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P.15.3 Effects of ZASP mutations on Z-disc proteins associated with myofibrillar myopathy in skeletal muscle
Abstracts / Neuromuscular Disorders 23 (2013) 738–852 We generated transgenic medakas expressing human BAG3 (WT or RAASPdel) in the skeletal muscle. Each transgenic fish was histologically analyzed. The RAASPdel BAG3 expressing fishes, but not WT, displayed lateral curvature of body axis and hardly swam. Histological analysis revealed a marked variation in muscle fiber size. Confocal microscopic analysis revealed that both WT and RAASPdel BAG3 were localized in the Z-line, however, additional scattered accumulations of mutant BAG3 were seen in many muscle fibers. Similar to the human patient muscle, the accumulations of mutant BAG3 were co-localized with other Z-line proteins such as filamin C and desmin but not a-actinin. Electron microscopic observations revealed widen Z-line in the mutant fish. We could reproduce myopathic phenotype pathologically consistent with MFM using the RAASPdel BAG3 fish. This result strongly suggests that the p.261_265RAASPdel in BAG3 can be a causative mutation of MFM. Transgenic medaka is a useful model for evaluating pathogenicity of human mutation. http://dx.doi:10.1016/j.nmd.2013.06.626
P.15.2 Elucidating the mechanism of disease in BAG3 related myofibrillar myopathy A.A. Ruparelia, R.J. Bryson-Richardson Monash University, School of Biological Sciences, Melbourne, Australia Myofibrillar myopathies are a group of chronic muscle diseases characterised at the cellular level by failure of the muscle fibre at the Z-disk and accumulation of protein aggregates. The vast majority of these diseases are late onset dominant disorders with symptoms typically evident between 35 and 50 years of age. Patients suffer progressive muscle weakness over many years and have reduced life expectancy due to respiratory muscle failure and cardiac complications. Whilst structural failure of the muscle fibre is a feature of myofibrillar myopathies not all of the proteins associated with myofibrillar myopathy have a structural role. Recently mutations in BAG3, a co-chaperone with no direct role in muscle function, were identified as a cause of this disorder. This discovery opened a new area of investigation into the role of chaperones in myopathies and the mechanism of disease in myofibrillar myopathy. In order to investigate the role of BAG3 in muscle development and disease we examined zebrafish lacking BAG3 and those with ectopic expression of GFP-tagged forms of either wildtype BAG3 or the dominant myofibrillar myopathy mutant form Bag3P209L. Examination of these fish identified both the fibre failure and the formation of protein aggregates characteristic of myofibrillar myopathy. Utilising the advantage of the zebrafish model system to examine the onset and progression of the phenotype in vivo, combined with detailed characterisation by immunolabelling and confocal microscopy, we were able to develop a novel model that explains the mechanism of disease in BAG3 related myofibrillar myopathy. We will present this model and discuss its implications for other forms of myofibrillar myopathy. http://dx.doi:10.1016/j.nmd.2013.06.627
P.15.3 Effects of ZASP mutations on Z-disc proteins associated with myofibrillar myopathy in skeletal muscle X. Lin 1, L. Brubaker 1, I. Bajraktari 1, R. Ohman 1, R. Griggs 2, K. Fischbeck 1, A. Mankodi 1 1 National Institutes of Health, National Institutes of Neurological Disorders and Stroke, Bethesda, United States; 2 University of Rochester Medical Center, Department of Neurology, Rochester, United States
819
Myofibrillar myopathies (MFM) are caused by mutations in at least 6 Z-disc associated proteins, including ZASP, myotilin, desmin, BAG3, abcrystallin, and filamin C. MFM are characterized by early and prominent disruption of the Z-disc with focal dissolution of myofibrils and ectopic accumulation of myofibrillar proteins. Similar morphological changes in skeletal muscle in MFM suggest a shared cellular and molecular disease mechanism. ZASP interacts with myotilin, and knock down of ZASP in mice results in alterations in the amounts of other MFM gene products. We hypothesize that ZASP associations with other MFM gene products play a role in the disease mechanism in zaspopathy. To examine the effects of ZASP mutations on the other MFM gene products in skeletal muscle, WT and mutant (A147T and A165V) ZASP-GFP proteins were expressed by electroporation in the tibialis anterior muscles of opposite limbs in wild type mice (n = 18). Muscle