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Laminopathies are a wide and heterogeneous group of rare human diseases caused by mutations of the LMNA gene or related nuclear envelope genes. The variety of clinical phenotypes and the wide spectrum of histopathological changes among patients carrying an identical mutation in the LMNA gene make the prognostic process rather difficult, and classical genetic screens appear to have limited predictive value for disease development. The aim of this study was to evaluate whether a comprehensive profile of circulating cytokines may be a useful tool to differentiate and stratify disease subgroups, support clinical follow-ups and contribute to new therapeutic approaches. Serum levels of 51 pro- and anti-inflammatory molecules, including cytokines, chemokines and growth factors, were quantified by a Luminex multiple immune-assay in 53 patients with muscular laminopathy (Musc-LMNA), 10 with non-muscular laminopathy, 22 with other muscular disorders and in 35 healthy controls. Interleukin-17 (IL-17), granulocyte colony-stimulating factor (G-CSF) and transforming growth factor beta (TGF-&beta, 2) levels significantly discriminated Musc-LMNA from controls, interleukin-1&beta, (IL-1&beta, ), interleukin-4 (IL-4) and interleukin-8 (IL-8) were differentially expressed in Musc-LMNA patients compared to those with non-muscular laminopathies, whereas IL-17 was significantly higher in Musc-LMNA patients with muscular and cardiac involvement. These findings support the hypothesis of a key role of the immune system in Musc-LMNA and emphasize the potential use of cytokines as biomarkers for these disorders.

Eukaryotic genomes contain a large number of transposable elements, part of which are still active and able to transpose in the host genome. Mobile element activation is repressed to avoid deleterious effects, such as gene mutations or chromosome rearrangements. Control of transposable elements includes a variety of mechanisms comprising silencing pathways, which are based on the production of small non-coding RNAs. Silencing can occur either through transposable element RNA degradation or through the targeting of DNA sequences by heterochromatin formation and consequent transcriptional inhibition. Since the important role of the heterochromatin silencing, the gradual loss of heterochromatin marks in constitutive heterochromatin regions during the aging process promotes derepression of transposable elements, which is considered a cause of the progressive increase in genomic instability and of the activation of inflammatory responses. This review provides an overview of the effects of heterochromatin loss on the activity of transposable elements during the aging process and the possible impact on genome function. In this context, we discuss the possible role of the nuclear lamina, a major player in heterochromatin dynamics, in the regulation of transposable element activity and potential implications in laminopathic diseases.

We introduce a mesoscopic model to predict the charge mobility of organic semiconductors characterized by a coexistence of crystalline and amorphous phases. First, we validate our scheme by reproducing the trends in charge mobility observed in thin films of poly(3-hexylthiophene) (P3HT) polymers. Next, we address the problem of predicting the morphologies that lead to the highest mobility. Our main finding is the identification of a region of the model's multidimensional parameter space, in which the charge mobility effectively depends on a single morphological feature: the average intercrystallite distance. This scaling behavior provides insight into the main physical mechanism limiting charge mobility in organic semiconductors. Our proposed framework can be adapted to study a wide class of polymeric systems and used to guide the manufacturing of new, high-performing organic semiconductor materials.

The mammalian target of rapamycin (mTOR) pathway is an highly conserved signal transduction axis involved in many cellular processes, such as cell growth, survival, transcription, translation, apoptosis, metabolism, motility and autophagy. Recently, this signalling pathway has come to the attention of the scientific community owing to the unexpected finding that inhibition of mTOR by rapamycin, an antibiotic with immunosuppressant and chemotherapeutic properties, extends lifespan in diverse animal models. Moreover, rapamycin has been reported to rescue the cellular phenotype in a progeroid syndrome [Hutchinson–Gilford Progeria syndrome (HGPS)] that recapitulates most of the traits of physiological ageing. The promising perspectives raised by these results warrant a better understanding of mTOR signalling and the potential applications of mTOR inhibitors to counteract ageing‐associated diseases and increase longevity. This review is focused on these issues.

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.

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A-linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A-Akt interaction and on the biological significance of their relationship. J. Cell. Physiol. 220: 553–561, 2009. © 2009 Wiley-Liss, Inc.

