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Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.

Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of β-amyloid (Aβ) peptides. Aβ is produced from amyloid precursor protein (APP) that is sequentially cleaved by β- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aβ production, we performed a gene microarray–based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aβ production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aβ production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3–binding protein (GAL3BP), which suppressed Aβ production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aβ production by directly interacting with APP and thereby inhibiting APP processing by β-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity., Fukutin-related protein (FKRP) catalyses the addition of ribitol-phosphate (RboP) to the O-mannosyl glycan of α-dystroglycan and mutations in FKRP cause dystroglycanopathy. Here the authors provide insights into its oligomerization and recognition of the substrates, CDP-Rbo and the RboP-(phospho-)core M3 glycan, by determining the crystal structures of human FKRP.

Objective: This study aimed to elucidate the molecular features of inclusion body myositis (IBM). Methods: We performed RNA sequencing analysis of muscle biopsy samples from 67 participants, consisting of 58 myositis patients with the pathological finding of CD8-positive T cells invading non-necrotic muscle fibers expressing major histocompatibility complex class I (43 IBM, 6 polymyositis, and 9 unclassifiable myositis), and 9 controls. Results: Cluster analysis, principal component analysis, and pathway analysis showed that differentially expressed genes and pathways identified in IBM and polymyositis were mostly comparable. However, pathways related to cell adhesion molecules were upregulated in IBM as compared with polymyositis and controls (p¿

Glycosylation is a major form of post-translational modification and plays various important roles in organisms by modifying proteins or lipids, which generates functional variability and can increase their stability. Because of the physiological importance of glycosylation, defects in genes encoding proteins involved in glycosylation or glycan degradation are sometimes associated with human diseases. A number of genetic neuromuscular diseases are caused by abnormal glycan modification or degeneration. Heterogeneous and complex modification machinery, and difficulties in structural and functional analysis of glycans have impeded the understanding of how glycosylation contributes to pathology. However, recent rapid advances in glycan and genetic analyses, as well as accumulating genetic and clinical information have greatly contributed to identifying glycan structures and modification enzymes, which has led to breakthroughs in the understanding of the molecular pathogenesis of various diseases and the possible development of therapeutic strategies. For example, studies on the relationship between glycosylation and muscular dystrophy in the last two decades have significantly impacted the fields of glycobiology and neuromyology. In this review, the basis of glycan structure and biosynthesis will be briefly explained, and then molecular pathogenesis and therapeutic concepts related to neuromuscular diseases will be introduced from the point of view of the life cycle of a glycan molecule.

Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disease that is characterized by varying degrees of cerebellar dysfunction and Parkinsonism. The neuropathological hallmark of MSA is alpha-synuclein (AS)-positive glial cytoplasmic inclusions (GCIs). Although severe neuronal loss (NL) is also observed in MSA, neuronal inclusions (NIs) are rare compared to GCIs, such that the pathological mechanism of NL in MSA is unclear. GCIs and NIs are late-stage pathology features relative to AS oligomers and may not represent early pathological changes in MSA. To reveal the early pathology of MSA, it is necessary to examine the early aggregation of AS, i.e., AS oligomers. Here, we adopted a proximity ligation assay (PLA) to examine the distribution of AS oligomers in brain tissue samples from patients with MSA and other diseases. Surprisingly, MSA brains showed a widespread distribution and abundant accumulation of oligomeric AS in neurons as well as oligodendrocytes of the neocortex. In several regions, oligomeric AS signal intensity was higher in cases with MSA than in cases with Parkinson’s disease. In contrast to previous studies, AS–PLA revealed abundant AS oligomer accumulation in Purkinje cells in MSA brains, identifying oligomeric AS accumulation as a possible cause of Purkinje cell loss. This wide distribution of AS oligomers in MSA brain neurons has not been described previously and indicates a pathological mechanism of NL in MSA.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuron loss. At present, there are no drugs that stop the progression of PD. As with other multifactorial genetic disorders, genome-wide association studies (GWASs) found multiple risk loci for PD, although their clinical significance remains uncertain. Here, we report the identification of candidate drugs for PD by a method using GWAS data and in silico databases. We identified 57 Food and Drug Administration-approved drug families as candidate neuroprotective drugs for PD. Among them, dabrafenib, which is known as a B-Raf kinase inhibitor and is approved for the treatment of malignant melanoma, showed remarkable cytoprotective effects in neurotoxin-treated SH-SY5Y cells and mice. Dabrafenib was found to inhibit apoptosis, and to enhance the phosphorylation of extracellular signal-regulated kinase (ERK), and inhibit the phosphorylation of c-Jun NH2-terminal kinase. Dabrafenib targets B-Raf, and we confirmed a protein–protein interaction between B-Raf and Rit2, which is coded by RIT2, a PD risk gene in Asians and Caucasians. In RIT2-knockout cells, the phosphorylation of ERK was reduced, and dabrafenib treatment improved the ERK phosphorylation. These data indicated that dabrafenib exerts protective effects against neurotoxicity associated with PD. By using animal model, we confirmed the effectiveness of this in silico screening method. Furthermore, our results suggest that this in silico drug screening system is useful in not only neurodegenerative diseases but also other common diseases such as diabetes mellitus and hypertension.

