Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

Lipodystrophies are a heterogeneous group of human disorders characterized by the anomalous distribution of body fat associated with insulin resistance and altered lipid metabolism. The pathogenetic mechanism of inherited lipodystrophies is not yet clear; at the molecular level they have been linked to mutations of lamin A/C, peroxisome proliferator-activated receptor (PPARgamma) and other seemingly unrelated proteins. In this study, we examined lamin A/C processing in three laminopathies characterized by lipodystrophic phenotypes: Dunnigan type familial partial lipodystrophy, mandibuloacral dysplasia and atypical Werner's syndrome. We found that the lamin A precursor was specifically accumulated in lipodystrophy cells. Pre-lamin A was located at the nuclear envelope and co-localized with the adipocyte transcription factor sterol regulatory element binding protein 1 (SREBP1). Using co-immunoprecipitation experiments, we obtained the first demonstration of an in vivo interaction between SREBP1 and pre-lamin A. Binding of SREBP1 to the lamin A precursor was detected in patient fibroblasts as well as in control fibroblasts forced to accumulate pre-lamin A by farnesylation inhibitors. In contrast, SREBP1 did not interact in vivo with mature lamin A or C in cultured fibroblasts. To gain insights into the effect of pre-lamin A accumulation in adipose tissue, we inhibited lamin A precursor processing in 3T3-L1 pre-adipocytes. Our results show that pre-lamin A sequesters SREBP1 at the nuclear rim, thus decreasing the pool of active SREBP1 that normally activates PPARgamma and causing impairment of pre-adipocyte differentiation. This defect can be rescued by treatment with troglitazone, a known PPARgamma ligand activating the adipogenic program.

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.

Abstract. Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fi broblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fi broblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectifi ed by drug treatment.

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleocytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.

by a mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence, chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA nuclei, consistent with the finding that heterochromatin-associated protein HP1␤ and histone H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR) are delocalized and solubilized. Both accumulation of lamin A precursor and chromatin defects become more severe in older patients. These results strongly suggest that altered chromatin remodeling is a key event in the cascade of epigenetic events causing MADA and could be related to the premature-aging phenotype.LMNA; heterochromatin; heterochromatin protein-1␤; prelamin A MANDIBULOACRAL DYSPLASIA type A [MADA; Online Mendelian Inheritance in Man (OMIM) no. 248370] is a rare and complex disease characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, mottled cutaneous pigmentation, partial lipodystrophy (type A pattern), and insulin resistance (11,17,49). MADA patients seem to be genetically homogeneous, since they show the same mutation (R527H) in the LMNA gene that encodes A-type lamins, lamins A and C (35, 42). In contrast, patients with generalized loss of subcutaneous fat involving the face, trunk, and extremities (type B pattern) carry mutations in the ZMPSTE24 gene (MADB; OMIM no. 608612) (1, 43).Lamins are type V intermediate filament proteins that display a central rod domain, an NH 2 -terminal head domain, and a COOH-terminal globular tail. Lamins A and C, together with B-type lamins, lamin B1 and B2, are the major components of the nuclear lamina, located between the inner nuclear membrane and the chromatin. A growing number of proteins are known to interact with lamins. Numerous experimental evidences suggest that nuclear lamins are involved in many functions including nuclear positioning and shape, chromatin organization, nuclear envelope assembly/disassembly, DNA replication, and regulation of gene transcriptional activity (18).

Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals. Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation.

Reduction of lamin A/C, which is evolutionarily required for the modulation of Polycomb group (PcG) protein–dependent transcriptional repression by sustaining PcG protein nuclear architecture, leads to PcG protein diffusion and to muscle differentiation.