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Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1−/− mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis., Highlights ► We study collagen VI alpha 5 and alpha 6 chains expression in human skeletal muscle. ► ► We show that alpha 5 chain is exclusively present at the myotendinous junctions. ► We show that alpha 6 chain is present in the ECM but not at the basement membranes. ► We show that alpha 6 chain increases in Duchenne muscular dystrophy muscle fibrosis. ► We hypothesize the possible formation of alpha1-2-6 or alpha1-2-5 chains trimers.

Collagen VI myopathies (Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), and myosclerosis myopathy) share a common pathogenesis, that is, mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). This effect was first identified in the Col6a1−/− mouse model and then in muscle cell cultures from UCMD and BM patients; the normalizing effect of cyclosporin A (CsA) confirmed the pathogenic role of PTP opening. In order to determine whether mitochondrial performance can be used as a criterion for inclusion in clinical trials and as an outcome measure of the patient response to therapy, it is mandatory to establish whether mitochondrial dysfunction is conserved in primary cell cultures from UCMD and BM patients. In this study we report evidence that mitochondrial dysfunction and the consequent increase of apoptotic rate can be detected not only, as previously reported, in muscle, but also in fibroblast cell cultures established from muscle biopsies of collagen VI-related myopathic patients. However, the mitochondrial phenotype is no longer maintained after nine passages in culture. These data demonstrate that the dire consequences of mitochondrial dysfunction are not limited to myogenic cells, and that this parameter can be used as a suitable diagnostic criterion, provided that the cell culture conditions are carefully established. J. Cell. Physiol. 227: 2927–2935, 2012. © 2011 Wiley Periodicals, Inc.

Collagen VI is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in collagen VI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by collagen VI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of collagen VI and altered distribution of collagen I and fibronectin. In patient's tenocyte culture, collagen VI web formation and cell surface association were severely impaired; large aggregates of collagen VI, which matched with collagen I labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that collagen VI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of collagen VI related myopathies.

The possibility of transplanting adult stem cells into damaged organs has opened new prospects for the treatment of several human pathologies. The purpose of this study was to develop a culture system for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free media formulation and ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy. Somatic stem cells were isolated from the human periodontium using a minimally invasive periodontal access flap surgery in healthy donors. Expanded hPDLSCs in a xeno-free culture showed the morphological features of stem cells, expressed the markers associated with pluripotency, and a normal karyotype. Under appropriate culture conditions, hPDLSCs presented adipogenic and osteogenic potential; indeed, a very high accumulation of lipid droplets was evident in the cytoplasm of adipogenic-induced cells, and indisputable evidence of osteogenic differentiation, investigated by transmission electron microscopy, and analyzed for gene expression analysis has been shown. Based on these data, the novel xeno-free culture method might provide the basis for Good Manufacturing Procedure culture of autologous stem cells, readily accessible from human periodontium, and can be a resource to facilitate their use in human clinical studies for potential therapeutic regeneration.

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.

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders., Highlights • Collagen VI is an ECM component of the human skeletal muscle. • We evaluated the α6 chain in collagen VI-related and other muscular dystrophies. • The α6 chain was reduced in collagen VI-related diseases but not in other myopathies. • A correlation between the α6 chain and fibrosis was demonstrated in MDC1A. • The α6 chain is involved in the pathogenesis of collagen VI diseases and fibrosis.

We have previously demonstrated that intraperitoneal injections of 2′-O-methyl-phosphorothioate (2′OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles ofmdxmice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were trackedin vivoas well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, inmdxmice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth, and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work, we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions and are therefore bona fide substrates of Akt. We report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. Our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamin physiology to therapeutic approaches for lamin A-linked disorders.

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.

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal–epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323–1331, 2013. © 2012 Wiley Periodicals, Inc.

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.

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.

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.

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.

