Dystrophin-associated Protein Complex Research Articles

Organisation of extracellular matrix proteins laminin and agrin in pericapillary basal laminae in mouse brain

Evidence suggests that extracellular matrix molecules of perivascular basal laminae help orchestrate the molecular assemblies at the gliovascular interface. Specifically, laminin and agrin are thought to tether the dystrophin-associated protein (DAP) complex to the astrocytic basal lamina. This complex includes α-syntrophin (α-Syn), which is believed to anchor aquaporin-4 (AQP4) to astrocytic endfoot membrane domains. We have previously shown that the size of the perivascular AQP4 pool differs considerably between brain regions in an α-Syn-dependent manner. Also, both AQP4 and α-Syn occur at higher densities in endfoot membrane domains facing pericytes than in endfoot membrane domains facing endothelial cells. The heterogeneous distribution of AQP4 at the regional and capillary level has been attributed to a direct interaction between AQP4 and α-Syn. This would be challenged (1) if the microdistributions of laminin and agrin fail to align with those of DAP and AQP4 and (2) if targeted deletion of α-Syn leads to a loss of laminin and/or agrin. Here, we provide the first detailed and quantitative analysis of laminin and agrin in brain basal laminae of mice. We show that the microdistributions of these molecules vary in a fashion that is well aligned with the previously reported microdistribution of AQP4. We also demonstrate that the expression patterns of laminin and agrin are insensitive to targeted deletion of α-Syn, suggesting that α-Syn deletion affects AQP4 directly and not indirectly via laminin or agrin. These data fill remaining voids in the current model of how key molecules are assembled and tethered at the gliovascular interface.

Restoration of Functional Full-Length Dystrophin After Intramuscular Transplantation of Foamy Virus-Transduced Myoblasts.

Stem cell therapy is a promising strategy to treat muscle diseases such as Duchenne muscular dystrophy (DMD). To avoid immune rejection of donor cells or donor-derived muscle, autologous cells, which have been genetically modified to express dystrophin, are preferable to cells derived from healthy donors. Restoration of full-length dystrophin (FL-dys) using viral vectors is extremely challenging, due to the limited packaging capacity of the vectors, but we have recently shown that either a foamy viral or lentiviral vector is able to package FL-dys open-reading frame and transduce myoblasts derived from a DMD patient. Differentiated myotubes derived from these transduced cells produced FL-dys. Here, we transplanted the foamy viral dystrophin-corrected DMD myoblasts intramuscularly into mdx nude mice, and showed that the transduced cells contributed to muscle regeneration, expressing FL-dys in nearly all the muscle fibers of donor origin. Furthermore, we showed that the restored FL-dys recruited members of the dystrophin-associated protein complex and neuronal nitric oxide synthase within donor-derived muscle fibers, evidence that the restored dystrophin protein is functional. Dystrophin-expressing donor-derived muscle fibers expressed lower levels of utrophin than host muscle fibers, providing additional evidence of functional improvement of donor-derived myofibers. This is the first in vivo evidence that foamy virus vector-transduced DMD myoblasts can contribute to muscle regeneration and mediate functional dystrophin restoration following their intramuscular transplantation, representing a promising therapeutic strategy for individual small muscles in DMD.

P.145Optimisation of a high–throughput digital script for multiplexed immunofluorescent analysis of sarcolemmal dystrophin - associated protein complex (DPC) and myofibre regeneration in entire transverse sections of muscle biopsies in Duchenne muscular dystrophy

P.145Optimisation of a high–throughput digital script for multiplexed immunofluorescent analysis of sarcolemmal dystrophin - associated protein complex (DPC) and myofibre regeneration in entire transverse sections of muscle biopsies in Duchenne muscular dystrophy

Regenerative biomarkers for Duchenne muscular dystrophy.