Background information. Emerin is a nuclear envelope protein that contributes to nuclear architecture, chromatin structure, and gene expression through its interaction with various nuclear proteins. In particular, emerin is molecularly connected with the nuclear lamina, a protein meshwork composed of lamins and lamin-binding proteins underlying the inner nuclear membrane. Among nuclear lamina components, lamin A is a major emerin partner. Lamin A, encoded by the LMNA gene (lamin A/C gene), is produced as a precursor protein (prelamin A) that is post-transcriptionally modified at its C-terminal region where the CaaX motif triggers a sequence of modifications, including farnesylation, carboxymethylation, and proteolytic cleavage by ZMPSTE 24 (zinc metalloproteinase Ste24) metalloproteinase. Impairment of the lamin A maturation pathway causing lamin A precursor accumulation is linked to the development of rare diseases such as familial partial lipodystrophy, MADA (mandibuloacral dysplasia), the Werner syndrome, Hutchinson—Gilford progeria syndrome and RD (restrictive dermopathy). Results. In the present study, we show that emerin and different prelamin A forms influence each other's localization. We show that the accumulation of non-farnesylated as well as farnesylated carboxymethylated lamin A precursors in human fibroblasts modifies emerin localization. On the contrary, emerin absence at the inner nuclear membrane leads to unprocessed (non-farnesylated) prelamin A aberrant localization only. Moreover, we observe that the restoration of emerin expression in emerin-null cells induces the recovery of non-farnesylated prelamin A localization. Conclusion. These results indicate that emerin—prelamin A interplay influences nuclear organization. This finding may be relevant to the understanding of laminopathies.

Lamin A is a nuclear lamina constituent implicated in a number of human disorders including Emery-Dreifuss muscular dystrophy. Since increasing evidence suggests a role of the lamin A precursor in nuclear functions, we investigated the processing of prelamin A during differentiation of C2C12 mouse myoblasts. We show that both protein levels and cellular localization of prelamin A are modulated during myoblast activation. Similar changes of lamin A-binding proteins emerin and LAP2alpha were observed. Furthermore, prelamin A was found in a complex with LAP2alpha in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affected LAP2alpha and PCNA amount and increased caveolin 3 mRNA and protein levels, while accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor appeared to inhibit caveolin 3 expression. Our data provide evidence for a critical role of the lamin A precursor in the early steps of muscle cell differentiation.

Osteoclast differentiation is a complex process involving cytoskeleton and nuclear reorganization. Osteoclasts regulate bone homeostasis and have a key role in bone degenerative processes. Osteolysis and osteoporosis characterize a subset of laminopathies, inherited disorders due to defects in lamin A/C. Laminopathies featuring bone resorption are characterized, at the molecular level, by anomalous accumulation of the unprocessed lamin A precursor, called prelamin A. To obtain a suitable cell model to study prelamin A effects on osteoclasts, prelamin A processing inhibitors FTI-277 or AFCMe were applied to peripheral blood monocytes induced to differentiate towards the osteoclastic lineage. Previous studies have shown that treatment with FTI-277 causes accumulation of non-farnesylated prelamin A, while AFCMe inhibition of prelamin A maturation causes accumulation of a farnesylated form. We demonstrate that monocytes subjected to FTI-277 treatment and mostly those subjected to AFCMe administration, differentiate towards the osteoclastic lineage more efficiently than untreated monocytes, in terms of number of multinucleated giant cells, mRNA expression of osteoclast-related genes and TRACP 5b activity. On the other hand, the bone resorption activity of osteoclasts obtained in the presence of high prelamin A levels is lower with respect to control osteoclasts. This finding may help the understanding of the osteolytic and osteoporotic processes that characterize progeroid laminopathies.

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.

