Distal Anterior Compartment Myopathy Research Articles

The muscle protein dysferlin accumulates in the Alzheimer brain

Dysferlin is a transmembrane protein that is highly expressed in muscle. Dysferlin mutations cause limb-girdle dystrophy type 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin has also been described in neural tissue. We studied dysferlin distribution in the brains of patients with Alzheimer disease (AD) and controls. Twelve brains, staged using the Clinical Dementia Rating were examined: 9 AD cases (mean age: 85.9 years and mean disease duration: 8.9 years), and 3 age-matched controls (mean age: 87.5 years). Dysferlin is a cytoplasmic protein in the pyramidal neurons of normal and AD brains. In addition, there were dysferlin-positive dystrophic neurites within Aβ plaques in the AD brain, distinct from tau-positive neurites. Western blots of total brain protein (RIPA) and sequential extraction buffers (high salt, high salt/Triton X-100, SDS and formic acid) of increasing protein extraction strength were performed to examine solubility state. In RIPA fractions, dysferlin was seen as 230–272 kDa bands in normal and AD brains. In serial extractions, there was a shift of dysferlin from soluble phase in high salt/Triton X-100 to the more insoluble SDS fraction in AD. Dysferlin is a new protein described in the AD brain that accumulates in association with neuritic plaques. In muscle, dysferlin plays a role in the repair of muscle membrane damage. The accumulation of dysferlin in the AD brain may be related to the inability of neurons to repair damage due to Aβ deposits accumulating in the AD brain.

P.P.6 09 Damage-induced dysferlin translocation in C2C12 myotubes requires the full length protein

Recently, a novel pathomechanism, defective repair of the plasma membrane, was demonstrated in dysferlin deficient myofibres. Dysferlin is the protein involved in limb-girdle MD type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. So in dysferlinopathy it appears that rather than an inherent problem with the structural integrity of the plasma membrane, it is a defect in its maintenance process which is disease causing. Dysferlin is a homologue of the Caenorhabditis elegans fer-1 gene which mediates vesicle fusion in spermatids. It is characterised by the presence of six C2 domains which are known to be involved in vesicle trafficking and membrane fusion events. To investigate the role of dysferlin in membrane maintenance and its subcellular expression pattern in response to membrane damage, we established two in vivo wounding assays in differentiated C2C12 myotubes: (a) glass beads were dispersed on a monolayer of cells in the presence of a Texas Red conjugated dextran. These 10 kDa dextran molecules are excluded from cells under normal circumstances, but dispersion of beads leads to breaches in the myotube membranes which can be identified by intracellular uptake of fluorescing dextran. (b) A UV nitrogen laser was utilised to create membrane disruptions on single C2C12 myotubes. GFP fusion constructs coding for full length dysferlin, truncated dysferlin mutants coding for distinct C2 domains, caveolin 3 and a Golgi marker were transiently transfected into C2C12 cells and their cellular localisation identified via immunofluorescence in response to the different methods of membrane damage. Both wounding assays showed distinct translocation of full length dysferlin to the plasma membrane after injury. This translocation was dependent on the expression of all C2 domains of dysferlin and the presence of extracellular calcium. Translocation of dysferlin to the plasma membrane in C2C12 myotubes supports a role for dysferlin in plasma membrane maintenance. This response requires the presence of the full length protein and extracellular calcium.

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.

Dysferlin in a hyperCKaemic patient with caveolin 3 mutation and in C2C12 cells after p38 MAP kinase inhibition.

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies.

Molecular analysis of LGMD-2B and MM patients: identification of novel DYSF mutations and possible founder effect in the Italian population

Dysferlin, the protein product of the dysferlin gene ( DYSF), has been shown to have a role in calcium-induced membrane fusion and repair. Dysferlin is absent or drastically reduced in patients with the following autosomal recessive disorders: limb-girdle muscular dystrophy type 2B (LGMD-2B), Miyoshi myopathy (MM) and distal anterior compartment myopathy. To date, less than 45 mutations have been described in DYSF and a wide inter- and intra-familial variation in clinical phenotype has been associated with the same mutation. This observation underlines the relevance of any new report describing genotype/phenotype correlations in dysferlinopathic patient and families. Here we present the results of clinical, biochemical and genetic analysis performed on one MM and three LGMD Italian families. By screening the entire coding region of DYSF, we identified three novel mutations (two missense substitutions and one frame shift microdeletion). The possible existence of a founder effect for the Arg959Trp mutation in the Italian population is discussed.