Protein dynamics at invadopodia control invasion-migration transitions in melanoma cells.

Cell invasion is a highly complex process that requires the coordination of cell migration and degradation of the extracellular matrix. In melanoma cells, as in many highly invasive cancer cell types these processes are driven by the regulated formation of adhesives structures such as focal adhesions and invasive structures like invadopodia. Structurally, focal adhesion and invadopodia are quite distinct, yet they share many protein constituents. However, quantitative understanding of the interaction of invadopodia with focal adhesion is lacking, and how invadopodia turn-over is associated with invasion-migration transition cycles remains unknown. In this study, we investigated the role of Pyk2, cortactin and Tks5 in invadopodia turnover and their relation with focal adhesions. We found that active Pyk2 and cortactin are localised at both focal adhesions and invadopodia. At invadopodia, localisation of active Pyk2 is correlated with ECM degradation. During invadopodia disassembly, Pyk2 and cortactin but not Tks5 are often relocated at nearby nascent adhesions. We also show that during ECM degradation, cell migration is reduced which is likely related to the sharing of common molecules within the two structures. Finally, we found that the dual FAK/Pyk2 inhibitor PF-431396 inhibits both focal adhesion and invadopodia activities thereby reducing both migration and ECM degradation.

Inhibiting FAK-Paxillin Interaction Reduces Migration and Invadopodia-Mediated Matrix Degradation in Metastatic Melanoma Cells.

The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. Investigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells' invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK-Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK-paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK-paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers.

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.

Expression/activation of α5ß1 integrin is linked to the ß-catenin signaling pathway to drive migration in glioma cells.

The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that α5ß1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between α5ß1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of α5ß1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that α5ß1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of α5ß1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that α5ß1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma.

CD47 activation-induced UHRF1 over-expression is associated with silencing of tumor suppressor gene p16INK4A in glioblastoma cells.

CD47, an integrin-associated protein is over-expressed in several tumors including glioblastomas. Activation of CD47 induces proliferation of human astrocytoma cells but not normal astrocytes via an Akt-dependent way. However, the pathways mediating this process are still unknown. The epigenetic integrator UHRF1 (Ubiquitin-like containing PHD and RING Finger 1) is over-expressed in various cancers and plays a vital role in the silencing of numerous tumor suppressor genes including p16(INK4A), thereby promoting cell proliferation. The aim of the present study was to investigate the role of UHRF1 and p16(INK4A) in CD47-induced effects. Herein we showed that activation of CD47 in human astrocytoma cell lines U87 and CCF- STTG1 (Grade IV), up-regulated the expression of UHRF1 with subsequent down-regulation of p16(INK4A), thus promoting cell proliferation. Blockage of CD47 using a blocking antibody down-regulated UHRF1 expression, accompanied by a re-expression of p16(INK4A), conducting to decreased cell proliferation in both cancer cell lines. Neither CD47 activation nor its blocking has any effect on UHRF1/p16(INK4A) expression in normal human astrocytes. Depletion of CD47 in the U87 cell line resulted in down-regulation of UHRF1. We also found that CD47 activated the inflammatory genes IL-6, IL-7 and MCP-1 by a NF-κB-dependent mechanism in human astrocytoma but not in normal astrocytes. In conclusion, the present findings indicate that CD47 activation increases expression of UHRF1 and suggest, for the first time, that CD47 regulates the epigenetic code by targeting UHRF1. This could represent a new pathway towards cell proliferation and metastasis.

Pro-inflammatory and pro-oxidant status of pancreatic islet in vitro is controlled by TLR-4 and HO-1 pathways.