Context: Mandibuloacral dysplasia type A (MADA; OMIM 248370) is a rare progeroid syndrome characterized by dysmorphic craniofacial and skeletal features, lipodystrophy, and metabolic complications. Most Italian patients carry the same homozygous missense mutation (p.R527H) in the C-terminal tail domain of the LMNA gene, which encodes lamin A/C, an intermediate filament component of the nuclear envelope.Objective: The objective of the study was to identify novel LMNA mutations in individuals with clinical characteristics (bird-like facies, mandibular and clavicular hypoplasia, acroosteolysis, lipodystrophy, alopecia) observed in other well-known patients.Design: The LMNA gene was sequenced. Functional properties of the mutant alleles were investigated.Patient: We report a 27-yr-old Italian woman showing a MADA-like phenotype. Features include a hypoplastic mandible, acroosteolysis, pointed nose, partial loss of sc fat, and a progeric appearance. Due to the absence of clavicular dysplasia and normal metabolic profiles, generally associated with muscle hyposthenia and generalized hypotonia, this phenotype can be considered an atypical laminopathy.Results: We identified a patient compound heterozygote for the p.R527H and p.V440M alleles. The patient’s cells showed nuclear shape abnormalities, accumulation of pre-lamin A, and irregular lamina thickness. Lamins A and C showed normal expression and localization. The electron microscopy detected heterochromatin defects with a pattern similar to those observed in other laminopathies. However, chromatin analysis showed a normal distribution pattern of the major heterochromatin proteins: heterochromatin protein-1β and histone H3 methylated at lysine 9.Conclusions: The clinical and cellular features of this patient show overlapping laminopathy phenotypes that could be due to the combination of p.R527H and p.V440M alleles.

Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

Objectives: Our aim was to conduct a comparative study in a large cohort of myopathic patients carrying LMNA gene mutations to evaluate clinical and molecular features associated with different phenotypes. Methods: We performed a retrospective cohort study of 78 myopathic patients with LMNA mutation and 30 familial cases with LMNA mutation without muscle involvement. We analyzed features characterizing the various forms of LMNA -related myopathy through correlation statistics. Results: Of the 78 patients, 37 (47%) had limb-girdle muscular dystrophy 1B (LGMD1B), 18 (23%) congenital muscular dystrophy (MDCL), 17 (22%) autosomal dominant Emery-Dreifuss muscular dystrophy 2 (EDMD2), and 6 (8%) an atypical myopathy. The myopathic phenotypes shared a similar cardiac impairment. Cardioverter defibrillator or pacemaker was implanted in 41 (53%) myopathic patients compared to 7 (23%) familial cases without muscle involvement ( p = 0.005). Heart transplantation was performed in 8 (10.3%) myopathic patients and in none of the familial cases. Ten (12.8%) myopathic patients died; there were no deaths among the familial cases ( p = 0.032). Missense mutations were found in 14 patients (82%) with EDMD2 and 14 patients (78%) with MDCL compared to 17 patients (45%) with LGMD1B and 4 (67%) atypical patients. Frameshift mutations were detected in 17 (45%) LGMD1B compared to 3 (18%) EDMD2, 1 (6%) MDCL, and 2 (33%) with atypical myopathy ( p = 0.021). Furthermore, frameshift mutations were found in 30 of 73 patients (41%) with heart involvement compared to 4 of 35 (11%) without heart involvement ( p = 0.004). Conclusions: Our data provided new insights in LMNA -related myopathies, whose natural history appears to be dominated by cardiac involvement and related complications.

The photodissociation and geminate recombination of nitric oxide in myoglobin, under continuous illumination, is modeled computationally. The relaxation of the photon energy into the protein matrix is also considered in a single simulation scheme that mimics a complete experimental setup. The dynamic approach to non-equilibrium molecular dynamics is used, starting from a steady state, to compute its relaxation to equilibrium. Simulations are conducted for the native form of sperm whale myoglobin and for two other mutants, V68W and L29F, illustrating a fair diversity of spatial and temporal geminate recombination processes. Energy flow to the heme and immediate protein environment provide hints to allostery. In particular, a pathway of energy flow between the heme and the FG loop is illustrated. Although the simulations were conducted for myoglobin only, the thermal fluctuations of the FG corner are in agreement with the large structural shifts of FG during the allosteric transition of tetrameric hemoglobin.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00418-012-0977-5) contains supplementary material, which is available to authorized users.

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.

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.