Skeletal muscle has an extraordinary capacity to regenerate after injury and trauma. The muscle repair mechanism is a complex process orchestrated by multiple steps. In neuromuscular disorders such as Duchenne muscular dystrophy (DMD), the pathological consequences of the lack of dystrophin and the loss of the dystrophin-associated protein complex are dramatic, with a progressive cascade of events, such as continual influx of inflammation, repeated cycles of degeneration and impaired regeneration. Thus, muscle regeneration is a hallmark of the disease and careful monitoring of regenerative processes with robust markers should provide useful information to the field. Since decades, several indices of regeneration such as centronucleation and fibre size have been commonly used. In the present review, we discuss the impaired regenerative process in DMD, the common and new indices of regeneration and their associated methodologies. We notably highlight the regenerative marker embryonic myosin as a robust indicator of muscle regeneration. We also describe new quantitative methodologies offering the possibility of using a panel of translational regenerative biomarkers to obtain a more complete view of the regeneration processes. Upregulation of utrophin, an autosomal and functional paralogue of dystrophin, is one of the most promising therapeutic strategies as it targets the primary cause of the disease and is applicable to all DMD patients regardless their genetic defects. As utrophin is a regeneration associated protein increased in dystrophic muscle, we discuss the correlation of utrophin levels after drug treatment with regeneration markers. The recent advances in technologies and complementary markers of muscle regeneration described in this review, provide an unprecedented opportunity to develop more robust utrophin DMD based strategies for all DMD patients.

Expression and localization of dystrophins and β-dystroglycan in the hypothalamic supraoptic nuclei of rat from birth to adulthood

Dystrophins (Dps) are the sub-membranous proteins that work via the dystrophin-associated proteins complex, which comprises β-dystroglycan (β-DG), a cell surface receptor for extracellular matrix. Recently, we have revealed β-DG decrease and central function impairment of supraoptic nucleus (SON) in Dp71 deficient adult mice, opening the question on the profiles of Dps and β-DG during SON development. At birth and the age of 10, 20 and 60 days, we examined the expression by RT-PCR and Western-blotting, and the distribution by immunohistochemistry of Dps and β-DG. Also, we analyzed, by immunohistochemistry and Western-blotting, the neuropeptide, arginine vasopressin (AVP), in the SON at the different ages.At birth, Dp71 and to a lesser extends, Dp140 and Dp427, and also β-DG are revealed in the SON. They are localized in the magnocellular neurons (MCNs), astrocytes and vessels.From birth to adulthood, the AVP raise in the SON coincides with the progressive increase of Dp71 level while the level of Dp140 and Dp427 increased only at D20, D10 post-natal development, respectively, and β-DG expression did not change. Moreover, the location of Dps or/and β-DG in the cell compartments was modified during development: at D10, Dps appeared in the astrocytes end-feet surrounding MCNs, and at D20, Dps and β-DG codistributed in the astrocytes end-feet, surrounding MCNs and vessels. Such a distribution marks the first steps of post-natal SON development and may be considered essential in the establishment of structural plasticity mechanisms in SON, where astrocyte end-feet, vessels, magnocellular neurons, are physiologically associated. The disappearance of β-DG in the MCNs nucleus marks the adulthood SON and suggests that the complex of Dps associating β-DG is required for the nucleoskeleton function in the post-natal development.

