Muscular dystrophy is a genetic disease that is characterized by progressive muscle weakness and degeneration. Currently, over 50 distinct disease forms and responsible genes are known. Dystroglycanopathy is a collective term for referring to a group of congenital and limb-girdle types of muscular dystrophy that are caused by abnormal glycosylation of dystroglycan.
Dystroglycan is a cellular receptor for basement membrane proteins or synaptic proteins such as laminins, agrin, and neurexin [ 29 ]. O-Mannosyl glycosylation is essential for the ligand-binding activity of dystroglycan, and the most genes responsible for dystroglycanopathy encode enzymes involved in the synthesis of O-Man glycan [ 30 ]. In this section, I will introduce the history of dystroglycanopathy research and then describe recent progress in identifying a novel glycan unit, ribitol-phosphate RboP , and several therapeutic strategies.
Schematic representation of the structures of O-Man glycans on dystroglycan and modification enzymes. This repeating unit and CoreM3 are linked by tandem RboP groups.
Enzymes responsible for these modifications and muscular dystrophy are illustrated with blue rectangle. In the early s, it was shown that abnormal glycosylation of dystroglycan with reduced ligand-binding activity was associated with muscle—eye—brain MEB disease, Fukuyama muscular dystrophy FCMD , and Walker—Warburg syndrome WWS [ 31 ]. These three diseases are characterized by severe congenital forms of muscular dystrophy with brain malformation micropolygyria of the cerebrum and cerebellum, and type II lissencephaly , mental retardation, and eye involvement myopia, cataracts, abnormal eye movement, pale optic discs, and retinal detachment.
Subsequently, less severe forms of muscular dystrophy with abnormal glycosylation of dystroglycan were reported such as limb-girdle type 2I. In the s, a novel glycan structure GalNAc-GlcNAc-Man-O namely CoreM3 glycan was identified and this finding led to the discovery that several dystroglycanopathy-related gene products function as glycosyltransferases [ 35, 36 ]. A GAG-like repeating structure consisting of a disaccharide unit glucuronic acid [GlcA]-Xyl was also identified and, importantly, this repeat was shown to serve as a ligand-binding domain.
This repeating unit is synthesized by the bifunctional enzyme LARGE, which has two glycosyltransferase activities e. Ribitol Rbo is a sugar alcohol that was previously not known to be used in mammals, but was known to be present in bacterial cell walls as an RboP polymer. For more details regarding sugar chain structures and modification enzymes, please refer to our previous reviews [ 29, 30 ]. Strictly speaking, Rbo is not a sugar, but a precursor of RboP modification is the nucleotide-linked compound CDP-Rbo, as is the case with other sugar precursors used for glycosylation.
CDP-Rbo is synthesized from cytosolic free Rbophosphate and CTP by theisoprenoid domain-containing protein ISPD — mutations in which were also identified in patients with dystroglycanopathy [ 38—40 ]. Defects in the synthesis pathway for the O-mannosylation precursor Dol-P-Man have also been implicated in dystroglycanopathy. Dystroglycan plays crucial roles in maintaining the physical strength of the muscle plasma membrane in a glycosylation-dependent manner [ 41 ].
Thus, abnormal glycosylation renders muscles prone to contraction-induced injuries disease-triggering membrane fragility. In addition, dystroglycan also plays important roles in the maintenance of viability of satellite cells stem cells required for muscle regeneration and the proliferation and differentiation activities of myoblasts [ 42 ]. Conditional deletion of mouse Fktn in muscle precursor cells impaired muscle regeneration, which underlies severe muscle pathology in dystroglycanopathy.
However, viral-mediated selective gene expression in mature myofibers ameliorated the pathology, suggesting that protecting disease-triggering membrane fragility is a potential target for therapeutic intervention [ 42 ].
Several types of dystroglycanopathy model mice exhibited pathological abnormalities such as neuron over-migration, basement membrane breakdown, and cerebral hemisphere fusion [ 43, 44 ], indicating that these abnormalities underlie the cause of brain malformation. We recently reported that gene rescue in the developing embryo brain suppressed the disease-associated phenotype, which opens new avenues of intervention for therapeutic strategies related to central nervous system abnormalities [ 44 ]. This insertion contains a strong splice-acceptor site, which induces an alternative donor site in the last exon, and thus causes abnormal mRNA splicing exon trapping [ 46 ].
Introduction of antisense oligonucleotide targeting to prevent pathogenic exon trapping restores normal FKTN protein production and dystroglycan glycosylation in FCMD patient cells and model mice. Thus, this exon trapping strategy may serve as a radical approach for clinically treating FCMD.
