Rimmed Vacuoles Research Articles

Peracetylated N-Acetylmannosamine, a Synthetic Sugar Molecule, Efficiently Rescues Muscle Phenotype and Biochemical Defects in Mouse Model of Sialic Acid-deficient Myopathy

Distal myopathy with rimmed vacuoles/hereditary inclusion body myopathy (DMRV/hIBM), characterized by progressive muscle atrophy, weakness, and degeneration, is due to mutations in GNE, a gene encoding a bifunctional enzyme critical in sialic acid biosynthesis. In the DMRV/hIBM mouse model, which exhibits hyposialylation in various tissues in addition to muscle atrophy, weakness, and degeneration, we recently have demonstrated that the myopathic phenotype was prevented by oral administration of N-acetylneuraminic acid, N-acetylmannosamine, and sialyllactose, underscoring the crucial role of hyposialylation in the disease pathomechanism. The choice for the preferred molecule, however, was limited probably by the complex pharmacokinetics of sialic acids and the lack of biomarkers that could clearly show dose response. To address these issues, we screened several synthetic sugar compounds that could increase sialylation more remarkably and allow demonstration of measurable effects in the DMRV/hIBM mice. In this study, we found that tetra-O-acetylated N-acetylmannosamine increased cell sialylation most efficiently, and in vivo evaluation in DMRV/hIBM mice revealed a more dramatic, measurable effect and improvement in muscle phenotype, enabling us to establish analysis of protein biomarkers that can be used for assessing response to treatment. Our results provide a proof of concept in sialic acid-related molecular therapy with synthetic monosaccharides.

P5.35 Inclusion is accumulated by CHMP2B in sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is a common inflammatory muscle disease in elderly person. Outstanding feature of sIBM is the presence of rimmed vacuoles (RV) revealed by modified Gomori trichrome staining and multiprotein aggregates made of amyloid β (Aβ) and ubiquitin in muscle fibers. RV are considered autophagic, since they often contain lysosomal membranous debris, an end product of muscle-fiber digestion, and increased immunoreactivity of some of the lysosomal enzymes. Autophagy is implicated in not only sIBM but also some frontotemporal dementia (FTD).

P4.47 Hereditary inclusion body myopathy therapy: Assessment of an adeno associated virus AAV2/8 based GNE gene delivery system

Hereditary inclusion body myopathy (HIBM) is a unique neuromuscular disorder characterized by adult onset and slowly progressive distal and proximal muscle and a typical histology including rimmed vacuoles and filamentous inclusions. The disease is caused by recessive mutations in the UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase gene (GNE), encoding the key enzyme in the biosynthetic pathway of sialic acid. In an attempt to develop a possible treatment for HIBM patients, we have establish a platform for gene therapy trials based on adeno associated virus (AAV 8) vectors harboring the wild type GNE gene.

Increase in number of sporadic inclusion body myositis (sIBM) in Japan

Sporadic inclusion body myositis (sIBM) is the most common form of myopathy with inflammation in those over the age of 50 years in Western countries [1, 3, 5, 7]. The prevalence in Caucasians is 4.9–14.9 per million, but 1.07 in Turkey [6]. The prevalence of sIBM in Asian people including Japanese has not been examined. Several mechanisms of sIBM are proposed, for example, betaamyloid accumulation, immune system abnormalities, viral infection, genetic background [1, 8]. However, none of these are concluded to be the specific cause of sIBM. We have now performed a retrospective survey of Japanese patients of sIBM diagnosed at the National Center of Neurology and Psychiatry (NCNP). The increasing numbers of sIBM patients may suggest the clue to elucidate the pathomechanism of sIBM. Only patients with ‘definite’ or ‘probable’ sIBM by the clinical and biopsy criteria [7] were included in the analysis. Biopsies were re-evaluated, and were confirmed the pathological diagnosis of sIBM. We also used revised Bohan and Peter criteria for diagnosis of polymyositis (PM) [5]. In NCNP, the first patient of sIBM was diagnosed in 1989, and the number of patients diagnosed has been increasing year by year, especially after 2002 (Fig. 1). A total of 77 sIBM patients were identified between 1990 and 2007. The average age of onset in sIBM in Japan was 63.4 years old. The numbers of patients with sIBM and PM between 1999 and 2007 were 69 and 165, respectively (Table 1). Accordingly, the number of sIBM patients is estimated to be half that of PM. Given the number of PM patients in the national survey in 2003 (approximately 3,000 patients) in Japan, the number of sIBM is estimated to be around 1,250. Therefore, we assess that the prevalence of sIBM in Japan is 9.83 per million in 2003. The numbers of sIBM and PM between 1990 and 1998 were 8 and 151 patients, respectively. As the number of PM patients in the national survey of 1991 was still around 3,000, the prevalence calculated by the same method was 1.28 per million in 1991, suggesting an increase in the number of sIBM in Japan. We also examined the relationship between birth year and the number of sIBM patients diagnosed in NCNP since 1978 (Fig. 1b). The numbers of sIBM patients are increasing in a linear manner among the individuals born after 1920s. The etiology of sIBM is not yet known and still under discussion in either a primary inflammatory myopathy or a primary degenerative myopathy with a secondary inflammatory disease. The lack of significant clinical response with various immunosuppressants is against sIBM being a primary autoimmune disorder. Accumulation of betaamyloid in rimmed vacuoles is interpreted as a primary N. Suzuki M. Aoki (&) Department of Neurology, Tohoku University School of Medicine, 1-1Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan e-mail: aokim@med.tohoku.ac.jp

