Elisabetta Mattioli

Mitochondrial dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins

Ullrich congenital muscular dystrophy is a severe genetically and clinically heterogeneous muscle disorder linked to collagen VI deficiency. The pathogenesis of the disease is unknown. To assess the potential role of mitochondrial dysfunction in the onset of muscle fiber death in this form of dystrophy, we studied biopsies and myoblast cultures obtained from patients with different genetic defects of collagen VI and variable clinical presentations of the disease. We identified a latent mitochondrial dysfunction in myoblasts from patients with Ullrich congenital muscular dystrophy that matched an increased occurrence of spontaneous apoptosis. Unlike those in myoblasts from healthy donors, mitochondria in cells from patients depolarized upon addition of oligomycin and displayed ultrastructural alterations that were worsened by treatment with oligomycin. The increased apoptosis, the ultrastructural defects, and the anomalous response to oligomycin could be normalized by Ca2+ chelators, by plating cells on collagen VI, and by treatment with cyclosporin A or with the specific cyclophilin inhibitor methylAla3ethylVal4-cyclosporin, which does not affect calcineurin activity. Here we demonstrate that mitochondrial dysfunction plays an important role in muscle cell wasting in Ullrich congenital muscular dystrophy. This study represents an essential step toward a pharmacological therapy of Ullrich congenital muscular dystrophy with cyclosporin A and methylAla3ethylVal4 cyclosporin.

Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment

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 fibroblasts, 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 fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.

Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.

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.

Association of emerin with nuclear and cytoplasmic actin is regulated in differentiating myoblasts.

Emerin is a nuclear envelope protein whose biological function remains to be elucidated. Mutations of emerin gene cause the Emery-Dreifuss muscular dystrophy, a neuromuscular disorder also linked to mutations of lamin A/C. In this paper, we analyze the interaction between emerin and actin in differentiating mouse myoblasts. We demonstrate that emerin and lamin A/C are bound to actin at the late stages of myotube differentiation and in mature muscle. The interaction involves both nuclear alpha and beta actins and cytoplasmic actin. A serine-threonine phosphatase activity markedly increases emerin-actin binding even in cycling myoblasts. This effect is also observed with purified nuclear fractions in pull-down assay. On the other hand, active protein phosphatase 1, a serine-threonine phosphatase known to associate with lamin A/C, inhibits emerin-actin interaction in myotube extracts. These data provide evidence of a modulation of emerin-actin interaction in muscle cells, possibly through differentiation-related stimuli.

Pre‐Lamin A processing is linked to heterochromatin organization

Pre‐lamin A undergoes subsequent steps of post‐translational modification at its C‐terminus, including farnesylation, methylation, and cleavage by ZMPSTE24 metalloprotease. Here, we show that accumulation of different intermediates of pre‐lamin A processing in nuclei, induced by expression of mutated pre‐lamin A, differentially affected chromatin organization in human fibroblasts. Unprocessed (non‐farnesylated) pre‐lamin A accumulated in intranuclear foci, caused the redistribution of LAP2alpha and of the heterochromatin markers HP1alpha and trimethyl‐K9‐histone 3, and triggered heterochromatin localization in the nuclear interior. In contrast, the farnesylated and carboxymethylated lamin A precursor accumulated at the nuclear periphery and caused loss of heterochromatin markers and Lap2alpha in enlarged nuclei. Interestingly, pre‐lamin A bound both HP1alpha and LAP2alpha in vivo, but the farnesylated form showed reduced affinity for HP1alpha. Our data show a link between pre‐lamin A processing and heterochromatin remodeling and have major implications for understanding molecular mechanisms of human diseases linked to mutations in lamins. J. Cell. Biochem. 102: 1149–1159, 2007.

Diverse lamin-dependent mechanisms interact to control chromatin dynamics Focus on laminopathies

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.

Laminopathies : Involvement of structural nuclear proteins in the pathogenesis of an increasing number of human diseases

Just at the beginning of the millennium the neologism laminopathies has been introduced in the scientific vocabulary. An exponential increase of interest on the subject started concomitantly, so that a formerly quite neglected group of rare human diseases is now widely investigated. This review will cover the history of the identification of the molecular basis for fourteen (since now) hereditary diseases arising from defects in genes that encode nuclear envelope and nuclear lamina‐associated proteins and will also consider the hypotheses that can account for the role of structural nuclear proteins in the pathogenesis of diseases affecting a wide spectrum of tissues.

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.

Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy

Background: Type 2 familial partial lipodystrophy (FPLD2) is characterised by loss of fat in the limbs and buttocks and results from mutations in the LMNA gene. Aim: To evaluate the role of several genes involved in adipogenesis in order to better understand the underlying mechanisms of regional loss of subcutaneous adipose tissue (scAT) in patients with FPLD2. Methods: In total, 7 patients with FPLD2 and 10 healthy control participants were studied. A minimal model was used to calculate the insulin sensitivity (IS). scAT was obtained from abdomen and thigh by biopsy. Relative gene expression was quantified by real-time reverse transcription PCR in a thermal cycler. Prelamin A western blot analysis was carried out on scAT and prelamin A nuclear localisation was determined using immunofluorescence. Adipocyte nuclei were examined by electron microscopy. Results: Patients with FPLD2 were found to have significantly lower IS. The expression of LMNA was similar in both groups. The expression of PPARG2, RB1, CCND3 and LPL in thigh but not in abdomen scAT was significantly reduced (67%, 25%, 38% and 66% respectively) in patients with FPLD2. Significantly higher levels of prelamin A were found in peripheral scAT of patients with FPLD2. Defects in the peripheral heterochromatin and a nuclear fibrous dense lamina were present in the adipocytes of patients with FPLD2. Conclusions: In FPLD2 participants, prelamin A accumulation in peripheral scAT is associated with a reduced expression of several genes involved in adipogenesis, which could perturb the balance between proliferation and differentiation in adipocytes, leading to less efficient tissue regeneration.