Since their isolation until implantation, pancreatic islets suffer a major stress leading to the activation of inflammatory reactions. The maintenance of controlled inflammation is essential to preserve survival and function of the graft. Identification and targeting of pathway(s) implicated in post-transplant detrimental inflammatory events, is mandatory to improve islet transplantation success. We sought to characterize the expression of the pro-inflammatory and pro-oxidant mediators during islet culture with a focus on Heme oxygenase (HO-1) and Toll-like receptors-4 signaling pathways. Rat pancreatic islets were isolated and pro-inflammatory and pro-oxidant status were evaluated after 0, 12, 24 and 48 hours of culture through TLR-4, HO-1 and cyclooxygenase-2 (COX-2) expression, CCL-2 and IL-6 secretion, ROS (Reactive Oxygen Species) production (Dihydroethidine staining, DHE) and macrophages migration. To identify the therapeutic target, TLR4 inhibition (CLI-095) and HO-1 activation (cobalt protoporphyrin,CoPP) was performed. Activation of NFκB signaling pathway was also investigated. After isolation and during culture, pancreatic islet exhibited a proinflammatory and prooxidant status (increase levels of TLR-4, COX-2, CCL-2, IL-6, and ROS). Activation of HO-1 or inhibition of TLR-4 decreased inflammatory status and oxidative stress of islets. Moreover, the overexpression of HO-1 induced NFκB phosphorylation while the inhibition of TLR-4 had no effect NFκB activation. Finally, inhibition of pro-inflammatory pathway induced a reduction of macrophages migration. These data demonstrated that the TLR-4 signaling pathway is implicated in early inflammatory events leading to a pro-inflammatory and pro-oxidant status of islets in vitro. Moreover, these results provide the mechanism whereby the benefits of HO-1 target in TLR-4 signaling pathway. HO-1 could be then an interesting target to protect islets before transplantation.

TiO2 photocatalysis damages lipids and proteins in Escherichia coli.

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO2) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO2 photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O2·(-)), inhibited this effect by half, showing us that O2·(-) radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO2, respectively, which is accounted for by the cytotoxic effect of TiO2. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO2 increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O2·(-) on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.

Limoniastrum guyonianum aqueous gall extract induces apoptosis in human cervical cancer cells involving p16 INK4A re-expression related to UHRF1 and DNMT1 down-regulation.

Several reports have described the potential effects of natural compounds as anti-cancer agents in vitro as well as in vivo. The aim of this study was to evaluate the anti-cancer effect of Limoniastrum guyonianum aqueous gall extract (G extract) and luteolin in the human cervical cancer HeLa cell line, and, if so, to clarify the underlying mechanism. Our results show that G extract and luteolin inhibited cell proliferation and induced G2/M cell cycle arrest in a concentration and time-dependent manner. Both natural products induced programmed cell death as confirmed by the presence of hypodiploid G0/G1 cells. These effects are associated with an up-regulation of the expression of the tumor suppressor gene p16INK4A and a down-regulation of the expression of the anti-apoptotic actor UHRF1 and its main partner DNMT1. Moreover, G extract- and luteolin-induced UHRF1 and DNMT1 down-regulation is accompanied with a global DNA hypomethylation in HeLa cell line. Altogether our results show that G extract mediates its growth inhibitory effects on human cervical cancer HeLa cell line likely via the activation of a p16INK4A-dependent cell cycle checkpoint signalling pathway orchestrated by UHRF1 and DNMT1 down-regulation.

On the use of capillary cytometry for assessing the bactericidal effect of TiO2. Identification and involvement of reactive oxygen species.

The photocatalytic antimicrobial properties of TiO2 were studied on Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa bacterial strains taken as model strains for pathogenic species mainly implied in nosocomial infections. Capillary cytometry, coupled to a double-staining method for visualizing membrane integrity as a cell viability indicator, was highlighted as a rapid, easy-to-use, and automated numeration technique for quantitative and reproducible determination of cellular viability and thus, was able to give an accurate evaluation of the bactericidal effect of UV-A photocatalysis. Cytometry also enabled the study of TiO2-bacteria interactions and aggregation in the dark as well as TiO2 cytotoxicity. Compared with the traditional agar plate cultivation method, a significatively weaker reduction in cell viability was recorded by cytometry whatever the bacteria, TiO2 concentration, and duration of the photocatalytic treatment. The mismatch between both numeration methods was attributed to: (i) the presence of mixed bacteria-TiO2 aggregates that could interfere with bacteria measurement on plates, (ii) prolonged contact of the bacteria with TiO2 during incubation, which could cause additional cytotoxic damage to the bacterial wall, and (iii) the counting of viable but non-culturable bacteria as live bacteria in cytometry, whereas they cannot grow on solid media. A more pronounced difference was observed for P. aeruginosa and S. aureus bacteria, for which 2.9 and 1.9 log10 survival reduction overestimations were measured by plate counting, respectively. Using chemiluminescence, full restoration of cell viability by controlled addition of the O2˙(-) scavenger superoxide dismutase enzyme suggests that O2˙(-) acts, in our conditions, as the main reactive oxygen species responsible for the photocatalytic attack towards the targeted bacteria.