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

Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI-knockout (Col6a1(-/-)) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B-interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1(-/-) mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as alpha-, beta-, gamma- and delta-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2′-O-methyl-phosphorothioate (2′OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

1 Department of Biomedical Sciences, 2 Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, 35121 Padova, Italy, 3 IGM-CNR, Unit of Bologna c/o IOR, 40136, Bologna, Italy and 4 Vollum Institute, Oregon Health and Science University, Portland, OR 97239-3098, USA Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy are inherited muscle disorders caused by mutations of genes encoding the extracellular matrix protein collagen VI (ColVI). Mice lacking ColVI (CoI6a1 -/- ) display a myopathic phenotype associated with ultrastructural alterations of mitochondria and sarcoplasmic reticulum, mitochondrial dysfunction with abnormal opening of the permeability transition pore (PTP) and increased apoptosis of muscle fibers. Treatment with cyclosporin (Cs) A, a drug that desensitizes the PTP by binding to cyclophilin (Cyp)-D, was shown to rescue myofiber alterations in Col6a1 -/- mice and in UCMD patients, suggesting a correlation between PTP opening and pathogenesis of ColVI muscular dystrophies. Here, we show that inactivation of the gene encoding for Cyp-D rescues the disease phenotype of ColVI deficiency. In the absence of Cyp-D, Col6a1 -/- mice show negligible myofiber degeneration, rescue from mitochondrial dysfunction and ultrastructural defects, and normalized incidence of apoptosis. These findings (i) demonstrate that lack of Cyp-D is equivalent to its inhibition with CsA at curing the mouse dystrophic phenotype; (ii) establish a cause-effect relationship between Cyp-D-dependent PTP regulation and pathogenesis of the ColVI muscular dystrophy and (iii) validate Cyp-D and the PTP as pharmacological targets for the therapy of human ColVI myopathies.

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.

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.

Ullrich congenital muscular dystrophy is a severe genetically and clinically heterogeneous muscle disorder linked to collagen VI deficiency. The pathogenesis of the disease is unknown. To assess the potential role of mitochondrial dysfunction in the onset of muscle fiber death in this form of dystrophy, we studied biopsies and myoblast cultures obtained from patients with different genetic defects of collagen VI and variable clinical presentations of the disease. We identified a latent mitochondrial dysfunction in myoblasts from patients with Ullrich congenital muscular dystrophy that matched an increased occurrence of spontaneous apoptosis. Unlike those in myoblasts from healthy donors, mitochondria in cells from patients depolarized upon addition of oligomycin and displayed ultrastructural alterations that were worsened by treatment with oligomycin. The increased apoptosis, the ultrastructural defects, and the anomalous response to oligomycin could be normalized by Ca 2+ chelators, by plating cells on collagen VI, and by treatment with cyclosporin A or with the specific cyclophilin inhibitor methylAla 3 ethylVal 4 -cyclosporin, which does not affect calcineurin activity. Here we demonstrate that mitochondrial dysfunction plays an important role in muscle cell wasting in Ullrich congenital muscular dystrophy. This study represents an essential step toward a pharmacological therapy of Ullrich congenital muscular dystrophy with cyclosporin A and methylAla 3 ethylVal 4 cyclosporin.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.

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.

Phosphoinositides play an essential role in diverse cellular functions such as cell proliferation, cytoskeletal regulation, intracellular vesicle trafficking, motility, cell metabolism and death. Alteration of these pathways is common to many diseases. In this study, we show that osteoblasts from patients affected by osteoarthritis (OA) and by rheumatoid arthritis (RA) present a decreased cell proliferation and a reduced expression of the key elements of polyphosphoinositide signal transduction such as phosphatidylinositol-3-kinase (PI 3K), phospholipase C gamma1 (PLCgamma1), and protein kinase C zeta (PKCzeta) compared to the post-traumatic (PT) patients. Our results suggest that a correlation may exist between the reduced osteoblast proliferation observed in OA and RA patients and the lowered expression of PI 3K, PLCgamma1, and PKCzeta enzymes. The reduced proliferation rate of osteoblasts in response to these signal transduction effectors could counteract the evolution of arthritic disease.

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.