A proteomic-based search for novel substrates of Akt was undertaken in C2C12 murine muscle cells. Our data demonstrate that Akt isoform 2 phosphorylates Ankrd2 at Serine 99 in response to H2O2 stimuli, regulating muscle differentiation rate., Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization databases in combination with an anti–phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C2C12 myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

Mandibuloacral dysplasia type A (MADA) is a rare disease caused by mutations in the LMNA gene encoding A type lamins. Patients affected by mandibuloacral dysplasia type A suffer from partial lipodystrophy, skin abnormalities and accelerated aging. Typical of mandibuloacral dysplasia type A is also bone resorption at defined districts including terminal phalanges, mandible and clavicles. Little is known about the biological mechanism underlying osteolysis in mandibuloacral dysplasia type A. In the reported study, we analyzed an osteoblast primary culture derived from the cervical vertebrae of a mandibuloacral dysplasia type A patient bearing the homozygous R527H LMNA mutation. Mandibuloacral dysplasia type A osteoblasts showed nuclear abnormalities typical of laminopathic cells, but they proliferated in culture and underwent differentiation upon stimulation with dexamethasone and beta-glycerophosphate. Differentiated osteoblasts showed proper production of bone mineral matrix until passage 8 in culture, suggesting a good differentiation activity. In order to evaluate whether mandibuloacral dysplasia type A osteoblast-derived factors affected osteoclast differentiation or activity, we used a conditioned medium from mandibuloacral dysplasia type A or control cultures to treat normal human peripheral blood monocytes and investigated whether they were induced to differentiate into osteoclasts. A higher osteoclast differentiation and matrix digestion rate was obtained in the presence of mandibuloacral dysplasia type A osteoblast medium with respect to normal osteoblast medium. Further, TGFbeta 2 and osteoprotegerin expression were enhanced in mandibuloacral dysplasia type A osteoblasts while the RANKL/osteoprotegerin ratio was diminished. Importantly, inhibition of TGFbeta 2 by a neutralizing antibody abolished the effect of mandibuloacral dysplasia type A conditioned medium on osteoclast differentiation. These data argue in favor of an altered bone turnover in mandibuloacral dysplasia type A, caused by upregulation of bone-derived stimulatory cytokines, which activate non-canonical differentiation stimuli. In this context, TGFbeta 2 appears as a major player in the osteolytic process that affects mandibuloacral dysplasia type A patients.

Mutations in A-type lamins or lamin-binding proteins are involved in the pathogenesis of diseases referred to as laminopathies (Worman et al., 2009). They include both tissue-specific disorders affecting striated muscle (muscular laminopathies), adipose tissue (lipodystrophic laminopathies), and syndromes in which many tissues undergo premature ageing (progeric laminopathies). Howmutations in LMNA cause diverse diseases is one of the most intriguing riddle in medical genetics. Two hypotheses have emerged to account for the molecular basis of the wide spectrum of these diseases. The mechanical stress hypothesis, mainly based on observations in cultured cells, proposes that mutations in A-type lamins lead to increased nuclear fragility and eventual nuclear disruption in tissues exposed to mechanical strain (Lammerding et al., 2005). The gene expression hypothesis suggests that mutations in A-type lamins lead to abnormal tissue-specific gene regulation. This model is based on findings that A-type lamins and associated proteins bind to chromatin and transcriptional regulators. The gene expression hypothesis does not exclude effects that mechanical stress may have on cells; in fact, both gene expression, as well as signalling pathways and mechanical integrity should be perturbed in cells carrying laminopathy mutations (Cohen et al., 2008). Furthermore, signalling pathways that culminate in transcription factor activity may also be regulated at the nuclear envelope and lamina, which act as a platform or scaffold necessary for the appropriate localization of factors important in specific tissue differentiation (Schirmer and Foisner, 2007; Pekovic and Hutchison, 2008; Andrés and Gonzalez, 2009).

