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.

Lamin A N-terminal phosphorylation is associated with myoblast activation: impairment in Emery-Dreifuss muscular dystrophy.

BACKGROUND: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. OBJECTIVE: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. METHODS: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery-Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. RESULTS: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. CONCLUSIONS: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.

Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies.

The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.

Emerin increase in regenerating muscle fibers.

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.

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.

Staurosporine treatment and serum starvation promote the cleavage of emerin in cultured mouse myoblasts: involvement of a caspase-dependent mechanism.

Emerin is a nuclear membrane-anchored protein which is absent or mutated in patients affected by Emery-Dreifuss muscular dystrophy. In this study, we induced apoptosis in cultured mouse myoblasts to evaluate emerin fate during the nuclear destabilization involved in programmed cell death. Emerin proteolysis was observed in myocytes during the apoptotic process. Myoblast apoptosis and emerin degradation were associated with chromatin compaction and detachment from the nuclear lamina, as detected by electron microscopy. In vivo specific inhibition of caspase 3 or caspase 6 activity completely abolished emerin proteolysis. These results show that the process of programmed cell death in muscle cells leads to emerin proteolysis, which appears to be related to caspase 6 activation and to cleavage of other nuclear envelope proteins, that share sequence homologies or functional features with emerin.

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Immunocytochemistry of nuclear domains and Emery-Dreifuss muscular dystrophy pathophysiology.

The present review summarizes recent cytochemical findings on the functional organization of the nuclear domains, with a particular emphasis on the relation between nuclear envelope-associated proteins and chromatin. Mutations in two nuclear envelope-associated proteins, emerin and lamin A/C cause the Emery-Dreifuss muscular dystrophy; the cellular pathology associated with the disease and the functional role of emerin and lamin A/C in muscle cells are not well established. On the other hand, a large body of evidence indicates that nuclear envelope-associated proteins are involved in tissue-specific gene regulation. Moreover, chromatin remodeling complexes trigger gene expression by utilizing the nuclear matrix-associated actin, which is known to interact with both emerin and lamin A/C. It is thus conceivable that altered expression of these nuclear envelope-associated proteins can account for an impairment of gene expression mainly during cell differentiation as suggested by recent experimental findings on the involvement of emerin in myogenesis. The possibility that Emery-Deifuss muscular dystrophy pathogenesis could involve alteration of the signaling pathway is considered.

A multiple technical approach to the study of apoptotic cell micronuclei.

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.

Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent.

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.

Calcific chronic lateral epicondylitis: a histological and ultrastructural study.

Fragments of insertion tissue from right arm common extensor muscle have been collected from a 25-year patient with chronic lateral epicondylitis. Specimens, processed for light (LM) and electron (EM) microscopy, evidentiated a variety of degenerative alterations, such as focal hyalinosis, lipoidosis, collagen fiber redistribution, calcifications and vascular changes. Evidence of collagen normal function maintenance and turnover have been also observed in tenocytes.

Ultrastructural changes in rat cornea after prolonged hypobaric hypoxia.

A study was performed to evaluate the effect of prolonged environmental hypobaric hypoxia on the ultrastructure of rat cornea. 60-day-old female Wistar rats were exposed to a simulated altitude of 5,500 m (350 mmHg) and pO2 of 76 mmHg for 30 days. Control rats were exposed to atmospheric sea level pressure (760 mmHg) and pO2 of 150 mmHg, for the same period. Ultrastructural analysis of the corneal epithelium did not reveal any relevant difference between control and treated rats. In contrast, the corneal stroma of rats subjected to hypoxia showed vascularization with advanced vessel differentiation and signs of active proliferation. The endothelium of hypoxic cornea showed swollen mitochondria and large empty cytoplasmic areas. The endothelial intercellular junctions could hardly be identified in the hypoxic condition. Nevertheless, the most evident change in hypoxic cornea was in Descemet's membrane, which was considerably thickened, to approximately twice that of the control specimen. These results suggest that environmental hypobaric hypoxia induces marked alterations in the corneal stroma and endothelium morphology, probably related to reduced oxygen tension in the aqueous humor, consequent to hypoxemia.

Prelamin A-mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle.

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 nucleo-cytoskeleton 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.

Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria.

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.

Prevalent cardiac phenotype resulting in heart transplantation in a novel LMNA gene duplication.

Mutations in the lamin A/C gene (LMNA) are known to be involved in several diseases such as Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy type 1B and dilated cardiomyopathies with conduction disease, with considerable phenotype heterogeneity. Here we report on a novel autosomal dominant mutation in LMNA in two direct relatives presenting with different clinical phenotypes, characterized by severe life-threatening limb-girdle muscle involvement and cardiac dysfunction treated with heart transplantation in the proband, and by ventricular tachyarrhythmias with preserved cardiac and skeletal muscle function in her young son. To our knowledge, this is the first report of a duplication in the LMNA gene. The two phenotypes described could reflect different clinical stages of the same disease. We hypothesize that early recognition and initiation of therapeutic manoeuvres in the younger patient may retard the rate of progression of the cardiomyopathy.

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

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.

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells.

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.

In vitro senescence and apoptotic cell death of human megakaryocytes.

To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of alpha(IIb)beta3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary alpha(IIb)beta3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, alpha(IIb)beta3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of alpha(IIb)beta3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.