Sarcoglycan Expression Research Articles

Sarcoglycans are enriched at the neuromuscular junction in a nerve-dependent manner

Sarcoglycanopathies are heterogeneous proximo-distal diseases presenting severe muscle alterations. Although there are 6 different sarcoglycan isoforms, sarcoglycanopathies are caused exclusively by mutations in genes coding for one of the four sarcoglycan transmembrane proteins (alpha, beta, gamma and delta) forming the sarcoglycan complex (SGC) in skeletal and cardiac muscle. Little is known about the different roles of the SGC beyond the dystrophin glycoprotein complex (DGC) structural role. Here, we show that SGC proteins are enriched at the post-synaptic membrane of neuromuscular junctions (NMJs). Using a mouse model lacking the beta-sarcoglycan subunit, we describe for the first time that the loss of the SGC in the NMJ area results in alterations of pre- and postsynaptic membrane, as well as a significant reduction of membrane potential. Moreover, using different denervated wild-type mouse models, we demonstrate that nerve presence precedes the sarcoglycan enrichment at NMJ, suggesting a nerve-dependent sarcoglycan expression. Altogether, our findings suggest that pathological decline should no longer be understood only in terms of sarcolemma damage but also in terms of sarcoglycans’ participation in the NMJ. Henceforth, our work paves the way for the identification of new mechanisms involving sarcoglycans and new approaches for the treatment of sarcoglycanopathies.

Clinical and Genetic Characteristics of Limb-Girdle Muscular Dystrophy in Iranian Patients

Background: Limb-girdle muscular dystrophy (LGMD) is a bothersome muscle disease associated with weakness of the shoulder and pelvic girdle. Objectives: The study aimed to determine the genetic diversity and relative frequency of various forms of LGMD in Iranian children. Methods: In this descriptive research, 60 children referred to the neurology or emergency department of the Pediatric Medical Center were studied from April 2019 to April 2020. Additional tests (muscle biopsy and genetic testing) were performed to confirm the diagnosis of LGMDs. Quantitative data such as disease level, motor, respiratory, and cardiac functions, and molecular data underwent statistical analysis. Results: A total of 41 patients with a mean age of 11.1 were studied. Twenty-two patients were diagnosed with genetic tests and 19 with muscle biopsies. Also, there were 26.8% cases of alpha sarcoglycanopathy, 24.4% beta sarcoglycanopathy, 17.1% gamma sarcoglycanopathy, 7.3% calpainopathy, 7.3% dysferlinopathy, 7.3% dystroglycanopathy, 7.3% titinopathy, and one case of laminopathy. Among genetically confirmed individuals, 27.3% had SGCB mutation, and 18.2% had SGCA mutation. A significant relationship was seen between the mutation type and creatine phosphokinase (CPK) levels (P < 0.05). Conclusions: The prevalence of alpha and beta sarcoglycanopathy phenotypes in the study population showed that the severity of clinical involvement may be predicted by SGCB gene mutation and sarcoglycan expression.

Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy.

Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies, as pathological control group, and four age-matched controls. Fluorescence data showed a significantly reduced expression of the mutated sarcoglycan in all patients when compared to their respective age-matched healthy controls, and a variable reduction of the other sarcoglycans. The reduction is due to the effect of gene mutation and not to the increasing age of controls. Fluorescence normalized data analyzed in relation to the age of onset of the disease, showed a negative correlation of a-sarcoglycan fluorescence signal vs fibrosis in patients with an early age of onset and a negative correlation between d-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful also in view of possible inferences on disease prognosis. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.

LGMD: EP.185 Safety -sarcoglycan expression, and functional outcomes from systemic gene transfer of rAAVrh74.MHCK7.hSGCB in LGMD2E/R4

LGMD: EP.185 Safety -sarcoglycan expression, and functional outcomes from systemic gene transfer of rAAVrh74.MHCK7.hSGCB in LGMD2E/R4

