Full-length Dystrophin Research Articles

Genomic integration of the full-length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells.

The plasmid vectors that express the full-length human dystrophin coding sequence in human cells was developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower-copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single-copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid-mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.

Lentiviral vectors can be used for full-length dystrophin gene therapy

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Mechanism of Deletion Removing All Dystrophin Exons in a Canine Model for DMD Implicates Concerted Evolution of X Chromosome Pseudogenes

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder caused by mutations in the large, 2.4-Mb dystrophin gene. The majority of DMD-causing mutations are sporadic, multi-exon, frameshifting deletions, with the potential for variable immunological tolerance to the dystrophin protein from patient to patient. While systemic gene therapy holds promise in the treatment of DMD, immune responses to vectors and transgenes must first be rigorously evaluated in informative preclinical models to ensure patient safety. A widely used canine model for DMD, golden retriever muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to alternative splicing around an intronic point mutation, thereby confounding the interpretation of immune responses to dystrophin-derived gene therapies. Here we characterize a naturally occurring deletion in a dystrophin-null canine, the German shorthaired pointer. The deletion spans 5.6 Mb of the X chromosome and encompasses all coding exons of the DMD and TMEM47 genes. The sequences surrounding the deletion breakpoints are virtually identical, suggesting that the deletion occurred through a homologous recombination event. Interestingly, the deletion breakpoints are within loci that are syntenically conserved among mammals, yet the high homology among this subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific concerted evolution of these atypical sequence elements.

Uniform low-level dystrophin expression in the heart partially preserved cardiac function in an aged mouse model of Duchenne cardiomyopathy

Dystrophin deficiency results in Duchenne cardiomyopathy, a primary cause of death in Duchenne muscular dystrophy (DMD). Gene therapy has shown great promise in ameliorating the cardiac phenotype in mouse models of DMD. However, it is not completely clear how much dystrophin is required to treat dystrophic heart disease. We and others have shown that mosaic dystrophin expression at the wild-type level, depending on the percentage of dystrophin positive cardiomyocytes, can either delay the onset of or fully prevent cardiomyopathy in dystrophin-null mdx mice. Many gene therapy strategies will unlikely restore dystrophin to the wild-type level in a cardiomyocyte. To determine whether low-level dystrophin expression can reduce the cardiac manifestations in DMD, we examined heart histology, ECG and hemodynamics in 21-m-old normal BL6 and two strains of BL6-background dystrophin-deficient mice. Mdx3cv mice show uniform low-level expression of a near full-length dystrophin protein in every myofiber while mdx4cv mice have no dystrophin expression. Immunostaining and western blot confirmed marginal level dystrophin expression in the heart of mdx3cv mice. Although low-level expression did not reduce myocardial histopathology, it significantly ameliorated QRS prolongation and normalized diastolic hemodynamic deficiencies. Our study demonstrates for the first time that low-level dystrophin can partially preserve heart function.

Duchenne Muscular Dystrophy Myogenic Cells from Urine-Derived Stem Cells Recapitulate the Dystrophin Genotype and Phenotype.

A ready source of autologous myogenic cells is of vital importance for drug screening and functional genetic studies in Duchenne muscular dystrophy (DMD), a rare disease caused by a variety of dystrophin gene mutations. As stem cells (SCs) can be easily and noninvasively obtained from urine specimens, we set out to determine whether they could be myogenically induced and useful in DMD research. To this end, we isolated stem cells from the urine of two healthy donors and from one patient with DMD, and performed surface marker characterization, myogenic differentiation (MyoD), and then transfection with antisense oligoribonucleotides to test for exon skipping and protein restoration. We demonstrated that native urine-derived stem cells express the full-length dystrophin transcript, and that the dystrophin mutation was retained in the cells of the patient with DMD, although the dystrophin protein was detected solely in control cells after myogenic transformation according to the phenotype. Notably, we also showed that treatment with antisense oligoribonucleotide against dystrophin exon 44 induced skipping in both native and MyoD-transformed urine-derived stem cells in DMD, with a therapeutic transcript-reframing effect, as well as visible protein restoration in the latter. Hence MyoD-transformed cells may be a good myogenic model for studying dystrophin gene expression, and native urine stem cells could be used to study the dystrophin transcript, and both diagnostic procedures and splicing modulation therapies in both patients and control subjects, without invasive and costly collection methods. New, bankable bioproducts from urine stem cells, useful for prescreening studies and therapeutic applications alike, are also foreseeable after further, more in-depth characterization.

