Takahito Niiyama

Induction of apoptosis signal regulating kinase 1 (ASK1) after spinal cord injury in rats: possible involvement of ASK1-JNK and -p38 pathways in neuronal apoptosis.

The aims of this study were to clarify the mechanism of cell death by apoptosis in the spinal cord after traumatic injury, and to examine the role of the mitogen-activated protein kinase (MAPK) pathways in the transmission of apoptosis signals. The rat spinal cord, experimentally injured by extradural static weight-compression, was studied by hematoxylin and eosin staining, Nissl-staining, terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) staining, and immunostaining using polyclonal antibodies against Apoptosis Signal-regulating Kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38 MAPK. TUNEL-positive cells were present at all stages studied until 7 days after injury, and percentage positivity for these cells was maximal at 3 days after injury. Electron microscopic analysis revealed the occurrence of apoptosis in both neuronal cells and glial cells. TUNEL-positive glial cells were stained by oligodendrocyte-specific maker. Expression of ASK1 was maximal at 24 h after injury in the gray matter and at 3 days after injury in the white matter. Following the expression of ASK1, activated forms of JNK and p38 were observed in apoptotic cells detected by the TUNEL method. Colocalization of ASK1 and activated JNK or activated p38 was observed in the same cell. These findings suggest the involvement of the stress-activated MAPK pathways including ASK1 in the transmission of apoptosis signals after spinal cord injury.

Frameshift mutation in the collagen VI gene causes Ullrich's disease

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in two patients with Ullrichs disease. In this study, we detected a homozygous 26 bp deletion in exon 14 of the collagen VI alpha 2 gene (COL6A2) in one patient. This mutation causes a frameshift and a premature termination codon, and results in a truncated collagen VI alpha 2 chain. Our data suggest that at least some cases of Ullrichs disease result from recessive mutations in COL6A2.

Multiple episodes of thrombosis in a patient with Becker muscular dystrophy with marked expression of utrophin on the muscle cell membrane

Abstract We previously reported a patient with Becker muscular dystrophy (BMD) who exhibited a benign clinical phenotype and marked expression of utrophin on the muscle cell membrane. The patient developed multiple episodes of thrombosis (middle cerebral and femoral arteries) in the course of the disease. We re-examined the biopsy muscle specimen from the patient immunohistochemically as to the expression of procoagulant or anticoagulant factors. We found a lower expression of thrombomodulin on the muscle cell membrane in the BMD patient compared with other BMD or Duchenne muscular dystrophy (DMD) patients. Although utrophin up-regulation in muscle is thought to prevent the muscle wasting in dystrophin-deficient DMD or BMD, the data obtained in the present study indicate that up-regulated utrophin may have an unexpected influence on the function of the vascular or coagulation system.

Electron microscopic abnormalities of skeletal muscle in patients with collagen VI deficiency in Ullrich's disease

Abstract. By electron microscopy, we examined the skeletal muscle from two patients with Ullrichs disease. One patient had a deletion in the collagen VI alpha 2 gene. The muscle biopsy specimens showed no collagen VI immunoreaction, while the expression of collagen IV was increased. Collagen VI is a microfibrillar protein in the extracellular matrix with cell adhesive properties, and collagen IV is a principal component of the basal lamina. Electron microscopy revealed unevenness, extension, and folding of the muscle plasma membrane, and showed thickening and overproduction of the basal lamina. The data show that type VI collagen is certainly one of the important extracellular matrix components maintaining the structural integrity of skeletal muscle, and a defect of the collagen VI protein causes abnormalities of the muscle plasma membrane, dystrophic muscle changes, and up-regulation of collagen IV.

Pathological characteristics of skeletal muscle in Ullrich's disease with collagen VI deficiency

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints and hyperextensibility of the distal joints. Recently, we found a deficiency of collagen VI protein in skeletal muscle from two patients with Ullrichs disease. In this study, we investigated immunohistochemically the expression of extracellular matrix proteins and various proteins, which are markers for regenerating muscle fibers. Although we have detected the reduction of collagen VI in Ullrichs disease with the two kinds of monoclonal antibodies for the different domains of collagen VI, the remaining immunoreactive material was different between them. This might suggest the presence of incomplete collagen VI protein in the muscle fibers. Furthermore, we found that very small muscle fibers in the patients with Ullrichs disease showed marked expression of desmin, neural cell adhesion molecule and neonatal myosin heavy chain, which is a characteristic finding of regenerating fibers, however, they showed poor expression of developmental myosin heavy chain and thrombomodulin. The present findings suggest that abnormal regeneration or maturation processes are involved in the pathogenesis of dystrophic muscle changes at least in the advanced stage of Ullrichs disease.

An overexpression of fibroblast growth factor (FGF) and FGF receptor 4 in a severe clinical phenotype of facioscapulohumeral muscular dystrophy.

