Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved ...in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.
Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are ...currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.
Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of ...AONs with highest
in vitro
efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs
in vitro
.
The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to ...stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 μm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 μm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 μm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 μm concavities, and osteocalcin staining predominantly for 440 and 800 μm concavities, but not for 1800 μm concavities and only slightly for planar surface controls.
DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine–threonine protein kinases, whose members play a role in actin-based cell ...morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast–myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells.
► Expression of DMPK in the cytosol induces formation of stellar actomyosin stress fibers. ► Stellar stress fiber formation is accompanied by augmented phosphorylation of MLC2. ► Myoblasts expressing active cytosolic DMPK display altered cell morphology and motility. ► Early steps in myogenesis are delayed upon DMPK activity expression in the cytosol. ► High cytosolic DMPK activity during myogenesis supports formation of myosacs.
Polycystic liver disease (PCLD) is an inherited disorder caused by mutations in either
PRKCSH
(hepatocystin) or
SEC63
(Sec63p). However, expression patterns of the implicated proteins in diseased and ...normal liver are unknown. We analyzed subcellular and cellular localization of hepatocystin and Sec63p using cell fractionation, immunofluorescence, and immunohistochemical methods. Expression patterns were assessed in fetal liver, PCLD liver, and normal adult liver. We found hepatocystin and Sec63p expression predominantly in the endoplasmic reticulum. In fetal tissue, there was intense expression of hepatocystin in ductal plate, bile ducts, and hepatocytes. However, Sec63p staining was prominent in early hepatocytes only and weak in bile ducts throughout development. In PCLD tissue, hepatocystin was expressed in hepatocytes, bile ducts, and in cyst epithelium of patients negative for
PRKCSH
mutation. In contrast, the majority of cysts from
PRKCSH
mutation carriers did not express hepatocystin. Sec63p expression was observed in all cyst epithelia regardless of mutational state. We conclude that hepatocystin is probably required for development of bile ducts and does not interact with Sec63p. The results support the hypothesis that cyst formation in PCLD results from a cellular recessive mechanism involving loss of hepatocystin. Cystogenesis in
SEC63
-associated PCLD occurs via a different mechanism.
Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi ...apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.
Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillogenesis. Several developmental stages ...of these cultures were double stained with antibodies to titin in combination with antibodies to alpha-actin, alpha-actinin, myosin heavy chain (MHC), nebulin, desmin, and beta-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing alpha-actinin and sarcomeric alpha-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, alpha-actin and alpha-actinin. Thereafter a more periodic localization of titin, MHC, alpha-actin and alpha-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and alpha-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.
The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by ...the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.
In the present study the microscopic localization of polyethylene glycol (PEG) liposomes in infected tissues was studied with both light microscopy (LM) and transmission electron microscopy (TEM) in ...rats with focal intramuscular
Staphylococcus aureus infection. PEG-liposomes containing colloidal gold were prepared and injected intravenously in rats with focal
S. aureus infection and tissues were dissected at 24 h post injection. Sections were cut and liposomes were visualized for microscopic evaluation using silver enhancement. Uptake of PEG-liposomes was visualized by both scintigraphy and LM in the abscess, liver and spleen. In the infected area, the liposomes were mainly found in the vicinity of blood vessels. TEM showed that the liposomes were localized in the macrophages and to a lesser extent in endothelial cells in the infectious tissue. In the liver, the liposomes appeared mainly localized in Kupffer cells. In the spleen, uptake was only seen in cells of the red pulp and in cells around the central arteries. Our microscopic observations indicate that uptake and retention of PEG-liposomes in the infectious focus is a result of enhanced extravasation due to increased vascular permeability and subsequent phagocytosis of PEG-liposomes by macrophages in the infected tissue.
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