The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se

Background:The dystrophin protein is part of the dystrophin associated protein complex (DAPC) linking the intracellular actin cytoskeleton to the extracellular matrix. Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy (D/BMD). Neuronal nitric oxide synthase associates with dystrophin in the DAPC to generate the vasodilator nitric oxide (NO). Systemic dystrophin deficiency, such as in D/BMD, results in muscle ischemia, injury and fatigue during exercise as dystrophin is lacking, affecting NO production and hence vasodilation. The role of neuregulin 1 (NRG) signaling through the epidermal growth factor family of receptors ERBB2 and ERBB4 in skeletal muscle has been controversial, but it was shown to phosphorylate α-dystrobrevin 1 (α-DB1), a component of the DAPC. The aim of this investigation was to determine whether NRG signaling had a functional role in muscular dystrophy.Methods:Primary myoblasts (muscle cells) were isolated from conditional knock-out mice containing lox P flanked ERBB2 and ERBB4 receptors, immortalized and exposed to Cre recombinase to obtainErbb2/4double knock-out (dKO) myoblasts where NRG signaling would be eliminated. Myotubes, thein vitroequivalent of muscle fibers, formed by fusion of the lox P flankedErbb2/4myoblasts as well as theErbb2/4dKO myoblasts were then used to identify changes in dystrophin expression.Results:Elimination of NRG signaling resulted in the absence of dystrophin demonstrating that it is essential for dystrophin expression. However, unlike the DMD mouse model mdx, with systemic dystrophin deficiency, lack of dystrophin in skeletal muscles ofErbb2/4dKO mice did not result in muscular dystrophy. In these mice, ERBB2/4, and thus dystrophin, is still expressed in the smooth muscle of blood vessels allowing normal blood flow through vasodilation during exercise.Conclusions:Dystrophin deficiency in smooth muscle of blood vessels, rather than in skeletal muscle, is the main cause of disease progression in DMD.

Significant Association Between Variant in SGCD and Age-Related Macular Degeneration.

CFH and HTRA1 genes are traditional markers of increased risk of age-related macular degeneration (AMD) across populations. Recent findings suggest that additional genes—for instance, in the dystrophin-associated protein complex—might be promising markers for AMD. Here, we performed a case-control study to assess the effect of SGCD single nucleotide polymorphisms (SNPs), a member of this protein family, on AMD diagnosis and phenotype. We performed a case-control study of an under-studied population from Hispanics in Mexico City, with 134 cases with 134 unpaired controls. Cases were 60 years or older (Clinical Age-Related Maculopathy Staging (CARMS) grade 4–5, as assessed by experienced ophthalmologists following the American Association of Ophthalmology (AAO) guidelines), without other retinal disease or history of vitreous-retinal surgery. Controls were outpatients aged 60 years or older, with no drusen or retinal pigment epithelium (RPE) changes on a fundus exam and a negative family history of AMD. We examined SNPs in the SGCD gene (rs931798, rs140617, rs140616, and rs970476) by sequencing and real-time PCR. Genotyping quality checks and univariate analyses were performed with PLINK v1.90b3.42. Furthermore, logistic regression models were done in SAS v.9.4 and haplotype configurations in R v.3.3.1. After adjusting for clinical covariates, the G/A genotype of the SGCD gene (rs931798) significantly increases the odds of being diagnosed with AMD in 81% of cases (1.81; 95% CI 1.06–3.14; p = 0.031), especially the geographic atrophy phenotype (1.82; 95% CI 1.03–3.21; p = 0.038) compared to the G/G homozygous genotype. Moreover, the GATT haplotype in this gene (rs931798, rs140617, rs140616, and rs970476) is associated with lower odds of AMD (adjusted odds ratio (OR) 0.13; 95% CI 0.02–0.91; p = 0.041). SGCD is a promising gene for AMD research. Further corroboration in other populations is warranted, especially among other Hispanic ethnicities.

Nanotopography-responsive myotube alignment and orientation as a sensitive phenotypic biomarker for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal genetic disorder currently having no cure. Here we report that culture substrates patterned with nanogrooves and functionalized with Matrigel (or laminin) present an engineered cell microenvironment to allow myotubes derived from non-diseased, less-affected DMD, and severely-affected DMD human induced pluripotent stem cells (hiPSCs) to exhibit prominent differences in alignment and orientation, providing a sensitive phenotypic biomarker to potentially facilitate DMD drug development and early diagnosis. We discovered that myotubes differentiated from myogenic progenitors derived from non-diseased hiPSCs align nearly perpendicular to nanogrooves, a phenomenon not reported previously. We further found that myotubes derived from hiPSCs of a dystrophin-null DMD patient orient randomly, and those from hiPSCs of a patient carrying partially functional dystrophin align approximately 14° off the alignment direction of non-diseased myotubes. Substrates engineered with micron-scale grooves and/or cell adhesion molecules only interacting with integrins all guide parallel myotube alignment to grooves and lose the ability to distinguish different cell types. Disruption of the interaction between the Dystrophin-Associated-Protein-Complex (DAPC) and laminin by heparin or anti-α-dystroglycan antibody IIH6 disenables myotubes to align perpendicular to nanogrooves, suggesting that this phenotype is controlled by the DAPC-mediated cytoskeleton-extracellular matrix linkage.