tissues were harvested at 1, 2, and 4 weeks after the electroporation. The amount and distribution of MFM gene products, F-actin, and a-actinin-2 were examined in the electroporated mouse muscle fibers and in skeletal muscle fibers of patients with zaspopathy. Wild type and mutant ZASP interactions with other MFM gene products were examined by co-IP and pulldown assays. Preliminary data suggests that wild type and mutant ZASP associate with myotilin, desmin, and BAG3. The effects of ZASP mutations on the other MFM gene products and the effects of the other MFM gene defects on ZASP will provide insight into shared disease mechanisms in this group of degenerative myopathies. http://dx.doi:10.1016/j.nmd.2013.06.628
P.15.4 ZASP –sZM mutations in myofibrillar myopathy cause skeletal muscle Zdisc disruption by disassembling a-actinin cross-linked skeletal actin filaments1 X. Lin , J. Ruiz 1, I. Bajraktari 1, S. Banerjee 1, K. Gribble 1, R. Griggs 2, K. Fischbeck 1, A. Mankodi 1 1 NINDS/NIH, Neurogenetics Branch, Bethesda, United States; 2 University of Rochester Medical Center, Neurology, Rochester, United States Myofibrillar myopathies (MFM) are characterized by early and prominent disruption of the Z-disc with focal dissolution of myofibrils and ectopic accumulation of myofibrillar proteins. The molecular mechanisms underlying the Z-disc disruption in MFM are not yet delineated. The Z-disc is a key element for structural integrity of the sarcomere, the basic contractile unit in skeletal muscle. The core of the Z-disc consists of actin filaments (F-actin) from adjacent sarcomeres cross-linked by a-actinin. We explored the mechanism of skeletal muscle Z-disc disruption in zaspopathy, a prototype MFM caused by ZASP mutations (A147T and A165V) at or within a highly conserved motif (sZM) that is expressed in striated muscle. Our studies show that ZASP is a novel skeletal actin binding protein and that the sZM domain is important for ZASP-actin interaction in skeletal muscle. Both wild type and mutant ZASP interact with skeletal actin, but only mutant proteins cause disassembly of actinin-crosslinked F-actin in vitro and the Z-disc disruption with F-actin accumulation in electroporated mouse skeletal muscle, as in the human disease. These results show that ZASP–sZM mutations have deleterious effects on the core structure of the Z-discs in striated muscle and support a toxic gain-of-function disease mechanism. Other MFM gene products (myotilin, desmin, BAG3, abCrystallin, and filamin C) are known to regulate the organization and stability of F-actin in skeletal muscle. It is possible that the molecular pathways leading to skeletal muscle Z-disc disruption are shared by these myopathies. Alteration of F-actin dynamics, by either direct or indirect association of mutant proteins with actin filaments, may emerge as a unifying disease mechanism in this group of degenerative myopathies. http://dx.doi:10.1016/j.nmd.2013.06.629
P.15.2 Elucidating the mechanism of disease in BAG3 related myofibrillar myopathy A.A. Ruparelia, R.J. Bryson-Richardson Monash University, School of Biological Sciences, Melbourne, Australia Myofibrillar myopathies are a group of chronic muscle diseases characterised at the cellular level by failure of the muscle fibre at the Z-disk and accumulation of protein aggregates. The vast majority of these diseases are late onset dominant disorders with symptoms typically evident between 35 and 50 years of age. Patients suffer progressive muscle weakness over many years and have reduced life expectancy due to respiratory muscle failure and cardiac complications. Whilst structural failure of the muscle fibre is a feature of myofibrillar myopathies not all of the proteins associated with myofibrillar myopathy have a structural role. Recently mutations in BAG3, a co-chaperone with no direct role in muscle function, were identified as a cause of this disorder. This discovery opened a new area of investigation into the role of chaperones in myopathies and the mechanism of disease in myofibrillar myopathy. In order to investigate the role of BAG3 in muscle development and disease we examined zebrafish lacking BAG3 and those with ectopic expression of GFP-tagged forms of either wildtype BAG3 or the dominant myofibrillar myopathy mutant form Bag3P209L. Examination of these fish identified both the fibre failure and the formation of protein aggregates characteristic of myofibrillar myopathy. Utilising the advantage of the zebrafish model system to examine the onset and progression of the phenotype in vivo, combined with detailed characterisation by immunolabelling and confocal microscopy, we were able to develop a novel model that explains the mechanism of disease in BAG3 related myofibrillar myopathy. We will present this model and discuss its implications for other forms of myofibrillar myopathy. http://dx.doi:10.1016/j.nmd.2013.06.627