Muscle Biopsy as a Diagnostic Tool in Pompe Disease

Today, myopathological, biochemical and genetic characteristics of Pompe disease are well established, and many reports have been published following the first very precisely documented myopatholgical description of adultonset Pompe disease in 1968. The clinical awareness and need for diagnostic precision of Pompe disease has increased since enzyme replacement therapy (ERT) has become widely available. Primary characteristic myopathological findings of Pompe disease are rounded vacuoles that are irregular in shape and size. All infantile biopsies show pronounced vacuolisation of almost all fibres. In juvenile biopsies, the percentage of vacuolated fibres decreases to 75%, and in most adult patients only 10–50% of the fibres reveal vacuolisation. In infantile cases, both fibre types are equally affected, while in juvenile and adult cases a predominant vacuolisation of type 2 fibres occurs. The second myopathological hallmark is periodic acid-Schiff (PAS)– positive material packed in these vacuoles, sometimes lost due to fixation procedures and therefore sometimes only notable as optically empty vacuolar spaces. Thirdly, acid phosphates histochemistry reveals positive staining in vacuolated fibres, indicating lysosomal dysfunction. In addition to these classic findings, autophagic vacuoles, sometimes containing heterogeneous material including cytoplasmic degradation products and myeloid structures, are seen at all ages, especially in type 2 fibres. Ultra-structurally, these autophagic vacuoles contained particulate glycogen and/or autophagic debris, including myelin-like figures. Almost all samples show lipofuscin accumulation in the area of lysosomal residual bodies and rarely lipid droplets as a sign of lysosomal ageing. Infrequently, single rimmed vacuoles are notable. Fibre splitting is an uncommon aspect. Due to excessive fibre vacuolisation, a subsarcolemmal packing of mitochondria is notable in most cases. Single fibre necrosis, basophilia and phagocytosis may be present in biopsies of all ages. Furthermore, mild fibrosis and fatty degeneration may be found in about 10% of the biopsies. Immunohistochemistry does not normally present signs of an inflammatory myopathy. In selected biopsies, some macrophages may be noticed, and vacuoles may contain caveolin-3 and dysferlin positive membrane debris. Although all classic hallmarks of Pompe disease are well documented over several decades, Pompe disease in adult-onset patients may still be missed histologically, especially when a clinically unaffected muscle is chosen for biopsy. Consequently, in modern diagnostics, dry blood spot (DBS) testing followed by -glucosidase (GAA) gene nalysis helps to overcome this muscle biopsy neglect. However, in some adult patients presenting with a blurred imb girdle weakness and atrophy, combined muscle MRI, light and electron microscopy, and biochemical analysis ay be still essential for making an accurate diagnosis of Pompe disease.

Asian patients with limb girdle muscular dystrophy 2I (LGMD2I)

Limb girdle muscular dystrophy type 2I (LGMD2I) is caused by defects in the fukutin-related protein (FKRP) gene. In most Caucasian patients with LGMD2I, the condition is associated with a missense mutation – c.826C>A (p.Leu276Ile). We describe two Chinese brothers with progressive shoulder and pelvic muscle weakness. They had muscle stiffness and myalgia after exercise, but lacked obvious hypertrophy of the calves. Muscle biopsy showed dystrophic features with many rimmed vacuoles in the fibers. Immunohistochemistry and immunoblot analyses revealed reductions of alpha-(α)-dystroglycan (VIA4-1) and laminin-α2 (80-kDa C-terminal and 300-kDa N-terminal). Two novel heterozygous mutations (c.208T>A and c.1030G>T) in the FKRP gene were identified in these patients. In addition, we summarise the clinical features of patients with LGMD2I in the Asian region. Our findings might indicate that the pathogenic FKRP mutations in Asian patients with LGMD2I are sporadic compound heterozygous mutations rather than the hot-spot c.826C>A mutation seen in Caucasian populations.