Thymoquinone reduces migration and invasion of human glioblastoma cells associated with FAK, MMP-2 and MMP-9 down-regulation.

Glioblastoma represent the most frequent primary tumors of the central nervous system and remain among the most aggressive human cancers as available therapeutic approaches still fail to contain their invasiveness. Many studies have reported elevated expression of the Focal Adhesion Kinase (FAK) protein in glioblastoma, associated with an increase in the rates of both migration and invasion. This designates FAK as a promising target to limit invasiveness in glioblastoma. Thymoquinone (TQ), the main phytoactive compound of Nigella sativa has shown remarkable anti-neoplasic activities on a variety of cancer cells. Here, we studied the anti-invasive and anti-migratory effects of TQ on human glioblastoma cells. The results obtained indicated that TQ treatment reduced migration, adhesion and invasion of both U-87 and CCF-STTG1 cells. This was accompanied by a drastic down-regulation of FAK, associated with a reduction of ERK phosphorylation as well as MMP-2 and MMP-9 secretion. This study provides new data on FAK regulation by a natural product (TQ) which could be of a great value for the development of novel therapies in glioblastoma.

Anti-neoplastic agent thymoquinone induces degradation of α and ß tubulin proteins in human cancer cells without affecting their level in normal human fibroblasts.

The microtubule-targeting agents derived from natural products, such as vinca-alkaloids and taxanes are an important family of efficient anti-cancer drugs with therapeutic benefits in both haematological and solid tumors. These drugs interfere with the assembly of microtubules of α/ß tubulin heterodimers without altering their expression level. The aim of the present study was to investigate the effect of thymoquinone (TQ), a natural product present in black cumin seed oil known to exhibit putative anti-cancer activities, on α/ß tubulin expression in human astrocytoma cells (cell line U87, solid tumor model) and in Jurkat cells (T lymphoblastic leukaemia cells). TQ induced a concentration- and time-dependent degradation of α/ß tubulin in both cancer cell types. This degradation was associated with the up-regulation of the tumor suppressor p73 with subsequent induction of apoptosis. Interestingly, TQ had no effect on α/ß tubulin protein expression in normal human fibroblast cells, which were used as a non-cancerous cell model. These data indicate that TQ exerts a selective effect towards α/ß tubulin in cancer cells. In conclusion, the present findings indicate that TQ is a novel anti-microtubule drug which targets the level of α/ß tubulin proteins in cancer cells. Furthermore, they highlight the interest of developing anti-cancer therapies that target directly tubulin rather than microtubules dynamics.

Complement receptor 3 (CD11b/CD18) is implicated in the elimination of ß-amyloid peptides.

Microglia are the professional phagocytes of the brain and express phagocytic receptors such as complement receptor 3 (CR3 or CD11b/CD18). Using mimics of the amyloid deposit made of heat-killed yeasts coated with either Aß 1-40 or Aß 1-42, we were able to study how microglia interacted with and ingested these particles in vitro. We have shown previously that the low density lipoprotein receptor-related protein (LRP) is largely implied in the phagocytosis of Aß 1-42-opsonized heat-killed yeasts and partly in that of Aß 1-40-opsonized heat-killed yeasts. Here, we report that antibodies against CD11b or CD18 reduced the uptake of the artificial amyloid deposit by microglial cell showing that CR3 is involved in the mechanism. Moreover, a concomitant inhibition of LRP and CR3 completely blocked the ingestion of both kinds of particles suggesting that no other receptors participate to this mechanism.

Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt-dependent pathway.

