Autophagy in a Mouse Model of Distal Myopathy with Rimmed Vacuoles or Hereditary Inclusion Body Myopathy

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion body myopathy (hIBM) is an autosomal recessive disorder clinically characterized by weakness that initially involves the distal muscles, although other muscles can be affected as well. Pathological hallmarks include the presence of rimmed vacuoles (RVs) and intracellular Congo red-positive depositions in vacuolated or non-vacuolated fibers. Mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which encodes the rate-limiting enzyme in sialic acid biosynthesis, are causative of DMRV/hIBM. Recently, we have generated a mouse model (Gne^-/-hGNEV572L-Tg ) for this disease, and have shown that these mice exhibit hyposialylation and intracellular amyloid deposition before the characteristic RVs are detected, indicating that autophagy is a downstream phenomenon to hyposialylation and amyloid deposition in DMRV/hIBM.

Addendum to:

A Gne Knockout Mouse Expressing Human V572L Mutation Develops Features Similar to Distal Myopathy with Rimmed Vacuoles or Hereditary Inclusion Body Myopathy

M.C. Malicdan, S. Noguchi, I. Nonaka, Y.K. Hayashi and I. Nishino

Hum Mol Genet 2007; 16:115-28     

Autophagy in a Mouse Model of Distal Myopathy with Rimmed Vacuoles or Hereditary Inclusion Body Myopathy

In the article Autophagy in a Mouse Model of Distal Myopathy with Rimmed Vacuoles or Hereditary Inclusion Body Myopathy by May Christine V. Malicdan, Satoru Noguchi and Ichizo Nishino (Autophagy 2007; 3:396-8) the authors describe a transgenic mouse that displays the human distal myopathy with rimmed vacuoles (DMRV) phenotype. In this report, the mutation used to generate the mutation was incorrectly written as “V572L” when it should have been “D176V”. Accordingly, the transgenic mice should be named “Gne^-/- hGNED176V-Tg”. All of the other data have been verified by repeated experiments. Furthermore, the finding that the mouse phenotype resembles human DMRV, including the presence of autophagy, remains valid. We apologize for any inconvenience this may have caused.     

Molecular Markers for Granulovacuolar Degeneration Are Present in Rimmed Vacuoles

Background

Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.

Methods

Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.

Results

GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.

Conclusions

These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.     

缺失型Duchenne/Becker 肌营养不良家系女性携带者及新发突变的确认

应用多重PCR（multiple polymerase reaction／mPCR）技术,联合DMD基因内部及附近11个短串连重复序列（short tandem repeats,STRs）位点连锁分析,对缺失型Duchenne／Beeker肌营养不良（Duchenne／Becker Muscular Dystrophy,DMD／BMD）家系成员进行DMD基因分型,确定家系中女性成员是否携带者,并进行产前诊断。3个家系中的4名缺失型患者,其中2例为新发突变：4位女性成员中,1名为携带者。应用mPCR和11个STRs的连锁分析,能快速、准确、客观判断家系中女性成员是否携带者身份,适于DMD／BMD临床研究机构遗传咨询、基因诊断和产前诊断常规应用。但在mPCR分析过程中,发现45号外显子扩增产物在不同凝胶中电泳迁移率不同。聚丙烯酰胺凝胶电泳（Polyacrylamide Gels Electrophoresis／PAGE）对mPCR产物分析快速、清晰,但需要注意片段迁移率,以防止分析错误。     

Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

Creatine Phosphokinase in Facioscapulohumeral Muscular Dystrophy

Study of the serum creatine kinase levels in young patients with facioscapulohumeral muscular dystrophy suggests that enzyme assay may be valuable as a screening procedure for assessing the status of relatives of an affected individual who have no previous clinical history, and that consequently it may be of use in genetic counselling.     

The Identification of Carriers in Duchenne Muscular Dystrophy

Following recurrent reports in the literature that it is possible to detect carriers of the gene for progressive muscular dystrophy (Duchenne), a survey of nine families was performed. No unequivocal results could be obtained in comparison of clinical data with circulation-time measurements or serum enzyme studies. It is concluded that carrier-labelling with respect to progressive muscular dystrophy is not yet at a satisfactory stage, although progress is being made.     

Laminins in Peripheral Nerve Development and Muscular Dystrophy

Laminins are extracellular matrix (ECM) proteins that play an important role in cellular function and tissue morphogenesis. In the peripheral nervous system (PNS), laminins are expressed in Schwann cells and participate in their development. Mutations in laminin subunits expressed in the PNS and in skeleton muscle may cause peripheral neuropathies and muscular dystrophy in both humans and mice. Recent studies using gene knockout technology, such as cell-type specific gene targeting techniques, revealed that laminins and their receptors mediate Schwann cell and axon interactions. Schwann cells with disrupted laminin expression exhibit impaired proliferation and differentiation and also undergo apoptosis. In this review, we focus on the potential molecular mechanisms by which laminins participate in the development of Schwann cells.