P.15.3 Effects of ZASP mutations on Z-disc proteins associated with myofibrillar myopathy in skeletal muscle X. Lin 1, L. Brubaker 1, I. Bajraktari 1, R. Ohman 1, R. Griggs 2, K. Fischbeck 1, A. Mankodi 1 1 National Institutes of Health, National Institutes of Neurological Disorders and Stroke, Bethesda, United States; 2 University of Rochester Medical Center, Department of Neurology, Rochester, United States
819
Myofibrillar myopathies (MFM) are caused by mutations in at least 6 Z-disc associated proteins, including ZASP, myotilin, desmin, BAG3, abcrystallin, and filamin C. MFM are characterized by early and prominent disruption of the Z-disc with focal dissolution of myofibrils and ectopic accumulation of myofibrillar proteins. Similar morphological changes in skeletal muscle in MFM suggest a shared cellular and molecular disease mechanism. ZASP interacts with myotilin, and knock down of ZASP in mice results in alterations in the amounts of other MFM gene products. We hypothesize that ZASP associations with other MFM gene products play a role in the disease mechanism in zaspopathy. To examine the effects of ZASP mutations on the other MFM gene products in skeletal muscle, WT and mutant (A147T and A165V) ZASP-GFP proteins were expressed by electroporation in the tibialis anterior muscles of opposite limbs in wild type mice (n = 18). Muscle tissues were harvested at 1, 2, and 4 weeks after the electroporation. The amount and distribution of MFM gene products, F-actin, and a-actinin-2 were examined in the electroporated mouse muscle fibers and in skeletal muscle fibers of patients with zaspopathy. Wild type and mutant ZASP interactions with other MFM gene products were examined by co-IP and pulldown assays. Preliminary data suggests that wild type and mutant ZASP associate with myotilin, desmin, and BAG3. The effects of ZASP mutations on the other MFM gene products and the effects of the other MFM gene defects on ZASP will provide insight into shared disease mechanisms in this group of degenerative myopathies. http://dx.doi:10.1016/j.nmd.2013.06.628
P.15.4 ZASP –sZM mutations in myofibrillar myopathy cause skeletal muscle Zdisc disruption by disassembling a-actinin cross-linked skeletal actin filaments1 X. Lin , J. Ruiz 1, I. Bajraktari 1, S. Banerjee 1, K. Gribble 1, R. Griggs 2, K. Fischbeck 1, A. Mankodi 1 1 NINDS/NIH, Neurogenetics Branch, Bethesda, United States; 2 University of Rochester Medical Center, Neurology, Rochester, United States Myofibrillar myopathies (MFM) are characterized by early and prominent disruption of the Z-disc with focal dissolution of myofibrils and ectopic accumulation of myofibrillar proteins. The molecular mechanisms underlying the Z-disc disruption in MFM are not yet delineated. The Z-disc is a key element for structural integrity of the sarcomere, the basic contractile unit in skeletal muscle. The core of the Z-disc consists of actin filaments (F-actin) from adjacent sarcomeres cross-linked by a-actinin. We explored the mechanism of skeletal muscle Z-disc disruption in zaspopathy, a prototype MFM caused by ZASP mutations (A147T and A165V) at or within a highly conserved motif (sZM) that is expressed in striated muscle. Our studies show that ZASP is a novel skeletal actin binding protein and that the sZM domain is important for ZASP-actin interaction in skeletal muscle. Both wild type and mutant ZASP interact with skeletal actin, but only mutant proteins cause disassembly of actinin-crosslinked F-actin in vitro and the Z-disc disruption with F-actin accumulation in electroporated mouse skeletal muscle, as in the human disease. These results show that ZASP–sZM mutations have deleterious effects on the core structure of the Z-discs in striated muscle and support a toxic gain-of-function disease mechanism. Other MFM gene products (myotilin, desmin, BAG3, abCrystallin, and filamin C) are known to regulate the organization and stability of F-actin in skeletal muscle. It is possible that the molecular pathways leading to skeletal muscle Z-disc disruption are shared by these myopathies. Alteration of F-actin dynamics, by either direct or indirect association of mutant proteins with actin filaments, may emerge as a unifying disease mechanism in this group of degenerative myopathies. http://dx.doi:10.1016/j.nmd.2013.06.629