The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se

Background:The dystrophin protein is part of the dystrophin associated protein complex (DAPC) linking the intracellular actin cytoskeleton to the extracellular matrix. Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy (D/BMD). Neuronal nitric oxide synthase associates with dystrophin in the DAPC to generate the vasodilator nitric oxide (NO). Systemic dystrophin deficiency, such as in D/BMD, results in muscle ischemia, injury and fatigue during exercise as dystrophin is lacking, affecting NO production and hence vasodilation. The role of neuregulin 1 (NRG) signaling through the epidermal growth factor family of receptors ERBB2 and ERBB4 in skeletal muscle has been controversial, but it was shown to phosphorylate α-dystrobrevin 1 (α-DB1), a component of the DAPC. The aim of this investigation was to determine whether NRG signaling had a functional role in muscular dystrophy.Methods:Primary myoblasts (muscle cells) were isolated from conditional knock-out mice containing lox P flanked ERBB2 and ERBB4 receptors, immortalized and exposed to CRE recombinase to obtainErbb2/4double knock-out (dKO) myoblasts where NRG signaling would be eliminated. Myotubes, thein vitroequivalent of muscle fibers, formed by fusion of the lox P flankedErbb2/4myoblasts as well as theErbb2/4dKO myoblasts were then used to identify changes in dystrophin expression.Results:Elimination of NRG signaling resulted in the absence of dystrophin demonstrating that it is essential for dystrophin expression. However, unlike the DMD mouse model mdx, with systemic dystrophin deficiency, lack of dystrophin in skeletal muscles ofErbb2/4dKO mice did not result in muscular dystrophy. In these mice, ERBB2/4, and thus dystrophin, is expressed in the smooth muscle of blood vessels allowing normal blood flow through vasodilation during exercise.Conclusions:Dystrophin deficiency in smooth muscle of blood vessels, rather than in skeletal muscle, is the main cause of disease progression in DMD.

Role of α-Dystrobrevin in the differentiation process of HL-60 cells

The α-Dystrobrevin gene encodes at least five different protein isoforms, expressed in diverse tissues. The α-Dystrobrevin-1 isoform (α-Db-1) is a member of the cytoplasmic dystrophin-associated protein complex, which has a C-terminal extension comprising at least three tyrosine residues susceptible to phosphorylation in vivo. We previously described α-Db in stem-progenitor cells and blood neutrophils as playing a scaffolding role and, in association with kinesin and microtubules, α-Db promotes platelet-granule trafficking. Additionally, the microtubules must establish a balanced interaction with the lamina A/C network for appropriate nuclear morphology. Considering that the most outstanding feature during neutrophil differentiation is nuclei lobulation, we hypothesized that α-Db might possess a pivotal function during the neutrophil differentiation process. Western Blot (WB) and confocal microscope assays evidenced a differential pattern expression and a subcellular redistribution of α-Db in neutrophils derived from HL-60 cells. At the end of the differentiation process, we detected an important diminution in the expression of tubulin, kinesin, and α-Db-1. Knockdown of α-Db prevented nuclei lobulation, increased Lamin A/C and syne1 expression and augmented the roughness of derived neutrophil membrane and disturbed filopodia assembly.Our results suggest that HL-60 cells undergo extensive cytoskeletal reorganization including α-Db in order to possess lobulated nuclei when they further differentiate into neutrophils.