CD47 is a membrane receptor that plays pivotal roles in many pathophysiological processes, including infection, inflammation, cell spreading, proliferation, and apoptosis. We show that activation of CD47 increases proliferation of human U87 and U373 astrocytoma cells but not normal astrocytes. CD47 function-blocking antibodies inhibit proliferation of untreated U87 and U373 cells but not normal astrocytes, suggesting that CD47 may be constitutively activated in astrocytoma. CD47 expression levels were similar in our three cell types. CD47 couples to G-proteins in astrocytes and astrocytoma and especially to the Gßγ dimer. Downstream signaling following CD47 activation involves Gßγ dimer-dependent activation of the PI3K/Akt pathway in astrocytoma cells but not in normal astrocytes. This pathway is known to be deregulated in astrocytoma, leading to cell proliferation and enhanced survival signals. Putative PLIC-1 interaction with CD47 in astrocytoma cells but not astrocytes may contribute to the proliferative effect observed upon activation of CD47. Our data indicate that CD47 receptors have a stimulatory role in cell proliferation and demonstrate for the first time that CD47 signals via the PI3K/Akt pathway in cancerous cells but not normal cells.

Cardiac muscarinic receptor overexpression in sudden infant death syndrome.

BACKGROUND: Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [(3)H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled B(max) value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). CONCLUSIONS: In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism.

Constitutive overexpression of muscarinic receptors leads to vagal hyperreactivity.

BACKGROUND: Alterations in muscarinic receptor expression and acetylcholinesterase (AchE) activity have been observed in tissues from Sudden Infant Death Syndrome (SIDS). Vagal overactivity has been proposed as a possible cause of SIDS as well as of vasovagal syncopes. The aim of the present study was to seek whether muscarinic receptor overexpression may be the underlying mechanism of vagal hyperreactivity. Rabbits with marked vagal pauses following injection of phenylephrine were selected and crossed to obtain a vagal hyperreactive strain. The density of cardiac muscarinic receptors and acetylcholinesterase (AchE) gene expression were assessed. Blood markers of the observed cardiac abnormalities were also sought. METHODOLOGY/PRINCIPAL FINDINGS: Cardiac muscarinic M(2) and M(3) receptors were overexpressed in hyperreactive rabbits compared to control animals (2.3-fold and 2.5-fold, respectively) and the severity of the phenylephrine-induced bradycardia was correlated with their densities. A similar overexpression of M(2) receptors was observed in peripheral mononuclear white blood cells, suggesting that cardiac M(2) receptor expression can be inferred with high confidence from measurements in blood cells. Sequencing of the coding fragment of the M(2) receptor gene revealed a single nucleotide mutation in 83% of hyperreactive animals, possibly contributing for the transcript overexpression. Significant increases in AchE expression and activity were also assessed (AchE mRNA amplification ratio of 3.6 versus normal rabbits). This phenomenon might represent a compensatory consequence of muscarinic receptors overexpression. Alterations in M(2) receptor and AchE expression occurred between the 5th and the 7th week of age, a critical period also characterized by a higher mortality rate of hyperreactive rabbits (52% in H rabbits versus 13% in normal rabbits) and preceeded the appearance of functional disorders. CONCLUSIONS/SIGNIFICANCE: The results suggest that cardiac muscarinic receptor overexpression plays a critical role in the development of vagal hyperreactivity, whereas AchE hyperactivity appears as a compensatory consequence of it. Since similar vagal disorders were observed recently by us in SIDS, muscarinic receptor overexpression could become a marker of risk of vasovagal syncopes and SIDS.

Drugs and their molecular targets: an updated overview.

