2a, andS2b provide 3D rotations of the end plates and the equivalent objects

Fig. 2

Muscles from wild-type (wt) and wobbler (wr) mice were analyzed at 35 days of age. a Diameters were measured for 10 fibers per muscle from each genotype (n = 3/group) and were found 30% reduced in the wobbler. b The volumes were determined for every end plate in each biceps muscle sample (n = 4/group) and were found decreased by 75% in the wobbler mice (*p < 0.05)

Fig. 3

CGRP and CALCRL expression in spinal cord and biceps. a CGRP mRNA was significantly increased (*p <0.05) in wr spinal cords at 35 days of age (n = 9) compared to wt controls (n = 8). CGRP mRNA expression was not detected in biceps. CALCRL mRNA expression analysis revealed a trend toward higher expression in wr spinal cord and lower expression in wr biceps, though this was not statically significant. b In contrast, CALCRL protein was found elevated in the biceps. Western Blots were performed using an antibody for CALCRL on tissue harvested from wr and wt mice at 35 days of age. Expression of CALCRL was found to be increased in wr mice in both a spinal cord (n = 9) and b biceps (n = 5) (***p <0.0001)

Fig. 4

The mRNA expression of several acetylcholine receptor subtypes was evaluated by qRT-PCR of RNA isolated from the biceps. There were no detectable changes for AChRβ₁, AChRε, and AChE (not shown). Significant differences were only found for AChRα₁ and AChRδ, and these mRNAs were both elevated in the wr biceps (p < 0.05)