Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

Distinct roles of the dystrophin-glycoprotein complex: α-dystrobrevin and α-syntrophin in the maintenance of the postsynaptic apparatus of the neuromuscular synapse.

α-syntrophin (α-syn) and α-dystrobrevin (α-dbn), two components of the dystrophin-glycoprotein complex, are essential for the maturation and maintenance of the neuromuscular junction (NMJ) and mice deficient in either α-syn or α-dbn exhibit similar synaptic defects. However, the functional link between these two proteins and whether they exert distinct or redundant functions in the postsynaptic organization of the NMJ remain largely unknown. We generated and analyzed the synaptic phenotype of double heterozygote (α-dbn+/-, α-syn+/-), and double homozygote knockout (α-dbn-/-; α-syn-/-) mice and examined the ability of individual molecules to restore their defects in the synaptic phenotype. We showed that in double heterozygote mice, NMJs have normal synaptic phenotypes and no signs of muscular dystrophy. However, in double knockout mice (α-dbn-/-; α-syn-/-), the synaptic phenotype (the density, the turnover and the distribution of AChRs within synaptic branches) is more severely impaired than in single α-dbn-/- or α-syn-/- mutants. Furthermore, double mutant and single α-dbn-/- mutant mice showed more severe exercise-induced fatigue and more significant reductions in grip strength than single α-syn-/- mutant and wild-type. Finally, we showed that the overexpression of the transgene α-syn-GFP in muscles of double mutant restores primarily the abnormal extensions of membrane containing AChRs that extend beyond synaptic gutters and lack synaptic folds, whereas the overexpression of α-dbn essentially restores the abnormal dispersion of patchy AChR aggregates in the crests of synaptic folds. Altogether, these data suggest that α-syn and α-dbn act in parallel pathways and exert distinct functions on the postsynaptic structural organization of NMJs.

Generation of dystrophin short product-specific tag-insertion mouse: distinct Dp71 glycoprotein complexes at inhibitory postsynapse and glia limitans.

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA-tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71, although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinß (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevinα (Dtna). Thus, our data indicate that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

The α-dystrobrevins play a key role in maintaining the structure and function of the extracellular matrix-significance for protein elimination failure arteriopathies.

The extracellular matrix (ECM) of the cerebral vasculature provides a pathway for the flow of interstitial fluid (ISF) and solutes out of the brain by intramural periarterial drainage (IPAD). Failure of IPAD leads to protein elimination failure arteriopathies such as cerebral amyloid angiopathy (CAA). The ECM consists of a complex network of glycoproteins and proteoglycans that form distinct basement membranes (BM) around different vascular cell types. Astrocyte endfeet that are localised against the walls of blood vessels are tethered to these BMs by dystrophin associated protein complex (DPC). Alpha-dystrobrevin (α-DB) is a key dystrophin associated protein within perivascular astrocyte endfeet; its deficiency leads to a reduction in other dystrophin associated proteins, loss of AQP4 and altered ECM. In human dementia cohorts there is a positive correlation between dystrobrevin gene expression and CAA. In the present study, we test the hypotheses that (a) the positive correlation between dystrobrevin gene expression and CAA is associated with elevated expression of α-DB at glial-vascular endfeet and (b) a deficiency in α-DB results in changes to the ECM and failure of IPAD. We used human post-mortem brain tissue with different severities of CAA and transgenic α-DB deficient mice. In human post-mortem tissue we observed a significant increase in vascular α-DB with CAA (CAA vrs. Old p < 0.005, CAA vrs. Young p < 0.005). In the mouse model of α-DB deficiency, there was early modifications to vascular ECM (collagen IV and BM thickening) that translated into reduced IPAD efficiency. Our findings highlight the important role of α-DB in maintaining structure and function of ECM, particularly as a pathway for the flow of ISF and solutes out of the brain by IPAD.

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

PKCα-mediated phosphorylation of the diacylglycerol kinase ζ MARCKS domain switches cell migration modes by regulating interactions with Rac1 and RhoA.

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.

Essential roles of the dystrophin-glycoprotein complex in different cardiac pathologies.