Independent variability of microtubule perturbations associated with dystrophinopathy.

Absence of the protein dystrophin causes Duchenne muscular dystrophy. Dystrophin directly binds to microtubules in vitro, and its absence in vivo correlates with disorganization of the subsarcolemmal microtubule lattice, increased detyrosination of α-tubulin, and altered redox signaling. We previously demonstrated that the dystrophin homologue utrophin neither binds microtubules in vitro nor rescues microtubule lattice organization when overexpressed in muscles of dystrophin-deficient mdx mice. Here, we fine-mapped the dystrophin domain necessary for microtubule binding to spectrin-like repeats 20–22. We show that transgenic mdx mice expressing a full-length dystrophin/utrophin chimera completely lacking microtubule binding activity are surprisingly rescued for all measured dystrophic phenotypes, including full restoration of microtubule lattice organization. Conversely, despite the presence of dystrophin at the sarcolemma, β-sarcoglycan-deficient skeletal muscle presents with a disorganized and densified microtubule lattice. Finally, we show that the levels of α-tubulin detyrosination remain significantly elevated to that of mdx levels in transgenic mdx mice expressing nearly full-length dystrophin. Our results demonstrate that the microtubule-associated perturbations of mdx muscle are distinct, separable, and can vary independently from other parameters previously ascribed to dystrophin deficiency.

Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy.

Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE).Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats.Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls.Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated in human TLE, but not in AK rats, possibly indicating a compensatory mechanism in the chronic epileptic human brain.

Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules.

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease characterized by progressive physical disability, immobility, and premature death in affected boys. Underlying the devastating symptoms of DMD is the loss of dystrophin, a structural protein that connects the extracellular matrix to the cell cytoskeleton and provides protection against contraction-induced damage in muscle cells, leading to chronic peripheral inflammation. However, dystrophin is also expressed in neurons within specific brain regions, including the hippocampus, a structure associated with learning and memory formation. Linked to this, a subset of boys with DMD exhibit nonprogressing cognitive dysfunction, with deficits in verbal, short-term, and working memory. Furthermore, in the genetically comparable dystrophin-deficient mdx mouse model of DMD, some, but not all, types of learning and memory are deficient, and specific deficits in synaptogenesis and channel clustering at synapses has been noted. Little consideration has been devoted to the cognitive deficits associated with DMD compared with the research conducted into the peripheral effects of dystrophin deficiency. Therefore, this review focuses on what is known about the role of full-length dystrophin (Dp427) in hippocampal neurons. The importance of dystrophin in learning and memory is assessed, and the potential importance that inflammatory mediators, which are chronically elevated in dystrophinopathies, may have on hippocampal function is also evaluated.

