Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

National Center of Neurology and Psychiatry

Protein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes.

Localization of ubiquitin C-terminal hydrolase L1 in mouse ova and its function in the plasma membrane to block polyspermy.

Several investigators have used murine models to investigate the pathophysiology of brain ischemia. The focal ischemic model is a closer approximation to human stroke which includes a necrotic core, penumbra, and undamaged tissue. Occlusion of a unilateral artery, especially the middle cerebral artery (MCA), is performed in this model, but collateral circulation often induces variation of ischemic lesions both qualitatively and quantitatively. It is likely that the more proximal the artery which is unilaterally occluded is, the more inconsistent the outcomes. The present study was designed to examine the reproducibility of infarct lesion by distal or proximal artery occlusion. Direct occlusion of the distal MCA was performed and compared with unilateral common carotid artery occlusion (CCAO) in C57BL/6 mice. Direct MCA occlusion (MCAO) consistently induced ischemic lesions in cortical areas. All model animals (n=14) survived 24 h after occlusion, and exhibited a maximum infarct volume (20.0 +/- 5.0%). In contrast, permanent and transient unilateral CCAO models had mortality rates of 62.5 and 25.0%, and showed severe to absent lesions with the infarct volumes of 29.0 +/- 20.8 and 33.2 +/- 24.2%, respectively. In conclusion, distal MCAO produces high reproducibility of ischemic insults and survivability compared to unilateral CCAO. Thus, distal MCAO is a useful method for the focal ischemic model.

Direct experimental occlusion of the distal middle cerebral artery induces high reproducibility of brain ischemia in mice.

The molecular mechanism of muscle degeneration in a lethal muscle disorder Duchene muscular dystrophy (DMD) has not been fully elucidated. The dystrophic dog, a model of DMD, shows a high mortality rate with a marked increase in serum creatine kinase (CK) levels in the neonatal period. By measuring serum CK levels in cord and venous blood, we found initial pulmonary respiration resulted in massive diaphragm damage in the neonates and thereby lead to the high serum CK levels. Furthermore, molecular biological techniques revealed that osteopontin was prominently upregulated in the dystrophic diaphragm prior to the respiration, and that immediate-early genes (c-fos and egr-1) and inflammation/immune response genes (IL-6, IL-8, COX-2, and selectin E) were distinctly overexpressed after the damage by the respiration. Hence, we segregated dystrophic phases at the molecular level before and after mechanical damage. These molecules could be biomarkers of muscle damage and potential targets in pharmaceutical therapies.

Initial pulmonary respiration causes massive diaphragm damage and hyper-CKemia in Duchenne muscular dystrophy dog.

High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein-protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.

Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction.

Duchenne muscular dystrophy is a lethal X-linked muscle disorder. We have already reported that osteopontin (OPN), an inflammatory cytokine and myogenic factor, is expressed in the early dystrophic phase in canine X-linked muscular dystrophy in Japan, a dystrophic dog model. To further explore the possibility of OPN as a new biomarker for disease activity in Duchenne muscular dystrophy, we monitored serum OPN levels in dystrophic and wild-type dogs at different ages and compared the levels to other serum markers, such as serum creatine kinase, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Serum OPN levels in the dystrophic dogs were significantly elevated compared with those in wild-type dogs before and 1 hour after a cesarean section birth and at the age of 3 months. The serum OPN level was significantly correlated with the phenotypic severity of dystrophic dogs at the period corresponding to the onset of muscle weakness, whereas other serum markers including creatine kinase were not. Immunohistologically, OPN was up-regulated in infiltrating macrophages and developmental myosin heavy chain-positive regenerating muscle fibers in the dystrophic dogs, whereas serum OPN was highly elevated. OPN expression was also observed during the synergic muscle regeneration process induced by cardiotoxin injection. In conclusion, OPN is a promising biomarker for muscle regeneration in dystrophic dogs and can be applicable to boys with Duchenne muscular dystrophy.

Mutsuki Kuraoka

Department of Molecular Therapy,
National Institute of Neuroscience,
Department of Molecular Therapy, National Institute of Neuroscience

National Center of Neurology and Psychiatry

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Mutsuki Kuraoka

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Therapy,National Institute of Neuroscience,Department of Molecular Therapy, National Institute of Neuroscience

National Center of Neurology and Psychiatry

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Department of Molecular Therapy,
National Institute of Neuroscience,
Department of Molecular Therapy, National Institute of Neuroscience