The dystrophin-glycoprotein complex (DGC), situated at the sarcolemma dynamically remodels during cardiac disease. This review examines DGC remodeling as a common denominator in diseases affecting heart function and health. Dystrophin and the DGC serve as broad cytoskeletal integrators that are critical for maintaining stability of muscle membranes. The presence of pathogenic variants in genes encoding proteins of the DGC can cause absence of the protein and/or alterations in other complex members leading to muscular dystrophies. Targeted studies have allowed the individual functions of affected proteins to be defined. The DGC has demonstrated its dynamic function, remodeling under a number of conditions that stress the heart. Beyond genetic causes, pathogenic processes also impinge on the DGC, causing alterations in the abundance of dystrophin and associated proteins during cardiac insult such as ischemia-reperfusion injury, mechanical unloading, and myocarditis. When considering new therapeutic strategies, it is important to assess DGC remodeling as a common factor in various heart diseases. The DGC connects the internal F-actin-based cytoskeleton to laminin-211 of the extracellular space, playing an important role in the transmission of mechanical force to the extracellular matrix. The essential functions of dystrophin and the DGC have been long recognized. DGC based therapeutic approaches have been primarily focused on muscular dystrophies, however it may be a beneficial target in a number of disorders that affect the heart. This review provides an account of what we now know, and discusses how this knowledge can benefit persistent health conditions in the clinic.

Oncogenic microRNA-301b regulates tumor repressor dystrobrevin alpha to facilitate cell growth, invasion and migration in esophageal cancer.

BACKGROUND: Esophageal cancer (EC) ranks the eighth in morbidity and the sixth in mortality around the whole world, which is an aggressive malignancy. To authenticate potential therapeutic targets for EC is therefore imperative. Although miR-301b might display changed expression in esophageal adenocarcinoma by utilizing Taqman miRNA profiling analysis, much less is known about the impact of miR-301b in EC. METHODS AND RESULTS: By analyzing the data of 187 cancer tissues and 13 normal samples from TCGA database, we discovered that miR-301b was highly expressed in EC tissues. Then, RT-qPCR determined that miR-301b was up-regulated in EC cell lines (ECA109, JAR, TE-1 and OE33). Besides, miR-301b expression level was higher in ESCC cell line-TE-1 cells and lower in ESCC cell line-ECA109 cells compared to other EC cell lines. Hence, ECA109 cell line was used to up-regulate miR-301b expression while TE-1 cell line was applied to down-regulate miR-301b expression in the subsequent experiments. Additionally, OE33, as an ECA cell line, was applied to upregulate miR-301b expression to reflect the influence of miR-301b overexpression on EC progression. More interestingly, miR-301b appeared to act as a promoting effect on the proliferation of EC cells, which was tested by CCK8. Dystrobrevin alpha (DTNA) was a targeting gene of miR-301b, which was predicted by the websites of miRanda, miRWalk and TargetScan. Additionally, DTNA was low expressed in EC tissues and was an independent predictor of EC. Meanwhile, the low expression of DTNA was related to worse overall survival in EC patients. The Pearson correlation coefficient analyzed that DTNA expression was negatively correlated with miR-301b. Furthermore, RT-qPCR and western blotting assays ulteriorly indicated that DTNA was negatively modulated by miR-301b. The facilitating impact of miR-301b re-expression on ECA109 and OE33 cell growth, invasion and migration was receded by DTNA over-expression, whilst the repressive effect of miR-301b ablation on TE-1 cell growth, invasion and migration was inversed by DTNA silencing. Overexpression of miR-301b accelerated EC cell growth, migration and invasion through targeting DTNA. CONCLUSIONS: Above all, we concluded that miR-301b was concerned with the progression of EC via regulating DTNA, suggesting that miR-301b and its target gene, DTNA, might serve as predictive biomarkers for EC therapy.

Loss of Dysbindin Implicates Synaptic Vesicle Replenishment Dysregulation as a Potential Pathogenic Mechanism in Schizophrenia.