Dystrophin contains multiple independent membrane-binding domains

Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. It has long been thought that membrane binding of dystrophin depends on its cysteine-rich (CR) domain. Here, we provide in vivo evidence suggesting that dystrophin contains three additional membrane-binding domains including spectrin-like repeats (R)1-3, R10-12 and C-terminus (CT). To systematically study dystrophin membrane binding, we split full-length dystrophin into ten fragments and examined subcellular localizations of each fragment by adeno-associated virus-mediated gene transfer. In skeletal muscle, R1-3, CR domain and CT were exclusively localized at the sarcolemma. R10-12 showed both cytosolic and sarcolemmal localization. Importantly, the CR-independent membrane binding was conserved in murine and canine muscles. A critical function of the CR-mediated membrane interaction is the assembly of the dystrophin-associated glycoprotein complex (DGC). While R1-3 and R10-12 did not restore the DGC, surprisingly, CT alone was sufficient to establish the DGC at the sarcolemma. Additional studies suggest that R1-3 and CT also bind to the sarcolemma in the heart, though relatively weak. Taken together, our study provides the first conclusive in vivo evidence that dystrophin contains multiple independent membrane-binding domains. These structurally and functionally distinctive membrane-binding domains provide a molecular framework for dystrophin to function as a shock absorber and signaling hub. Our results not only shed critical light on dystrophin biology and DMD pathogenesis, but also provide a foundation for rationally engineering minimized dystrophins for DMD gene therapy.

Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.

628. Transposons Expressing Full-Length Human Dystrophin Enable Genetic Correction of Dystrophic Mesoangioblasts and iPS-Derived Mesoangioblast-Like Cells

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. Alternatively, dystrophic mesoangioblast-like cells were generated from iPS of DMD patients. These iPS-derived mesoangioblasts, constitute an essentially unlimited supply of stem/progenitor cells that could be genetically corrected using PB transposons expressing dystrophin. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

389. Muscular Dystrophy Genes and the Early Metazoan Transition from Dynein- to Myosin-Powered Locomotion

During early animal evolution, the primary source of locomotive mechanical power shifted from axonemal (flagellar) dynein to densely arrayed class II myosin. Although the emergence of muscle conferred the advantage of three-dimensional scalability, the transition brought with it the evolutionarily unprecedented problem of safely transmitting huge forces across rapidly deforming cell membranes. This task in vertebrates is accomplished in part through the dystrophin-dystroglycan-sarcoglycan complex (DGC), which is implicated in most forms of muscular dystrophy. We present the inferred earliest steps in the molecular evolution of these cell surface and cortical cytoskeletal proteins, using genome sequence data from all early branching metazoan phyla and a broad sampling of unicellular taxa. Results: Surprisingly, the phylogeny suggests that a DGC emerged before the “sarcomeric” clade of myosins, implying conserved function(s) among unicellular lineages closely related to the Metazoa. Furthermore, linkage of the DGC to the cytoskeleton occurred before the massive tandem-repeat expansions (>200) seen in sarcomeric scaffolding proteins of the titin superfamily. The rod-like domains of dystrophin and utrophin were coopted at their full length from a rapidly lengthening cytoskeletal protein with an unrelated function, that of connecting actin filaments to microtubules at large physical distances. Finally, intron positions in the inferred dystrophin gene in a common ancestor to all Bilateria provide clues to the molecular basis of Duchenne muscular dystrophy and to emerging therapies. Conclusions: Our reconstruction suggests that evolution of membrane-spanning “muscular dystrophy protein complexes” was an essential process as one geometric constraint on power transduction was traded for another. 80% of the length of dystrophin is attributable to tandem DNA duplications that pre-dated the evolutionary emergence of sarcomeres. This chronology implies that escalating power output from striated muscle played no role in providing selective pressure for the serial expansion of “spectrin-like” repeats. This finding has therapeutic implications, as it suggests that minimal length dystophins or utrophins may fully complement the physiological role of full length dystrophin in vertebrates as long as the deletional juxtaposition of triple helical repeats does not disrupt force transmission. This interpretation is consistent with the clinical findings of Becker Muscular Dystrophy in selected patients with in-frame exon duplications. In this view, the evolutionary stability of the length of the dystrophin rod is driven by the incompatibility, when placed in close physical proximity by deletion, of divergent triple helical repeats. The evolutionary translocation of the protein to the cytoskeletal cortex as part of the DGC may have greatly reduced the metabolic load associated with synthesis of the strong but scarce protein, also facilitating evolutionary drift to the enormous size of the gene in a common mammalian ancestor.