Altered somatosensory neurovascular response in patients with Becker muscular dystrophy.

IntroductionPatients with dystrophinopathies show low levels of neuronal nitric oxide synthase (nNOS), due to reduced or absent dystrophin expression, as nNOS is attached to the dystrophin‐associated protein complex. Deficient nNOS function leads to functional ischemia during muscle activity. Dystrophin‐like proteins with nNOS attached have also been identified in the brain. This suggests that a mechanism of cerebral functional ischemia with attenuation of normal activation‐related vascular response may cause changes in brain function.MethodsThe aim of this study was to investigate whether the brain response of patients with Becker muscular dystrophy (BMD) is dysfunctional compared to that of healthy controls. To investigate a potential change in brain activation response in patients with BMD, median nerve somatosensory evoked stimulation, with stimulation durations of 2, 4, and 10 s, was performed while recording electroencephalography and blood oxygen level‐dependent (BOLD) functional magnetic resonance imaging.ResultsResults in 14 male patients with BMD (36.2 ± 9.9 years) were compared with those of 10 healthy controls (34.4 ± 10.9 years). Compared to controls, the patients with BMD showed sustained cortical electrical activity and a significant smaller BOLD activation in contralateral primary somatosensory cortex and bilaterally in secondary somatosensory cortex. In addition, significant activation differences were found after long duration (10 s) stimuli in thalamus.ConclusionAn altered neurovascular response in patients with BMD may increase our understanding of neurovascular coupling and the pathogenesis related to dystrophinopathy and nNOS.

IMPACT OF BETA‐DYSTROGLYCAN DEFICIENCY ON EMERIN FUNCTIONS

Beta‐dystroglycan (b‐DG) is a plasma membrane protein that belongs to the dystrophin‐associated protein complex (DAPC), the latter provides a link between the extracellular matrix and the cytoskeleton. Previously we reported that b‐DG is translocated localized to the nucleus via recognition of its nuclear localization signal (NLS), located in the yuxtamembrane domain, by importins α2/β1. Nuclear b‐DG is involved in the maintenance of nuclear envelope structure through its interaction with emerin and lamins A/C and B1. Emerin is an inner nuclear membrane protein involved in nuclear morphology, myogenesis and cell proliferation.In this study, we analyzed the impact of β‐dystroglycan deficiency on emerin function, using C2C12 cells as model. We found that depletion of β‐DG resulted in decreased emerin levels, as shown by immunofluorescence and western blot assays with anti‐emerin antibodies. Furthermore, emerin‐mediated processes, including nuclear morphology and nucleus‐centrosome distance are altered in such cells. The analysis of emerin‐dependent nuclear signaling in β‐DG‐depleted cells is currently in progress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dystrophin 71 and α1syntrophin in morpho-functional plasticity of rat supraoptic nuclei: Effect of saline surcharge and reversibly normal hydration

Dystrophin (Dp) is a multidomain protein that links the actin cytoskeleton to the extracellular matrix through the dystrophin associated proteins complex (DAPC). Dp of 71 kDa (Dp71), corresponding to the COOH-terminal domain of dystrophin, and α1-syntrophin (α1Syn) as the principal component of the DAPC, are strongly expressed in the brain. To clarify their involvement in the central control of osmotic homeostasis, we investigated the effect of 14 days of salt loading (with drinking water containing 2% NaCl) and then reversibly to 30 days of normal hydration (with drinking water without salt), first on the expression by western-blotting and the distribution by immunochemistry of Dp71 and α1Syn in the SON of the rat and, second, on the level of some physiological parameters, as the plasma osmolality, natremia and hematocrit.Dp71 is the most abundant form of dystrophin revealed in the supraoptic nucleu (SON) of control rat. Dp71 was localized in magnocellular neurons (MCNs) and astrocytes, when α1Syn was observed essentially in astrocytes end feet. After 14 days of salt-loading, Dp71 and α1Syn signals decreased and a dual signal for these two proteins was revealed in the astrocytes processes SON surrounding blood capillaries. In addition, salt loading leads to an increase in plasma osmolality, natremia and hematocrit. Reversibly, after 30 days of normal hydration, the intensity of the signal for the two proteins, Dp71 and α1Syn, increased and approached that of control. Furtheremore, the levels of the physiological parameters decreased and approximated those of control.This suggests that Dp71 and α1Syn may be involved in the functional activity of the SON. Their localization in astrocyte end feet emphasizes their importance in neuronal–vascular–astrocyte interactions for the central detection of osmolality. In the SON, Dp71 and α1Syn may be involved in osmosensitivity.

