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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.

Given a current point, a final destination, and visiting data point categories on a trip, a sequenced route query finds the shortest route which meets the query condition. Several algorithms have been proposed targeting to this query. When this query is used in real applications, the popularity of the visiting points (for example, restaurant, shopping center, etc.) in a trip are also important besides the length of the route. Therefore, a recommendation query method that evaluates the goodness by multiple criteria, such as the length and the popularity, is requested. This type of queries has been called Top-k query. This paper proposes a fast Top-k query method targeting to sequenced route queries.

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell-membrane receptor (neuropilin2-plexinA3) → myogenin-myocyte enhancer factor 2D → slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2-Sema3Afl°x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key “commitment factor” for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease.

Mutations in the gene MAPT encoding tau, a microtubules-associated protein, cause a subtype of familial neurodegenerative disorder, known as frontotemporal lobar degeneration tauopathy (FTLD-Tau), which presents with dementia and is characterized by atrophy in the frontal and temporal lobes of the brain. Although induced pluripotent stem cell (iPSC) technology has facilitated the investigation of phenotypes of FTLD-Tau patient neuronal cells in vitro, it remains unclear how FTLD-Tau patient neurons degenerate. Here, we established neuronal models of FTLD-Tau by Neurogenin2-induced direct neuronal differentiation from FTLD-Tau patient iPSCs. We found that FTLD-Tau neurons, either with an intronic MAPT mutation or with an exonic mutation, developed accumulation and extracellular release of misfolded tau followed by neuronal death, which we confirmed by correction of the intronic mutation with CRISPR/Cas9. FTLD-Tau neurons showed dysregulation of the augmentation of Ca[2+] transients evoked by electrical stimulation. Chemogenetic or pharmacological control of neuronal activity-relevant Ca[2+] influx by the introduction of designer receptors exclusively activated by designer drugs (DREADDs) or by the treatment with glutamate receptor blockers attenuated misfolded tau accumulation and neuronal death. These data suggest that neuronal activity may regulate neurodegeneration in tauopathy. This FTLD-Tau model provides mechanistic insights into tauopathy pathogenesis and potential avenues for treatments., 患者さん由来iPS細胞とゲノム編集技術を用いて, 認知症・パーキンソニズムを来す前頭側頭葉変性症のメカニズムの一端を解明. 京都大学プレスリリース. 2016-11-15.

Caveolin-3, the principal scaffold protein in sarcolemmal caveolae, regulates signal transduction and vesicular trafficking. Dominant-negative mutations in the caveolin-3 gene (CAV3) cause autosomal dominant limb-girdle muscular dystrophy 1C (LGMD1C) and autosomal dominant rippling muscle disease (AD-RMD). Myostatin, a member of the muscle-specific transforming growth factor (TGF)-β family, negatively regulates muscle growth and volume. We recently showed that wild-type caveolin-3 binds to and inhibits TGF-β type I receptor (TβRI), thereby suppressing intracellular TGF-β signaling. In contrast, LGMD1C-causing mutant caveolin-3 activates TβRI, resulting in muscle atrophy. Recently, small-molecule compounds suppressing activation of TβRI, also known as activin receptor-like kinase 5 (ALK5), have been developed as anticancer agents. Oral administration of a TβRI inhibitor, Ki26894, ameliorates muscle atrophy and weakness in a caveolin-3-deficient LGMD1C mouse model. The therapeutic effect of Ki26894 is associated with a reduction in TGF-β signaling and an increase in the number of muscle precursor satellite cells. This suggests that the caveolin-3/TβRI signaling pathway plays an important role in the pathogenesis of LGMD1C and that it regulates skeletal muscle size by controlling the number of muscle precursor cells. Consequently, drugs that target the TGF-β pathway may have therapeutic potential for diseases characterized by muscle atrophy.

This paper proposes a fast safe-region generation method for several kinds of vicinity queries including distance range queries, set k nearest neighbor (NN) queries, and ordered kNN queries. When a user is driving a car on a road network, he/she wants to know objects located in a vicinity of the car. However, the result is changing according to the movement of the car, and therefore, the up-to-date result is always expected, and requested to the server. On the other hand, frequent requests for updating results to the server cause heavy loading. To cope with this problem efficiently, the idea of safe-region has been proposed. This paper proposes a fast generation method of the safe-region applicable to several types of vicinity queries. Through experimental evaluations, the proposed algorithm achieves a great performance in terms of processing times, and is one or two orders of magnitude faster than existing algorithms.

Given a current point q, a final destination point d, and a set of data points categories to be visited during a trip, a trip route planning query (TRPQ) determines the shortest route from q to d that includes data points one each from given data points categories. After the optimal route is determined, a user may sometimes deviate from the route. In such cases, a new route is needed from the new current position \(q'\). For simple continuous queries, a method to calculate the safe-region with the query result has been proposed. The safe-region is the area where the query result is not changed. This paper proposes two efficient methods called preceeding rival addition (PRA) and tardy rival addition (TRA) algorithms to obtain the safe-region for TRPQ queries, and a basic method as a comparison method. In two proposed algorithms, PRA gives an accurate safe-region, and TRA gives an approximate safe-region very fast. We evaluate the efficiency of the proposed methods experimentally comparing to the basic method for TRPQ query.

Myostatin, an endogenous negative regulator of skeletal muscle mass, is a therapeutic target for muscle atrophic disorders. Here, we identified minimum peptides 2 and 7 to effectively inhibit myostatin activity, which consist of 24 and 23 amino acids, respectively, derived from mouse myostatin prodomain. These peptides, which had the propensity to form α-helix structure, interacted to myostatin with KD values of 30-36 nM. Moreover, peptide 2 significantly increased muscle mass in Duchenne muscular dystrophy model mice.

Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

This paper proposes a bichromatic homogeneous vicinity query method and its efficient algorithm. Several query algorithms have been proposed individually including set k nearest neighbor (NN) query, ordered NN query, bichromatic reverse k NN query, and distance range query. When these types of queries are performed in the road network distance, all take long processing time. The algorithm proposed in this paper gives a unified procedure that can be applied to these queries with a different query condition for each, and it reduces the processing time drastically. The basic idea of the algorithm is to expand the region on the road network gradually while verifying the query condition. It is the most time consuming process, and to improve the deficiency in verification step, an efficient road network distance search method is proposed. Through extensive experiments, the proposed algorithm significantly improves the performance in terms of processing time by nearly two orders of magnitude.