In the latter case, surprisingly, abnormal glycosylation of dystroglycan and dystrophic pathology were improved [ 47 ]. Together, these findings suggest that the metabolic pathway for CDP-Rbo production could also be a therapeutic target, but more careful analyses are required. For example, it is necessary to clarify players in the biosynthesis pathway for Rbophosphate a CDP-Rboprecursor.
In , another form of muscular dystrophy associated with abnormal glycosylation was reported. POGLUT1 encodes the protein O-glucosyltransferase 1, which catalyzes the O-glucosylation of Notch, a famous signaling molecule that is widely involved in animal development. Notch is post-translationally modified with O-Glc and O-Fuc, and alteration of glycosylation affected Notch activities. The Notch pathway played roles in satellite cells, and disruption of Notch signaling in satellite cells recapitulated the muscular dystrophic phenotype in mice [ 49 ].
The disease-causing mutation reduced POGLUT1 activity, which impaired Notch signaling and consequently decreased myoblast proliferation and differentiation, as well as the number of satellite cells [ 48 ]. Very recently, mutations in the membrane-trafficking proteins TRAPPC11 and GOSR2 were reported in muscular dystrophy patients that were associated with abnormal dystroglycan glycosylation [ 50 ].
Since the Golgi is the major site for protein glycosylation, defects in the Golgi morphology and trafficking may result in abnormal protein glycosylation. However, in the latest case, glycosylation analysis was normal, suggesting other functional roles for TRAPPC11 [ 50 ]. In the recent report, the GOSR2 mutation was shown not to alter membrane trafficking. It is widely thought that defects in Golgi homeostasis via abnormal architecture or trafficking are associated with glycosylation diseases [ 54 ], but recent examination of the mutations in TRAPPC11 and GOSR2 indicate that they may selectively affect dystroglycan glycosylation.
The determinant mechanism explaining how mutations in these membrane-trafficking proteins in some cases selectively affect glycosylation of dystroglycan or in other cases affect global N-and O-glycosylation is unknown. A possible explanation is that trafficking proteins may also play specific roles in glycosylation, for example, by assuring proper trafficking of enzymes or by forming a zone where they function appropriately for the O-Man-glycosylation pathway e. Thus, some mutations could selectively affect dystroglycan, and other mutations could deteriorate trafficking functions more severely, thus affecting global glycosylation.
Glycans are turned over, like other components in living cells. Most glycan turnover occurs by endocytosis, and degradation is generally performed stepwise by multiple glycosidases in the lysosome. Exoglycosidases, which cleave the glycosidic bond from the non-reducing end, basically recognize one monosaccharide in a specific anomeric linkage. Modifications on the non-reducing terminal sugar e. In contrast to exoglycosidases, endoglycosidases cleave internal glycosidic linkages and thus release long oligosaccharides from conjugated molecules, including proteins or lipids.
These variations in the properties of degradation enzymes drive complexity in glycan degradation and synthesis. Monosaccharide units arising from degradation are typically exported from the lysosome to the cytosol and then reutilized for producing glycan precursors. Lysosomal enzymes are N-glycosylated, bearing Manphosphate on the sugar chain. This machinery constitutes the Manphosphate pathway.
There is a group of genetic diseases associated with decreased activities of lysosomal enzymes, consequently leading to accumulation of their substrates as undigested fragments in the lysosome; thus, this group of diseases is referred to collectively as lysosomal storage diseases LSDs [ 1, 55 ].
Currently, approximately 50 types of LSDs are known. Not only lysosomal enzymes, but also defects in lysosomal structural proteins can cause LSDs, such as Danon disease [ 56 ]. The accumulation of undigested macromolecules causes enlargement of the lysosomes and reduces the supply of products necessary for biosynthesis and energy metabolism. In addition, defects in lysosome functions can alter many cellular processes, including lysosomal pH regulation, synaptic release, endocytosis, exocytosis, and vesicle maturation [ 1, 55 ].
Clinical manifestations vary among LSD types. Many LSDs present with a range of severity, but hepatomegaly and splenomegaly are the most common symptoms. Over half of all LSDs are accompanied by central nervous system involvement, such as progressive cognitive and motor decline, and neuronal degeneration. The variation in pathology is thought to depend on the cell type and the cellular balance of synthesis and turnover of the macromolecule or its precursors. For example, sialylated glycolipid gangliosides are abundant in neurons, and thus LSDs caused by accumulated gangliosides are preliminary brain disorders GM1 gangliosidosis and GM2 gangliosidosis.
