Motor Axonal Sprouting and Neuromuscular Junction Loss in an Animal Model of Charcot-Marie-Tooth Disease

Abstract

Muscle weakness in Charcot-Marie-Tooth Type 1A disease (CMT1A) caused by mutations in peripheral myelin protein 22 (PMP22) has been attributed to an axonopathy that results in denervation and muscle atrophy. The underlying pathophysiological mechanisms involved are not understood. We investigated motor performance, neuromuscular junctions (NMJs), physiological parameters, and muscle morphometry of PMP22 transgenic mice. Neuromuscular junctions were progressively lost in hindlimb muscles of PMP22 transgenic mice, but their motor performance did not completely deteriorate during the observation period. There was considerable variability, including in laterality, in deficits among the animals. Cross-sectional areas and mean fiber size measurements indicated variable myofiber atrophy in hindlimb muscles. There was substantial concomitant axonal sprouting, and loss of neuromuscular junctions was inversely correlated with the accumulated length of axonal branches. Synaptic transmission studied in isolated nerve/muscle preparations indicated variable partial muscle denervation. Acetylcholine sensitivity was higher in the mutant muscles, and maximum tetanic force evoked by direct or indirect stimulation, specific force, and wet weights were markedly reduced in some mutant muscles. In summary, there is partial muscle denervation, and axons may retain some regenerative capacity but fail to reinnervate muscles in PMP22 transgenic mice.

Structural and Functional Abnormalities of the Neuromuscular Junction in the Trembler-J Homozygote Mouse Model of Congenital Hypomyelinating Neuropathy

“…These findings are in contrast to congenital models of motor neuron disease, such as spinal muscular atrophy (SMA), which exhibit anatomical as well as functional evidence of synaptic degeneration at the NMJ (37). They are also different than results obtained from adult, heterozygote Tr J mice as well as other CMT1 mouse models, which exhibit a modest but significant loss of neuromuscular synaptic maintenance (7, 8, 21). Together with the absence of severe muscle atrophy, the lack of peripheral denervation in Tr J mice suggests that the early lethality in this mouse is not caused by a failure to maintain neuromuscular synaptic connections.…”

“…Consistent with this idea, animal models of CMT1 exhibit a loss of large-diameter axons and impaired axonal transport even before impairment of MCV (6). In addition to these signs of axonal dysfunction, neuromuscular junctions (NMJs) show evidence of denervation in CMT1 mutant mice (7, 8). Therefore, peripheral dysfunction of motor nerve terminals represents an important early step in the pathophysiology of CMT1.…”

Long-term Analyses of Innervation and Neuromuscular Integrity in the Trembler-J Mouse Model of Charcot-Marie-Tooth Disease

“…2C). Because terminal axon sprouting has been reported in skeletal muscle of neuropathic mice (28), as well as in aged rodents (23), we also studied the expression of GAP43 (34), which in some samples appeared elevated. However, quantitative analyses of independent samples did not confirm this to be significant.…”

“…Tissue sections were imaged using either a Spot camera attached to a Nikon Eclipse E800 microscope, or an Olympus DSU spinning disc confocal camera using Slidebook software and deconvoluted using the standard nearest neighbor algorithm. For semiquantitative assessment, NMJs were identified and analyzed by a blind evaluator, as described (23, 28, 29). Briefly, intact NMJs included α-BTX in close proximity to pNFH-like immunoreactivity, while denervated NMJs were classified as α-BTX labeling without at least 50% of the junction closely adjacent to neurofilament heavy chain (NFH)-like staining.…”

Dissection of the <em>Transversus Abdominis</em> Muscle for Whole-mount Neuromuscular Junction Analysis

“…Typical symptoms include denervation, where the motor endplate becomes devoid of a presynaptic innervation, swelling of the presynaptic terminal, and a reduction in the complexity of the NMJ morphology [4][5][6][7][8][9][10][11] . Compensatory responses can also be noted, which include terminal and nodal sprouting, where axonal processes extend from remaining synaptic terminals or internodes to reinnervated denervated endplates 12,13 . Due to the tight correlation between synaptic activity and NMJ morphology, a great deal of information can be gained about the functional status of motor neurons from analysis of NMJ morphology.…”