倉岡 睦季

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder characterized by primary muscle degeneration. Therapeutic strategies for DMD have been extensively explored, and some are in the stage of human clinical trials. Along with the development of new therapies, sensitive outcome measures are needed to monitor the effects of new treatments. Therefore, we investigated outcome measures such as biomarkers and motor function evaluation in a dystrophic model of beagle dogs, canine X-linked muscular dystrophy in Japan (CXMDJ). Osteopontin (OPN), a myogenic inflammatory cytokine, was explored as a potential biomarker in dystrophic dogs over the disease course. The serum OPN levels of CXMDJ dystrophic dogs were elevated, even in the early disease phase, and this could be related to the presence of regenerating muscle fibers; as such, OPN would be a promising biomarker for muscle regeneration. Next, accelerometry, which is an efficient method to quantify performance in validated tasks, was used to evaluate motor function longitudinally in dystrophic dogs. We measured three-axis acceleration and angular velocity with wireless hybrid sensors during gait evaluations. Multiple parameters of acceleration and angular velocity showed notedly lower values in dystrophic dogs compared with wild-type dogs, even at the onset of muscle weakness. These parameters accordingly decreased with exacerbation of clinical manifestations along with the disease course. Multiple parameters also indicated gait abnormalities in dystrophic dogs, such as a waddling gait. These outcome measures could be applicable in clinical trials of patients with DMD or other muscle disorders.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are potentially therapeutic for muscle disease because they can accumulate at the sites of injury and act as immunosuppressants. MSCs are attractive candidates for cell-based strategies that target diseases with chronic inflammation, such as Duchenne muscular disease (DMD). We focused on the anti-inflammatory properties of IL-10 and hypothesized that IL-10 could increase the typically low survival of MSCs by exerting a paracrine effect after transplantation. METHODS: We developed a continuous IL-10 expression system of MSCs using an adeno-associated virus (AAV) vector. To investigate the potential benefits of IL-10 expressing AAV vector-transduced MSCs (IL-10-MSCs), we examined the cell survival rates in the skeletal muscles after intramuscular injection into mice and dogs. Systemic treatment with IL-10-MSCs derived from dental pulp (DPSCs) was comprehensively analyzed using the canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients. RESULTS: In vivo bioluminescence imaging analysis revealed higher retention of IL-10-MSCs injected into the hindlimb muscle of mice. In the muscles of dogs, myofiber-like tissue was formed after the stable engraftment of IL-10-MSCs. Repeated systemic administration of IL-10-DPSCs into the CXMDJ model resulted in long-term engraftment of cells and slightly increased the serum levels of IL-10. IL-10-hDPSCs showed significantly reduced expression of pro-inflammatory MCP-1 and upregulation of stromal-derived factor-1 (SDF-1). MRI and histopathology of the hDPSC-treated CXMDJ indicated the regulation of inflammation in the muscles, but not myogenic differentiation from treated cells. hDPSC-treated CXMDJ showed improved running capability and recovery in tetanic force with concomitant increase in physical activity. Serum creatine kinase levels, which increased immediately after exercise, were suppressed in IL-10-hDPSC-treated CXMDJ. CONCLUSIONS: In case of local injection, IL-10-MSCs could maintain the long-term engraftment status and facilitate associated tissue repair. In case of repeated systemic administration, IL-10-MSCs facilitated the long-term retention of the cells in the skeletal muscle and also protected muscles from physical damage-induced injury, which improved muscle dysfunction in DMD. We can conclude that the local and systemic administration of IL-10-producing MSCs offers potential benefits for DMD therapy through the beneficial paracrine effects of IL-10 involving SDF-1.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an inherited progressive disorder that causes skeletal and cardiac muscle deterioration with chronic inflammation. Dental pulp stem cells (DPSCs) are attractive candidates for cell-based strategies for DMD because of their immunosuppressive properties. Therefore, we hypothesized that systemic treatment with DPSCs might show therapeutic benefits as an anti-inflammatory therapy. METHODS: To investigate the potential benefits of DPSC transplantation for DMD, we examined disease progression in a DMD animal model, mdx mice, by comparing them with different systemic treatment conditions. The DPSC-treated model, a canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients, also underwent comprehensive analysis, including histopathological findings, muscle function, and locomotor activity. RESULTS: We demonstrated a therapeutic strategy for long-term functional recovery in DMD using repeated DPSC administration. DPSC-treated mdx mice and CXMDJ showed no serious adverse events. MRI findings and muscle histology suggested that DPSC treatment downregulated severe inflammation in DMD muscles and demonstrated a milder phenotype after DPSC treatment. DPSC-treated models showed increased recovery in grip-hand strength and improved tetanic force and home cage activity. Interestingly, maintenance of long-term running capability and stabilized cardiac function was also observed in 1-year-old DPSC-treated CXMDJ. CONCLUSIONS: We developed a novel strategy for the safe and effective transplantation of DPSCs for DMD recovery, which included repeated systemic injection to regulate inflammation at a young age. This is the first report on the efficacy of a systemic DPSC treatment, from which we can propose that DPSCs may play an important role in delaying the DMD disease phenotype.