The schizophrenia-susceptibility gene, dystrobrevin-binding protein 1 (DTNBP1), encodes the dysbindin protein and mediates neurotransmission and neurodevelopment in normal subjects. Functional studies show that DTNBP1 loss may cause deficient presynaptic vesicle transmission, which is related to multiple psychiatric disorders. However, the functional mechanism of dysbindin-mediated synaptic vesicle transmission has not been investigated systematically. In this study, we performed electrophysiological recordings in calyx of Held synapses. We found that excitatory postsynaptic current (EPSC) and miniature EPSC (mEPSC) amplitudes were unchanged in dysbindin-deficient synapses, but readily releasable pool (RRP) size and calcium dependent vesicle replenishment were affected during high-frequency stimulation. Moreover, dysbindin loss accompanied slightly decreases in Munc18-1 and snapin expression levels, which are associated with vesicle priming and synaptic homeostasis under high-frequency stimulation. Together, we inferred that dysbindin directly interacts with Munc18-1 and snapin to mediate calcium dependent RRP replenishment. Dysbindin loss may lead to RRP replenishment dysregulation during high-frequency stimulation, potentially causing cognitive impairment in schizophrenia.

DTNA promotes HBV-induced hepatocellular carcinoma progression by activating STAT3 and regulating TGFß1 and P53 signaling.

OBJECTIVE: Hepatitis B virus (HBV) infection causes liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) development, but the underlying mechanism remains poorly understood. This study aimed to investigate the roles and molecular mechanisms of Dystrobrevin-α (DTNA) in HBV-induced liver cirrhosis and HCC pathogenesis. METHODS: DTNA expression was bioinformatically analyzed using the GEO database. DTNA expression was silenced by transfection with shRNAs. Cell proliferation and apoptosis were evaluated by MTT and flow cytometry respectively. The expression of genes in mRNA or protein levels was assessed by quantitative RT-PCR and western blotting. The interaction between proteins was predicted with the String and GCBI online softwares, and then confirmed by co-immunoprecipitation. Animal models were established by injecting nude mice with AVV8-HBV1.3 vector. RESULTS: Bioinformatics analysis showed a significantly increase in DTNA expression in HBV-positive liver cirrhosis and HCC patients. HBV infection caused a significantly increase in DTNA expression in HCC cell lines HepAD38 and HepG2.2.15. DTNA knockdown suppressed proliferation and promoted apoptosis of HBV-infected HepAD38 and HepG2.2.15 cells. HBV induced elevated expression of fibrosis-related genes Collagen II and TGFß1 in LO-2 cells, which were suppressed by DTNA knockdown. DTNA directly binded with STAT3 protein to promote STAT3 phosphorylation and TGFß1 expression and repress P53 expression in HBV-infected HepAD38 and LO-2 cells. The DTNA/STAT3 axis was activated during HBV-induced fibrosis, cirrhosis and HCC development in mouse model. CONCLUSION: DTNA binds with and further activates STAT3 to induce TGFß1 expression and repress P53 expression, thus promoting HBV-induced liver fibrosis, cirrhosis and hepatocellular carcinoma progression.

Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability.

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap-α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap-α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Functional specialization of retinal Müller cell endfeet depends on an interplay between two syntrophin isoforms.

Retinal Müller cells are highly polarized macroglial cells with accumulation of the aquaporin-4 (AQP4) water channel and the inwardly rectifying potassium channel Kir4.1 at specialized endfoot membrane domains abutting microvessels and corpus vitreum. Proper water and potassium homeostasis in retina depends on these membrane specializations. Here we show that targeted deletion of ß1-syntrophin leads to a partial loss of AQP4 from perivascular Müller cell endfeet and that a concomitant deletion of both α1- and ß1-syntrophin causes a near complete loss of AQP4 from both perivascular and subvitreal endfoot membranes. α1-syntrophin is normally very weakly expressed in Müller cell endfeet but ß1-syntrophin knockout mice display an increased amount of α1-syntrophin at these sites. We suggest that upregulation of perivascular α1-syntrophin restricts the effect of ß1-syntrophin deletion. The present findings indicate that ß1-syntrophin plays an important role in maintaining the functional polarity of Müller cells and that α1-syntrophin can partially substitute for ß1-syntrophin in AQP4 anchoring. Functional polarization of Müller cells thus depends on an interplay between two syntrophin isoforms.

nNOS/GSNOR interaction contributes to skeletal muscle differentiation and homeostasis.

