The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Tse, Nigel; Morsch, Marco; Ghazanfari, Nazanin; Cole, Louise; Visvanathan, Archunan; Leamey, Catherine A.; Phillips, William D.

doi:10.3791/52220

Cited by 28 publications

(29 citation statements)

References 34 publications

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“…En face Z‐projection images of motor endplates (7–45 per muscle) were randomly sampled and ImageJ software was used to assess the area of postsynaptic AChRs, MuSK‐GFP/rapsyn‐GFP and presynaptic synaptophysin, using methods described previously (Tse et al . ). The average endplate areas for each muscle are plotted as symbols in Fig.…”

Section: Methodsmentioning

confidence: 97%

Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction‐induced loss of force‐producing capacity

Trajanovska

Ban

Huang

et al. 2019

The Journal of Physiology

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Key points Adeno‐associated viral vector was used to elevate the expression of muscle specific kinase (MuSK) and rapsyn (a cytoplasmic MuSK effector protein) in the tibialis anterior muscle of wild‐type and dystrophic (mdx) mice. In mdx mice, enhanced expression of either MuSK or rapsyn ameliorated the acute loss of muscle force associated with strain injury. Increases in sarcolemmal immunolabelling for utrophin and β‐dystroglycan suggest a mechanism for the protective effect of MuSK in mdx muscles. MuSK also caused subtle changes to the structure and function of the neuromuscular junction, suggesting novel roles for MuSK in muscle physiology and pathophysiology. Abstract Muscle specific kinase (MuSK) has a well‐defined role in stabilizing the developing mammalian neuromuscular junction, but MuSK might also be protective in some neuromuscular diseases. In the dystrophin‐deficient mdx mouse model of Duchenne muscular dystrophy, limb muscles are especially fragile. We injected the tibialis anterior muscle of 8‐week‐old mdx and wild‐type (C57BL10) mice with adeno‐associated viral vectors encoding either MuSK or rapsyn (a cytoplasmic MuSK effector protein) fused to green fluorescent protein (MuSK‐GFP and rapsyn‐GFP, respectively). Contralateral muscles injected with empty vector served as controls. One month later mice were anaesthetized with isoflurane and isometric force‐producing capacity was recorded from the distal tendon. MuSK‐GFP caused an unexpected decay in nerve‐evoked tetanic force, both in wild‐type and mdx muscles, without affecting contraction elicited by direct electrical stimulation of the muscle. Muscle fragility was probed by challenging muscles with a strain injury protocol consisting of a series of four strain‐producing eccentric contractions in vivo. When applied to muscles of mdx mice, eccentric contraction produced an acute 27% reduction in directly evoked muscle force output, affirming the susceptibility of mdx muscles to strain injury. mdx muscles overexpressing MuSK‐GFP or rapsyn‐GFP exhibited significantly milder force deficits after the eccentric contraction challenge (15% and 14%, respectively). The protective effect of MuSK‐GFP in muscles of mdx mice was associated with increased immunolabelling for utrophin and β‐dystroglycan in the sarcolemma. Elevating the expression of MuSK or rapsyn revealed several distinct synaptic and extrasynaptic effects, suggesting novel roles for MuSK signalling in muscle physiology and pathophysiology.

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Section: Methodsmentioning

confidence: 97%

Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction‐induced loss of force‐producing capacity

Trajanovska

Ban

Huang

et al. 2019

The Journal of Physiology

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“…The following day, the muscles were frozen for sectioning as described (Tse et al. ). Sections were preincubated for 1 h in 2% bovine serum albumin (BSA) in PBS.…”

Section: Methodsmentioning

confidence: 99%

“…ImageJ 1.31 v software was used to measure the relative fluorescence intensities using recently published protocols (Tse et al. ). A minimum of 20 endplates from each muscle were averaged.…”

