The Implication of Hinge 1 and Hinge 4 in Micro-Dystrophin Gene Therapy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins (µDys) are currently in clinical trials. They all carry H1 and H4. In this study, we investigated whether these two hinges are essential for µDy function in murine DMD models. Three otherwise identical µDys were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ΔH1 (without H1), and ΔH4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent µDys expression in total muscle lysate. However, only H1/H4 and ΔH1 showed correct sarcolemmal localization. ΔH4 mainly existed as sarcoplasmic aggregates. H1/H4 and ΔH1, but not ΔH4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by ΔH1, but not by ΔH4. Next, we compared H1/H4 and ΔH1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months postinjection, H1/H4 significantly outperformed ΔH1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable electrocardiography improvements. We conclude that H4 is essential for µDys function and H1 facilitates force production. Our findings will help develop next-generation µDys gene therapy.

Ketogenic diet containing medium-chain triglyceride ameliorates transcriptome disruption in skeletal muscles of rat models of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a myopathy characterized by progressive muscle weakness caused by a mutation in the dystrophin gene on the X chromosome. We recently showed that a medium-chain triglyceride-containing ketogenic diet (MCTKD) improves skeletal muscle myopathy in a CRISPR/Cas9 gene-edited rat model of DMD. We examined the effects of the MCTKD on transcription profiles in skeletal muscles of the model rats to assess the underlying mechanism of the MCTKD-induced improvement in DMD. DMD rats were fed MCTKD or normal diet (ND) from weaning to 9 months, and wild-type rats were fed with the ND, then tibialis anterior muscles were sampled for mRNA-seq analysis. Pearson correlation heatmaps revealed a one-node transition in the expression profile between DMD and wild-type rats. A total of 10,440, 11,555 and 11,348 genes were expressed in the skeletal muscles of wild-type and ND-fed DMD rats the MCTKD-fed DMD rats, respectively. The MCTKD reduced the number of DMD-specific mRNAs from 1624 to 1350 and increased the number of mRNAs in common with wild-type rats from 9931 to 9998. Among 2660 genes were differentially expressed in response to MCTKD intake, the mRNA expression of 1411 and 1249 of them was respectively increased and decreased. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses suggested that the MCTKD significantly suppressed the mRNA expression of genes associated with extracellular matrix organization and inflammation. This suggestion was consistent with our previous findings that the MCTKD significantly suppressed fibrosis and inflammation in DMD rats. In contrast, the MCTKD significantly increased the mRNA expression of genes associated with oxidative phosphorylation and ATP production pathways, suggesting altered energy metabolism. The decreased and increased mRNA expression of Sln and Atp2a1 respectively suggested that Sarco/endoplasmic reticulum Ca2+-ATPase activation is involved in the MCTKD-induced improvement of skeletal muscle myopathy in DMD rats. This is the first report to examine transcription profiles in the skeletal muscle of CRISPR/Cas9 gene-edited DMD model rats and the effect of MCTKD feeding on it.

Incomplete Assembly of the Dystrophin-Associated Protein Complex in 2D and 3D-Cultured Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are increasingly used to study genetic diseases on a human background. However, the lack of a fully mature adult cardiomyocyte phenotype of hiPSC-CM may be limiting the scope of these studies. Muscular dystrophies and concomitant cardiomyopathies result from mutations in genes encoding proteins of the dystrophin-associated protein complex (DAPC), which is a multi-protein membrane-spanning complex. We examined the expression of DAPC components in hiPSC-CM, which underwent maturation in 2D and 3D culture protocols. The results were compared with human adult cardiac tissue and isolated cardiomyocytes. We found that similarly to adult cardiomyocytes, hiPSC-CM express dystrophin, in line with previous studies on Duchenne's disease. ß-dystroglycan was also expressed, but, contrary to findings in adult cardiomyocytes, none of the sarcoglycans nor α-dystroglycan were, despite the presence of their mRNA. In conclusion, despite the robust expression of dystrophin, the absence of several other DAPC protein components cautions for reliance on commonly used protocols for hiPSC-CM maturation for functional assessment of the complete DAPC.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Natural products, PGC-1 α , and Duchenne muscular dystrophy.