About 330 targets bind approved drugs, 270 encoded by the human genome and 60 belonging to pathogenic organisms. A large number of druggable targets have been recently proposed from preclinical and first clinical data, but a huge reservoir of putative drug targets, possibly several thousands, remains to be explored. This overview considers the different types of ligands and their selectivity in the main superfamilies of drug targets, enzymes, membrane transporters and ion channels, and the various classes of membrane and nuclear receptors with their signalling pathway. Recently approved drugs such as monoclonal antibodies, tyrosine kinase and proteasome inhibitors, and major drugs under clinical studies are reviewed with their molecular target and therapeutic interest. The druggability of emerging targets is discussed, such as multidrug resistance transporters and cystic fibrosis transmembrane conductance regulator (CFTR), hyperpolarization-activated cyclic nucleotides-gated (HCN), cyclic nucleotide-gated (CNG) and transient receptor potential (TRP) ion channels, tumour necrosis factor (TNF) and receptor activator of NFkappaB (RANK) receptors, integrins, and orphan or recently deorphanized G-protein-coupled and nuclear receptors. Large advances have been made in the therapeutical use of recombinant cytokines and growth factors (i.e. tasonermin, TNFalpha-1a; becaplermin, platelet-derived growth factor (PDGF); dibotermin-alpha, bone morphogenetic proteins (BMP)2; anakinra, interleukin-1 receptor antagonist protein (IRAP), and in enzyme replacement therapy, i.e. algasidase (alpha-galactosidase) and laronidase (alpha-l-iduronidase). New receptor classes are emerging, e.g. membrane aminopeptidases, and novel concepts are stimulating drug research, e.g. epigenetic therapy, but the molecular target of some approved drugs, such as paracetamol and imidazolines, still need to be identified.

Comparing reagents for efficient transfection of human primary myoblasts: FuGENE 6, Effectene and ExGen 500.

This study compared three different synthetic reagents (FuGENE 6, Effectene and ExGen 500) for the transfection of human primary myoblasts. We examined the efficiency, cytotoxicity and size of the complexes formed in the presence of different amounts of vector and DNA and with variable amounts of serum. Transfection rates were relatively high for primary cells, especially with FuGENE 6 (20%), which appeared to be the best transfection reagent for these cells, even in the presence of 10% serum. Cultured human myoblasts are an interesting tool for studying neuromuscular diseases and are potentially useful for myoblast transfer therapy studies. Moreover, the efficiency of these transfection reagents in a medium containing 10% serum is promising for possible gene therapy protocols for muscle diseases.

Reduced expression of nicotinic AChRs in myotubes from spinal muscular atrophy I patients.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of motoneurons and skeletal muscle atrophy. In its most severe form, it leads to death before the age of 2 years. While primary degeneration of motor neurons is well established in this disease, and this results in neurogenic atrophy of skeletal muscle, we have previously reported evidence for a primary muscle defect. In this study, we used primary cultures of embryonic human skeletal muscle cells from patients with SMA and from controls to examine the effects of muscle fiber differentiation in the absence of a nerve component. Cultured SMA skeletal muscle cells are unable to fuse correctly to form multinuclear myotubes, the precursors of the myofibers. We also show that agrin-induced aggregates of nicotinic acetylcholine receptors, one of the earliest steps of neuromuscular junction formation, cannot be visualized by confocal microscopy on cells from SMA patients. In binding experiments, we demonstrate that this lack of clustering is due to defective expression of the nicotinic acetylcholine receptors in the myotubes of SMA patients whereas the affinity of alpha-bungarotoxin for its receptor remains unchanged regardless of muscle cell type (SMA or control). These observations suggest that muscle cells from SMA patients have intrinsic abnormalities that may affect proper formation of the neuromuscular junction.

Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP.

We describe here the construction of plasmid pEGFP-C3/SMN, bearing the human SMN gene coupled to the green fluorescent protein (GFP) sequence. The mutation of the SMN gene is responsible for spinal muscular atrophy (SMA), a frequent human infantile genetic disease. We introduced the SMN cDNA into the multiple cloning site of pEGFP-C3. This plasmid bears the neomycin-resistance sequence and the enhanced green fluorescent protein (EGFP). It results in the expression of a fusion protein bearing SMN coupled to a carboxy-terminal GFP tag, used for fluorescence localization studies. Transfection of primary human myoblasts with pEGFP-C3 or pEGFP-C3/SMN revealed that EGFP is intracellularly localized within the cytosol as well as in the nucleus, while the fusion protein EGFP-SMN localized within the nucleus in prominent dot-like structures termed "gems." These data demonstrate that human primary muscle cells can be efficiently transfected and may have important implications for the development of therapeutic strategies in SMA.