Immunocytochemistry is the most diffuse technique to visualize and localize specific biochemical components in cell compartments and tissues. With this method the antigens are tagged by antibodies that can be visualized with appropriate markers attached directly (direct method) or indirectly (indirect method) to them. The colloidal gold marker system (1) is the most widespread immunoelectron microscopy label for postembedding immunocytochemistry. Sections of embedded tissue are incubated with a primary antibody against a particular antigen, which is exposed on the surface of the section, and then with a secondary antibody conjugated to colloidal gold particles of a particular size (indirect method). It is possible to enlarge the colloidal gold size with silver enhancement. This technique is ideal for labeling both intracellular and surface antigens and it can be used for multiple labeling experiments and quantitative studies (2).

A case of leiomyosarcoma arising in the spermatic cord is described. A 83-year-old man required medical care for an irreducible inguinal hernia. The patient underwent herniorraphy and transinguinal radical orchiectomy. Macroscopically, the spermatic cord was enlarged by a gray-tan and ill-defined neoplasm measuring 4 x 4 x 3 cm. Histologically, this proliferation was composed of atypical spindle cells with blunted end nuclei and eosinophilic cytoplasm. Immunohistochemistry and ultrastructure confirmed the smooth muscle nature of the neoplastic cells. The diagnosis of leiomyosarcoma of the spermatic cord was made. To improve the assignment of this rare lesion to its specific anatomic location, we analyzed the immunohistochemical and ultrastructural characteristics of the smooth muscle tumoral cells and in particular those of the intracellular filament aggregates.

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 α and β 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.

The post-receptor pathway that leads to nuclear factor kappaB (NF-kappaB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-kappaB (IkappaB) by the IkappaB kinases releases NF-kappaB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by co-immunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-kappaB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-kappaB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-kappaB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1beta production in cells expressing dominant-negative Akt. However, NF-kappaB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-kappaB at a level distinct from the dissociation of p65 from IkappaBalpha and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain.

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.

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.

Interleukin 1 (IL-1) is a proinflammatory cytokine which can elicit proliferative, differentiative, or metabolic responses. The molecular mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus, although extensively studied, have not been completely elucidated. We previously demonstrated that human osteosarcoma Saos-2 cells incubated with IL-1 presented a rapid and transient increase of phospholipase C activity exclusively at the nuclear level. Moreover, we presented evidence that not only the canonical inositol lipid signalling pathway was involved, but also the D3-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-kinase) were affected. The results of this study indicate that in Saos-2 cells PI 3-kinase is recruited and activated by IL-1 receptor I (IL-1RI) through binding of the SH2 domains to the consensus sequence on the C-terminal tail of the receptor, and that Tyr-479 is essential for PI 3-kinase activation. Moreover, IL-1 treatment triggers PI 3-kinase translocation to the nucleus; this event is rapid and transient in cells expressing high levels of IL-1RI (Saos-2/IL-1R) as well as in untransfected cells, although to a lesser extent. The data, based on immunochemical and immunocytochemical quantitative methods, indicate that PI 3-kinase translocation to the nucleus depends on PI 3-kinase activation. In fact, inactivation by two independent mechanisms, addition of specific PI 3-kinase inhibitors, or overexpression of a mutant form of IL-1RI, resulted in a substantial inhibition of PI 3-kinase translocation to the nucleus. These data suggest that PI 3-kinase recruitment by the activated receptor is a limiting step in PI 3-kinase activation and nuclear translocation. This early event in the IL-1 signalling mechanisms confirms that D3 inositides, as well as canonical inositides produced by nuclear phospholipase C isoforms, are involved in this pathway of activation of transcription factors.

Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Nuclei isolated from rat liver, incubated in the presence of liposomes of different phospholipids, undergo typical modifications: chromatin dispersion and reduction of the interchromatin granules in nuclei incubated with negatively charged liposomes and increase of the chromatin density and of the number and size of the interchromatin granules in nuclei incubated with neutral liposomes. The possibility that the observed modifications are caused by an impairment of the transport and translocation of ribonucleoproteins belonging to the inner nuclear matrix, is suggested by the results obtained by radiotracer techniques on the release of RNA from liposome-incubated nuclei.