A novel PNPLA2 mutation causes neutral lipid storage disease with myopathy (NLSDM) presenting muscular dystrophic features with lipid storage and rimmed vacuoles

Neutral lipid storage disease with myopathy (NLSDM) is a type of lipid storage myopathy arising due to a mutation in the PNPLA2 gene encoding an adipose triglyceride lipase responsible for the degradation of intracellular triglycerides. Herein, we report the cases of two siblings manifesting slowly progressive proximal and distal limb weakness in adulthood. One of the patients had dilated cardiomyopathy, hearing loss and short stature. Muscle specimens of the 2 patients revealed muscular dystrophic features with massive lipid droplets and numerous rimmed vacuoles in the fibers. A novel homozygous mutation IVS2+1G > A in the PNPLA2 gene was identified in the 2 cases, but not in the healthy familial individuals. The presence of massive lipid droplets with muscular dystrophic changes and rimmed vacuoles in muscle fibers might be one of the characteristic pathological changes of NLSDM.

PONM14 A novel homozygous missense mutation in the GNE gene of two brothers with hereditary inclusion body myopathy type 2

Hereditary inclusion body myopathy type 2 (HIBM2) is an autosomal recessive neuromuscular disorder presenting in early adulthood with foot drop and progressive muscle weakness that involves all limbs but typically...

P2.42 Clinical and imaging features of inclusion body myositis

Objective: Sporadic inclusion body myositis (sIBM) is the most common myopathy presenting over the age of 50 years. The diagnosis of sIBM is frequently delayed being initial errors common, as a consequent of the variation of clinical phenotype at presentation and frequent false negative results of initial muscle biopsy because of lack of rimmed vacuoles. For better diagnosis, we evaluate the clinical and radiological characteristics of Japanese patients with sIBM. Patients and methods: Thirteen patients (11 male and 2 female, being mean age of onset and diagnosis 58±8 years old and 65±8 years old, respectively) with sIBM based on muscle biopsy in the Department of Neurology, the University of Tsukuba Hospital over a 33-year period from 1976 to 2009 were included in this study.

P1.31 Genetic mutations in sarcoglycanopathies in a Malaysian population

defects of the fukutin related protein (FKRP) gene. In Caucasus patients, themajority of LGMD2I cases are frequently associatedwith a single missense mutation of the FKRP gene, namely c.826C > A (p.Leu276Ile). We reported two Chinese siblings with adult-onset progressive shoulder and pelvic muscle weakness. They suffered from evident muscle stiffness and myalgia after exercise, but absence of obvious calves hypertrophy. Muscle biopsy showed dystrophic features with numerous rimmed vacuoles in fibers. Immunostaining and immunoblot analysis revealed the reductions of a-dystroglycan (VIA4-1) and laminin-a2 (80-kDa C-terminal and 300-kDa N-terminal). Genetic analysis of the FKRP gene revealed novel compound heterozygous mutations (c.208T > A and c.1030G > T). Meanwhile, we summarized the reported cases with LGMD2I in Asian region. To our knowledge, this is first report in Chinese mainland population. Our findings expand the genetic spectrum and geographic distribution of LGMD2I. The findings suggest the pathogenic FKRP mutations might be sporadic compound heterogeneous in Asian LGMD2I patients instead of hot-spot c.826C > A mutation in Caucasian population.

A preclinical trial of sialic acid metabolites on distal myopathy with rimmed vacuoles/ hereditary inclusion body myopathy, a sugar-deficient myopathy: a review

Distal myopathy with rimmed vacuoles (DMRV), also called hereditary inclusion body myopathy (hIBM), is a moderately progressive hereditary muscle disorder affecting young adults. DMRV/hIBM is characterized clinically by muscle atrophy and weakness initially involving the distal muscles, and pathologically by the presence of small angular fibers, formation of rimmed vacuoles and deposition of various proteins in the muscle fibers. This disease is known to be caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, which encodes the essential enzyme in sialic acid biosynthesis, leading to a reduction of sialic acid levels in the serum and skeletal muscles of affected patients. As it is a metabolic disease, metabolite supplementation is theoretically one of the therapeutic options. In this review, recent animal models for DMRV/hIBM are briefly characterized followed by a focus on the administration of sialic acid metabolites as a reliable therapeutic option to DMRV/hIBM with the following points highlighted: the property of compounds, the pharmacokinetic metabolism in vivo, and the therapeutic effects on the DMRV/hIBM mouse model.