Limb-Girdle Muscular Dystrophy Type 2C: Case Report

Limb-Girdle muscular dystrophy (LGMD) is a group of inherited disorders that lead to muscle weakness and skeletal muscle wasting involving the muscles around the hips and shoulders. This can cause a gait disturbance, difficulty running or even a complete loss of the ability to walk. The appearance of the disorder, the course and the muscles affected are variable between the different subtypes of the disease. Typically, patients with type 2C Limb-Girdle dystrophy (LGMD2C) start having symptoms early in infancy and lose their ability to walk around the age of 12. Others have less symptomatology and have late expression in adulthood. This disorder can affect the heart muscle in some patients. They can also have osteoarticular and vertebral deformations. The prognosis depends on the muscles often affected by respiratory failure.LGMD2C is caused by a pathogenic mutation in the SGCG gene. We report the case of a 13-year-old child, with a notion of femoral fracture at the age of 5 years and first degree consanguinity in the parents, no similar case in the family, and who presents since 3 years difficulty walking Clinical examination: walking, positive sign of Gowers, scapula alta, enlarged calves. On the biological level: CPK 6380, LDH 482, PAL 219, ASAT 68, ALAT 103. The electromyogram shows a slowing of the motor conduction speed of the external popliteal sciatic nerve with a slight loss of amplitude. Muscle biopsy objective a discreet dystrophic formula with a total absence of expression of gamma sarcoglycans and proteins of the muscle membrane. Gamma glycanopathy is genetically confirmed by the mutation of the δ-SG gene. A cardiac ultrasound was without abnormality.

An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative treatment for any of the sarcoglycanopathies. A first clinical trial to evaluate the safety of a recombinant AAV2/1 vector expressing γ-sarcoglycan using an intramuscular route of administration showed limited expression of the transgene and good tolerance of the approach. In this report, we undertook a dose-effect study in mice to evaluate the efficiency of an AAV2/8-expressing γ-sarcoglycan controlled by a muscle-specific promoter with a systemic mode of administration. We observed a dose-related efficiency with a nearly complete restoration of gamma sarcoglycan (SGCG) expression, histological appearance, biomarker level, and whole-body strength at the highest dose tested. In addition, our data suggest that a high expression threshold level must be achieved for effective protection of the transduced muscle, while a suboptimal transgene expression level might be less protective in the context of mechanical stress.

Clinical and genetic spectrum of sarcoglycanopathies in a large cohort of Chinese patients

BackgroundSarcoglycanopathies comprise four subtypes of autosomal recessive limb-girdle muscular dystrophy (LGMD2C, LGMD2D, LGMD2E, and LGMD2F) that are caused, respectively, by mutations in the SGCG, SGCA, SGCB, and SGCD genes. Knowledge about the clinical and genetic features of sarcoglycanopathies in Chinese patients is limited. The aims of this study were to investigate in detail the clinical manifestations, sarcoglycan expression, and gene mutations in Chinese patients with sarcoglycanopathies and to identify possible correlations between them.ResultsOf 3638 patients for suspected neuromuscular diseases (1733 with inherited myopathies, 1557 with acquired myopathies, and 348 unknown), 756 patients had next-generation sequencing (NGS) diagnostic panel. Twenty-five patients with sarcoglycanopathies (11.5%) were identified from 218 confirmed LGMDs, comprising 18 with LGMD2D, 6 with LGMD2E, and one with LGMD2C. One patient with LGMD2D also had Charcot-Marie-Tooth 1A. The clinical phenotypes of the patients with LGMD2D or LGMD2E were markedly heterogeneous. Muscle biopsy showed a dystrophic pattern in 19 patients and mild myopathic changes in 6. The percentage of correct prediction of genotype based on expression of sarcoglycan was 36.0% (4 LGMD2D, 4 LGMD2E, and one LGMD2C). There was a statistically significant positive correlation between reduction of α-sarcoglycan level and disease severity in LGMD2D. Thirty-five mutations were identified in SGCA, SGCB, SGCG, and PMP22, 16 of which were novel. Exon 3 of SGCA was a hotspot region for mutations in LGMD2D. The missense mutation c.662G > A (p.R221H) was the most common mutation in SGCA. Missense mutations in both alleles of SGCA were associated with a relative benign disease course. No obvious clinical, sarcoglycan expression, and genetic correlation was found in LGMD2E.ConclusionsThis study expands the clinical and genetic spectrum of sarcoglycanopathies in Chinese patients and provides evidence that disease severity of LGMD2D may be predicted by α-sarcoglycan expression and SGCA mutation.

Analysis on sarcoglycans expression as markers of septic cardiomyopathy in sepsis-related death.