Lamin A, a protein component of the nuclear lamina, is synthesized as a precursor named prelamin A, whose multi-step maturation process involves different protein intermediates. As demonstrated in laminopathies such as familial partial lipodystrophy, mandibuloacral dysplasia, Werner syndrome, Hutchinson-Gilford progeria syndrome and restrictive dermopathy, failure of prelamin A processing results in the accumulation of lamin A protein precursors inside the nucleus which dominantly produces aberrant chromatin structure. To understand if nuclear lamina components may be involved in prelamin A chromatin remodeling effects, we investigated barrier-to-autointegration factor (BAF) localization and expression in prelamin A accumulating cells. BAF is a DNA-binding protein that interacts directly with histones, lamins and LEM-domain proteins and has roles in chromatin structure, mitosis and gene regulation. In this study, we show that the BAF heterogeneous localization between nucleus and cytoplasm observed in HEK293 cycling cells changes in response to prelamin A accumulation. In particular, we observed that the accumulation of lamin A, non-farnesylated prelamin A and farnesylated carboxymethylated lamin A precursors induce BAF nuclear translocation. Moreover, we show that the treatment of human fibroblasts with prelamin A interfering drugs results in similar changes. Finally, we report that the accumulation of progerin, a truncated form of farnesylated and carboxymethylated prelamin A identified in Hutchinson-Gilford progeria syndrome cells, induces BAF recruitment in the nucleus. These findings are supported by coimmunoprecipitation of prelamin A or progerin with BAF in vivo and suggest that BAF could mediate prelamin A-induced chromatin effects.

Satellite cells (SCs) are skeletal muscle progenitor cells located between the basal lamina and the sarcolemma of muscle fibers. They are responsible for muscle growth and repair. In humans, aging results in the depletion of the SC population and in its proliferative activity, but not in its function. It has not yet been determined whether under conditions of massive muscle fiber death in vivo, the regenerative potential of SCs is totally or partially compromised in old muscle. No studies have yet tested whether advanced age is a factor that restrains the response of SCs to muscle denervation in humans; this is also due to difficulties in the isolation and in the culture of SCs from a small human surgery fragment. The aim of this study was to study in depth muscle regeneration analysing the SC ability of SCs to proliferate and differentiate in aging human patients.In order to study in more detail the molecular mechanism, the proliferative and differentiative ability of aging SCs, we isolated SCs from aging human muscle biopsies and analysed their morphology by transmission electron microscopy and immunocytochemical analysis (antibodies against desmin, N-CAM and M-cadherin) and their capacity to grow and to expand in vitro. Moreover, in order to evaluate gene expression of myogenic regulatory factors Myf5, MyoD and myogenin (Myf4), RT-PCR was performed.SCs isolated from aging human muscle biopsies and plated into favorable proliferation and differentiation conditions were able to proceed through the myogenic program and actively form myotubes, although taking longer than the young control sample. The RT-PCR analysis together with the ultrastructural SC features showed that the myogenic potential seemed to be compromised during the aging human muscle proliferation in vitro.

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.

The nuclear envelope (NE) is the most important border in the eukaryotic cells, essential in maintaining the identity of the nuclear and cytoplasmic compartments and in allowing additional levels of regulation and control of the gene expression. Previous studies of our group provided new insights in the complex mechanisms that are involved in the pathogenesis of a group of human diseases, collectively referred to as laminopathies (Maraldi et al., 2003, 2004). In particular, we investigated the influences of disease-causing mutations in A-type lamins on the nuclear morphology and chromatin arrangement (Maraldi et al., 2005, 2006), on the involvement of prelamin A accumulation in progeric laminopathies (Maraldi and Lattanzi, 2007; Maraldi et al., 2007), and on the possible effects of altered lamin A forms on the availability of specific transcription factors (Maraldi et al., 2008). The outer and inner nuclear membranes (ONM and INM, respectively) are characterized by different sets of proteins; in fact, whilst most of the ONM proteins are likely to perform endoplasmic reticulumrelated functions, the roles of INM proteins depend on their interactions with a variety of nuclear proteins, which include lamins, nesprins and chromatin-associated proteins. The INM is associated with two types of lamins, lamin B and lamin A/C, which form the network of filaments of the nuclear lamina. The interactions of many INM proteins with lamins are essential to maintain the stability of the NE. On the other hand, the correct positioning of the nucleus within each cell type is mediated by interaction of nesprins, that span the NE lumen and interact with cytoskeletal elements, and SUNs that interact with the nuclear lamina. Specific interactions of INM proteins and lamins with chromatinassociated proteins are conceivably involved in the organization of the chromatin rrangement. Additional putative functions of the INM proteins and lamins, which are emerging from recent insights, include specific interactions with transcription factors, and modulation of signal transduction pathways. This issue is more than just academic, because defects in lamin and INM protein expression are linked to several human genetic diseases, whose pathogenic mechanisms can be elucidated taking into account the complex functional roles of the NE. Here we discuss the experimental evidence that reveal that the NE functions as a signalling node with an active role in mechanotransduction and suggest a more direct role in gene regulation than previously anticipated