The role of AMP-activated protein kinase in the expression of the dystrophin-associated protein complex in skeletal muscle.

Stimulation of AMPK induces the expression of dystrophin-associated protein complex (DAPC) components in skeletal muscle, whereas reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast, glycolytic extensor digitorum longus (EDL) and slow, oxidative soleus (Sol) muscles from wild-type mice and from littermates with skeletal muscle-specific knockout of the AMPK β1 and β2 subunits (AMPK β1 β2M-KO; MKO) were analyzed. DAPC mRNA and protein expression were similar between genotypes, with the exception of elevated neuronal nitric oxide synthase expression at the sarcolemma in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC was also not affected by the loss of AMPK. However, MyoD and myogenin expression was diminished in MKO muscles, consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO Sol muscles, likely due to the adaptive accumulation of peroxisome proliferator-activated receptor γ coactivator-1α at the sarcolemma of MKO EDL muscles. Collectively, the evidence indicates that AMPK is sufficient but not essential for the maintenance of DAPC expression in skeletal muscle, yet it is required for preserving extrasynaptic utrophin levels in slow oxidative muscles.-Dial, A. G., Rooprai, P., Lally, J. S., Bujak, A. L., Steinberg, G. R., Ljubicic, V. The role of AMP-activated protein kinase in the expression of the dystrophin-associated protein complex in skeletal muscle.

The molecular cross talk of the dystrophin-glycoprotein complex.

The proper function of skeletal muscles relies on their ability to process signals derived from motor neurons, transmit stimuli along the muscle fibers, contract, and regenerate efficiently after injury. The dystrophin-glycoprotein complex (DGC; also called the dystrophin-associated protein complex) plays a central role in all of these processes. It acts as a transmembrane platform that anchors the extracellular matrix (ECM) to the intracellular cytoskeleton and makes muscle fibers more resistant to injury. The DGC also contributes to the transmission of contraction-evoked force from the sarcomere to the ECM. The dysfunction of DGC-associated proteins can lead to myopathies, including Duchenne's muscular dystrophy, manifested by progressive muscle damage and impairments in regeneration. The DGC also plays a pivotal role in the organization of neuromuscular junctions (NMJs), where it stabilizes postsynaptic machinery, including receptors for the neurotransmitter acetylcholine (AChRs). Here, we focus on the role of the DGC complex in NMJ and skeletal muscle physiology and discuss the novel components that are associated with the complex.

Effects of (-)-epicatechin on frontal cortex DAPC and dysbindin of the mdx mice.

Multiple components of the dystrophin-associated protein complex (DAPC) are expressed in numerous tissues including the brain. Members of the DAPC and dysbindin are abnormally expressed in the brain of Duchenne Muscular Dystrophy (DMD) patients, which has been associated with cognitive impairments. However, little is known about the expression pattern of individual members of the DAPC in animal models of DMD and their relationship with dysbindin. Ten mdx mice were randomly allocated into a control and intervention group [(-)-epicatechin (Epi) 1mg/kg/day for four weeks] and results compared to a wild-type mice. After sacrifice, brain pre-frontal cortices were collected for Western blotting and immunoprecipitation assays, and sagittal sections processed for immunohistochemistry. Epi promotes a partial recovery of DAPC members [α1-Syntrophin, sarcoglycans (SG), dystrophin 71 (Dp71)], dysbindin, and utrophin protein levels. Epi also appears to restore the association of DAPC between dysbindin, and utrophin with Dp71 and ε-SG. Co-immunostaining evidence increased protein levels of dysbindin, dystrophin, and ε-SG and their colocalization. Altogether, results suggest that Epi is capable of restoring pre-frontal cortex DAPC and dysbindin levels of mdx mice towards that of healthy brains. The functional implications of such studies warrant further investigation.

Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes

ABSTRACTKir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current IK1, which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential IK1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that IK1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

Myoclonus dystonia and muscular dystrophy: ɛ-sarcoglycan is part of the dystrophin-associated protein complex in brain.

ABSTRACTBackgroundMyoclonus‐dystonia is a neurogenic movement disorder caused by mutations in the gene encoding ɛ‐sarcoglycan. By contrast, mutations in the α‐, β‐, γ‐, and δ‐sarcoglycan genes cause limb girdle muscular dystrophies. The sarcoglycans are part of the dystrophin‐associated protein complex in muscle that is disrupted in several types of muscular dystrophy. Intriguingly, patients with myoclonus‐dystonia have no muscle pathology; conversely, limb‐girdle muscular dystrophy patients have not been reported to have dystonia‐associated features. To gain further insight into the molecular mechanisms underlying these differences, we searched for evidence of a sarcoglycan complex in the brain.MethodsImmunoaffinity chromatography and mass spectrometry were used to purify ubiquitous and brain‐specific ɛ‐sarcoglycan directly from tissue. Cell models were used to determine the effect of mutations on the trafficking and assembly of the brain sarcoglycan complex.ResultsUbiquitous and brain‐specific ɛ‐sarcoglycan isoforms copurify with β‐, δ‐, and ζ‐sarcoglycan, β‐dystroglycan, and dystrophin Dp71 from brain. Incorporation of a muscular dystrophy‐associated β‐sarcoglycan mutant into the brain sarcoglycan complex impairs the formation of the βδ‐sarcoglycan core but fails to abrogate the association and membrane trafficking of ɛ‐ and ζ‐sarcoglycan.Conclusionsɛ‐Sarcoglycan is part of the dystrophin‐associated protein complex in brain. Partial preservation of ɛ‐ and ζ‐sarcoglycan in brain may explain the absence of myoclonus dystonia‐like features in muscular dystrophy patients. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

In-silico structural analysis of E509K mutation in LARGE and T192M mutation in Alpha Dystroglycan in the inhibition of glycosylation of Alpha Dystroglycan by LARGE

Impaired glycosylation of cellular receptor Alpha Dystroglycan (α-DG) leads to dystroglycanopathy. Glycoprotein α-DG is the receptor protein in the Dystrophin Associated Protein Complex (DAPC), a macromolecular gathering on muscle cell membrane to form a bridge between extracellular matrix (ECM) and cellular actin cytoskeleton. Proper glycosylation of α-DG is mediated by the glycosylating enzyme LARGE. Mutations either in α-DG or in LARGE lead to improper glycosylations of α-DG thereby hampering the formation of final Laminin binding form α-DG resulting in dystroglycanopathy. In our current work, we explored the structural changes associated with the presence of mutations in α-DG as well as in the enzyme LARGE. We further extended our research to understand the effect of the mutations onto protein-enzyme interactions. Moreover, since LARGE transfers the sugar moiety (glucuronic acid; GlcA) onto α-DG, we tried to analyze what effect the mutation in LARGE confers on this enzyme ligand interaction. This work for the first time addressed the molecular changes occurring in the structures α-DG, LARGE and their interactions and shed lights on the as yet poorly understood mechanism behind the dystroglycanopathy onset.

Characterization of a Dmd (EGFP) reporter mouse as a tool to investigate dystrophin expression.

BackgroundDystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.ResultsWe generated a DmdEGFP reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.ConclusionsThe novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0095-5) contains supplementary material, which is available to authorized users.