Motoi Kanagawa

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix–like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

The genetic and molecular basis of muscular dystrophy: roles of cell-matrix linkage in the pathogenesis

AbstractMuscular dystrophies are a heterogeneous group of genetic disorders. In addition to genetic information, a combination of various approaches such as the use of genetic animal models, muscle cell biology, and biochemistry has contributed to improving the understanding of the molecular basis of muscular dystrophys etiology. Several lines of evidence confirm that the structural linkage between the muscle extracellular matrix and the cytoskeleton is crucial to prevent the progression of muscular dystrophy. The dystrophin-glycoprotein complex links the extracellular matrix to the cytoskeleton, and mutations in the component of this complex cause Duchenne-type or limb-girdle-type muscular dystrophy. Mutations in laminin or collagen VI, muscle matrix proteins, are known to cause a congenital type of muscular dystrophy. Moreover, it is not only the primary genetic defects in the structural or matrix proteins, but also the primary mutations of enzymes involved in the protein glycosylation pathway that are now recognized to disrupt the matrix-cell interaction in a certain group of muscular dystrophies. This group of diseases is caused by the secondary functional defects of dystroglycan, a transmembrane matrix receptor. This review considers recent advances in understanding the molecular pathogenesis of muscular dystrophies that can be caused by the disruption of the cell-matrix linkage.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.

Ribitol-phosphate—a newly identified posttranslational glycosylation unit in mammals: structure, modification enzymes and relationship to human diseases

Glycosylation is a crucial posttranslational modification that is involved in numerous biological events. Therefore, abnormal glycosylation can impair the functions of glycoproteins or glycolipids and is occasionally associated with cell dysfunction and human diseases. For example, aberrant glycosylation of dystroglycan (DG), a cellular receptor for matrix and synaptic proteins, is associated with muscular dystrophy and lissencephaly. DG sugar chains are required for high-affinity binding to ligand proteins, and thus disruption of DG-ligand linkages underlies disease conditions. Although their biological significance is well recognized, the sugar-chain structure of DG and its modification enzymes have long remained incompletely elucidated. However, recent seminal studies have finally revealed a highly regulated mechanism for DG glycosylation and have discovered a posttranslational unit, ribitol-phosphate, that was not previously known to be used in mammals. This review article introduces the structure, modification enzymes and functions of the sugar chains of DG, and then discusses their relationship to human diseases and therapeutic strategiesn.

Cell endogenous activities of fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5

Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.

Fukutin and Fukuyama Congenital Muscular Dystrophy

Recent genetic and biochemical studies have revealed that mutations in (putative) glycosyltransferases and subsequent abnormal glycosylation of dystroglycan are associated with several forms of congenital muscular dystrophies (Kanagawa and Toda 2006). Fukuyama congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is one of the congenital muscular dystrophies displaying glycosylation defects of α-dystroglycan. The gene responsible for this disease is fukutin, and its protein product is assumed to participate in cellular glycosylation events. FCMD is characterized by severe congenital muscular dystrophy, abnormal neuronal migration associated with mental retardation and epilepsy, and frequent eye abnormalities. Therefore, fukutin-dependent glycosylation of α-dystroglycan plays crucial roles in structural/functional maintenance of skeletal muscle, central/peripheral nervous system, and eye. α-Dystroglycan, a highly glycosylated protein, forms a protein complex with β-dystroglycan, and the dystroglycan complex links laminin in the extracellular matrix to the cellular actin cytoskeleton. Abnormal glycosylation of α-dystroglycan results in a severe reduction in laminin-binding activity, and thus disruption of the interaction between dystroglycan and laminin caused by fukutin mutations is believed to be the major cause of FCMD.