Research Article: Expression of a Mutant SEMA3A Protein with Diminished Signalling Capacity Does Not Alter ALS-Related Motor Decline, or Confer Changes in NMJ Plasticity after BotoxA-Induced Paralysis of Male Gastrocnemic Muscle

Date Published: January 19, 2017

Publisher: Public Library of Science

Author(s): Elizabeth B. Moloney, Barbara Hobo, Fred De Winter, Joost Verhaagen, Brian Key.


Terminal Schwann cells (TSCs) are specialized cells that envelop the motor nerve terminal, and play a role in the maintenance and regeneration of neuromuscular junctions (NMJs). The chemorepulsive protein semaphorin 3A (SEMA3A) is selectively up-regulated in TSCs on fast-fatigable muscle fibers following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve) or in the motor neuron disease amyotrophic lateral sclerosis (ALS). Re-expression of SEMA3A in this subset of TSCs is thought to play a role in the selective plasticity of nerve terminals as observed in ALS and following BotoxA-induced paralysis. Using a mouse model expressing a mutant SEMA3A with diminished signaling capacity, we studied the influence of SEMA3A signaling at the NMJ with two denervation paradigms; a motor neuron disease model (the G93A-hSOD1 ALS mouse line) and an injury model (BotoxA-induced paralysis). ALS mice that either expressed 1 or 2 mutant SEMA3A alleles demonstrated no difference in ALS-induced decline in motor behavior. We also investigated the effects of BotoxA-induced paralysis on the sprouting capacity of NMJs in the K108N-SEMA3A mutant mouse, and observed no change in the differential neuronal plasticity found at NMJs on fast-fatigable or slow muscle fibers due to the presence of the SEMA3A mutant protein. Our data may be explained by the residual repulsive activity of the mutant SEMA3A, or it may imply that SEMA3A alone is not a key component of the molecular signature affecting NMJ plasticity in ALS or BotoxA-induced paralysis. Interestingly, we did observe a sex difference in motor neuron sprouting behavior after BotoxA-induced paralysis in WT mice which we speculate may be an important factor in the sex dimorphic differences seen in ALS.

Partial Text

The development, maintenance and regeneration of neuromuscular junctions (NMJs) is highly dependent on the function of terminal Schwann cells (TSCs), specialized cells that envelop the motor nerve terminal [1–3]. Semaphorin 3A (SEMA3A) expression is up-regulated in TSCs at the NMJ following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve) or in the motor neuron disease amyotrophic lateral sclerosis [4]. This expression is selective to the TSCs which envelop the NMJ of fast-fatigable (TypeIIb) muscle fibers. The TypeIIb fibers are the first to undergo denervation in ALS, and show limited plasticity in sprouting and forming functional ectopic synapses upon denervation [5–9]. Re-expression of SEMA3A at the NMJs of TypeIIb fibers is thought to play a role in the selective degeneration of these junctions by creating a growth-inhibitory environment around the motor nerve terminal.

In the current study we used the K108N-SEMA3A mutant mouse to directly study the influence of SEMA3A on the ALS phenotype and NMJ morphology in an effort to specifically impair SEMA3A function without affecting the ability of NRP1 to interact with its additional ligands. The K108N-SEMA3A mutant retains the ability to bind to its receptor, NRP1, but the interaction between K108N-SEMA3A-NRP1 and the signal-transducing component of the holoreceptor, Plexin A, is impaired. The mutation in SEMA3A creates a protein with diminished signaling properties [approximately 85% less potent than WT SEMA3A [28]]. It is important to note, however, that the K108N-SEMA3A protein is capable of eliciting a full repulsive effect on neurons in culture when applied at higher concentrations [28]. Keeping this in mind, the K108N-SEMA3A mouse therefore is a “partial” SEMA3A knockout. Previous attempts to create a true SEMA3A knockout to study its role in adulthood failed. Behar and colleagues described that ~70% of SEMA3A homozygote knockouts die within the first 3 days [10], and Taniguichi and colleagues describe their homozygote knockouts as “largely viable” but give no indication to the numbers surviving into adulthood [39]. The K108N-SEMA3A mutant mice display a similar developmental neuronal phenotype to the SEMA3A-null mice, i.e. aberrant defasciculation of cranial and spinal nerves, and overgrowth of ulnar and radial nerves into their target areas, but importantly, are described as “homozygote viable”compared to the SEMA3A-null mice [28]. The K108N-SEMA3A mouse (homozygote and heterozygote) displays a normal behavioral phenotype compared to wild-type mice and remains fertile throughout adulthood (our observations). The discrepancy in viability between the traditional SEMA3A knockouts and the K108N-SEMA3A line may be explainable by the residual SEMA3A function attributed to the mutant K108N-SEMA3A.




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