Cristina Capanni

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.

Laminopathies and lamin-associated signaling pathways

Laminopathies are genetic diseases due to mutations or altered post‐translational processing of nuclear envelope/lamina proteins. The majority of laminopathies are caused by mutations in the LMNA gene, encoding lamin A/C, but manifest as diverse pathologies including muscular dystrophy, lipodystrophy, neuropathy, and progeroid syndromes. Lamin‐binding proteins implicated in laminopathies include lamin B2, nuclear envelope proteins such as emerin, MAN1, LBR, and nesprins, the nuclear matrix protein matrin 3, the lamina‐associated polypeptide, LAP2alpha and the transcriptional regulator FHL1. Thus, the altered functionality of a nuclear proteins network appears to be involved in the onset of laminopathic diseases. The functional interplay among different proteins involved in this network implies signaling partners. The signaling effectors may either modify nuclear envelope proteins and their binding properties, or use nuclear envelope/lamina proteins as platforms to regulate signal transduction. In this review, both aspects of lamin‐linked signaling are presented and the major pathways so far implicated in laminopathies are summarized. J. Cell. Biochem. 112: 979–992, 2011.

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.

Nuclear alterations in autosomal‐dominant Emery‐Dreifuss muscular dystrophy

Electron microscopy study of muscle biopsies from patients with autosomal‐dominant Emery‐Dreifuss muscular dystrophy revealed nuclear alterations in about 10% of the preserved muscle fibers. The major findings consisted of peripheral heterochromatin loss or detachment from the nuclear envelope, and of interchromatin texture alterations. These abnormalities are similar to those reported in an animal model of the disease and to those found in the X‐linked form of Emery‐Dreifuss muscular dystrophy. These results suggest that an abnormal ultrastructural arrangement of the nuclear periphery is a common feature in the known forms of Emery‐Dreifuss muscular dystrophy, and that several proteins of the nuclear scaffold are necessary in muscle cells to maintain the nuclear structural/functional integrity and a normal muscle cell metabolism.

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.

Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50-pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

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.

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 LAP2α 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.

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.