Pompe disease is caused by an accumulation of glycogen in the lysosome, and its clinical phenotypes particularly in cardiac and skeletal muscles are supported by the importance of glycogen for muscle tissues. It is proposed that lysosomal glycogen clearance probably has no genuine metabolic function, but serves to dispose of glycogen that is accidentally taken up into lysosomes through autophagy [ 57 ].
The clinical spectrum of Pompe disease can be categorized into classic infantile, childhood, and adult forms. The infantile form presents with generalized hypotonia, muscle weakness, hypertrophic cardiomyopathy, and respiratory failure. The adult form is characterized by a relatively slow progressive proximal and axial muscle weakness. Histopathologically, most infantile and childhood forms exhibit fibers occupied by huge vacuoles that contain basophilic amorphous periodic acid Schiff-positive materials, but in late-onset form, such vacuoles may be present only in a few fibers [ 58, 59 ].
Other glycogen storage diseases with myopathy include McArdle disease glycogenosis type V and Cori disease glycogenosis type III , among others.
The responsible gene products for McArdle disease and Cori disease function as a glycogen phosphorylase and a glycogen-debranching enzyme, respectively, both of which are coordinately involved in glycogenolysis in the cytoplasm [ 60 ]. Several strategies have been developed for treating LSDs. Enzyme-replacement therapy ERT is based on replacement of the mutant protein with its normal version to restore function. ERT relies on the Manphosphate pathway for efficient delivery of recombinant enzymes, which are modified with Manphosphate on the N-glycans, from outside of cells to the lysosome via cell surface Manphosphate receptors.
A limitation of ERT is that the enzymes cannot penetrate the blood-brain barrier so that ERTs do not affect symptoms of the central nervous system. The effectiveness of ERTs also depends on the severity of the disease when treatment is initiated [ 1, 59 ].
Substrate reduction therapy SRT using small molecules aims at the inhibition of enzymes located upstream of the mutated disease-causing protein to reduce production of substrates of the mutated proteins. Other strategies using small molecules include enzyme-enhancement therapy and pharmacological chaperone therapy aiming at enhancing the residual enzyme activity of the mutated proteins [ 1, 61 ].
Independent of the type of accumulated substances or mutated genes, enhancing lysosomal function can be a therapeutic option with broader applicability across LSDs. For example, it has been reported that overexpression of transcription factor EB TFEB , which regulates the coordinated transcriptional behavior of most lysosomal genes, induced lysosomal biogenesis and increased the degradation of complex molecules [ 62 ].
Very recently, fetal intracranial adeno-associated viral vector-mediated gene delivery for glucocerebrosidase was reported to ameliorate neurodegeneration seen in a mouse model for neuronopathic Gaucher disease [ 64 ]. This group also demonstrated the feasibility of ultrasound-guided global gene transfer to fetal macaque brains. Together, not only ERT, but also other strategies with gene replacement or small molecules hopefully will be developed and approved to overcome the hurdles of neurological problems of LSDs in the future. Understanding the molecular basis of the life cycle of glycans can help in clarifying disease pathogenesis and developing therapeutic strategies.
Many diseases directly caused by abnormal glycosylation are single-gene disorders, which can feasibly treated with gene therapy. However, many hurdles impede clinical applications in gene therapy, although progress has certainly been made. A systematic and integrated understanding of glycosylation pathways related to disease-causing gene products or biological characteristics of mutated enzymes may yield unexpected therapeutic approaches. Sometimes therapeutic target molecules may be safe and low-cost natural or metabolic products.
There are some established treatments for neuromuscular diseases such as LSDs, and the contributions of basic research in glycobiology were no doubt immeasurable. Although the significance of glycans is widely recognized, it is unfortunately true that difficulties in structural and functional analyses have limited advances. However, considering recent rapid technological advances in glycan and glycome analysis and accumulation of large information such as genetic and clinical database, the challenges of glycoscience are no longer high obstacles.
Rather, I believe, there is big potential for new breakthroughs in understanding the molecular mechanisms of pathogenesis and developing therapeutic interventions.
The author thanks Dr. Tamao Endo for fruitful comments and discussion. The regulatory designation impacts Chemistry, Manufacturing, and Controls CMC within a regulatory filing, in which the common and expected molecule structures and characterization methods differ widely for glycans when compared to small molecule drugs. While surmountable, the regulatory designation illustrates just one example of institutional challenge, and perhaps an opportunity for future change to facilitate development of glycan therapeutics.