Shuuichi Mori

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.

Intracellular and Extracellular ATP Coordinately Regulate the Inverse Correlation between Osteoclast Survival and Bone Resorption

Background: Mature osteoclasts with a spontaneous tendency toward apoptosis resorb bone efficiently during their short lifespan. Results: Released ATP from intracellular stores has a negative impact on the bone resorption activity of osteoclasts by altering their cytoskeletal structures. Conclusion: ATP depletion leads to osteoclastic bone resorption. Significance: This study provides a new direction for investigating the mechanisms involved in physiological and pathological 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-xL 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.

Ezetimibe decreases SREBP-1c expression in liver and reverses hepatic insulin resistance in mice fed a high-fat diet

Ezetimibe inhibits intestinal cholesterol absorption, thereby reducing serum cholesterol. Recent studies suggest that ezetimibe affects liver steatosis and insulin resistance. We investigated the impact of ezetimibe on insulin sensitivity and glucose metabolism in C57BL/6 mice. We analyzed 4 mouse groups fed the following diets: normal chow (4% fat) for 12 weeks, normal chow for 10 weeks followed by normal chow plus ezetimibe for 2 weeks, high-fat chow (32% fat) for 12 weeks, and high-fat chow for 10 weeks followed by high-fat chow plus ezetimibe for 2 weeks. In the normal chow + ezetimibe group, ezetimibe had no impact on body weight, fat mass, lipid metabolism, liver steatosis, glucose tolerance, or insulin sensitivity. In the high-fat chow + ezetimibe group, ezetimibe had no impact on body weight or fat mass but significantly decreased serum low-density lipoprotein cholesterol, triglyceride, and glutamate pyruvate transaminase levels; liver weight; hepatic triglyceride content; and hepatic cholesterol content and increased the hepatic total bile acid content. In association with increases in IRS-2 and Akt phosphorylation, ezetimibe ameliorated hepatic insulin resistance in the high-fat chow + ezetimibe group, but had no effect on insulin sensitivity in primary cultured hepatocytes. A DNA microarray and Taqman polymerase chain reaction revealed that ezetimibe up-regulated hepatic SREBP2 and SHP expression and down-regulated hepatic SREBP-1c expression. SHP silencing mainly in the liver worsened insulin resistance, and ezetimibe protected against insulin resistance induced by down-regulation of SHP. Ezetimibe down-regulated SREBP-1c in the liver and reversed hepatic insulin resistance in mice fed a high-fat diet.

3,4-Diaminopyridine improves neuromuscular transmission in a MuSK antibody-induced mouse model of myasthenia gravis

This study investigated the effect of 3,4-diaminopyridine (3,4-DAP), a potent potentiator of transmitter release, on neuromuscular transmission in vivo in a mouse model of myasthenia gravis (MG) caused by antibodies against muscle-specific kinase (MuSK; MuSK-MG) and ex vivo in diaphragm muscle from these mice. 3,4-DAP significantly improved neuromuscular transmission, predominantly by increasing acetylcholine (ACh) release, supporting presynaptic potentiation as an effective treatment strategy for MuSK-MG patients who have defective transmitter release. In MuSK-MG, we suggest that only low-dose acetylcholinesterase (AChE) inhibitors be used to avoid side effects, and we propose that 3,4-DAP may be effective as a symptomatic therapy.

Divalent and monovalent autoantibodies cause dysfunction of MuSK by distinct mechanisms in a rabbit model of myasthenia gravis.

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is required for the formation and maintenance of neuromuscular junctions (NMJs). Although autoantibodies against MuSK have been demonstrated to cause myasthenia gravis (MG), the underlying pathogenic mechanism remains unclear because a major subclass of these antibodies is functionally monovalent. We investigated the pathogenic role of MuSK antibodies in the onset of MG in vivo and in vitro. Ultrastructural visualization of NMJs in paretic rabbits with MuSK antibodies indicated that postsynaptic membranes were preserved, despite a significant loss of complexity in the convoluted synaptic folds. In addition, an in vitro assay indicated that both divalent and monovalent antibodies from paretic rabbits could interfere with agrin-induced acetylcholine receptor (AChR) clustering in cultured myotubes. Furthermore, in the absence of agrin, divalent antibodies induced MuSK phosphorylation and accelerated downregulation of Dok-7, an essential intracellular MuSK binding protein, while monovalent antibodies inhibited agrin-induced phosphorylation of MuSK, thus demonstrating distinct molecular mechanisms underlying the MuSK dysfunction induced by these two types of antibodies. Taken together, these findings suggest that complement activation is not necessary for the MG onset and that both divalent and monovalent antibodies may cause MG in vivo by inducing MuSK dysfunction.

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.

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.

Role of the liver in glucose homeostasis in PI 3-kinase p85α-deficient mice

Phosphoinositide 3-kinase (PI3K) p85alpha-deficient mice exhibit hypoglycemia as a result of increased insulin sensitivity and glucose uptake in peripheral tissues. Although PI3K is central to the metabolic actions of insulin, its mechanism of action in liver is not well understood. In the present study, we investigated hepatic insulin signaling and glucose homeostasis in p85alpha-deficient and wild-type mice. In the livers of p85alpha-deficient mice, p50alpha played a compensatory role in insulin-stimulated PI3K activation by binding to insulin receptor substrate (IRS)-1/2. In p85alpha-deficient mice, the ratio of p50alpha over p110 catalytic subunit of PI3K in the liver was higher than in the muscles. PI3K activity associated with IRS-1/2 was not affected by the lack of p85alpha in the liver. Insulin-stimulated Akt and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activities in the liver were similar in p85alpha-deficient and wild-type mice. A hyperinsulinemic-euglycemic clamp study revealed that the glucose infusion rate and the rate of disappearance were higher in p85alpha-deficient mice than in wild-type mice but that endogenous glucose production tended to be higher in p85alpha-deficient mice than in wild-type mice. Consistent with this finding, the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in livers after fasting was higher in p85alpha-deficient mice than in wild-type mice. After mice were fasted, the intrahepatic glucose-6-phosphate level was almost completely depleted in p85alpha-deficient mice. The glycogen content fell to nearly zero as a result of glycogenolysis shortly after the initiation of fasting in p85alpha-deficient mice. The absence of an increase in insulin-stimulated PI3K activation in the liver of p85alpha-deficient mice, unlike the muscles, may be associated with the molecular balance between the regulatory subunit and the catalytic subunit of PI3K. Gluconeogenesis was rather elevated in p85alpha-deficient mice, compared with in wild-type mice, and the liver seemed to partially compensate for the increase in glucose uptake in peripheral tissues.

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.

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.