Duchenne muscular dystrophy (DMD) is a severe congenital disease associated with mutation of the dystrophin gene. Supplementation of dystrophin using recombinant adeno-associated virus (rAAV) has promise as a treatment for DMD, although vector-related general toxicities, such as liver injury, neurotoxicity, and germline transmission, have been suggested in association with the systemic delivery of high doses of rAAV. Here, we treated normal or dystrophic dogs with rAAV9 transduction in conjunction with multipotent mesenchymal stromal cell (MSC) injection to investigate the therapeutic effects of an rAAV expressing microdystrophin (μDys) under conditions of immune modulation. Bone-marrow-derived MSCs, rAAV-CMV-μDys, and a rAAV-CAG-luciferase (Luc) were injected into the jugular vein of a young dystrophic dog to induce systemic expression of μDys. One week after the first injection, the dog received a second intravenous injection of MSCs, and on the following day, rAAV was intravenously injected into the same dog. Systemic injection of rAAV9 with MSCs pretreatment improves gene transfer into normal and dystrophic dogs. Dystrophic phenotypes significantly improved in the rAAV-μDys-injected dystrophic dog, suggesting that an improved rAAV-μDys treatment including immune modulation induces successful long-term transgene expression to improve dystrophic phenotypes.

Histidine decarboxylase (HDC), histamine synthase, is expressed in hematopoietic stem cells and in lineage-committed progenitors in the bone marrow (BM). However, the role of histamine in hematopoiesis is not well described. To evaluate the role of histamine in hematopoiesis, we analyzed the changes in HDC expression at hematopoietic sites, the BM, spleen, and liver of 2-, 3-, and 6-week old wild-type mice. We also performed morphological analyses of the hematopoietic sites using HDC-deficient (HDC-KO) mice. In wild-type adults, HDC expression in the BM was higher than that in the spleen and liver and showed an age-dependent increase. Histological analysis showed no significant change in the adult BM and spleen of HDC-KO mice compared to wild-type mice. In the liver, HDC expression was temporarily increased at 3 weeks, and decreased at 6 weeks of age. Morphological analysis of the liver revealed more numerous hematopoietic colonies and megakaryocytes in HDC-KO mice compared to wild-type mice at 2 and 3 weeks of age, whereas no changes were observed in adults. Most of these hematopoietic colonies consisted of B220-positive B-lymphocytes and TER119-positive erythroblasts and were positive for the cell proliferation marker PCNA. Notably, these hematopoietic colonies declined in HDC-KO mice upon N-acetyl histamine treatment. A significant increase in the expression of hematopoiesis-related cytokines, IL3, IL7, EPO, G-CSF, and Cxcl12 was observed in the liver of 3-week old HDC-KO mice compared to wild-type mice. These results suggest that histamine-deficiency may maintain an environment suitable for hematopoiesis by regulating hematopoiesis-related cytokine expression in the liver of postnatal mice. This article is protected by copyright. All rights reserved.

Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68(+) monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68(+) cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68(+) cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.

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.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

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. (C) 2015 The Authors. Published by Elsevier Inc.

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.

Axon-dendrite polarity of neurons is essential for information processing in the nervous system. Here we studied the functions of chondroitin sulfate (CS) and heparan sulfate (HS) in neuronal polarization using cultured dissociated hippocampal neurons. Immunohistochemical analyses of early cultured neurons indicated the distribution of these glycosaminoglycans to be quite different. While CS epitopes were accumulated in the focal contacts present in axons and cell bodies, those of HS were detected ubiquitously on the cell surface including on dendrites and axons. Treatment with chondroitinase (CHase) ABC, which degrades CS, and knockdown of a CS sulfotransferase, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST), which is involved in the biosynthesis of oversulfated structures, induced the formation of multiple axons in hippocampal neurons. Time-lapse recordings revealed the multiple axons of CHase ABC-treated neurons to be highly unstable, extending and retracting, repeatedly. CHase ABC-treatments suggested that CS is involved in the formation of phosphorylated focal adhesion kinase-positive focal contacts. Thus, CS may enhance integrin signaling in the nascent axons, supporting axon specification. On the other hand, when neurons were treated with heparitinases that specifically degrade HS, neurons with a single axon increased. The axons of HSase-treated neurons extended steadily and showed almost no retraction. These results suggest that CS stabilizes and HS destabilizes the growth of axons in an opposing manner, contributing to early neuronal polarization. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

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.

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.