Serum albumin redox state as an indicator of dietary protein intake among community-dwelling older adults.

BACKGROUND AND AIMS: Serum markers capable of detecting mild levels of undernutrition, such as insufficient dietary protein intake (IDPI), have not been established among community-dwelling older adults. Although the serum albumin redox state, expressed as the ratio of reduced albumin (Alb) to total Alb (the reduced albumin ratio), has the potential to overcome this challenge, empirical epidemiological data are lacking. This study aimed to investigate the association between a serum reduced Alb ratio and dietary protein intake among community-dwelling older adults. METHODS: This study analyzed cross-sectional data from 1,005 community dwelling population (572 males and 433 females) aged 70-84 years who participated in the Itabashi Longitudinal Study on Aging. Exclusion criteria included participants with incomplete data, individuals with a history of kidney disease and high C-reactive protein (CRP) levels. The dietary protein intake was estimated using validated food frequency questionnaires. The IDPI was defined as not meeting the level recommended by the Dietary Reference Intakes for Japanese (Men ≥60 g/day, Women ≥50 g/day). RESULTS: IDPI was observed in 14.1% of the study population. Logistic regression analyses adjusted for sex, age, body weight and malnutrition showed that a serum reduced Alb ratio was significantly associated with IDPI (odds ratio = 0.962, 95% confidence interval = 0.926-0.999), whereas serum albumin concentration was not (odds ratio = 0.549, 95% confidence interval = 0.285-1.061). CONCLUSIONS: A serum reduced Alb ratio would be a useful indicator of protein insufficiency among community-dwelling older adults.

[Pathogenesis of MuSK Antibody-positive Myasthenia Gravis].

Long-term remission is rare in patients with myasthenia gravis (MG), and health-related quality of life is lower in patients with MG than in healthy individuals. Approximately 5% of patients with MG show positive results on muscle-specific kinase (MuSK) antibody testing and usually have severe symptoms, refractory disease, residual muscle atrophy, and poor prognosis. Recent studies that have investigated the pathogenesis of MuSK antibody-positive MG have reported contributors to treatment refractoriness in cases of MG. In this article, we review the most recent findings.

Sympathetic modulation of hindlimb muscle contractility is altered in aged rats.

It has recently been demonstrated that reflex excitation of muscle sympathetic nerves triggered by muscle contraction contributes to the maintenance of tetanic force (TF) in rat hindlimb muscles. We hypothesized that this feedback mechanism between the contraction of hindlimb muscles and the lumbar sympathetic nerves declines during aging. In this study, we examined the contribution of sympathetic nerves on skeletal muscle contractility in young adult (4-9 months old, n = 11) and aged (32-36 months old, n = 11) male and female rats. The tibial nerve was electrically stimulated to measure the TF of the triceps surae muscles resulting from motor nerve activation before and after cutting or stimulating (at 5-20 Hz) the lumbar sympathetic trunk (LST). The TF amplitude decreased by cutting the LST in the young and aged groups; however, the magnitude of the decrease in TF following transection of the LST in the aged rats (6.2%) was significantly (P = 0.02) smaller compared with that in the young rats (12.9%). The TF amplitude was increased by LST stimulation at ≥ 5 Hz in the young and ≥ 10 Hz in the aged groups. The overall TF response to LST stimulation was not significantly different between the two groups; however, an increase in muscle tonus resulting from LST stimulation, independent of motor nerve stimulation, was significantly (P = 0.03) greater in aged compared with young rats. The sympathetic contribution to support motor nerve-induced muscle contraction declined, whereas sympathetic-mediated muscle tonus, independent of motor nerve activity, was augmented in aged rats. These changes in sympathetic modulation of hindlimb muscle contractility may underlie the reduction of skeletal muscle strength during voluntary contraction and rigidity of motion during senescence.

Proteolytic ectodomain shedding of muscle-specific tyrosine kinase in myasthenia gravis.

Autoantibodies to muscle-specific tyrosine kinase (MuSK) proteins at the neuromuscular junction (NMJ) cause refractory generalized myasthenia gravis (MG) with dyspnea more frequently than other MG subtypes. However, the mechanisms via which MuSK, a membrane protein locally expressed on the NMJ of skeletal muscle, is supplied to the immune system as an autoantigen remains unknown. Here, we identified MuSK in both mouse and human serum, with the amount of MuSK dramatically increasing in mice with motor nerve denervation and in MG model mice. Peptide analysis by liquid chromatography-tandem-mass spectrometry (LC-MS/MS) confirmed the presence of MuSK in both human and mouse serum. Furthermore, some patients with MG have significantly higher amounts of MuSK in serum than healthy controls. Our results indicated that the secretion of MuSK proteins from muscles into the bloodstream was induced by ectodomain shedding triggered by neuromuscular junction failure. The results may explain why MuSK-MG is refractory to treatments and causes rapid muscle atrophy in some patients due to the denervation associated with Ab-induced disruption of neuromuscular transmission at the NMJ. Such discoveries pave the way for new MG treatments, and MuSK may be used as a biomarker for other neuromuscular diseases in preclinical studies, clinical diagnostics, therapeutics, and drug discovery.