OBJECTIVE Transient receptor potential vanilloid channel 2 (TRPV2) is a Ca2+ -permeable channel and plays a role in mediating intracellular Ca2+ current via mechanical stimuli. This study was undertaken to examine the expression and role of TRPV2 in adult articular cartilage and the development of osteoarthritis (OA). METHODS We examined TRPV2 expression in mouse and human articular cartilage. We analyzed the development of OA in Col2a1-CreERt2 ;Trpv2fl/fl mice and Trpv2fl/fl littermates in the resection of the medial meniscus and medial collateral ligament model (n = 5 each), the destabilization of the medial meniscus model (n = 5 each), and the aging mouse model (n = 8-9 each). We examined marker protein expression in these joints, Ca2+ influx by mechanical stimuli, and downstream pathways in vitro. RESULTS TRPV2 was expressed in mouse and human articular cartilage and ectopic ossification lesions. In all mouse models of OA examined, Col2a1-CreERt2 ;Trpv2fl/fl mice were observed to have enhanced degradation of articular cartilage accompanied by decreased expression of lubricin/Prg4, and marked formation of periarticular ectopic ossification. Mechanical stress-induced Ca2+ influx was decreased by Trpv2 knockout (KO). Prg4 induction by fluid-flow shear stress was diminished in Trpv2-KO mouse chondrocytes, and this was mediated by the Ca2+ /calmodulin-dependent protein kinase kinase-cyclic AMP response element binding protein axis. Hypertrophic differentiation was enhanced in Trpv2-KO mouse chondrocytes. Increased activity of calcineurin and nuclear translocation of nuclear factor in activated T cells 1 induced by fluid-flow shear stress or TRP agonist treatment was reversed by Trpv2 knockout. CONCLUSION Our findings demonstrate regulation of articular cartilage by TRPV2 through Prg4 induction and suppression of ectopic ossification.

Hearing loss (HL) is one of the most common sensory impairments and etiologically and genetically heterogeneous disorders in humans. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. Aberrant glycosylation of α-dystroglycan causes at least eighteen subtypes of MD, now categorized as MD-dystroglycanopathy (MD-DG), with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes and increasing evidence regarding their molecular pathogeneses, no comprehensive study has investigated sensorineural HL (SNHL) in MD-DG. Here, we found that two mouse models of MD-DG, Largemyd/myd and POMGnT1-KO mice, exhibited congenital, non-progressive, and mild-to-moderate SNHL in auditory brainstem response (ABR) accompanied by extended latency of wave I. Profoundly abnormal myelination was found at the peripheral segment of the cochlear nerve, which is rich in the glycosylated α-dystroglycan–laminin complex and demarcated by “the glial dome.” In addition, patients with Fukuyama congenital MD, a type of MD-DG, also had latent SNHL with extended latency of wave I in ABR. Collectively, these findings indicate that hearing impairment associated with impaired Schwann cell-mediated myelination at the peripheral segment of the cochlear nerve is a notable symptom of MD-DG., Author summary Hearing loss (HL) is one of the most common sensory impairments and heterogeneous disorders in humans. Up to 60% of HL cases are caused by genetic factors, and approximately 30% of genetic HL cases are syndromic. Although 400–700 genetic syndromes are associated with sensorineural HL (SNHL), caused due to problems in the nerve pathways from the cochlea to the brain, only about 45 genes are known to be associated with syndromic HL. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. MD-dystroglycanopathy (MD-DG), caused by aberrant glycosylation of α-dystroglycan, is an MD subtype with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes (at least 18), no comprehensive study has investigated SNHL in MD-DG. Here, we found that hearing impairment was associated with abnormal myelination of the peripheral segment of the cochlear nerve caused by impaired dystrophin–dystroglycan complex in two mouse models (type 3 and 6) of MD-DG and in patients (type 4) with MD-DG. This is the first comprehensive study investigating SNHL in MD-DG. Our findings may provide new insights into understanding the pathogenic characteristics and mechanisms underlying inherited syndromic hearing impairment.

