Rapamycin treatment of Mandibuloacral dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics.

Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.

Lamins are rapamycin targets that impact human longevity: a study in centenarians.

The dynamic organisation of the cell nucleus is profoundly modified during growth, development and senescence as a result of changes in chromatin arrangement and gene transcription. A plethora of data suggests that the nuclear lamina is a key player in chromatin dynamics and argues in favour of a major involvement of prelamin A in fundamental mechanisms regulating cellular senescence and organism ageing. As the best model to analyse the role of prelamin A in normal ageing, we used cells from centenarian subjects. We show that prelamin A is accumulated in fibroblasts from centenarians owing to downregulation of its specific endoprotease ZMPSTE24, whereas other nuclear envelope constituents are mostly unaffected and cells do not enter senescence. Accumulation of prelamin A in nuclei of cells from centenarians elicits loss of heterochromatin, as well as recruitment of the inactive form of 53BP1, associated with rapid response to oxidative stress. These effects, including the prelamin-A-mediated increase of nuclear 53BP1, can be reproduced by rapamycin treatment of cells from younger individuals. These data identify prelamin A and 53BP1 as new targets of rapamycin that are associated with human longevity. We propose that the reported mechanisms safeguard healthy ageing in humans through adaptation of the nuclear environment to stress stimuli.

Oct-1 recruitment to the nuclear envelope in adult-onset autosomal dominant leukodystrophy.

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterised by pyramidal, cerebellar, and autonomic disturbances. Duplication of the LMNB1 gene is the genetic cause of ADLD, yet the pathogenetic mechanism is not defined. In this study, we analysed cells and muscle tissue from three patients affected by ADLD, carrying an extra copy of the LMNB1 gene. Lamin B1 levels were dramatically increased in ADLD nuclei, both in skin fibroblasts and skeletal muscle fibres. Since lamin B1 is known to bind Oct-1, a transcription factor involved in the oxidative stress pathway, we investigated Oct-1 fate in ADLD. Oct-1 recruitment to the nuclear periphery was increased in ADLD cells, while nucleoplasmic localisation of the transcription factor under oxidative stress conditions was reduced. Importantly, lamin B1 degradation occurring in some, but not all ADLD cell lines, slowed down lamin B1 and Oct-1 accumulation. In skeletal muscle, focal disorganisation of sarcomeres was observed, while IIB-myosin heavy chain, an Oct-1 target gene, was under-expressed and rod-containing fibres were formed. These data show that a high degree of regulation of lamin B1 expression is implicated in the different clinical phenotypes observed in ADLD and show that altered Oct-1 nuclear localisation contributes to the disease phenotype.

The empowerment of translational research: lessons from laminopathies.

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.

Altered chromatin organization and SUN2 localization in mandibuloacral dysplasia are rescued by drug treatment.

Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane-centrosome interplay and nuclear movement may be affected in MADA fibroblasts.

Suppression of procollagen {alpha}1 type 1 by long-term low-dose exposure to 2-hydroxyethylmethacrylate in human gingival fibroblasts in vitro.

The purpose of this study was to evaluate the cytotoxicity of low doses and long-term exposure to 2-hydroxyethylmethacrylate (HEMA) on the protein expression of human gingival fibroblasts (HGFs). Human gingival fibroblasts were exposed to different concentrations of HEMA ranging from 0.5 mmol/L to 3 mmol/L for periods of time from 72 hours to 2 weeks. A significant decrease in the expression of procollagen α1 type I protein was observed 72 hours after treatment of cells with 3 mmol/L HEMA. Although low concentrations of the monomer after 2 weeks of exposure to HEMA did not appear to induce any marked changes in the morphology or viability of cells, the expression of procollagen α1 type I protein and its messenger RNA (mRNA) markedly decreased. In conclusion, our data demonstrated that cell viability and morphology assays could be deficient parameters in evaluating the biocompatibility of dental resin materials.

HEMA down-regulates procollagen alpha1 type I in human gingival fibroblasts.

2-Hydroxyethyl methacrylate (HEMA) can be released from restorative materials and diffused into the tooth pulp over long periods of time. Although cytotoxicity due to high concentrations of monomers has been well studied, little is known about the risk of chronic toxicity resulting from low concentrations. The purpose of the study was to evaluate the effects of a minor toxic concentration of HEMA in the synthesis and expression of procollagen alpha1 type I produced by human gingival fibroblasts (HGF). HGF were exposed to 3 mM HEMA from 24 to 96 h. An MTT assay was performed to evaluate cell viability while reverse-transcriptase polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), and Western-blot analysis were carried out to evaluate the variability in the expression and synthesis of procollagen alpha1. Immunofluorescence was performed to detect the protein inside the cells. The results showed that there was a strong reduction of procollagen alpha 1 type I expression at 72 and 96 h. These findings demonstrate that, even if it does not reduce cell viability, 3 mM HEMA interferes both with the synthesis of the procollagen alpha 1 type I protein and its mRNA expression, suggesting that normal cell production and activity are modified by HEMA at concentrations below those which cause acute cytotoxicity.

Expression of procollagen alpha1 type I and tenascin proteins induced by HEMA in human pulp fibroblasts.

In the dental pulp extracellular matrix, the main macromolecules are collagenous proteins, non-collagenous proteins and proteoglycans. Regulated synthesis of the interstitial collagens, in particular, type I collagen, is important during development and wound healing but also in a number of pathological conditions. Tenascin is also a matrix protein highly expressed during development while it decreases in mature organs. Under pathological conditions such as infections and inflammation, during tumorigenesis and mechanical stress applied to cells in culture or tissue in vivo, the expression of tenascin is increased. In this study, HEMA, widely used in dentistry, ophthalmology and drug delivery, has been used to study its influence on the expression of procollagen alpha1 type I and tenascin proteins in the primary cultures of human pulp fibroblasts. Different concentrations of the resin monomer and different times of exposition were tested. The influence of HEMA on the cell viability was evaluated by means of an MTT assay while immunofluorescence and western blotting analysis were performed to detect possible interference with the presence and the synthesis of these proteins. We observed a strong reduction in cell viability in specimens treated for 96 h and 168 h, especially at concentrations of 1 and 3 mmol/L HEMA. Both immunofluorescence and western blotting analysis demonstrated a reduction of procollagen alpha1 type I protein and an overexpression of tenascin protein. Our results showed that long-term exposure and low concentrations of HEMA influence normal cell activity, such as the synthesis of some of the dental pulp extracellular matrix proteins.