Impaired viability of muscle precursor cells in muscular dystrophy with glycosylation defects and amelioration of its severe phenotype by limited gene expression

Abstract

A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model-myofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] mice-showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model-muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice-exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

Contribution of Dysferlin Deficiency to Skeletal Muscle Pathology in Asymptomatic and Severe Dystroglycanopathy Models: Generation of a New Model for Fukuyama Congenital Muscular Dystrophy

“…Our previous data and those of others suggest that muscle cell membrane fragility due to loss of DG or its functional glycosylation triggers disease manifestation [24], [29]. However, we have not observed evidence indicating membrane fragility in fukutin Hp/− skeletal muscle [23].…”

“…Therefore, these mice are not suitable for testing effectiveness of the antisense oligonucleotide therapy for FCMD. Although skeletal muscle-selective fukutin conditional knock-out mice, namely MCK-fukutin-cKO and Myf5-fukutin-cKO, show dystrophic phenotype [24], they are not applicable for the examination of the antisense oligonucleotide therapy because they do not possess the retrotransposal insertion. We previously reported that the small amount of normally glycosylated α-DG remaining in the skeletal muscle of the knock-in mice prevents muscular dystrophy [23].…”

Déficits intrinsèques des cellules satellites dans la dystrophie musculaire de Duchenne

“…En effet, sa délétion spécifique uniquement dans les fibres musculaires entraîne une dégénérescence musculaire moindre que celle observée suite à sa délétion géné-ralisée dans les fibres musculaires et les cellules satellites [9]. La glycosylation de Dag1 par deux glycosyltransférases, fukutine et LARGE (like-glycosyltransferase), est également essentielle pour permettre l'interaction du DGC avec la matrice extracellulaire [10]. Là encore, la délétion spécifique de la fukutine uniquement dans les fibres musculaires entraîne une dégénérescence musculaire moins importante que sa délétion dans les progéniteurs myogéniques [10].…”

“…La glycosylation de Dag1 par deux glycosyltransférases, fukutine et LARGE (like-glycosyltransferase), est également essentielle pour permettre l'interaction du DGC avec la matrice extracellulaire [10]. Là encore, la délétion spécifique de la fukutine uniquement dans les fibres musculaires entraîne une dégénérescence musculaire moins importante que sa délétion dans les progéniteurs myogéniques [10]. Enfin, les cellules satellites déficientes pour LARGE présentent une capacité prolifé-rative diminuée [11].…”

Dystroglycan Glycosylation and Its Involvement in Muscular Dystrophy

“…Therefore, the therapeutic benefits of rescuing fukutin expression that is limited in myofibers were tested. This gene therapy study was conducted by using recombinant adeno-associated virus 9 (AAV9) vectors containing the mouse fukutin cDNA under the control of the MCK promoter, which allowed fukutin expression in differentiated myofibers (74). Results…”