Neuronal nitric oxide synthase (nNOS) plays a crucial role in the maintenance of correct skeletal muscle function due, at least in part, to S-nitrosylation of specific protein targets. Similarly, we recently provided evidence for a muscular phenotype in mice lacking the denitrosylase S-nitrosoglutathione reductase (GSNOR). Here, we demonstrate that nNOS and GSNOR are concomitantly expressed during differentiation of C2C12. They colocalizes at the sarcolemma and co-immunoprecipitate in cells and in myofibers. We also provide evidence that GSNOR expression decreases in mouse models of muscular dystrophies and of muscle atrophy and wasting, i.e., aging and amyotrophic lateral sclerosis, suggesting a more general regulatory role of GSNOR in skeletal muscle homeostasis.

Adipocyte Hypertrophy and Improved Postprandial Lipid Response in Beta 2 Syntrophin Deficient Mice.

BACKGROUND/AIMS: Adipocyte hypertrophy in obesity is associated with inflammation and adipose tissue fibrosis which both contribute to metabolic diseases. Mechanisms regulating lipid droplet expansion are poorly understood. Knock down of the scaffold protein beta 2 syntrophin (SNTB2) increases lipid droplet size of 3T3-L1 adipocytes and the physiological relevance of SNTB2 in adipose tissue morphology and metabolic health was analyzed herein. METHODS: Wild type and SNTB2-/- mice were challenged with 24 weeks high fat diet. Adipose tissue morphology and expression of various genes / proteins including collagens and caveolin-1 was examined. Glucose, insulin, fasting and fed free fatty acids were measured in serum. SNTB2 expression was determined in adipose tissues of patients. RESULTS: Upon high fat diet SNTB2-/- mice displayed reduced adiposity and adipocyte hypertrophy. Expression of various proteins was normal in the different white fat depots of SNTB2-/- mice while caveolin-1 protein and collagen mRNA levels were diminished. Null mice had reduced systemic glucose while fasting and postprandial insulin and insulin response were normal. Fatty acid clearance in the fed state and after insulin injection was enhanced. SNTB2 and caveolin-1 were increased in fat of ob/ob mice. However, no correlation between body mass index and SNTB2 protein in adipose tissues of seven patients was found. In subcutaneous but not in visceral fat the ratio of SNTB2 to alpha syntrophin protein, which affects lipid droplet size in the opposite manner, was associated with BMI. In subcutaneous fat of extremely obese patients SNTB2 mRNA levels were not correlated with weight loss after bariatric surgery. CONCLUSION: Current study shows that high SNTB2 in obese adipose tissues restricts adipocyte growth and thereby may contribute to metabolic diseases.

Dystrobrevin is required postsynaptically for homeostatic potentiation at the Drosophila NMJ.

Evolutionarily conserved homeostatic systems have been shown to modulate synaptic efficiency at the neuromuscular junctions of organisms. While advances have been made in identifying molecules that function presynaptically during homeostasis, limited information is currently available on how postsynaptic alterations affect presynaptic function. We previously identified a role for postsynaptic Dystrophin in the maintenance of evoked neurotransmitter release. We herein demonstrated that Dystrobrevin, a member of the Dystrophin Glycoprotein Complex, was delocalized from the postsynaptic region in the absence of Dystrophin. A newly-generated Dystrobrevin mutant showed elevated evoked neurotransmitter release, increased bouton numbers, and a readily releasable pool of synaptic vesicles without changes in the function or numbers of postsynaptic glutamate receptors. In addition, we provide evidence to show that the highly conserved Cdc42 Rho GTPase plays a key role in the postsynaptic Dystrophin/Dystrobrevin pathway for synaptic homeostasis. The present results give novel insights into the synaptic deficits underlying Duchenne Muscular Dystrophy affected by a dysfunctional Dystrophin Glycoprotein complex.

The utrophin-beta 2 syntrophin complex regulates adipocyte lipid droplet size independent of adipogenesis.