BACKGROUND Amyloid-β (Aβ) induces cerebrovascular damage and is reported to stimulate endothelial cell senescence. We previously demonstrated that angiotensin II (Ang II)-promoted vascular senescence. We examined the possible cross-talk between Ang II and Aβ in regulating brain vascular smooth muscle cell (BVSMC) senescence. METHODS BVSMCs were prepared from adult male mice and stimulated with Ang II (0, 0.1, 1, 10, and 100 nmol/l) and/or Aβ 1–40 (0, 0.1, 0.3, 0.5, 1, 3, and 5 µmol/l) for the indicated times. Cellular senescence was evaluated by senescence-associated β-galactosidase staining. RESULTS Treatment with Ang II (100 nmol/l) or Aβ (1 µmol/l) at a higher dose increased senescent cells compared with control at 6 days. Treatment with Ang II (10 nmol/l) or Aβ (0.5 µmol/l) at a lower dose had no effect on senescence whereas a combined treatment with lower doses of Ang II and Aβ significantly enhanced senescent cells. This senescence enhanced by lower dose combination was markedly blocked by valsartan (Ang II type 1 receptor inhibitor) or TAK-242 (Aβ receptor TLR4 inhibitor) treatment. Moreover, lower dose combination caused increases in superoxide anion levels and p-ERK expression for 2 days, NF-κB activity, p-IκB, p-IKKα/β, p16 and p53 expression for 4 days, and an obvious decrease in pRb expression. These changes by lower dose combination, except in p-IκB expression and NF-κB activity, were significantly inhibited by pretreatment with U0126 (ERK inhibitor). CONCLUSIONS Ang II and Aβ synergistically promoted BVSMC senescence at least due to enhancement of the p-ERK–p16–pRb signaling pathway, oxidative stress, and NF-κB/IκB activity.

The anabolic effects of androgen on skeletal muscles are thought to be mediated by androgen receptor (AR). Although multiple studies concerning the effects of AR in males have been performed, the molecular mechanisms of AR in skeletal muscles remain unclear. Here we first confirmed that satellite cells from mouse hindlimb muscles express AR. We then generated satellite cell-specific AR knockout mice using Pax7CreERT2 and ARL2/Y mice to test whether AR in satellite cells is necessary for muscle regeneration. Surprisingly, we found that muscle regeneration was compromised in both Pax7CreERT2(Fan)/+ control mice and Pax7CreERT2(Fan)/+;ARL2/Y mice compared to ARL2/Y mice. However, Pax7CreERT2(Gaka)/+;ARL2/Y;R26tdTomato/+ mice showed no significant differences between control and mutant muscle regeneration. These findings indicate that AR in satellite cells is not essential for muscle regeneration. We propose that Pax7CreERT2(Fan)/+ control mice should be included in all experiments, because these mice negatively affect the muscle regeneration and show the mild regeneration phenotype.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.

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 strategies .

Congenital muscular dystrophies (CMDs) are characterized by progressive weakness and degeneration of skeletal muscle. In several forms of CMD, abnormal glycosylation of α-dystroglycan (α-DG) results in conditions collectively known as dystroglycanopathies, which are associated with central nervous system involvement. We recently demonstrated that fukutin, the gene responsible for Fukuyama congenital muscular dystrophy, encodes the ribitol-phosphate transferase essential for dystroglycan function. Brain pathology in patients with dystroglycanopathy typically includes cobblestone lissencephaly, mental retardation, and refractory epilepsy; however, some patients exhibit average intelligence, with few or almost no structural defects. Currently, there is no effective treatment for dystroglycanopathy, and the mechanisms underlying the generation of this broad clinical spectrum remain unknown. Here, we analysed four distinct mouse models of dystroglycanopathy: two brain-selective fukutin conditional knockout strains (neuronal stem cell-selective Nestin-fukutin-cKO and forebrain-selective Emx1-fukutin-cKO), a FukutinHp strain with the founder retrotransposal insertion in the fukutin gene, and a spontaneous Large-mutant Largemyd strain. These models exhibit variations in the severity of brain pathology, replicating the clinical heterogeneity of dystroglycanopathy. Immunofluorescence analysis of the developing cortex suggested that residual glycosylation of α-DG at embryonic day 13.5 (E13.5), when cortical dysplasia is not yet apparent, may contribute to subsequent phenotypic heterogeneity. Surprisingly, delivery of fukutin or Large into the brains of mice at E12.5 prevented severe brain malformation in Emx1-fukutin-cKO and Largemyd/myd mice, respectively. These findings indicate that spatiotemporal persistence of functionally glycosylated α-DG may be crucial for brain development and modulation of glycosylation during the fetal stage could be a potential therapeutic strategy for dystroglycanopathy.

