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

Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: altered intermolecular interaction with emerin and implications for gene transcription

Familial partial lipodystrophy is an autosomal dominant disease caused by mutations of the LMNA gene encoding alternatively spliced lamins A and C. Abnormal distribution of body fat and insulin resistance characterize the clinical phenotype. In this study, we analyzed primary fibroblast cultures from a patient carrying an R482L lamin A/C mutation by a morphological and biochemical approach. Abnormalities were observed consisting of nuclear lamin A/C aggregates mostly localized close to the nuclear lamina. These aggregates were not bound to either DNA-containing structures or RNA splicing intranuclear compartments. In addition, emerin did not colocalize with nuclear lamin A/C aggregates. Interestingly, emerin failed to interact with lamin A in R482L mutated fibroblasts in vivo, while the interaction with lamin C was preserved in vitro, as determined by coimmunoprecipitation experiments. The presence of lamin A/C nuclear aggregates was restricted to actively transcribing cells, and it was increased in insulin-treated fibroblasts. In fibroblasts carrying lamin A/C nuclear aggregates, a reduced incorporation of bromouridine was observed, demonstrating that mutated lamin A/C in FPLD cells interferes with RNA transcription

Extracellular matrix and nuclear abnormalities in skeletal muscle of a patient with Walker–Warburg syndrome caused by POMT1 mutation

AbstractWalker–Warburg syndrome (WWS) is an autosomal recessive disorder characterized by congenital muscular dystrophy, structural eye abnormalities and severe brain malformations. We performed an immunohistochemical and electron microscopy study of a muscle biopsy from a patient affected by WWS carrying a homozygous frameshift mutation in O-mannosyltransferase 1 gene (POMT1). α-Dystroglycan glycosylated epitope was not detected in muscle fibers and intramuscular peripheral nerves. Laminin α2 chain and perlecan were reduced in muscle fibers and well preserved in intramuscular peripheral nerves. The basal lamina in several muscle fibers showed discontinuities and detachment from the plasmalemma. Most nuclei, including myonuclei and satellite cell nuclei, showed detachment or complete absence of peripheral heterochromatin from the nuclear envelope. Apoptotic changes were detected in 3% of muscle fibers. The particular combination of basal lamina and nuclear changes may suggest that a complex pathogenetic mechanism, affecting several subcellular compartments, underlies the degenerative process in WWS muscle

Exon Skipping-Mediated Dystrophin Reading Frame Restoration for Small Mutations

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Collagen VI myopathies: pathogenic mechanism and therapeutic strategies

Collagen (Col) VI is a major component of the extracellular matrix which, in skeletal muscle, is localized just outside the basement membrane. Deficiency of ColVI in humans due to mutations of COL6 genes gives rise to Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD), and Myosclerosis Myopathy. About 70 different COL6 gene  mutations have been associated to ColVI myopathies which, although present  a wide range of clinical features, share a common pathogenesis. This mechanism, initially identified in the Col6a1-/- model (Irwin et al., 2003), and then in cultures from BM and UCMD patients (Angelin et al., 2007), involves a mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). The pat hogenic role of PTP opening, causing the release of proapoptotic factors, has been confirmed by the normalizing effect of cyclosporine A (CsA) on  the mitochondrial defect and on the increased apoptotic rate in both the mouse model and in a selected group of patients (Merlini et al., 2008). We have recently demonstrated that the persistence of abnormal mitochondria and apoptosis are amplified by defective autophagy (Grumati et al., 2010). In fact, forced activation of autophagy by genetic, dietary and pharmacological approaches restore myofiber survival and ameliorate the dystrophic phenotype in mice. Since also muscle cells of BM and UCMD patients  present a defective activation of the autophagic machinery, it will be possible to restore this activity by using  a low protein diet or drugs capable to reactivate autophagy. To monitor the effects of therapies on ColVI-related myopathies highly invasive muscle/skin biopsies have been so far utilized. We have recently obtained evidence that ColVI expression in blood macrophages from BM and UCMD patients can be detected at levels comparable to those observed in muscle biopsies and cultured skin fibroblasts (Gualandi et al., 2011). These data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing highly invasive biopsies in the diagnosis and monitoring of ColVI myopathies. 1. Irwin WA et al., Nat Genet 2003;35:267-71 2. Angelin A et al., PNAS USA 2007;104:991-6 3. Merlini L et al., PNAS USA 2008;105:5225-9 4. Gualandi F et al., Muscle Nerve 2011;44:80-

Autophagy is defective in collagen VI muscular dystrophies and its reactivation rescues myofiber degeneration

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A pathogenic mechanism leading to partial lipodistrophy and prospects for pharmacological treatment of insulin resistance syndrome

The understanding of a common complex phenotype such as insulin resistance can be favoured by evaluation of monogenic syndromes. Clinical definition, pathogenesis, and therapeutical strategies for the insulin resistance syndrome can thus be improved by the characterization at the molecular genetic level of monogenic forms of lipodystrophies. Here we report experimental evidence on the pathogenic mechanism underlying insulin resistance in a rare form of laminopathy, due to mutation of the LMNA gene coding for lamin A/C, the Dunnigan-type familial partial lipodystrophy (FPLD). The defect, consisting in the intranuclear accumulation of mutant unprocessed precursors of lamin A, reduces the amount of the DNA-bound adipocyte transcription factor sterol regulatory element binding protein 1 (SREBP1) and lowers the peroxisome proliferator-activated receptor (PPARgamma) expression, causing the impairment of pre-adipocyte differentiation. The treatment with the PPARgamma ligand troglitazone (TDZ) is able to rescue the adipogenic program. Since FPLD recapitulates the essential metabolic abnormalities of the common insulin resistance syndrome, the beneficial effects of TDZ on monogenic lipodystrophies might provide a clue as to the future treatment strategies also for the common syndrome of insulin resistance

Sleep disorders in patients with spinal cord injury

Sleep disturbances are globally more frequent in patients with spinal cord injury (SCI) than in the able-bodied population, and could contribute to dysfunction and poor quality of life in these patients. Specific sleep disorders may also contribute to negative health outcomes enhancing cardiovascular risk in a condition that per se increases heart disease related mortality. This review focuses on prevalence, features and treatment of sleep disorders in SCI. Although data on these subjects have been produced, reports on pathophysiology, consequences and treatment of sleep disorders are scarce or contradictory and more studies are required