P22 Clinical and pathological heterogeneity in partial merosin deficiency

P22 Clinical and pathological heterogeneity in partial merosin deficiency

Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA(+)-ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. Towards elucidating the pathogenic mechanism we have developed and characterized transgenic mice with ubiquitous expression of wild-type and disease-causing versions of human VCP/p97. Here, we report that mice expressing VCP/p97 harboring the mutations R155H or A232E develop pathology that is limited to muscle, brain and bone, recapitulating the spectrum of disease in humans with IBMPFD. The mice exhibit progressive muscle weakness and pathological examination of muscle shows classic characteristics of inclusion body myopathy including rimmed vacuoles and TDP-43 pathology. The mice exhibit abnormalities in behavioral testing and pathological examination of the brain shows widespread TDP-43 pathology. Furthermore, radiological examination of the skeleton reveals that mutant mice develop severe osteopenia accompanied by focal lytic and sclerotic lesions in vertebrae and femur. In vitro studies indicate that mutant VCP causes inappropriate activation of the NF-kappaB signaling cascade, which could contribute to the mechanism of pathogenesis in multiple tissues including muscle, bone and brain.

The prevalence of rimmed vacuoles in biopsy-proven dermatomyositis

The prevalence of rimmed vacuoles in biopsy-proven dermatomyositis

自己貪食空胞性ミオパチーの病態解明と治療法開発

Autophagic vacuolar myopathy (AVM) is an entity defined by the presence of autophagic vacuoles on muscle pathology. There are two emerging categories in AVM in addition to the best characterized Pompe disease. One is Danon disease and its related disorders, which are characterized by autophagic vacuoles with unique sarcolemmal features (AVSF). AVSF express virtually all sarcolemmal proteins, in addition to acetylcholinesterase, on their vacuolar membranes. Danon disease is caused by primary deficiency of a lysosomal membrane protein, LAMP-2. Interestingly, in this disease, the number of AVSF increases as the patients age. Other AVSF myopathies include X-linked myopathy with excessive autophagy which is now known to be caused by VMA21 mutations. The other AVM is typified by the presence of rimmed vacuoles, which are actually clusters of autophagic vacuoles on electron microscopy. One of the well known diseases in this group is distal myopathy with rimmed vacuoles (DMRV), also called hereditary inclusion body myopathy (HIBM). DMRV is caused by mutations in GNE gene that encode a rate-limiting enzyme in the sialic acid biosynthetic pathway. Interestingly, in DMRV model mice, sialic acid supplementation almost completely precluded the disease phenotype, indicating that decreased sialic acid is the cause of myopathic phenotype and sialic acid supplementation can prevent the disease process. Interestingly, both genetically diagnosable AVSF myopathies are primarily due to lysosomal dysfunctions. In contrast, rimmed vacuoles are secondarily caused by extra-lysosomal defects, such as hyposialylation in DMRV/HIBM, and are formed at later stages of the disease.

Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease

Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM), Paget's disease of the bone, and frontotemporal dementia (IBMPFD). Patient muscle has degenerating fibers, rimmed vacuoles (RVs), and sarcoplasmic inclusions containing ubiquitin and TDP-43 (TARDNA-binding protein 43). In this study, we find that IBMPFD muscle also accumulates autophagosome-associated proteins, Map1-LC3 (LC3), and p62/sequestosome, which localize to RVs. To test whether VCP participates in autophagy, we silenced VCP or expressed adenosine triphosphatase-inactive VCP. Under basal conditions, loss of VCP activity results in autophagosome accumulation. After autophagic induction, these autophagosomes fail to mature into autolysosomes and degrade LC3. Similarly, IBMPFD mutant VCP expression in cells and animals leads to the accumulation of nondegradative autophagosomes that coalesce at RVs and fail to degrade aggregated proteins. Interestingly, TDP-43 accumulates in the cytosol upon autophagic inhibition, similar to that seen after IBMPFD mutant expression. These data implicate VCP in autophagy and suggest that impaired autophagy explains the pathology seen in IBMPFD muscle, including TDP-43 accumulation.

Isolated inflammatory myopathy with rimmed vacuoles presenting with dropped head

We describe an unusual case of inflammatory myopathy with rimmed vacuoles associated with dropped head syndrome. Muscle biopsy in our patient revealed variations in fiber size with fiber necrosis and regeneration, accompanied by many rimmed vacuoles and areas of endomysial cell infiltration. Electron microscopy demonstrated autophagic vacuoles and tubulofilamentous inclusions. This myopathy can cause dropped head syndrome in a subgroup of patients.