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.

A 1-year-old Japanese female patient with motor retardation was brought to our outpatient clinic by her mother. The child was found to have high serum creatine kinase (CK) level according to the 1-year-old medical checkup and was recommended further evaluation. The girl, the second child in her family, was born at full term in a local hospital and, after birth, was transferred to a general hospital because of muscle hypotonus, poor eye opening and sucking, and a high serum CK level (5461 IU/L: normal ranges are between 40 and 310 IU/L). She was followed up because her doctors considered these symptoms to arise from neonatal asphyxia. The infant developed quickly after 2 weeks, and her serum CK level decreased to normal. She grew up slightly slowly and acquired head control at 5 months of age (commonly acquired at 3–4 months after birth); she could not roll over at 7 months and rehabilitation was started. At 1 year of age, her muscle tonus remained decreased, and her serum CK level was elevated again (7532 IU/L). She could not stand up without support.

The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose-type GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-β1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients.

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.

Muscular dystrophy is a group of genetic disorders characterized by progressive muscle weakness. In the early 2000s, a new classification of muscular dystrophy, dystroglycanopathy, was established. Dystroglycanopathy often associates with abnormalities in the central nervous system. Currently, at least eighteen genes have been identified that are responsible for dystroglycanopathy, and despite its genetic heterogeneity, its common biochemical feature is abnormal glycosylation of alpha-dystroglycan. Abnormal glycosylation of alpha-dystroglycan reduces its binding activities to ligand proteins, including laminins. In just the last few years, remarkable progress has been made in determining the sugar chain structures and gene functions associated with dystroglycanopathy. The normal sugar chain contains tandem structures of ribitol-phosphate, a pentose alcohol that was previously unknown in humans. The dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and isoprenoid synthase domain-containing protein (ISPD) encode essential enzymes for the synthesis of this structure: fukutin and FKRP transfer ribitol-phosphate onto sugar chains of alpha-dystroglycan, and ISPD synthesizes CDP-ribitol, a donor substrate for fukutin and FKRP. These findings resolved long-standing questions and established a disease subgroup that is ribitol-phosphate deficient, which describes a large population of dystroglycanopathy patients. Here, we review the history of dystroglycanopathy, the properties of the sugar chain structure of alpha-dystroglycan, dystroglycanopathy gene functions, and therapeutic strategies.

Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin α6β1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin α5 2688–2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.

A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1]n-3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of α-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. Although the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of α-dystroglycan have been reported, both in Japan and elsewhere. To understand better the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and α-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R, and Y371C instead accumulated in the endoplasmic reticulum. Protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) also mislocalizes when co-expressed with these missense mutants. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of α-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.

α-Dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.

SUMMARY Establishing synaptic contacts between neurons is paramount for nervous system function. This process involves transsynaptic interactions between a host of cell adhesion molecules that act in cooperation with the proteins of the extracellular matrix to specify uniquephysiological propertiesofindividual synaptic connections. However, understanding of the molecular mechanisms that generate functional diversity in an input-specific fashion is limited. In this study, we identify that major components of the extracellular matrix proteins present in the synaptic cleft—members oftheheparansulfateproteoglycan (HSPG) family—associate with the GPR158/179 group of orphan receptors. Using the mammalian retina as a model system, we demonstrate that the HSPG member Pikachurin, released by photoreceptors, recruits a key post-synaptic signaling complex of downstream ON-bipolar neurons in coordination with the presynaptic dystroglycan glycoprotein complex. We further demonstrate that this transsynaptic assembly plays an essential role in synaptic transmission of photoreceptor signals., Graphical Abstract, In Brief Orlandi et al. identify transsynaptic assembly at photoreceptor synapses involving pre-synaptic dystrophindystroglycan complex and the postsynaptic orphan receptor GPR179 bridged by HSPG protein Pikachurin in the cleft and demonstrate its role in shaping transmission of photoreceptor signals.

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation., Myotube formation by fusion of myoblasts is essential for skeletal muscle formation, but which molecules regulate this process remains elusive. Here authors identify the mechanosensitive PIEZO1 channel as a key element, whose activity is regulated by phosphatidylserine during myotube formation.

Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by α-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin α7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Large myd muscles, which have an intact dystrophin–glycoprotein complex and lack only the laminin globular domain-binding motif on α-dystroglycan. Compromised sarcolemmal integrity is directly shown in Large myd muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding α-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage.

Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large(myd) mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

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 recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large myd mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystro- glycan, laminin and perlecan. These data suggest functional dis- ruption of the trimolecular complex in glycosylation-deficient muscular dystrophy. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.