We evaluated the expression of a select panel of growth factors and their receptors, including fibroblast growth factor 1 (FGF‐1), fibroblast growth factor 2 (FGF‐2), platelet‐derived growth factor (PDGF), FGF receptor 1 (FGF‐R1), FGF receptor 3 (FGF‐R3), FGF receptor 4 (FGF‐R4), PDGF receptor α (PDGF‐Rα), PDGF receptor β (PDGF‐Rβ), and heparan sulfate proteoglycan (HSPG), in muscle biopsy specimens from nine facioscapulohumeral muscular dystrophy (FSHD) patients using immunohistochemistry. Two cases of Duchenne‐type muscular dystrophy (DMD), two of Becker‐type muscular dystrophy (BMD), and one of limb‐girdle‐type muscular dystrophy (LGMD) were also investigated. Widespread immunostaining for FGF‐1 and FGF‐2 on the sarcolemma and overexpression of FGF‐R4 in endomysial and perimysial connective tissue were seen in one patient with a severe clinical phenotype of FSHD who had respiratory failure. Standard histochemistry in this patient revealed marked interstitial fibrosis and lobulated fibers. The overexpression of FGF and FGF‐R4 in this severe FSHD case may be associated with the muscle fibrosis and disease severity.

Abnormal expression of proteoglycans in Ullrich's disease with collagen VI deficiency.

Patients with Ullrichs disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently we found a marked reduction of fibronectin receptors in the skin and cultured fibroblasts of two patients with Ullrichs disease with collagen VI deficiency, and speculated that an abnormality of cell adhesion may be involved in the pathogenesis of the disease. In this study, we investigated the expression of proteoglycans and adhesion molecules in Ullrichs disease and other muscle diseases. We found a reduction of NG2 proteoglycan in the membrane of skeletal muscle but not in the skin in Ullrichs disease. By contrast, we found the upregulation of tenascin C in the extracellular matrix of skeletal muscle in Ullrichs disease. Our findings suggest that abnormal expression of proteoglycans and adhesion molecules may be involved in the pathogenesis of the dystrophic muscle changes in Ullrichs disease. Muscle Nerve, 2006

Diagnosis of dystrophinopathy by skin biopsy.

We studied the expression of dystrophin in skin biopsy samples from 19 patients with neuromuscular diseases. Immunohistochemical procedures for dystrophin analyses were performed using monoclonal antibodies for three different domains. Arrector pili muscles, which are smooth muscles in the skin, expressed dystrophin in the patients with limb‐girdle muscular dystrophy (5), facioscapulohumeral muscular dystrophy (1), and spinal muscular atrophy (3), and in normal controls (2). The C‐terminus of dystrophin was slightly expressed in the patients with Duchenne muscular dystrophy, whereas the rod domain and N‐terminus were absent. In one patient with Becker muscular dystrophy, the expression of dystrophin was reduced. The mosaic of dystrophin positive and negative smooth muscle fibers was observed in a manifesting carrier of Duchenne muscular dystrophy. Our results suggest that skin biopsy is very useful for the diagnosis of Duchenne/Becker muscular dystrophy and manifesting carrier of Duchenne muscular dystrophy, and can be performed even at an advanced stage of the disease.

Fibronectin receptor reduction in skin and fibroblasts of patients with Ullrich's disease

Ullrichs disease is a congenital muscular dystrophy characterized clinically by generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recent studies have demonstrated that collagen VI is deficient in the muscles of patients with Ullrichs disease, and some cases result from recessive mutations of the collagen VIα2 gene (COL6A2). Fibronectin is one of the main components of the extracellular matrix (ECM) and associates with a variety of other matrix molecules including collagen. The behavior of fibronectin on cells is mediated by fibronectin receptors, members of the integrin family. We studied the expression of fibronectin receptors and fibronectin in patients with Ullrichs disease, and found a marked reduction of fibronectin receptors in the ECM of skin and cultured fibroblasts of these patients. These results suggest that collagen VI deficiency may lead to the reduction of fibronectin receptors and that an abnormality of cell adhesion may be involved in the pathogenesis of Ullrichs disease.

Adult onset limb-girdle type mitochondrial myopathy with a mitochondrial DNA np8291 A-to-G substitution.

AbstractWe analyzed mitochondrial DNA (mtDNA) from 7 patients in four families with adult onset limb-girdle type mitochondrial myopathy to clarify their genetic background. The patients, 2 men and 5 women, showed common clinical features, characterized by isolated skeletal myopathy, high serum creatine kinase level, ragged-red fibers and cytochrome c oxidase-defective fibers. Analysis of muscle biopsy specimens indicated that cytochrome c oxidase activity was decreased relative to that of citrate synthase in 5 of the 7 patients. Southern blotting and direct sequence analyses showed an A-to-G homoplasmic transition at np8291 and intergenic COII/tRNA(Lys) 9bp deletion in all patients. This substitution was detected in only 2 of 600 control individuals including healthy subjects and patients with other neuromuscular disorders; these 2 individuals had diabetes mellitus and myotonic dystrophy, respectively. Consequently, the mtDNA transition at np8291 was a rare polymorphism. However, the 7 patients we studied had identical clinical, pathological, biochemical, and genetic features. Therefore, limb-girdle type mitochondrial myopathy with this rare polymorphism may form a subgroup of adult onset mitochondrial myopathy.