The post-mortem assessment of sepsis-related death can be carry out by many methods recently suggested as microbiological and biochemical investigations. In these cases, the cause of death is a multiple organ dysfunction due to a dysregulated inflammatory response occurring after the failure of infection control process. It was highlighted also that the heart can be a target organ in sepsis which determines the so-called septic cardiomyopathy characterized by myocardial depression. Several mechanisms to explain the pathophysiology of septic cardiomyopathy were suggested, but very few studies about the structural alterations of cardiac cells responsible for myocardial depression were carried out. The aim of this study was to evaluate whether sarcoglycans (SG) were involved in septic cardiac damage analyzing their expression in sepsis-related deaths and, particularly, if these proteins can be used as markers of septic myocardial dysfunction. Cases of septic-related death confirmed by clinical and autopsy records were investigated and compared to a control group of traumatic deaths. Indirect immunofluorescence analysis was performed to analyze α-SG, β-SG, δ-SG, ζ-SG, ε-SG, and γ-SG. Decrease of fluorescence staining pattern for all tested sarcoglycans was observed in the septic-related deaths compared to normal fluorescence staining pattern of control group. These results provide new findings about the myocytes structural alterations due to sepsis and suggest that these proteins could be used in forensic assessment of septic cardiomyopathy.

Sarcoglycan sub-complex in the adipose organ: a molecular and immunofluorescence study

The sarcoglycan sub-complex (SGC) is made up of six glycoproteins which connect the cytoskeleton to the extracellular matrix in skeletal muscle. Recent data show that this complex is also expressed in epithelial tissue such as gingival, breast and prostatic ones [1]. The adipose organ is organized in multiple fat depots consisting of white and brown adipose cells. White adipocytes can store energy in triglycerides; brown adipocytes can dissipate energy for thermogenesis. It has been demonstrated that white adipocytes transdifferentiate to brown adipocytes after cold exposure [3]. In this study we examined the expression of sarcoglycans (SGs) in the adipose organ from two groups of mice: the first group was exposed at 25 C°, as control, and the second one was exposed at low temperature (4 C°) for 24 hours and 4 days. Fat depots from the visceral and interscapular region, but also from the mammary gland, have been examined by histological, immunofluorescence and molecular techniques. Results have shown that SGC is expressed in the adipose organ, both in brown and white adipocytes of mice exposed at 25 C°. The main results is that the expression level of all sarcoglycans increase in cold exposure experiment. For the first time the expression of all SGs in the adipose organ has been demonstrated, both at mRNA and protein levels. Since we found an increase in SGs expression after transdifferentiation from white to brown adipocytes cold exposure induced, we hypothesize that sarcoglycans could be associated with β3 adrenergic receptor; sarcoglycans associations with other receptors, as GABAr, has been already demonstrated in central nervous system. Although that, the function of these glycoproteins in the adipose organ remain still unclear and further investigation are required.

726. Prevention of Muscular Dystrophy by an AAV Vector Encoding a Nonimmunogenic Protein Based on the Evolution of Utrophin and Dystrophin

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions in the dystrophin gene. Expression of recombinant dystrophin in DMD therefore risks chronic immune recognition of the “non-self” protein. Early developmental expression of the paralogous protein utrophin in the thymus may confer central immunological tolerance to its peptide sequence. Here we address previously unanswered questions about the therapeutic efficacy and immunogenicity of “µutrophins” encoded by synthetic minigenes suitable for use in AAV. First we used a comparative phylogenomic approach to address a deceptively simple question with critical implications for this field: “which came first-dystrophin or the sarcomere”. This analysis provided evidence that the long rod-like domain of utrophin and dystrophin was “coopted” from a higher molecular weight protein in dynein-propelled animals lacking striated muscle. When considered in light of recent crystalographic studies, these results suggest that the full functionality of native dystrophin may be conferred by recombinant mini-utrophins with internally deleted rod domains that preserve inter-repeat folding and hence maximal tensile strength. We maximized recombinant µUtrophin (µU) expression from synthetic open reading frames by using the codon bias of striated myosin. Our blinded studies of mdx mice injected with a µU transgene are the first to show COMPLETE recovery of peripheral myofiber nucleation following a single systemic injection of an AAV vector. Sarcoglycan expression, as measured by western blot in the µU-treated mice, are restored to control levels throughout growth to skeletal maturity. To investigate the correlation between our histological findings and functionality, we validated a novel open field cage and attached running wheel system. Our blinded data show remarkable functional rescue in µU-treated mdx mice using this non-invasive test with relevance to the clinical assessment of disease progression in DMD. These data also strongly correlate with those derived from in vivo force grip and ex vivo force transducer measurements. Remarkably, a majority of mdx mice show complete normalization of serum creatine kinase (CK), a first for single-dose treatment by any clinically translatable modality. In dystrophic puppies, intravenous injection of a 30-fold lower relative dose of AAV9U fully restored sarcoglycan levels and normalized the myofiber size-distribution following a threefold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings suggest a rationale for neonatal gene therapy using utrophin as an internally deleted “self” protein in DMD to minimize the risk of chronic immunotoxicity. We outline a rigorous translational approach using escalating vector doses in GSHPMD dogs harboring a newly defined 5 megabase deletion encompassing the dystrophin ORF.