Clinical potential of ataluren in the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an autosomal dominant, X-linked neuromuscular disorder caused by mutations in dystrophin, one of the largest genes known to date. Dystrophin gene mutations are generally transmitted from the mother to male offspring and can occur throughout the coding length of the gene. The majority of the methodologies aimed at treating the disorder have focused on restoring a shorter, although partially functional, dystrophin protein. The approach has the potential of converting a severe DMD phenotype into a milder form of the disease known as Becker muscular dystrophy. Others have focused on ameliorating the disease by targeting secondary pathologies such as inflammation or loss of regeneration. Of great potential is the development of strategies that are capable of restoring full-length dystrophin expression due to their ability to produce a normal, fully functional protein. Among these strategies, the use of read-through compounds (RTCs) that could be administered orally represents an ideal option. Gentamicin has been previously tested in clinical trials for DMD with limited or no success, and its use in the clinic has been dismissed due to issues of toxicity and lack of clear benefits to patients. More recently, new RTCs have been identified and tested in animal models for DMD. This review will focus on one of those RTCs known as ataluren that has now completed Phase III clinical studies for DMD and at providing an overview of the different stages that have led to its clinical development for the disease. The impact that this new drug may have on DMD and its future perspectives will also be described, with an emphasis on the importance of further assessing the clinical benefits of this molecule in patients as it becomes available on the market in different countries.

501. In Vivo Gene Editing for Duchenne Muscular Dystrophy

Gene replacement therapies utilizing adeno-associated viral vector delivery of truncated versions of dystrophin (mini- and microdystrophins) hold great potential for the treatment of Duchenne muscular dystrophy (DMD). However, these truncated dystrophins are not fully functional and episomal AAV vectors are not permanently retained following delivery to muscle. A more long-lasting and effective approach to gene therapy could result from methods to modify the dystrophin gene using gene editing strategies. The CRISPR/Cas9 system has been shown to be a highly specific method for genetic modifications, and recently has been proven applicable to correct mutations responsible for DMD. Here we present in vivo approaches for targeted correction of the dystrophin gene in the mdx4cv mouse model of DMD using both single- and dual adeno-associated viral (AAV) vector delivery systems of Cas9 and DMD mutation specific targeting guide RNA's. Treated muscles demonstrated successful targeting of the dystrophin gene leading to a corrected open reading frame in the messenger RNA resulting in a restoration of near-to full-length dystrophin expression in up to 70% of treated muscle cross-sectional area. These results demonstrate that successful in vivo dystrophin correction can be achieved following AAV mediated delivery of CRISPR/Cas9, showing promise for the development of future clinical application of technologies for permanent correction of mutations leading to DMD and other genetic muscle disorders.

503. Adeno-Associated Virus Vector (AAV) Microdystrophin Gene Therapy Prolongs Survival and Restores Muscle Function in the Canine Model of Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD) is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno-Associated Virus vectors (rAAV) hold great promise and allow very efficient transduction of skeletal and cardiac muscles. However, full-length dystrophin cDNA exceeds the packaging capacity for a single rAAV gene-delivery cassette. Therefore, truncated versions namely micro-dystrophins have been designed and optimized to contain few clinically important regions of the dystrophin protein. We have tested a rAAV2/8 vector encoding a sequence optimised canine micro-dystrophin transgene, driven by a muscle-synthetic Spc512 promoter (rAAV2/8-Spc512-µDys) in a total of 12 Golden Retriever Muscular Dystrophy (GRMD) dogs, the canine model of DMD. Isolated limb perfusion studies using a single administration of vector induced high levels of micro-dystrophin expression in the treated limb (up to 90% dystrophin positive fibres) with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. Similarly, single-dose intravascular delivery of the same rAAV2/8-Spc512-µDys, in absence of immunosuppression, led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years). Profound improvement of multiple clinical features was observed, including gait and respiratory parameters and no toxicity or deleterious humoral and/or cell-mediated immune responses were observed. This study demonstrates the safety and long term efficacy of rAAV2/8-Spc5.12-µDys gene therapy in a relevant large-animal models of DMD and paves the way towards human clinical gene therapy using systemic peripheral vein administration of vector, and applicable to all DMD patients regardless of their genotype.

HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

Isolation and characterization of neural stem cells from dystrophic mdx mouse

The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.

Spectroscopic and Computational Analysis of Dystrophin Regulation of Actin Dynamics

Dystrophin is a filamentous muscle protein that links the cytoskeleton to the extracellular matrix, dampening the laterally transduced force of sarcomere shortening and lengthening and thus limiting the mechanical stress reaching the sarcolemma membrane. Dystrophin's homologue, utrophin, fulfills a similar role in developing muscle but is ultimately replaced by dystrophin. Previous spectroscopic measurements on full-length dystrophin and utrophin showed that each significantly restrict the amplitude of actin filament rotational dynamics upon binding, but utrophin restricts this motion to a much greater extent. We hypothesize that part of this functional difference between homologous proteins stems from distinct structural binding modes and dynamics in the first actin-binding domain (ABD1). Using double electron-electron resonance (DEER) and time-resolved FRET, we characterized the closed-to-open structural transition of dystrophin's ABD1 and compared it to the previously studied utrophin. Dystrophin exhibits more disorder and a greater propensity to be in a closed (compact) state. To understand and complement these spectroscopic findings, we tested the thermal stability of dystrophin's ABD1 and examined possible driving forces for ABD1 closure with molecular dynamics (MD) simulations. We found that dystrophin is only marginally stable, consistent with the structural disorder measured spectroscopically. In MD simulations, the ABD1 crystal structure relaxed to a closed compact structural state and thus protected several hydrophobic residues. This suggests that location of hydrophobic residues is an important dictator of dystrophin's open-closed structural equilibrium. Together these results indicate that dystrophin's ABD1 forms an unstable complex upon binding to F-actin, due to energetically unfavorable exposure of hydrophobic surfaces within ABD1. These features may contribute to the reduced overall restriction of actin dynamics in comparison to utrophin. Funding to DDT (NIH grant AR63007).

Dystrophin Dp71, a novel tumor suppressor?

Dystrophins belong to a group of proteins encoding by the Duchenne muscular dystrophy ( DMD ) gene. DMD gene generates full-length dystrophin (427 kDa) and different C-terminal truncated isoforms through alternative usage of internal promoters. Short isoforms are termed in accordance with their molecular mass as Dp140, Dp116, Dp71 and Dp40. Dystrophin Dp71, is ubiquitously expresses, with the exception of skeletal muscle, and is the predominant DMD gene product in nervous system. In addition, Dp71 undergoes alternative splicing of exons 71 and/or 78 to generate different splicing variants, being Dp71f (−71/−78) and Dp71d (−71/+78) the most studied isoforms. While deficiency of dystrophin causes muscle degeneration, absence of Dp71 has been implicated in cognitive impairment and abnormal retinal physiology, two well-defined non-muscular alterations of DMD patients, reviewed in (1). As dystrophin does, Dp71 associates with sarcoglycans, dystroglycans, syntrophins and dystrobrevin, to conform the plasma membrane-associated dystrophin associated protein complex. Such protein assembly provides stability to the plasma membrane and modulates cell signaling. Interestingly, Dp71 undergoes nuclear import trough recognition of an atypical nuclear localization signal by the importin α2/β1 system. Nuclear Dp71 in turn, is involved in the maintenance of nuclear architecture, via its interaction with nuclear envelope proteins, including emerin and lamins A/C and B1 (2). Thus, it appears that Dp71 is a multifunctional protein that interacts with different partners in both the cytoplasm and nucleus, which enables it to modulate a variety of cell functions.

PiggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.