Maintenance of contractile force of the hind limb muscles by the somato-lumbar sympathetic reflexes.

This study aimed to clarify whether the reflex excitation of muscle sympathetic nerves induced by contractions of the skeletal muscles modulates their contractility. In anesthetized rats, isometric tetanic contractions of the triceps surae muscles were induced by electrical stimulation of the intact tibial nerve before and after transection of the lumbar sympathetic trunk (LST), spinal cord, or dorsal roots. The amplitude of the tetanic force (TF) was reduced by approximately 10% at 20 min after transection of the LST, spinal cord, or dorsal roots. The recorded postganglionic sympathetic nerve activity from the lumbar gray ramus revealed that both spinal and supraspinal reflexes were induced in response to the contractions. Repetitive electrical stimulation of the cut peripheral end of the LST increased the TF amplitude. Our results indicated that the spinal and supraspinal somato-sympathetic nerve reflexes induced by contractions of the skeletal muscles contribute to the maintenance of their own contractile force.

Muscle fiber type specific alterations of mitochondrial respiratory function and morphology in aged female mice.

Mitochondrial dysfunction is considered to be a major cause of sarcopenia, defined as age-related muscle fiber atrophy and muscle weakness, as reduced mitochondrial respiration and morphological changes such as ragged red fibers (RRFs) are observed in aging muscles. However, the role of mitochondrial dysfunction in sarcopenia is not fully elucidated. Although previous studies have suggested that aging has a fiber type-specific effect on mitochondrial function, little is known about mitochondrial changes in individual fiber types. Here, we used C57BL/6NCr female mice to identify fiber type-specific pathological changes, examine the significance of pathological changes in sarcopenia, and identify possible mechanisms behind mitochondrial changes in slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL). We observed reduced type I fiber-specific mitochondrial respiratory enzyme activity, impaired respiration, and subsarcolemmal mitochondrial accumulation in aged SOL, which was different from RRFs. These pathological alterations were not directly associated with fiber atrophy. Additionally, we found increased oxidative stress markers in aged SOL, suggesting that oxidative stress is involved in the pathological and functional changes in mitochondria. Meanwhile, obvious mitochondrial changes were not seen in aged EDL. Thus, age-related mitochondrial dysfunction is specific to the fiber type and may correlate with the muscle quality rather than the muscle mass.

Tbx1 regulates inherited metabolic and myogenic abilities of progenitor cells derived from slow- and fast-type muscle.

Skeletal muscle is divided into slow- and fast-type muscles, which possess distinct contractile and metabolic properties. Myogenic progenitors associated with each muscle fiber type are known to intrinsically commit to specific muscle fiber lineage during embryonic development. However, it is still unclear whether the functionality of postnatal adult myogenic cells is attributable to the muscle fiber in which they reside, and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level. In this study, we isolated adult satellite cells from slow- and fast-type muscle individually and observed that satellite cells from each type of muscle generated myotubes expressing myosin heavy chain isoforms similar to their original muscle, and showed different metabolic features. Notably, we discovered that slow muscle-derived cells had low potential to differentiate but high potential to self-renew compared with fast muscle-derived cells. Additionally, cell transplantation experiments of slow muscle-derived cells into fast-type muscle revealed that slow muscle-derived cells could better contribute to myofiber formation and satellite cell constitution than fast muscle-derived cells, suggesting that the recipient muscle fiber type may not affect the predetermined abilities of myogenic cells. Gene expression analyses identified T-box transcriptional factor Tbx1 as a highly expressed gene in fast muscle-derived myoblasts. Gain- and loss-of-function experiments revealed that Tbx1 modulated muscle fiber types and oxidative metabolism in myotubes, and that Tbx1 stimulated myoblast differentiation, but did not regulate myogenic cell self-renewal. Our data suggest that metabolic and myogenic properties of myogenic progenitor cells vary depending on the type of muscle from which they originate, and that Tbx1 expression partially explains the functional differences of myogenic cells derived from fast-type and slow-type muscles.

Practical Anatomy of the Neuromuscular Junction in Health and Disease.

Neuromuscular junctions (NMJs) form between nerve terminals of spinal cord motor neurons and skeletal muscles, and perisynaptic Schwann cells and kranocytes cap NMJs. One muscle fiber has one NMJ, which is innervated by one motor nerve terminal. NMJs are excitatory synapses that use P/Q-type voltage-gated calcium channels to release the neurotransmitter acetylcholine. Acetylcholine receptors accumulate at the postsynaptic specialization called the end plate on the muscle fiber membrane, the sarcolemma. Proteins essential for the organization of end plates include agrin secreted from nerve terminals, Lrp4 and MuSK receptors for agrin, and Dok-7 and rapsyn cytosolic proteins in the muscle.

Immunization of mice with LRP4 induces myasthenia similar to MuSK-associated myasthenia gravis.

