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.

α-Dystrobrevin knockout mice have increased motivation for appetitive reward and altered brain cannabinoid receptor 1 expression.

α-Dystrobrevin (α-DB) is a major component of the dystrophin-associated protein complex (DAPC). Knockout (KO) of α-DB in the brain is associated with astrocytic abnormalities and loss of neuronal GABA receptor clustering. Mutations in DAPC proteins are associated with altered dopamine signaling and cognitive and psychiatric disorders, including schizophrenia. This study tested the hypothesis that motivation and associated underlying biological pathways are altered in the absence of α-DB expression. Male wildtype and α-DB KO mice were tested for measures of motivation, executive function and extinction in the rodent touchscreen apparatus. Subsequently, brain tissues were evaluated for mRNA and/or protein levels of dysbindin-1, dopamine transporter and receptor 1 and 2, mu opioid receptor 1 (mOR1) and cannabinoid receptor 1 (CB1). α-DB KO mice had significantly increased motivation for the appetitive reward, while measures of executive function and extinction were unaffected. No differences were observed between wildtype and KO animals on mRNA levels of dysbindin-1 or any of the dopamine markers. mRNA levels of mOR1were significantly decreased in the caudate-putamen and nucleus accumbens of α-DB KO compared to WT animals, but protein levels were unaltered. However, CB1 protein levels were significantly increased in the prefrontal cortex and decreased in the nucleus accumbens of α-DB KO mice. Triple-labelling immunohistochemistry confirmed that changes in CB1 were not specific to astrocytes. These results highlight a novel role for α-DB in the regulation of appetitive motivation that may have implications for other behaviours that involve the dopaminergic and endocannabinoid systems.

Region-based analysis of rare genomic variants in whole-genome sequencing datasets reveal two novel Alzheimer's disease-associated genes: DTNB and DLG2.

Alzheimer's disease (AD) is a genetically complex disease for which nearly 40 loci have now been identified via genome-wide association studies (GWAS). We attempted to identify groups of rare variants (alternate allele frequency <0.01) associated with AD in a region-based, whole-genome sequencing (WGS) association study (rvGWAS) of two independent AD family datasets (NIMH/NIA; 2247 individuals; 605 families). Employing a sliding window approach across the genome, we identified several regions that achieved association p values <10-6, using the burden test or the SKAT statistic. The genomic region around the dystobrevin beta (DTNB) gene was identified with the burden and SKAT test and replicated in case/control samples from the ADSP study reaching genome-wide significance after meta-analysis (pmeta = 4.74 × 10-8). SKAT analysis also revealed region-based association around the Discs large homolog 2 (DLG2) gene and replicated in case/control samples from the ADSP study (pmeta = 1 × 10-6). In conclusion, in a region-based rvGWAS of AD we identified two novel AD genes, DLG2 and DTNB, based on association with rare variants.

Protein Kinase B/Akt1 Phosphorylates Dysbindin-1A at Serine 10 to Regulate Neuronal Development.

Schizophrenia is a neurodevelopmental disorder with dendrite and dendritic spine dysfunction. Dysbindin-1, a protein decreased in the brains of schizophrenia patients, is involved in the development of dendrites and spines. However, it is still unclear how the role of dysbindin-1 in neuronal development is regulated. Here, we showed protein kinase B/Akt1, a serine/threonine kinase implicated in schizophrenia, phosphorylated dysbindin-1A at serine 10 (S10). S10 phosphorylation of dysbindin-1A was increased during postnatal neuronal and synapse development stage, and was enriched in postsynaptic densities (PSDs). Furthermore, overexpressing wild type or S10 phospho-mimic mutant (S10D), but not S10 phospho-dead mutant (S10A) of dysbindin-1A rescued the dendrite and spine deficits in dysbindin-1A knockdown neurons. These results indicate S10 phosphorylation of dysbindin-1A by Akt1 is essential for neuronal development, providing a potential regulation mechanism for dysbindin-1A in neuronal development.

Evaluating the detection ability of a range of epistasis detection methods on simulated data for pure and impure epistatic models.

BACKGROUND: Numerous approaches have been proposed for the detection of epistatic interactions within GWAS datasets in order to better understand the drivers of disease and genetics. METHODS: A selection of state-of-the-art approaches were assessed. These included the statistical tests, fast-epistasis, BOOST, logistic regression and wtest; swarm intelligence methods, namely AntEpiSeeker, epiACO and CINOEDV; and data mining approaches, including MDR, GSS, SNPRuler and MPI3SNP. Data were simulated to provide randomly generated models with no individual main effects at different heritabilities (pure epistasis) as well as models based on penetrance tables with some main effects (impure epistasis). Detection of both two and three locus interactions were assessed across a total of 1,560 simulated datasets. The different methods were also applied to a section of the UK biobank cohort for Atrial Fibrillation. RESULTS: For pure, two locus interactions, PLINK's implementation of BOOST recovered the highest number of correct interactions, with 53.9% and significantly better performing than the other methods (p = 4.52e - 36). For impure two locus interactions, MDR exhibited the best performance, recovering 62.2% of the most significant impure epistatic interactions (p = 6.31e - 90 for all but one test). The assessment of three locus interaction prediction revealed that wtest recovered the highest number (17.2%) of pure epistatic interactions(p = 8.49e - 14). wtest also recovered the highest number of three locus impure epistatic interactions (p = 6.76e - 48) while AntEpiSeeker ranked as the most significant the highest number of such interactions (40.5%). Finally, when applied to a real dataset for Atrial Fibrillation, most notably finding an interaction between SYNE2 and DTNB.

