Truncated dystrophins can influence neuromuscular synapse structure

Abstract

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and structural defects in the neuromuscular synapse that are caused by mutations in dystrophin. Whether aberrant neuromuscular synapse structure is an indirect consequence of muscle degeneration or a direct result of loss of dystrophin function is not known. Rational design of truncated dystrophins has enabled the design of expression cassettes highly effective at preventing muscle degeneration in mouse models of DMD using gene therapy. Here we examined the functional capacity of a minidystrophin (minidysGFP) and a microdystrophin (microdystrophin(DeltaR4-R23)) transgene on the maturation and maintenance of neuromuscular junctions (NMJ) in mdx mice. We found that minidysGFP prevents fragmentation and the loss of postsynaptic folds at the NMJ. In contrast, microdystrophin (DeltaR4-R23) was unable to prevent synapse fragmentation in the limb muscles despite preventing muscle degeneration, although fragmentation was observed to temporally correlate with the formation of ringed fibers. Surprisingly, microdystrophin(DeltaR4-R23) increased the length of synaptic folds in the diaphragm muscles of mdx mice independent of muscle degeneration or the formation of ringed fibers. We also demonstrate that the number and depth of synaptic folds influences the density of voltage-gated sodium channels at the neuromuscular synapse in mdx, microdystrophin(DeltaR4-R23)/mdx and mdx:utrophin double knockout mice. Together, these data suggest that maintenance of the neuromuscular synapse is governed through its lateral association with the muscle cytoskeleton, and that dystrophin has a direct role in promoting the maturation of synaptic folds to allow more sodium channels into the junction.

Figure 1

Structure of truncated dystrophins and their effect on neuromuscular synapse stabilization in mdx mice. A) ABD, actin binding domain. CR, cysteine-rich domain. CT, C-terminal domain. R, spectrin-like repeats. The numbers in the shaded boxes represent the hinge regions. GFP, enhanced green fluorescent protein. B) Topographic view of the acetylcholine receptor (AChR) clusters in the postsynaptic membrane of the neuromuscular junction stained with α-bungarotoxin (α-BTX; in red) on teased gastrocnemius muscles at 3 months of age. Note that synapses were fragmented in mdx fibers that have central nuclei (DAPI in blue), whereas synapses in microdystrophin^ΔR4–R23/mdx mice were fragmented on myofibers with peripheral nuclei. Dashed lines outline the muscle fiber. Scale bar = 15 μm.

Figure 2

Synapses fragmentation was coincident with ringed fiber formation in microdystrophin^ΔR4–R23/mdx mice. Shown are electron microscopy images of two adjacent muscle fibers from A) wild-type, B) mdx, C) MinidysGFP/mdx D) microdystrophin^ΔR4–R23/mdx mice. White arrows show the ringed fibers in microdystrophin^ΔR4–R23/mdx mice. E) Longitudinal section of a muscle fiber from microdystrophin^ΔR4–R23/mdx mice showing the instability of the sarcolemma (black arrows). F) Ringed fibers were not found in the diaphragm in microdystrophin^ΔR4–R23/mdx mice. Scale bars = 2 μm.

Figure 3

Synapses begin to fragment in microdystrophin^ΔR4–R23/mdx mice before mdx mice. A) Topographic view of AChR clusters stained with α-BTX at 2 weeks of age. Scale bar = 15 μm. B) A ringed fiber in microdystrophin^ΔR4–R23/mdx gastrocnemius muscle at 2 weeks of age. Arrowhead points to the sarcolemma. Arrow points to the interface between ringed fibers and normally oriented longitudinal fibers. Scale bar = 1 μm.

Figure 4

A) Ultrastructure of neuromuscular synapses in wild-type, mdx, microdystrophin^ΔR4–R23/mdx mice and minidysGFP/mdx mice. Scale bar = 2μm. B) The graph shows the mean +/− SD number of folds per μm of postsynaptic membrane juxtaposed to the presynaptic cleft. The number of folds/μm was significantly reduced in mdx mice compared to wild-type mice (P < 0.05). C) The mean +/− SD depth of the folds was significantly reduced in mdx diaphragm (*P < 0.05), and significantly increased in microdystrophin^ΔR4–R23/mdx diaphragms (***P < 0.001).

Figure 5

Localization of nNOS and utrophin in transverse sections of the gastrocnemius muscles. Antibody staining is shown in green and αBTX in red. Scale bar = 30 μm.

Figure 6

Synaptic folds influence the density of voltage gated sodium channels at the NMJ. A) Voltage gated sodium channels concentrate to the postsynaptic folds of wild-type, mdx, microdystrophin^ΔR4–R23/mdx mice and minidysGFP/mdx mice. Voltage gated sodium channels are shown in green or blue and AChR clusters are shown in red. B) Electron micrograph of a NMJ from mdx/utrophin double knockout mice (mdx:utrn^−/−) showing few folds in the postsynaptic membrane. Scale bar = 2μm. C) Mean +/− S.D. number of folds per μm of postsynaptic membrane in mdx:utrn^−/− mice. dko is mdx:utrn^−/− mice. ***P < 0.001. D) Synapses in mdx:utrn^−/− mice have a low density of voltage gated sodium channels. Sodium channels are in green and AChR are in red. Scale bar = 10 μm. E) Bars represent the mean +/− S.D. immunofluorescence intensity of sodium channels at the NMJs from wild-type, mdx, microdystrophin^ΔR4–R23/mdx and mdx:utrn^−/− gastrocnemius muscles. ***P < 0.001 compared to wild-type.