Since the first report of experimental animal models of myasthenia gravis (MG) with autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), there have not been any major reports replicating the pathogenicity of anti-LRP4 antibodies (Abs). Recent clinical studies have cast doubt on the specificity and pathogenicity of anti-LRP4 antibodies for MG, highlighting the need for further research. In this study, we purified antigens corresponding to the extracellular region of human LRP4 stably expressed with chaperones in 293 cells and used these antigens to immunize female A/J mice. Immunization with LRP4 protein caused mice to develop myasthenia having similar electrophysiological and histological features as are observed in MG patients with circulating Abs against muscle-specific kinase (MuSK). Our results clearly demonstrate that active immunization of mice with LRP4 proteins causes myasthenia similar to the MG induced by anti-MuSK Abs. Further experimental and clinical studies are required to prove the pathogenicity of anti-LRP4 Abs in MG patients.

Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice.

Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography.

Molecular characterization of a novel hepatitis E virus (HEV) strain obtained from a wild boar in Japan that is highly divergent from the previously recognized HEV strains.

Although a consensus classification system for hepatitis E virus (HEV) genotypes is currently unavailable, HEV variants (JBOAR135-Shiz09 and wbJOY_06) from wild boars (Sus scrofa leucomystax) have provisionally been classified into two novel genotypes (5 and 6). While performing a survey of HEV infections among 566 wild boars that were captured in Japan between January 2010 and August 2013, we found 24 boars (4.2%) with ongoing HEV infections: 13 had genotype 3 HEV, 10 had genotype 4 HEV and the remaining boar possessed a novel HEV variant (designated wbJNN_13). The entire wbJNN_13 genome comprised 7247 nucleotides excluding the poly(A) tail, and was highly divergent from known genotype 1 to 4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits by 22.4-28.2%, JBOAR135-Shiz09 and wbJOY_06 by 19.6-21.9% and rat, ferret, bat and avian HEV isolates by 40.9-46.1% over the entire genome. Phylogenetic trees confirmed that wbJNN_13 is distantly related to all known HEV isolates. A Simplot analysis revealed no significant recombination among the existing HEV strains. These results indicate the presence of at least three genetic lineages of presumably boar-indigenous HEV strains. Further studies to fully understand the extent of the genomic heterogeneity of HEV variants infecting wild boars are warranted.

Layer-specific dilation of penetrating arteries induced by stimulation of the nucleus basalis of Meynert in the mouse frontal cortex.

To clarify mechanisms through which activation of the nucleus basalis of Meynert (NBM) increases cerebral cortical blood flow, we examined whether cortical parenchymal arteries dilate during NBM stimulation in anesthetized mice. We used two-photon microscopy to measure the diameter of single penetrating arteries at different depths (~800 µm, layers I to V) of the frontal cortex, and examined changes in the diameter during focal electrical stimulation of the NBM (0.5 ms at 30 to 50 µA and 50 Hz) and hypercapnia (3% CO2 inhalation). Stimulation of the NBM caused diameter of penetrating arteries to increase by 9% to 13% of the prestimulus diameter throughout the different layers of the cortex, except at the cortical surface and upper part of layer V, where the diameter of penetrating arteries increased only slightly during NBM stimulation. Hypercapnia caused obvious dilation of the penetrating arteries in all cortical layers, including the surface arteries. The diameters began to increase within 1 second after the onset of NBM stimulation in the upper cortical layers, and later in lower layers. Our results indicate that activation of the NBM dilates cortical penetrating arteries in a layer-specific manner in magnitude and latency, presumably related to the density of cholinergic nerve terminals from the NBM.

RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

OBJECTIVE: Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. METHODS: Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. RESULTS: COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. CONCLUSION: We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases.

Mechanisms associated with the pathogenicity of antibodies against muscle-specific kinase in myasthenia gravis.

The presence of autoantibodies against muscle-specific kinase (MuSK) at the neuromuscular junction (NMJ) results in myasthenia gravis (MG). MuSK antibody-associated MG (MuSK MG) patients often have severe symptoms, including bulbar dysfunction, respiratory insufficiency and atrophy of the facial and tongue muscles. MuSK antibodies in MG patients predominantly belong to the IgG4 subclass, and the unique properties of IgG4 antibodies are directly associated with the pathogenic mechanisms of MuSK MG. Histopathological studies in animal models of MuSK MG have revealed that anti-MuSK antibodies cause contraction of motor terminals, significant loss of acetylcholine receptor (AChR) expression, and a reduction in synaptic folds at the postsynaptic membrane in the absence of complement involvement. Failure of neuromuscular transmission at pre- and postsynaptic membranes of the NMJs has been observed in both patients and animal models of MuSK MG. A murine model of MuSK-MG revealed the mechanisms underlying cholinergic hypersensitivity after administration of acetylcholinesterase inhibitors, which has also been observed in MuSK-MG patients. Further studies of this model have provided evidence suggesting that 3,4-diaminopyridine may be effective as a symptomatic therapy for MuSK MG.

[Aging of muscle].

Sarcopenia has attracted attention recently in various fields of clinical and basic research to understand the mechanisms and developing new methods for diagnosis and prevention. We review pathological features in motor neurons, synapses, muscle fibers, satellite cells associated with sarcopenia based on recent findings. The author believes that the pathological evidence comes to an important measure in the development of research on sarcopenia.