Whole-exome sequencing reveals a rare missense variant in DTNA in an Iranian pedigree with early-onset atrial fibrillation.

Atrial fibrillation (AF) is a morbid and heritable irregular cardiac rhythm that affects about 2%-3% of the population. Patients with early-onset AF have a strong genetic association with the disease; nonetheless, the exact underlying mechanisms need clarification. We herein present our evaluation of a 2-generation Iranian pedigree with early-onset AF. Whole-exome sequencing was applied to elucidate the genetic predisposition. Direct DNA sequencing was utilized to confirm and screen the variants in the proband and his available family members. The pathogenicity of the identified nucleotide variations was scrutinized via either segregation analysis in the family or in silico predictive software. The comprehensive variant analysis revealed a missense variant (c.G681C, p.E227D, rs1477078144) in the human α-dystrobrevin gene (DTNA), which is rare in genetic databases. Most in silico analyses have predicted this variant as a disease-causing variant, and the variant is co-segregated with the disease phenotype in the family. Previous studies have demonstrated the association between the DTNA gene and left ventricular noncompaction cardiomyopathy. Taken together, we provide the first evidence of an association between a nucleotide variation in the DTNA gene and early-onset AF in an Iranian family. However, the genetic testing of AF in the Iranian population is still limited. This finding not only further confirms the significant role of genetics in the incidence of early-onset AF but also expands the spectrum of the gene variations that lead to AF. Additionally, it may have further implications for the treatment and prevention of AF.

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.

Orchestrating aquaporin-4 and connexin-43 expression in brain: Differential roles of α1- and ß1-syntrophin.

Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether ß1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of ß1-syntrophin deletion is rather limited, double knockout of α1- and ß1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.

Re-evaluation of single nucleotide variants and identification of structural variants in a cohort of 45 sudden unexplained death cases.

Sudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.

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.

Association of SNTB1 with High Myopia.

PURPOSE: To investigate the associations of Single Nucleotide Polymorphisms (SNPs) in the SNTB1 gene with high myopia in a Han Chinese population. MATERIALS AND METHODS: Based on previous studies, four SNPs from the SNTB1 gene were chosen for genotyping. This is a case-control genetic association study comprising 193 high myopia participants and 135 normal emmetropic controls from a Han Chinese population. Allelic frequencies of the SNPs and haplotypes were compared to assess the associations of the SNPs with high myopia and axial length (AL). RESULTS: The SNPs rs7839488 (effect allele: A; OR = 0.685), rs4395927 (effect allele: T; OR = 0.692), and rs6469937 (effect allele: A; OR = 0.683) displayed significant associations with high myopia initially (P = .044, 0.049, and 0.035, respectively), but did not withstand permutation testing (all Ppermutation>0.05). rs6469937 displayed associations with high myopia in the dominant model (AG+AA: OR = 0.609) against GG (reference). rs6469937 was also associated with AL in the dominant model (AG+AA: Beta = -0.58) against GG (reference). The haplotype analysis demonstrated ATGA as the protective haplotype against high myopia, which remained statistically significant in permutation testing (Ppermutation = 0.045). CONCLUSIONS: Our findings are suggestive that SNTB1 is associated with high myopia in a Han Chinese population.

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.

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.

Characterization of the expression of dystrophins and dystrophin-associated proteins during embryonic neural stem/progenitor cell differentiation.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene. Dystrophin is required for the organization of a complex consisting of dystroglycans, sarcoglycans, dystrobrevins and syntrophins, known as the dystrophin-associated proteins complex (DAPC). In addition to muscle degeneration, cognitive impairment has been reported in DMD patients. To characterize a suitable model for studying the embryonic cerebral functions of dystrophin, we analyzed the expression patterns of dystrophins/DAPC in undifferentiated and differentiated embryonic neural stem/progenitor cells (NSPC). We found that NSPC express mRNAs for dystrophins Dp427, Dp140, Dp71 and Dp40; ß-dystroglycan; α- and ß-dystrobrevin; α1-, ß1-, ß2- and γ2-syntrophin; and ß-, γ-, δ- and ε-sarcoglycan. Some of these were differentially regulated during neuronal or astrocytic differentiation. Interestingly, the protein expression levels of Dp140, ß-dystroglycan and α2-dystrobrevin were also differentially regulated. Additionally, we found that proliferating NSPC and differentiated neurons and astrocytes show immuno-positive staining for dystrophins and ß-dystroglycan. Our results show that dystrophins and DAPC components are expressed and regulated during the neuronal or astrocytic differentiation of NSPC, suggesting that these proteins may have different roles in the brain development.

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.