Development of inclusion bodies late in the course of necrotizing myositis due to systemic lupus erythematosus

Background: The diagnosis of myositis and its nosological differentiation into polymyositis, inclusion body myositis (IBM) and myositis associated to systemic autoimmune diseases strongly relies on muscle biopsy findings. We report a case of necrotizing polymyositis associated to systemic lupus erythematosus (SLE) which 11 years later developed inclusion bodies and other histopathological features typical for IBM. Case report: A 45-year-old man presented in 1997 with progressive proximal symmetric tetraparesis pronounced for the upper extremities (grade 4/5) and discoid skin lesions. Further investigations were diagnostic for systemic lupus erythematosus. Creatine kinase (CK) of 438 IU/l (normal <80), myopathic EMG findings and deltoid muscle biopsy confirmed a severe necrotizing polymyositis. Under prednisolone and azathioprine complete remission was achieved. After three years tetraparesis insidiously returned and progressed with transiently increased CK up to 300 IU/l. Replacement of azathioprine by mycophenolate mofetil and later by methotrexate had a brief stabilizing effect. In 2008 muscle rebiopsy showed a severe inflammatory myositis with an advanced fibrinous and lipomatous conversion. The lymphoid infiltrate was unchanged with an intense perivascular cuffing predominated by CD20+ B-cells and CD4+ T-cells whereas CD8+ T-cells were sparse. Mononuclear infiltration involved nonnecrotic muscle fibers and MHC-I was generally upregulated. Contrasting to the first biopsy, advanced chronic neurogen changes, rimmed vacuoles, ubiquitine-positive intracellular aggregates and electromicroscopic abnormal filamentous inclusions were evidenced. Conclusions: The composition of the inflammatory infiltrate in both biopsies is well compatible with a SLE-associated myositis and atypical for classical polymyositis or inclusion body myositis. The second biopsy fulfilled all formal criteria for the definite diagnosis of inclusion body myositis. Only two cases of IBM-like myopathy decades after SLE diagnosis have been reported, but both received hydroxycholorquine which is neuromyotoxic and can induce some IBM-like myopathological features. We suggest that rimmed vacuoles and inclusion bodies can develop late in chronic autoimmune myositis as secondary degenerative changes although the combination of these findings is usually considered specific for IBM. The nature of the underlying autoimmune process rather determines the composition of the inflammatory infiltrate.

Hereditary rimmed vacuole myopathy showing interstitial amyloid deposition in muscle tissue

We describe a consanguineous family that had progressive myopathy with rimmed vacuole (RV) formation and amyloid deposition. Patient 1 is a 71-year-old woman with muscle atrophy in the lumbar girdle and lower extremities. Patient 2 is a 40-year-old man (the son of Patient 1) with fatty changes in the biceps femoris muscles. Muscle biopsies revealed myopathic and neurogenic degeneration with RV, necrotic fibers, and interstitial amyloid deposition. Amyloid deposition was detected only in the muscle tissue.

Role of ubiquitin–proteasome proteolysis in muscle fiber destruction in experimental chloroquine‐induced myopathy

Previous studies have documented the presence of rimmed vacuoles, atrophic fibers, and increased lysosomal cathepsin activity in skeletal muscle from animal models of chloroquine-induced myopathy, suggesting that muscle fibers in this type of myopathy may be degraded via the lysosomal-proteolysis pathway. Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, in this study we chose to examine the significance of the ubiquitin-proteasome proteolytic system in the process of muscle fiber destruction in experimental chloroquine myopathy. Expression of ubiquitin, 26S proteasome proteins, and ubiquitin ligases, such as muscle-specific RING finger-1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (MAFbx), was analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Abnormal accumulation of rimmed vacuoles was observed only in chloroquine-treated denervated muscles. Ubiquitin and proteasome immunostaining, and ubiquitin, MuRF-1, and atrogin-1/MAFbx mRNAs were significantly increased in denervated soleus muscles from saline- and chloroquine-treated rats when compared with contralateral innervated muscles. Further, ubiquitin and ubiquitin ligase mRNA levels were higher in denervated muscles from chloroquine-treated rats when compared with saline-treated rats. These data demonstrate increased proteasomes and ubiquitin in denervated muscles from chloroquine-treated rats and suggest that the ubiquitin-proteasome proteolysis pathway as well as the lysosomal-proteolysis pathway mediate muscle fiber destruction in experimental chloroquine myopathy.