Substantial deficiency of free sialic acid in muscles of patients with GNE myopathy and in a mouse model.

GNE myopathy (GNEM), also known as hereditary inclusion body myopathy (HIBM), is a late- onset, progressive myopathy caused by mutations in the GNE gene encoding the enzyme responsible for the first regulated step in the biosynthesis of sialic acid (SA). The disease is characterized by distal muscle weakness in both the lower and upper extremities, with the quadriceps muscle relatively spared until the late stages of disease. To explore the role of SA synthesis in the disease, we conducted a comprehensive and systematic analysis of both free and total SA levels in a large cohort of GNEM patients and a mouse model. A sensitive LC/MS/MS assay was developed to quantify SA in serum and muscle homogenates. Mean serum free SA level was 0.166 µg/mL in patients and 18% lower (p<0.001) than that of age-matched control samples (0.203 µg/mL). In biopsies obtained from patients, mean free SA levels of different muscles ranged from 0.046-0.075 µg/µmol Cr and were markedly lower by 72-85% (p<0.001) than free SA from normal controls. Free SA was shown to constitute a small fraction (3-7%) of the total SA pool in muscle tissue. Differences in mean total SA levels in muscle from patients compared with normal controls were less distinct and more variable between different muscles, suggesting a small subset of sialylation targets could be responsible for the pathogenesis of GNEM. Normal quadriceps had significantly lower levels of free SA (reduced by 39%) and total SA (reduced by 53%) compared to normal gastrocnemius. A lower SA requirement for quadriceps may be linked to the reported quadriceps sparing in GNEM. Analysis of SA levels in GneM743T/M743T mutant mice corroborated the human study results. These results show that serum and muscle free SA is severely reduced in GNEM, which is consistent with the biochemical defect in SA synthesis associated with GNE mutations. These results therefore support the approach of reversing SA depletion as a potential treatment for GNEM patients.

Myoclonus dystonia and muscular dystrophy: ɛ-sarcoglycan is part of the dystrophin-associated protein complex in brain.

BACKGROUND: Myoclonus-dystonia is a neurogenic movement disorder caused by mutations in the gene encoding ɛ-sarcoglycan. By contrast, mutations in the α-, ß-, γ-, and δ-sarcoglycan genes cause limb girdle muscular dystrophies. The sarcoglycans are part of the dystrophin-associated protein complex in muscle that is disrupted in several types of muscular dystrophy. Intriguingly, patients with myoclonus-dystonia have no muscle pathology; conversely, limb-girdle muscular dystrophy patients have not been reported to have dystonia-associated features. To gain further insight into the molecular mechanisms underlying these differences, we searched for evidence of a sarcoglycan complex in the brain. METHODS: Immunoaffinity chromatography and mass spectrometry were used to purify ubiquitous and brain-specific ɛ-sarcoglycan directly from tissue. Cell models were used to determine the effect of mutations on the trafficking and assembly of the brain sarcoglycan complex. RESULTS: Ubiquitous and brain-specific ɛ-sarcoglycan isoforms copurify with ß-, δ-, and ζ-sarcoglycan, ß-dystroglycan, and dystrophin Dp71 from brain. Incorporation of a muscular dystrophy-associated ß-sarcoglycan mutant into the brain sarcoglycan complex impairs the formation of the ßδ-sarcoglycan core but fails to abrogate the association and membrane trafficking of ɛ- and ζ-sarcoglycan. CONCLUSIONS: ɛ-Sarcoglycan is part of the dystrophin-associated protein complex in brain. Partial preservation of ɛ- and ζ-sarcoglycan in brain may explain the absence of myoclonus dystonia-like features in muscular dystrophy patients. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Absence of post-phosphoryl modification in dystroglycanopathy mouse models and wild-type tissues expressing non-laminin binding form of α-dystroglycan.

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-ß1,4-GlcNAc-ß1,2-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.

Post-Natal knockdown of fukutin-related protein expression in muscle by long-termRNA interference induces dystrophic pathology [corrected].

Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.

Fukutin-related protein is essential for mouse muscle, brain and eye development and mutation recapitulates the wide clinical spectrums of dystroglycanopathies.

Mutations in fukutin-related protein (FKRP) cause a common subset of muscular dystrophies characterized by aberrant glycosylation of alpha-dystroglycan (α-DG), collectively known as dystroglycanopathies. The clinical variations associated with FKRP mutations range from mild limb-girdle muscular dystrophy type 2I with predominantly muscle phenotypes to severe Walker-Warburg syndrome and muscle-eye-brain disease with striking structural brain and eye defects. In the present study, we have generated animal models and demonstrated that ablation of FKRP functions is embryonic lethal and that the homozygous-null embryos die before reaching E12.5. The homozygous knock-in mouse carrying the missense P448L mutation almost completely lacks functional glycosylation of α-DG in muscles and brain, validating the essential role of FKRP in the functional glycosylation of α-DG. However, the knock-in mouse survives and develops a wide range of structural abnormalities in the central nervous system, characteristics of neuronal migration defects. The brain and eye defects are highly reminiscent of the phenotypes seen in severe dystroglycanopathy patients. In addition, skeletal muscles develop progressive muscular dystrophy. Our results confirm that post-translational modifications of α-DG are essential for normal development of the brain and eyes. In addition, both the mutation itself and the levels of FKRP expression are equally critical for the survival of the animals. The exceptionally wide clinical spectrums recapitulated in the P448L mice also suggest the involvement of other factors in the disease progression. The mutant mouse represents a valuable model to further elucidate the functions of FKRP and develop therapies for FKRP-related muscular dystrophies.

Mutations alter secretion of fukutin-related protein.

Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases.

Acute nerve compression and the compound muscle action potential.

Detecting acute nerve compression using neurophysiologic studies is an important part of the practice of clinical intra-operative neurophysiology. The goal of this paper was to study the changes in the compound muscle action potential (CMAP) during acute mechanical compression. This is the type of injury most likely to occur during surgery. Thus, understanding the changes in the CMAP during this type of injury will be useful in the detection and prevention using intra-operative neurophysiologic monitoring. The model involved compression of the hamster sciatic nerve over a region of 1.3 mm with pressures up to 2000 mmHg for times on the order of 3 minutes. In this model CMAP amplitude dropped to 50% of its baseline value when a pressure of roughly 1000 mmHg is applied while, at the same time, nerve conduction velocities decline by only 5%. The ability to detect statistically significant changes in the CMAP at low force levels using other descriptors of the CMAP including duration, latency variation, etc alone or in conjunction with amplitude and velocity measures was investigated. However, these other parameters did not allow for earlier detection of significant changes. This study focused on a model in which nerve injury on a short time scale is purely mechanical in origin. It demonstrated that a pure compression injury produced large changes in CMAP amplitude prior to large changes in conduction velocity. On the other hand, ischemic and stretch injuries are associated with larger changes in conduction velocity for a given value of CMAP amplitude reduction.