Utrophin is a widely expressed cytoskeleton protein and is associated with lipid droplets (LDs) in adipocytes. The scaffold protein beta 2 syntrophin (SNTB2) controls signaling events by recruiting distinct membrane and cytoskeletal proteins, and binds to utrophin. Here we show that SNTB2 forms a complex with utrophin in adipocytes. SNTB2 protein is strongly diminished when utrophin is low. Of note, knock-down of utrophin or SNTB2 enhances LD growth during adipogenesis. SNTB2 reduction has no effect on basal and induced lipolysis, and insulin-stimulated phosphorylation of Akt is normal. The antilipolytic activity of insulin is enhanced in adipocytes with low SNTB2, while knock-down of utrophin has no effect. Uptake of exogenously supplied oleate and linoleate is comparable in scrambled and SNTB2 siRNA-treated cells. In the fibroblasts, diminished SNTB2 is associated with lower proliferation. CCAAT/enhancer-binding protein alpha and sterol regulatory element-binding proteins which are critical transcription factors for adipogenesis are normally expressed. Consequently, maturation of cells with SNTB2 knock-down is not grossly impaired. In fibroblasts, SNTB2 is localized to filamentous and vesicular structures which are distinct from beta actin, alpha tubulin, endoplasmic reticulum, early endosomes, lysosomes and mitochondria. Collectively, our data provide evidence that the utrophin-SNTB2 complex regulates LD size without affecting adipogenesis.

Syntrophins entangled in cytoskeletal meshwork: Helping to hold it all together.

Syntrophins are a family of 59 kDa peripheral membrane-associated adapter proteins, containing multiple protein-protein and protein-lipid interaction domains. The syntrophin family consists of five isoforms that exhibit specific tissue distribution, distinct sub-cellular localization and unique expression patterns implying their diverse functional roles. These syntrophin isoforms form multiple functional protein complexes and ensure proper localization of signalling proteins and their binding partners to specific membrane domains and provide appropriate spatiotemporal regulation of signalling pathways. Syntrophins consist of two PH domains, a PDZ domain and a conserved SU domain. The PH1 domain is split by the PDZ domain. The PH2 and the SU domain are involved in the interaction between syntrophin and the dystrophin-glycoprotein complex (DGC). Syntrophins recruit various signalling proteins to DGC and link extracellular matrix to internal signalling apparatus via DGC. The different domains of the syntrophin isoforms are responsible for modulation of cytoskeleton. Syntrophins associate with cytoskeletal proteins and lead to various cellular responses by modulating the cytoskeleton. Syntrophins are involved in many physiological processes which involve cytoskeletal reorganization like insulin secretion, blood pressure regulation, myogenesis, cell migration, formation and retraction of focal adhesions. Syntrophins have been implicated in various pathologies like Alzheimer's disease, muscular dystrophy, cancer. Their role in cytoskeletal organization and modulation makes them perfect candidates for further studies in various cancers and other ailments that involve cytoskeletal modulation. The role of syntrophins in cytoskeletal organization and modulation has not yet been comprehensively reviewed till now. This review focuses on syntrophins and highlights their role in cytoskeletal organization, modulation and dynamics via its involvement in different cell signalling networks.

Dystrophin R16/17-syntrophin PDZ fusion protein restores sarcolemmal nNOSµ.

BACKGROUND: Loss of sarcolemmal nNOSµ is a common manifestation in a wide variety of muscle diseases and contributes to the dysregulation of multiple muscle activities. Given the critical role sarcolemmal nNOSµ plays in muscle, restoration of sarcolemmal nNOSµ should be considered as an important therapeutic goal. METHODS: nNOSµ is anchored to the sarcolemma by dystrophin spectrin-like repeats 16 and 17 (R16/17) and the syntrophin PDZ domain (Syn PDZ). To develop a strategy that can independently restore sarcolemmal nNOSµ, we engineered an R16/17-Syn PDZ fusion construct and tested whether this construct alone is sufficient to anchor nNOSµ to the sarcolemma in three different mouse models of Duchenne muscular dystrophy (DMD). RESULTS: Membrane-associated nNOSµ is completely lost in DMD. Adeno-associated virus (AAV)-mediated delivery of the R16/17-Syn PDZ fusion construct successfully restored sarcolemmal nNOSµ in all three models. Further, nNOS restoration was independent of the dystrophin-associated protein complex. CONCLUSIONS: Our results suggest that the R16/17-Syn PDZ fusion construct is sufficient to restore sarcolemmal nNOSµ in the dystrophin-null muscle.