Section: Methodsmentioning

confidence: 99%

Forced expression of muscle specific kinase slows postsynaptic acetylcholine receptor loss in a mouse model of MuSK myasthenia gravis

et al. 2015

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We investigated the influence of postsynaptic tyrosine kinase signaling in a mouse model of muscle‐specific kinase (MuSK) myasthenia gravis (MG). Mice administered repeated daily injections of IgG from MuSK MG patients developed impaired neuromuscular transmission due to progressive loss of acetylcholine receptor (AChR) from the postsynaptic membrane of the neuromuscular junction. In this model, anti‐MuSK‐positive IgG caused a reduction in motor endplate immunolabeling for phosphorylated Src‐Y418 and AChR β‐subunit‐Y390 before any detectable loss of MuSK or AChR from the endplate. Adeno‐associated viral vector (rAAV) encoding MuSK fused to enhanced green fluorescent protein (MuSK‐EGFP) was injected into the tibialis anterior muscle to increase MuSK synthesis. When mice were subsequently challenged with 11 daily injections of IgG from MuSK MG patients, endplates expressing MuSK‐EGFP retained more MuSK and AChR than endplates of contralateral muscles administered empty vector. Recordings of compound muscle action potentials from myasthenic mice revealed less impairment of neuromuscular transmission in muscles that had been injected with rAAV‐MuSK‐EGFP than contralateral muscles (empty rAAV controls). In contrast to the effects of MuSK‐EGFP, forced expression of rapsyn‐EGFP provided no such protection to endplate AChR when mice were subsequently challenged with MuSK MG IgG. In summary, the immediate in vivo effect of MuSK autoantibodies was to suppress MuSK‐dependent tyrosine phosphorylation of proteins in the postsynaptic membrane, while increased MuSK synthesis protected endplates against AChR loss. These results support the hypothesis that reduced MuSK kinase signaling initiates the progressive disassembly of the postsynaptic membrane scaffold in this mouse model of MuSK MG.

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“…Images were analyzed using NIH ImageJ (http://rsb.info.nih.gov/iJ/) (Tse et al, ), and figures were prepared using Adobe Photoshop CC 2018 and Adobe Illustrator CC 2018 system (Adobe Systems, San Jose, CA). For quantitative analyses, muscle sections from 3 to 6 animals from each genotype were processed, and at least 15 random sections and totaling >100 NMJs lying en face were analyzed per muscle.…”

Section: Methodsmentioning

confidence: 99%

Gpr126/Adgrg6 contributes to the terminal Schwann cell response at the neuromuscular junction following peripheral nerve injury

Jablonka‐Shariff

Campbell

et al. 2019

Glia

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Gpr126/Adgrg6 is an adhesion G protein‐coupled receptor essential for Schwann cell (SC) myelination with important contributions to repair after nerve crush injury. Despite critical functions in myelinating SCs, the role of Gpr126 within nonmyelinating terminal Schwann cells (tSCs) at the neuromuscular junction (NMJ), is not known. tSCs have important functions in synaptic maintenance and reinnervation, and after injury tSCs extend cytoplasmic processes to guide regenerating axons to the denervated NMJ. In this study, we show that Gpr126 is expressed in tSCs, and that absence of Gpr126 in SCs (SC‐specific Gpr126 knockout, cGpr126) results in a NMJ maintenance defect in the hindlimbs of aged mice, but not in young adult mice. After nerve transection and repair, cGpr126 mice display delayed NMJ reinnervation, altered tSC morphology with decreased S100β expression, and reduced tSC cytoplasmic process extensions. The immune response promoting reinnervation at the NMJ following nerve injury is also altered with decreased macrophage infiltration, Tnfα, and anomalous cytokine expression compared to NMJs of control mice. In addition, Vegfa expression is decreased in muscle, suggesting that cGpr126 non‐cell autonomously modulates angiogenesis after nerve injury. In sum, cGpr126 mice demonstrated delayed NMJ reinnervation and decreased muscle mass following nerve transection and repair compared to control littermates. The integral function of Gpr126 in tSCs at the NMJ provides the framework for new therapeutic targets for neuromuscular disease.

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The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Cited by 28 publications

References 34 publications

Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction‐induced loss of force‐producing capacity

Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction‐induced loss of force‐producing capacity

Forced expression of muscle specific kinase slows postsynaptic acetylcholine receptor loss in a mouse model of MuSK myasthenia gravis

Gpr126/Adgrg6 contributes to the terminal Schwann cell response at the neuromuscular junction following peripheral nerve injury

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