The expression of the chemorepellent Semaphorin 3A is selectively induced in terminal Schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease

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Neuromuscular synapses differ markedly in their plasticity. Motor nerve terminals innervating slow muscle fibers sprout vigorously following synaptic blockage, while those innervating fast-fatigable muscle fibers fail to exhibit any sprouting. Here, we show that the axon repellent Semaphorin 3A is differentially expressed in terminal Schwann cells (TSCs) on different populations of muscle fibers: postnatal, regenerative and paralysis induced remodeling of neuromuscular connections is accompanied by increased expression of Sema3A selectively in TSCs on fast-fatigable muscle fibers. To our knowledge, this is the first demonstration of a molecular difference between TSCs on neuromuscular junctions of different subtypes of muscle fibers. Interestingly, also in a mouse model for amyotrophic lateral sclerosis (ALS), Sema3A is expressed at NMJs of fast-fatigable muscle fibers. We propose that expression of Sema3A by TSCs not only suppresses nerve terminal plasticity at specific neuromuscular synapses, but may also contribute to their early and selective loss in the motor neuron disease ALS.

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Results

Since there are no reliable antibodies available specifically recognizing Sema3A protein, Sema3A expression was examined by in situ hybridization and real time quantitative PCR (RT-qPCR). In situ hybridization and RT-qPCR were performed on gastrocnemic muscles from 1, 4, 7, 14, 21, 28, and 60 day old rats, and on adult gastrocnemic muscles at 4, 7, 14, 21, 36, and 60 days following sciatic nerve crush.

Discussion

Here, we show that TSCs located on NMJs of type IIb/x muscle fibers, but not on type I and type IIa muscle fibers, upregulate the expression of the chemorepellent Sema3A after mechanical denervation and following pharmacological blockage of neurotransmission. In addition, we demonstrate that TSCs positioned at the NMJs of type IIb/x muscle fibers in the G93A-hSOD1 mouse model for ALS selectively up-regulate the chemorepellent Sema3A. In contrast to motor nerve terminals on type I and type IIa

Animals and surgical procedures

All surgical and animal care procedures were carried out according to the local guidelines of the experimental animal care committee. Wistar rats (Harlan CPB-Zeist, Netherlands) were housed in group cages, maintained on a 12 h light/dark cycle with ad libitum access to food and water.

Statistical analysis

For the quantitative polymerase chain reaction every third cryostat section (60 μm) of the complete (i.c. postnatal time course study) or the medial (i.c. sciatic nerve crush study) gastrocnemic muscle was used for total RNA isolation. Three animals were used per time point and RT-qPCR reaction was carried out in duplet. The mRNA levels of the housekeeping gene L37a (this gene was chosen because it was stably expressed in the gastrocnemic muscle, while other housekeeping genes were not) were

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    Currently working in the Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92186, USA.

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