Sarcoglycan complex in masseter and sternocleidomastoid muscles of baboons: an immunohistochemical study.

The sarcoglycan complex consists of a group of single-pass transmembrane glycoproteins that are essential to maintain the integrity of muscle membranes. Any mutation in each sarcoglycan gene causes a series of recessive autosomal dystrophin-positive muscular dystrophies. Negative fibres for sarcoglycans have never been found in healthy humans and animals. In this study, we have investigated whether the social ranking has an influence on the expression of sarcoglycans in the skeletal muscles of healthy baboons. Biopsies of masseter and sternocleidomastoid muscles were processed for confocal immunohistochemical detection of sarcoglycans. Our findings showed that baboons from different social rankings exhibited different sarcoglycan expression profiles. While in dominant baboons almost all muscles were stained for sarcoglycans, only 55% of muscle fibres showed a significant staining. This different expression pattern is likely to be due to the living conditions of these primates. Sarcoglycans which play a key role in muscle activity by controlling contractile forces may influence the phenotype of muscle fibres, thus determining an adaptation to functional conditions. We hypothesize that this intraspecies variation reflects an epigenetic modification of the muscular protein network that allows baboons to adapt progressively to a different social status.

G.P.225: Concomitant alpha and gamma sarcoglycan deficiencies in a Turkish boy with a novel deletion in the alpha sarcoglycan gene

Limb-girdle muscular dystrophy type 2D (LGMD-2D) is caused by autosomal recessive defects in the alpha-sarcoglycan gene located on chromosome 17q21. In this study, we present a child with alpha-sarcoglycanopathy and describe a novel deletion in the alpha-sarcoglycan gene. A 5-year-old boy had a very high serum creatinine phosphokinase level, which was determined incidentally, and a negative molecular test for the dystrophin gene. Muscle biopsy showed dystrophic features. Immunohistochemistry showed that, there was diminished expression of alpha and gamma sarcoglycans. DNA analysis revealed a novel 7bp homozygous deletion in exon 3 of the alpha-sarcoglycan gene. His parents were consanguineous heterozygous carriers of the same deletion. We believe this is the first confirmed case of primary alpha-sarcoglycanopathy with a novel deletion in Turkey. In addition, this study demonstrated that, both muscle biopsy and DNA analysis remain important methods for the differential diagnosis of muscular dystrophies because dystrophinopathies and sarcoglycanopathies are so similar.

Map of the sarcoglycan sub-complex in rat brain

The sarcoglycan-sub-complex is made up of six glycoproteins which play mechanosignaling functions, connecting the extracellular matrix to cytoskeleton. This protein complex has been identified in different kind of tissues; in central nervous system, instead, only the ζ-sarcoglycans and the e-sarcoglycans are considered to be present, where they seem to play a different role from the role played in muscle. Although that, previous our study have shown the expression of the entire sarcoglycan sub-complex in some region of the rat brain and the colocalization of this complex with post-synaptic receptors as GABA and DOPA receptors. Since we found that each sarcoglycan changes in staining pattern level among the brain regions, in the present study we performed, for the first time, a map of sarcoglycans expression in whole brain of rat and we examined which kind of post-synaptic receptor colocalizes with sarcoglycans in each part of brain. Results have shown that in rat brain the staining pattern level for each sarcoglycan and the different kind of colocalization between sarcoglycans and post-synaptic receptors, sarcoglycan/GABA or sarcoglycan/ DOPA, are characteristic of each brain region. These results support a role of the sarcoglycan sub-complex in post-synaptic neurotransmission, maybe modulating post-synaptic receptor assembly and stabilization.

Sarcoglycan subcomplex: state of the art

sarcoglycan subcomplex: state of the art

Sub-physiological sarcoglycan expression contributes to compensatory muscle protection in mdx mice.