The organization of chromosomes into euchromatin and heterochromatin is amongst the most important and enigmatic aspects of genome evolution. Constitutive heterochromatin is a basic yet still poorly understood component of eukaryotic chromosomes, and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Although recent evidence indicates that the presence of transcribed genes in constitutive heterochromatin is a conserved trait that accompanies the evolution of eukaryotic genomes, the term heterochromatin is still considered by many as synonymous of gene silencing. In this paper, we comprehensively review data that provide a clearer picture of transcribed sequences within constitutive heterochromatin, with a special emphasis on Drosophila and humans.

Increasing interest in drugs acting on prelamin A has derived from the finding of prelamin A involvement in severe laminopathies. Amelioration of the nuclear morphology by inhibitors of prelamin A farnesylation has been widely reported in progeroid laminopathies. We investigated the effects on chromatin organization of two drugs inhibiting prelamin A processing by an ultrastructural and biochemical approach. The farnesyltransferase inhibitor FTI-277 and the non-peptidomimetic drug N-acetyl-S-farnesyl- l -cysteine methylester (AFCMe) were administered to cultured control human fibroblasts for 6 or 18 h. FTI-277 interferes with protein farnesylation causing accumulation of non-farnesylated prelamin A, while AFCMe impairs the last cleavage of the lamin A precursor and is expected to accumulate farnesylated prelamin A. FTI-277 caused redistribution of heterochromatin domains at the nuclear interior, while AFCMe caused loss of heterochromatin domains, increase of nuclear size and nuclear lamina thickening. At the biochemical level, heterochromatin-associated proteins and LAP2α were clustered at the nuclear interior following FTI-277 treatment, while they were unevenly distributed or absent in AFCMe-treated nuclei. The reported effects show that chromatin is an immediate target of FTI-277 and AFCMe and that dramatic remodeling of chromatin domains occurs following treatment with the drugs. These effects appear to depend, at least in part, on the accumulation of prelamin A forms, since impairment of prelamin A accumulation, here obtained by 5-azadeoxycytidine treatment, abolishes the chromatin effects. These results may be used to evaluate downstream effects of FTIs or other prelamin A inhibitors potentially useful for the therapy of laminopathies.

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.

In the last 5 years, an impressive series of genetic diseases (16 distinct diseased phenotypes have been so far identified), affecting metabolic and/or developmental processes, have been demonstrated to be caused by mutation of LMNA gene and collectively referred to as laminopathies. Most of these diseases are characterized by dystrophic/degenerative processes affecting different tissues and organs. Nuclear defects, consisting in heterochromatin focal or total loss and in nuclear lamina thickening or invagination, characterize the cells from laminopathic patients. Furthermore, accumulation of pre-lamin A has been demonstrated to occur, at least in a large group of laminopathies. These findings suggest a possible pathogenic mechanism for laminopathies. The cells bearing mutated pre-lamin A could not be able to maintain the chromatin organization required by differentiation programs, which represents an epigenetic marker of each cell lineage undergoing a differentiation process. These alterations have been demonstrated to be due to a lack of mutant lamin A to physiologically interact with heterochromatin-associated proteins, including HP1. The use of specific drugs that interfere with the maturation of lamin A allowed us to demonstrate that the accumulation of farnesylated pre-lamin A induces the appearance of nuclear phenotypes similar to those occurring in progeric syndromes. Furthermore, LMNA mutations found in several laminopathies reduce the transcriptional capability of the cells accumulating pre-lamin A. Therefore, the pathogenic mechanism of laminopathies appears to involve alterations of the chromatin organization and transcriptional capability required by differentiation programs, not necessarily during early embryogenesis, in which cells lack lamin A/C, but in adult cell populations capable of differentiating under specific stimuli. These cells, that onstitute the stem reservoir of several adult tissues, and that are typically represented by mesenchymal stromal cells, in the presence of mutant lamin A/C, and of accumulating prelamin A, present phenotypic alterations of the chromatin pattern and impairment of gene expression mechanisms. This could result in an accelerated cellular senescence, which could be characterized by the presence of progerin in progeric laminopathies, and of pre-lamin A in other laminopathies, in which not all tissues but specific tissues are involved. In fact, lamins and lamin-associated proteins, by constituting a platform for the interaction with transcription factors, may contribute to a fine modulation of gene expression programs, typical of each cell lineage.

