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.

[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.

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.

Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption.

Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption.

Antibodies against muscle-specific kinase impair both presynaptic and postsynaptic functions in a murine model of myasthenia gravis.

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

[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.

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.

Induction of myasthenia by immunization against muscle-specific kinase.

Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.

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.

A mitotic kinase TOPK enhances Cdk1/cyclin B1-dependent phosphorylation of PRC1 and promotes cytokinesis.

A MAPKK-like mitotic kinase, TOPK, implies the formation of mitotic spindles and spindle midzone and accomplishing cytokinesis, however, its underlying mechanism remains unclear. A microtubule bundling protein, PRC1, plays a pivotal role in the formation of mitotic spindles and spindle midzone. Because of their functional resemblance, we attempted to clarify the links between these two molecules. TOPK supported mitotic advance via the cdk1/cyclin B1-dependent phosphorylation of PRC1. TOPK induced the phosphorylation of PRC1 at T481 in vivo, however, TOPK did not phosphorylate PRC1 in vitro. TOPK induced the phosphorylation of PRC1 at T481 only when the cdk1/cyclin B1 existed simultaneously in vitro. Both the enzymatic activity of TOPK and association competence of TOPK with PRC1 were mandatory for this phosphorylation. TOPK binds to cdk1/cyclin B1, microtubules and PRC1 via its unique region near the C terminus. TOPK co-localized closely with cdk1 throughout the cell cycle in vivo. Collectively, these data indicate that TOPK, which makes a kinase-substrate complex with cdk1/cyclin B1 and PRC1 on microtubules during mitosis, enhances the cdk1/cyclin B1-dependent phosphorylation of PRC1 and thereby strongly promotes cytokinesis.

Myasthenia gravis experimentally induced with muscle-specific kinase.

Here we present the first evidence that muscle-specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG-like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin-independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin-independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin-independent AChR clustering. MuSK plays multiple roles in pre-patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.

[Myasthenia gravis induced by autoantibodies against MuSK].

Myasthenia gravis (MG) is caused by the failure of neuromuscular transmission mediated by autoantibodies. That is, the binding of autoantibodies to postsynaptic membranes in neuromuscular junctions (NMJ) results in weakening of the ocular, bulbar and limb muscles and produces the characteristic syndrome of MG. About 80 to 85% of patients witth MG have autoantibodies against acetylcholine receptors (AChR). Antibodies against muscle-specific kinase (MuSK) have been found in 30% of MG patients without AChR antibodies. Here we describe recent progress toward understanding the pathogenic role of MuSK antibodies in the decline of muscle strength that typifies MG.

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.

Anti-alkaline phosphatase antibody positive myasthenia gravis.

Anti-alkaline phosphatase antibody (AP Ab) was specific in 9% of 249 anti-acetylcholine receptor (AChR) Ab-positive myasthenia gravis (MG) (SPMG) patients but not in patients with AChR Ab-negative MG (SNMG), other neurological and immunological diseases, or healthy volunteers. No cross-reactivity and no significant titer correlation were found between AP Ab and AChR Ab. We confirmed immunologically by radioimmunoassay and western blot analysis the presence of antibodies directed against AP. AP Ab-positive SPMG patients were characterized clinically as having female predominance and a more severe form of generalized MG than AP Ab-negative SPMG patients, and about half required artificial ventilation at maximum severity. AP Ab's pathogenic role in MG is yet unclarified, but our findings show AP to be a novel antigen among the various autoantigens present in MG patients and in whom AP Ab may modify clinical symptoms.

Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting.

The ribosomal frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed -1 frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29, 4079-4088]. Here, we have employed site-directed mutagenesis and RNA structure probing to characterize the Edr signal. We began by confirming the functionality and magnitude of the signal and the role of a GGGAAAC motif as the slippery sequence. Subsequently, we derived a model of the Edr stimulatory RNA and assessed its similarity to those stimulatory RNAs found at viral frameshift sites. We found that the structure is an RNA pseudoknot possessing features typical of retroviral frameshifter pseudoknots. From these experiments, we conclude that the Edr signal and by inference, the human orthologue PEG10, do not represent a novel 'cellular class' of programmed -1 ribosomal frameshift signal, but rather are similar to viral examples, albeit with some interesting features. The similarity to viral frameshift signals may complicate the design of antiviral therapies that target the frameshift process.

[Pathogenic mechanisms of myasthenia gravis induced by antibodies against muscle-specific kinase].

Antibodies to acetylcholine receptor (AChR) are major cause of the human autoimmune disease, myasthenia gravis (MG). Additionally, autoantibodies against Muscle-specific kinase (MuSK) were found in a proportion of patients with generalized MG. After the identification of MuSK antibodies in MG patients, laboratory test for measuring antibodies to MuSK is now required to confirm the diagnosis of MG and the clinical treatment as well as AChR antibodies. MuSK is critical to the clustering of AChR and plays multiple roles at neuromuscular junctions (NMJ). However, it has been dispute concerning the pathogenicity of MuSK antibodies in muscle weakness of MG, as the experimental autoimmune MG caused by MuSK antibodies was absent. Here we describe the recent progress to understand the pathogenic roles of MuSK antibodies in muscle weakness of experimental animals induced by MuSK protein.