Peroxisome proliferator-activated receptor γ (PPARγ) is a transcriptional coactivator that binds to a diverse range of transcription factors. PPARγ coactivator 1 (PGC-1) coactivators possess an extensive range of biological effects in different tissues, and play a key part in the regulation of the oxidative metabolism, consequently modulating the production of reactive oxygen species, autophagy, and mitochondrial biogenesis. Owing to these findings, a large body of studies, aiming to establish the role of PGC-1 in the neuromuscular system, has shown that PGC-1 could be a promising target for therapies targeting neuromuscular diseases. Among these, some evidence has shown that various signaling pathways linked to PGC-1α are deregulated in muscular dystrophy, leading to a reduced capacity for mitochondrial oxidative phosphorylation and increased reactive oxygen species (ROS) production. In the light of these results, any intervention aimed at activating PGC-1 could contribute towards ameliorating the progression of muscular dystrophies. PGC-1α is influenced by different patho-physiological/pharmacological stimuli. Natural products have been reported to display modulatory effects on PPARγ activation with fewer side effects in comparison to synthetic drugs. Taken together, this review summarizes the current knowledge on Duchenne muscular dystrophy, focusing on the potential effects of natural compounds, acting as regulators of PGC-1α.

biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

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.

Age-related Differences in Dystrophin: Impact on Force Transfer Proteins, Membrane Integrity, and Neuromuscular Junction Stability.

The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, ß1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious.

Distribution of Astrocytic Syntrophin in Hippocampus from Human Mesial Temporal Lobe Epilepsy / 中国康复理论与实践

Objective To investigate the expression changes of astrocytic syntrophin in hippocampus from human mesial temporal lobe epilepsy (MTLE). Methods From April, 2015 to July, 2016, 17 cases of hippocampus, collected from temporal lobectomy, were divided into MTLE group (n=13) and non-MTLE group (n=4) according to hematoxylin and eosin staining, glial fibrillary acidic protein and neuronal nu-clei immunohistochemical staining. Immunofluorescence double labeling and immunofluorescence histochemistry were used to observe the expression of syntrophin. Results The proliferation of astrocytes increased and neurons reduced in the hippocampus of MTLE group. Syntro-phin was found in the membrane and foot processes of astrocyte, that was enriched along perivascular astrocyte end-feet domain in non-MTLE group, but lost in MTLE group. While the whole expression of syntrophin was more in MTLE group than in non-MTLE group (t=5.421, P<0.001). Conclusion The distribution of syntrophin in hippocampus astrocytes may be related to the development of MTLE.

Α-Dystrobrevin-1 recruits Grb2 and α-catulin to organize neurotransmitter receptors at the neuromuscular junction.

Neuromuscular junctions (NMJs), the synapses made by motor neurons on muscle fibers, form during embryonic development but undergo substantial remodeling postnatally. Several lines of evidence suggest that α-dystrobrevin, a component of the dystrophin-associated glycoprotein complex (DGC), is a crucial regulator of the remodeling process and that tyrosine phosphorylation of one isoform, α-dystrobrevin-1, is required for its function at synapses. We identified a functionally important phosphorylation site on α-dystrobrevin-1, generated phosphorylation-specific antibodies to it and used them to demonstrate dramatic increases in phosphorylation during the remodeling period, as well as in nerve-dependent regulation in adults. We then identified proteins that bind to this site in a phosphorylation-dependent manner and others that bind to α-dystrobrevin-1 in a phosphorylation-independent manner. They include multiple members of the DGC, as well as α-catulin, liprin-α1, Usp9x, PI3K, Arhgef5 and Grb2. Finally, we show that two interactors, α-catulin (phosphorylation independent) and Grb2 (phosphorylation dependent) are localized to NMJs in vivo, and that they are required for proper organization of neurotransmitter receptors on myotubes.