CD99 was recently reported to be under control of the osteoblast-specific transcription factor Cbfa1 (RUNX2) in osteoblasts, suggesting a role in the phato-physiology of these cells. No extensive information is available on the role(s) of this molecule in malignant phenotype, and osteosarcoma, in particular, has never been studied. We report that in 11 different cell lines and 17 clinical samples CD99 expression is either undetectable or very low. Being expressed in the normal counterpart, we tested the hypothesis that CD99 down-regulation may have a role in osteosarcoma development and progression. CD99-forced expression in two osteosarcoma cell lines significantly reduced resistance to anoikis, inhibited growth in anchorage independence as well as cell migration, and led to abrogation of tumorigenic and metastatic ability. Therefore, the molecule acts as a potent suppressor of malignancy in osteosarcoma. CD99 gene transfection induces caveolin-1 up-regulation and the two molecules were found to colocalize on the cell surface. Treatment with antisense oligonucleotides to caveolin-1 abrogates the effects of CD99 on migration. The findings point to an antioncogenic role for CD99 in osteosarcoma, likely through the regulation of caveolin-1 and inhibition of c-Src kinase activity.

Two major events are emerging as being involved in the fine regulatory mechanisms that control gene expression through the nuclear envelope/lamina. The first is the linkage of envelope proteins to transcription factors, most of which function as transcriptional repressors. The second is the interplay between nuclear lamina/nuclear envelope proteins and chromatin, through more or less direct interactions with DNA-binding proteins. The first mechanism appears to play a major role in tissue-specific functions, whereas the latter mechanism likely modifies higher-order chromatin organization, thus regulating differentiation and aging. This review highlights the importance of the study of a group of genetic diseases, collectively referred to as laminopathies, to understand the actual role played by lamins in ensuring the fidelity of gene expression and in modulating cell differentiation.

Autosomal recessive mandibuloacral dysplasia [mandibuloacral dysplasia type A (MADA); Online Mendelian Inheritance in Man (OMIM) no. 248370 ] is caused 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 HP1β 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.

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.

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. Copyright 2004 Wiley-Liss, Inc.

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

We studied the nuclear topography of the replicating enzyme DNA polymerase alpha in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase alpha antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase alpha observed in G1- and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase alpha complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase alpha. The evidence of DNA polymerase alpha circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

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.

Lipodystrophy syndromes are rare diseases primarily affecting the development or maintenance of the adipose tissue but are also distressing indirectly multiple organs and tissues, often leading to reduced life expectancy and quality of life. Lipodystrophy syndromes are multifaceted disorders caused by genetic mutations or autoimmunity in the vast majority of cases. While many subtypes are now recognized and classified, the disease remains remarkably underdiagnosed. The European Consortium of Lipodystrophies (ECLip) was founded in 2014 as a non-profit network of European centers of excellence working in the field of lipodystrophies aiming at promoting international collaborations to increase basic scientific understanding and clinical management of these syndromes. The network has developed a European Patient Registry as a collaborative research platform for consortium members. ECLip and ECLip registry activities involve patient advocacy groups to increase public awareness and to seek advice on research activities relevant from the patients perspective. The annual ECLip congress provides updates on the research results of various network groups members., (Copyright © 2024.)