B. Edwards

The Caenorhabditis elegans lev‐8 gene encodes a novel type of nicotinic acetylcholine receptor α subunit

We have cloned Caenorhabditis elegans lev‐8 and demonstrated that it encodes a novel nicotinic acetylcholine receptor (nAChR) subunit (previously designated ACR‐13), which has functional roles in body wall and uterine muscles as part of a levamisole‐sensitive receptor. LEV‐8 is an α subunit and is the first to be described from the ACR‐8‐like group, a new class of nAChR with atypical acetylcholine‐binding site (loop C) and channel‐lining motifs. A single base pair change in the first intron of lev‐8 in lev‐8(x15) mutants leads to alternative splicing and the introduction of a premature stop codon. lev‐8(x15) worms are partially resistant to levamisole‐induced egg laying and paralysis, phenotypes rescued by expression of the wild‐type gene. lev‐8(x15) worms also show reduced rates of pharyngeal pumping. Electrophysiological recordings from body wall muscle show that currents recorded in response to levamisole have reduced amplitude in lev‐8(x15) compared with wild‐type animals. Consistent with these phenotypic observations, green fluorescent protein fused to LEV‐8 is expressed in body wall and uterine muscle, motor neurons and epithelial‐derived socket cells. Thus, LEV‐8 is a levamisole receptor subunit and exhibits the most diverse expression pattern of any invertebrate nAChR subunit studied to date.

Intermediate filament-like protein syncoilin in normal and myopathic striated muscle.

The intermediate filament-like protein syncoilin is a member of the dystrophin protein complex, and links the complex to the cytoskeleton through binding alpha-dystrobrevin and desmin in muscle. Here, we identify further sites of syncoilin location in normal muscle: at the perinuclear space, myotendinous junction, and enrichment in the sarcolemma and sarcoplasm of oxidative muscle fibers in mice. To understand the importance of the dystrophin protein complex-syncoilin-cytoskeletal link and its implication to disease, we analyzed syncoilin in mice null for alpha-dystrobrevin (adbn-/-) and desmin (des-/-). Syncoilin was upregulated in dystrophic muscles of adbn-/- mice, without alteration in its subcellular location. In des-/- mice, syncoilin was severely reduced in skeletal muscle; lost from sarcomeric Z-lines and neuromuscular junctions, and redistributed from the sub-sarcolemmal cytoskeleton to the cytoplasm. The data show that absence of alpha-dystrobrevin or desmin leads to dynamic changes in syncoilin that may compensate for, or participate in, different muscle myopathies.

Syncoilin modulates peripherin filament networks and is necessary for large-calibre motor neurons

Syncoilin is an atypical type III intermediate filament (IF) protein, which is expressed in muscle and is associated with the dystrophin-associated protein complex. Here, we show that syncoilin is expressed in both the central and peripheral nervous systems. Isoform Sync1 is dominant in the brain, but isoform Sync2 is dominant in the spinal cord and sciatic nerve. Peripherin is a type III IF protein that has been shown to colocalise and interact with syncoilin. Our analyses suggest that syncoilin might function to modulate formation of peripherin filament networks through binding to peripherin isoforms. Peripherin is associated with the disease amyotrophic lateral sclerosis (ALS), thus establishing a link between syncoilin and ALS. A neuronal analysis of the syncoilin-null mouse (Sync−/−) revealed a reduced ability in accelerating treadmill and rotarod tests. This phenotype might be attributable to the impaired function of extensor digitorum longus muscle and type IIb fibres caused by a shift from large- to small-calibre motor axons in the ventral root.

Analysis of skeletal muscle function in the C57BL6/SV129 syncoilin knockout mouse

Syncoilin is a 64-kDa intermediate filament protein expressed in skeletal muscle and enriched at the perinucleus, sarcolemma, and myotendinous and neuromuscular junctions. Due to its pattern of cellular localization and binding partners, syncoilin is an ideal candidate to be both an important structural component of myocytes and a potential mediator of inherited myopathies. Here we present a report of a knockout mouse model for syncoilin and the results of an investigation into the effect of a syncoilin null state on striated muscle function in 6–8-week-old mice. An analysis of proteins known to associate with syncoilin showed that ablation of syncoilin had no effect on absolute expression or spatial localization of desmin or alpha dystrobrevin. Our syncoilin-null animal exhibited no differences in cardiotoxin-induced muscle regeneration, voluntary wheel running, or enforced treadmill exercise capacity, relative to wild-type controls. Finally, a mechanical investigation of isolated soleus and extensor digitorum longus indicated a potential differential reduction in muscle strength and resilience. We are the first to present data identifying an increased susceptibility to muscle damage in response to an extended forced exercise regime in syncoilin-deficient muscle. This study establishes a second viable syncoilin knockout model and highlights the importance of further investigations to determine the role of syncoilin in skeletal muscle.

Utrophin influences mitochondrial pathology and oxidative stress in dystrophic muscle

BackgroundDuchenne muscular dystrophy (DMD) is a lethal X-linked muscle wasting disorder caused by the absence of dystrophin, a large cytoskeletal muscle protein. Increasing the levels of the dystrophin-related-protein utrophin is a highly promising therapy for DMD and has been shown to improve pathology in dystrophin-deficient mice. One contributing factor to muscle wasting in DMD is mitochondrial pathology that contributes to oxidative stress and propagates muscle damage. The purpose of this study was to assess whether utrophin could attenuate mitochondria pathology and oxidative stress.MethodsSkeletal muscles from wildtype C57BL/10, dystrophin-deficient mdx, dystrophin/utrophin double knockout (dko) and dystrophin-deficient mdx/utrophin over-expressing mdx-Fiona transgenic mice were assessed for markers of mitochondrial damage.ResultsUsing transmission electron microscopy, we show that high levels of utrophin ameliorate the aberrant structure and localisation of mitochondria in mdx mice whereas absence of utrophin worsened these features in dko mice. Elevated utrophin also reverts markers of protein oxidation and oxidative stress, elevated in mdx and dko mice, to wildtype levels. These changes were observed independently of a shift in oxidative phenotype.ConclusionThese findings show that utrophin levels influence mitochondrial pathology and oxidative stress. While utrophin deficiency worsens the pathology, utrophin over-expression in dystrophic muscle benefits mitochondria and attenuates the downstream pathology associated with aberrant mitochondrial function.

Micro-utrophin improves cardiac and skeletal muscle function of severely affected D2/mdx mice

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by mutations in the dystrophin gene. DMD boys are wheelchair-bound around 12 years and generally survive into their twenties. There is currently no effective treatment except palliative care, although personalized treatments such as exon skipping, stop codon read-through, and viral-based gene therapies are making progress. Patients present with skeletal muscle pathology, but most also show cardiomyopathy by the age of 10. A systemic therapeutic approach is needed that treats the heart and skeletal muscle defects in all patients. The dystrophin-related protein utrophin has been shown to compensate for the lack of dystrophin in the mildly affected BL10/mdx mouse. The purpose of this investigation was to demonstrate that AAV9-mediated micro-utrophin transgene delivery can not only functionally replace dystrophin in the heart, but also attenuate the skeletal muscle phenotype in severely affected D2/mdx mice. The data presented here show that utrophin can indeed alleviate the pathology in skeletal and cardiac muscle in D2/mdx mice. These results endorse the view that utrophin modulation has the potential to increase the quality life of all DMD patients whatever their mutation.