Studying the role of dystrophin-associated proteins in influencing Becker muscular dystrophy disease severity.

Becker muscular dystrophy is characterized by a variable disease course. Many factors have been implicated to contribute to this diversity, among which the expression of several components of the dystrophin associated glycoprotein complex. Together with dystrophin, most of these proteins anchor the muscle fiber cytoskeleton to the extracellular matrix, thus protecting the muscle from contraction induced injury, while nNOS is primarily involved in inducing vasodilation during muscle contraction, enabling adequate muscle oxygenation. In the current study, we investigated the role of three components of the dystrophin associated glycoprotein complex (beta-dystroglycan, gamma-sarcoglycan and nNOS) and the dystrophin homologue utrophin on disease severity in Becker patients. Strength measurements, data about disease course and fresh muscle biopsies of the anterior tibial muscle were obtained from 24 Becker patients aged 19 to 66. The designation of Becker muscular dystrophy in this study was based on the mutation and not on the clinical severity. Contrary to previous studies, we were unable to find a relationship between expression of nNOS, beta-dystroglycan and gamma-sarcoglycan at the sarcolemma and disease severity, as measured by muscle strength in five muscle groups and age at reaching several disease milestones. Unexpectedly, we found an inverse correlation between utrophin expression at the sarcolemma and age at reaching disease milestones.

Regional and genotypic differences in intrinsic electrophysiological properties of cerebellar Purkinje neurons from wild-type and dystrophin-deficient mdx mice.

Cerebellar subregions are recognized as having specialized roles, with lateral cerebellum considered crucial for cognitive processing, whereas vermal cerebellum is more strongly associated with motor control. In human Duchenne muscular dystrophy, loss of the cytoskeletal protein dystrophin is thought to cause impairments in cognition, including learning and memory. Previous studies demonstrate that loss of dystrophin causes dysfunctional signaling at γ-aminobutyric acid (GABA) synapses on Purkinje neurons, presumably by destabilization of GABAA receptors. However, potential differences in the intrinsic electrophysiological properties of Purkinje neurons, including membrane potential and action potential firing rates, have not been investigated. Here, using a 2×2 analysis of variance (ANOVA) experimental design, we employed patch clamp analysis to compare membrane properties and action potentials generated by acutely dissociated Purkinje neurons from vermal and lateral cerebellum in wild-type (WT) mice and mdx dystrophin-deficient mice. Compared to Purkinje neurons from WT mice, neurons from mdx mice exhibited more irregular action potential firing and a hyperpolarization of the membrane potential. Firing frequency was also lower in Purkinje neurons from the lateral cerebellum of mdx mice relative to those from WT mice. Several action potential waveform parameters differed between vermal and lateral Purkinje neurons, irrespective of dystrophin status, including action potential amplitude, slope (both larger in the vermal region), and duration (shorter in the vermal region). Moreover, the membrane potential of Purkinje neurons from the vermal region of WT mice exhibited a significant hyperpolarization and concurrent reduction in the frequency of spontaneous action potentials compared to Purkinje neurons from the lateral region. This regional hyperpolarization and reduction in spontaneous action potential frequency was abolished in mdx mice. These results from mice demonstrate the presence of differential electrophysiological properties between Purkinje neurons from different regions of the WT mouse cerebellum and altered intrinsic membrane properties in the absence of dystrophin. These findings provide a possible mechanism for the observations that absence of cerebellar dystrophin contributes to deficits in mental function observed in humans and mouse models of muscular dystrophy. Moreover, these results highlight the importance of distinguishing functional zones of the cerebellum in future work characterizing Purkinje neuron electrophysiology and studies using the model of dissociated Purkinje neurons from mice.