Sarcoglycans are a group of single-pass transmembrane glycoproteins. In striated muscle, sarcoglycans interact with dystrophin and other dystrophin-associated proteins (DAPs) to form the dystrophin-associated glycoprotein complex (DGC). The DGC protects the sarcolemma from contraction-induced injury. Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations. In the absence of dystrophin, the DGC is disassembled from the sarcolemma. This initiates a chain reaction of muscle degeneration, necrosis, inflammation and fibrosis. In contrast to human patients, dystrophin-null mdx mice are only mildly affected. Enhanced muscle regeneration and the up-regulation of utrophin and integrin are thought to protect mdx muscle. Interestingly, trace amounts of sarcoglycans and other DAPs can be detected at the mdx sarcolemma. It is currently unclear whether sub-physiological sarcoglycan expression also contributes to the mild phenotype in mdx mice. To answer this question, we generated delta-sarcoglycan/dystrophin double knockout mice (delta-Dko) in which residual sarcoglycans were completely eliminated from the sarcolemma. Interestingly, utrophin levels were further increased in these mice. However, enhanced utrophin expression did not mitigate disease. The clinical manifestation of delta-Dko mice was worse than that of mdx mice. They showed characteristic dystrophic signs, body emaciation and more macrophage infiltration. Their lifespan was reduced by 60%. Furthermore, delta-Dko muscle generated significantly less absolute muscle force and became more susceptible to contraction-induced injury. Our results suggest that sub-physiological sarcoglycan expression plays a critical role in ameliorating muscle disease in mdx mice. We speculate that low-level sarcoglycan expression may represent a useful strategy to palliate DMD.

Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy.

Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.

Sarcoglycanopathies: Can muscle immunoanalysis predict the genotype?

Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (alpha-, beta-, gamma-, delta-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed alpha-sarcoglycan deficient LGMD2D, 7 patients beta-sarcoglycan deficient LGMD2E, 3 patients gamma-sarcoglycan deficient LGDM2C, and one patient delta-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and beta-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD.

Muscular dystrophy associated with α-dystroglycan deficiency in Sphynx and Devon Rex cats

Recent studies have identified a number of forms of muscular dystrophy, termed dystroglycanopathies, which are associated with loss of natively glycosylated alpha-dystroglycan. Here we identify a new animal model for this class of disorders in Sphynx and Devon Rex cats. Affected cats displayed a slowly progressive myopathy with clinical and histologic hallmarks of muscular dystrophy including skeletal muscle weakness with no involvement of peripheral nerves or CNS. Skeletal muscles had myopathic features and reduced expression of alpha-dystroglycan, while beta-dystroglycan, sarcoglycans, and dystrophin were expressed at normal levels. In the Sphynx cat, analysis of laminin and lectin binding capacity demonstrated no loss in overall glycosylation or ligand binding for the alpha-dystroglycan protein, only a loss of protein expression. A reduction in laminin-alpha2 expression in the basal lamina surrounding skeletal myofibers was also observed. Sequence analysis of translated regions of the feline dystroglycan gene (DAG1) in affected cats did not identify a causative mutation, and levels of DAG1 mRNA determined by real-time QRT-PCR did not differ significantly from normal controls. Reduction in the levels of glycosylated alpha-dystroglycan by immunoblot was also identified in an affected Devon Rex cat. These data suggest that muscular dystrophy in Sphynx and Devon Rex cats results from a deficiency in alpha-dystroglycan protein expression, and as such may represent a new type of dystroglycanopathy where expression, but not glycosylation, is affected.

Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle

The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation.One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 × 1011 vector genomes of rAAV2/1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the null background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.

The sarcoglycan complex in Schwann cells and its role in myelin stability

Sarcoglycans are originally identified in muscle for their involvement in limb-girdle muscular dystrophies. They form a multi-meric complex (alpha-, beta-, gamma-, delta-sarcoglycan) that associates with dystrophin, dystroglycan and other proteins to constitute the larger dystrophin-glycoprotein complex at the muscle membrane. Three sarcoglycan subunits (epsilon-, beta-, delta-sarcoglycan) were previously identified in Schwann cells and shown to associate with dystroglycan and a Schwann cell-specific dystrophin isoform (Dp116) at the outermost Schwann cell membrane. Currently, little is known about the exact composition and function of the sarcoglycan complex in the peripheral nervous system. In this study, we showed that the Schwann cell sarcoglycan complex consists of epsilon-, beta-, delta-sarcoglycan and the newly identified zeta-sarcoglycan subunit. The expression of sarcoglycans precedes the onset of myelination and is induced by neurons. In sarcoglycan-deficient BIO14.6 hamsters, loss of the Schwann cell sarcoglycan complex reduces the steady state levels of alpha-dystroglycan and Dp116. Ultrastructural analysis of sciatic nerves from the mutant animals revealed altered myelin sheaths and disorganized Schmidt-Lanterman incisures indicative of myelin instability. The disruption in myelin structure increased in severity with age. Nerve conduction studies also showed subtle electrophysiological abnormalities in the BIO14.6 hamsters consistent with reduced myelin stability. Together, these findings suggest an important role of sarcoglycans in the stability of peripheral nerve myelin.