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Muscle phosphorylase b kinase (PHK) deficiency is a rare mild metabolic disorder caused by mutations of the PHKA1 gene encoding the αM subunit of PHK. A 16-year-old boy experienced myalgia during the maximal multistage 20-m shuttle run test targeting the maximal oxygen consumption. Although an ischemic forearm exercise test was normal, a muscle biopsy revealed subsarcolemmal glycogen accumulation. He harbored a novel insertion mutation in the PHKA1 gene that resulted in premature termination of the αM subunit close to the C-terminus. Compared with previously reported cases, his reduction in PHK activity was relatively mild.

Mutations of the caveolin 3 gene cause autosomal dominant limb-girdle muscular dystrophy (LGMD)1C. In mice, overexpression of mutant caveolin 3 leads to loss of caveolin 3 and results in myofiber hypotrophy in association with activation of neuronal nitric oxide synthase (nNOS) at the sarcolemma. Here, we show that caveolin 3 directly bound to nNOS and suppressed its phosphorylation-dependent activation at a specific residue, Ser1412 in the nicotinamide adenine dinucleotide phosphate (NADPH)-flavin adenine dinucleotide (FAD) module near the C-terminus of the reduction domain, in vitro. Constitutively active nNOS enhanced myoblast fusion, but not myogenesis, in vitro. Phosphorylation-dependent activation of nNOS occurred in muscles from caveolin 3-mutant mice and LGMD1C patients. Mating with nNOS-mutant mice exacerbated myofiber hypotrophy in the caveolin 3-mutant mice. In nNOS-mutant mice, regenerating myofibers after cardiotoxin injury became hypotrophic with reduced myoblast fusion. Administration of NO donor increased myofiber size and the number of myonuclei in the caveolin 3-mutant mice. Exercise also increased myofiber size accompanied by phosphorylation-dependent activation of nNOS in wild-type and caveolin 3-mutant mice. These data indicate that caveolin 3 inhibits phosphorylation-dependent activation of nNOS, which leads to myofiber hypertrophy via enhancing myoblast fusion. Hypertrophic signaling by nNOS phosphorylation could act in a compensatory manner in caveolin 3-deficient muscles.

ObjectiveThe aim of this study was to evaluate the efficacy and safety of high-dose taurine supplementation for prevention of stroke-like episodes of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), a rare genetic disorder caused by point mutations in the mitochondrial DNA that lead to a taurine modification defect at the first anticodon nucleotide of mitochondrial tRNALeu(UUR), resulting in failure to decode codons accurately.MethodsAfter the nationwide survey of MELAS, we conducted a multicentre, open-label, phase III trial in which 10 patients with recurrent stroke-like episodes received high-dose taurine (9 g or 12 g per day) for 52 weeks. The primary endpoint was the complete prevention of stroke-like episodes during the evaluation period. The taurine modification rate of mitochondrial tRNALeu(UUR)was measured before and after the trial.ResultsThe proportion of patients who reached the primary endpoint (100% responder rate) was 60% (95% CI 26.2% to 87.8%). The 50% responder rate, that is, the number of patients achieving a 50% or greater reduction in frequency of stroke-like episodes, was 80% (95% CI 44.4% to 97.5%). Taurine reduced the annual relapse rate of stroke-like episodes from 2.22 to 0.72 (P=0.001). Five patients showed a significant increase in the taurine modification of mitochondrial tRNALeu(UUR)from peripheral blood leukocytes (PConclusionsThe current study demonstrates that oral taurine supplementation can effectively reduce the recurrence of stroke-like episodes and increase taurine modification in mitochondrial tRNALeu(UUR)in MELAS.Trial registration numberUMIN000011908.

Familial amyloid polyneuropathy (FAP) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of the transthyretin (TTR) gene, and usually shows sensory-dominant polyneuropathy and autonomic neuropathy at the initial stage. The pathogenesis of this neuropathy remains unknown, although several mechanisms, including mechanical compression, vessel occlusion, TTR toxicity and Schwann cell dysfunction have been proposed. We describe a patient with late-onset FAP caused by a TTR E61K mutation. Amyloid deposits were not detected in the endoneurium or perineurium of the sural nerve 7years after the onset of the disease, but a marked loss of nerve fibers was observed in the sural nerve. TTR-derived amyloid deposits were confirmed in the peroneus brevis muscle, salivary gland and heart tissue. DNA analysis revealed a heterozygous mutation in TTR. These findings suggest that proximal parts of the peripheral nervous system might be strongly affected by TTR aggregates or amyloid fibrils, and that the blood-nerve barrier in distal parts of peripheral nerves are initially preserved in this patient. This case indicates that several biopsy sites other than nerves may be helpful and necessary for the diagnosis of TTR amyloidosis in mild or late-onset FAP.

Background The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning β-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa β-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved β-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of β-dystroglycan. However, this has remained uninvestigated in vivo. Methods We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of β-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. Results Unexpectedly, β-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave β-dystroglycan. Conclusions Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of β-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to β-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.

We developed a simplified and sensitive method to identify Alzheimer's disease (AD) biomarker candidates by a quantitative and targeted proteomic analysis (combination of liquid chromatography tandem mass spectrometry and multiplexed-multiple reaction monitoring/selected reaction monitoring analysis) of culture media from neurons differentiated from induced pluripotent stem cells (iPSCs) established from AD patients. We found that alpha-1-acid glycoprotein (ORM1) was decreased in the culture media of AD-iPSC-derived neurons, consistent with previous observations for AD patient cerebrospinal fluid, thus validating our new strategy. Moreover, our method is applicable for identifying biomarker candidates for other neurodegenerative disorders using patient-derived iPSCs.

Previously, we reported that MyoD, a master gene for myogenic cells, could efficiently convert primary skin fibroblasts into myoblasts and myotubes, thereby effecting direct reprogramming. In this study, we further demonstrated that MyoD-expressing primary fibroblasts displayed rapid movement in culture, with a movement velocity that was significantly faster, almost four times, than mouse primary myoblasts. MyoD-transduced cells obtained the characteristics of Ca2 + release and electrically-stimulated contraction, which was comparable to C2C12 myotubes, suggesting that the essential features of muscle were observed in the transduced cells. Furthermore, the ability to fuse to the host myoblasts means that gene transfer from MyoD-transduced cells to host muscle cells could be obtained by cell fusion. In comparison with the iPS method (indirect reprogramming), our transduction method has a low risk for tumorigenesis and carcinogenesis because the starting cells are fibroblasts and the transduced cells are myoblasts, both normal and mortal cells. Accordingly, MyoD transduction of human skin fibroblasts using the adenoviral vector is a simple, inexpensive and promising candidate as a new cell transplantation therapy for patients with muscular disorders., Highlights • Adenoviral MyoD vector transduced fibroblasts directly to myoblasts. • Myoblast cells were well differentiated into functional muscle cells. • Direct reprogramming is cost-effective and safe compared to iPS method. • Cell fusion and high motility were observed in MyoD-transduced cells. • Transduced cells are candidates for cell transplantation in muscle disorders.

A 59-year-old woman developed early-onset, slowly progressive dementia and spastic paraplegia. positron emission tomography (PET) imaging revealed a large reduction in the level of glucose uptake without amyloid deposition in the cerebral cortex. We identified a homozygous microdeletion within the amyloid β (Aβ) coding sequence in the amyloid precursor protein (APP) gene (c.2080_2082delGAA, p.E693del) in three affected siblings and a heterozygous microdeletion in an unaffected sibling. The identical mutation was previously reported in the first Alzheimer's pedigree without amyloid deposits. Furthermore, an increase in high-molecular-weight Aβ-reactive bands was detected in the patient's CSF. Our findings suggest that soluble Aβ-oligomers induce neuronal toxicity, independent of insoluble Aβ fibrils.