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Dystrophin Dp71 is expressed in skeletal muscle
Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249 and DMD human myoblasts (DMDd45-52). First, we showed a decrease of nNOS expression when miR-708 or -34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed the same characteristics as in BMDd45-55 patients. In this DMDd45-52 cells we demonstrated that an inhibitor of miR-708 increases nNOS expression, validating that this miRNA acts on the nNOS-3’UTR modulating its expression in human myoblasts. Altogether, these results strongly suggest that miR-708 is a new actor involved in the regulation of nNOS expression in dystrophic muscle. http://dx.doi.org/10.1016/j.nmd.2017.06.267
P.238 Dystrophin Dp71 is expressed in skeletal muscle T. Kawaguchi 1, E. Niba 2, A. Rani 2, S. Yoshida 1, S. Sakakibara 3, N. Maeda 3, O. Sato 3, M. Matsuo 2 1 Daiichi Sankyo RD Novare co., Ltd., Tokyo, Japan; 2 Kobe Gakuin University, Kobe, Japan; 3 Daiichi Sankyo co., Ltd., Tokyo, Japan Duchenne muscular dystrophy (DMD) is a common inherited neuromuscular disorder characterized by progressive muscular degeneration. DMD is caused by a mutation in the DMD gene, which encodes dystrophin protein generated from a 14 kb transcript containing 79 exons. The DMD gene has at least four internal promoters that give rise to shorter dystrophin transcripts. One of them is Dp71, which is a 71 kDa protein transcribed from the promoter located in intron 62 of the DMD gene. Dp71 is known to be expressed ubiquitously in various tissues, except for skeletal muscle. However, our PCR analysis indicated that Dp71 mRNA was expressed in human skeletal muscle. Simple Western® is a fully automated capillary electrophoresis based immunodetection technology with high quantitativeness and reproducibility. Using this method, the expression of Dp71 protein in skeletal muscle was quantified. Both signal peaks of full-length dystrophin (Dp427) and Dp71 were identified in human skeletal muscle. In C57BL/6 mice, both Dp427 and Dp71 were shown to be expressed. On the other hand, in mdx mice (a mouse model of DMD), Dp427 was not detected, and the expression level of Dp71 protein was higher than that of the C57BL/6 mice. As we had confirmed the expression of Dp71 in skeletal muscle, these results raised the possibility that the expression of the Dp71 protein, which is controlled by a different promoter on the DMD gene, may be activated in mdx mice because of the Dp427 deficiency in skeletal muscle. These findings can aid in accelerating the understanding of the physiological role of Dp71. We considered that in future exon skipping treatment for DMD patients, the state of skeletal muscle could be evaluated by quantifying the expression of Dp71 in addition to that of Dp427. http://dx.doi.org/10.1016/j.nmd.2017.06.268
P.239 Identification of serum protein biomarkers for utrophin based DMD therapy S. Guiraud, B. Edwards, S. Squire, A. Babbs, N. Shah, A. Berg, H. Chen, K. Davies Medical Research Council Functional Genomics Unit, University of Oxford, Oxford, UK Despite promising therapeutic avenues, there is currently no effective treatment for Duchenne muscular dystrophy (DMD), a lethal, X-linked recessive disorder caused by the loss of the cytoskeletal protein dystrophin. A highly promising approach to therapy, applicable to all DMD patients irrespective to their genetic defect, is to modulate utrophin, a functional paralogue of dystrophin, able to compensate for the primary defects of DMD restoring sarcolemmal stability. One of the major difficulties in assessing the effectiveness of therapeutic strategies is to define appropriate outcome
S167
measures. The identification of non-invasive biomarkers will accelerate evaluation of utrophin modulators and translation to patients. In the present study, we utilised an aptamer based proteomics approach to profile 1,310 proteins in plasma of wild-type, mdx and Fiona (mdx overexpressing utrophin) mice. Comparison of the C57 and mdx sera revealed 83 proteins with statistically significant >2 fold changes in dystrophic serum abundance. A large majority of previously described biomarkers (ANP32B, THBS4, CAMK2A/ B/D, CYCS, CAPNI) were normalised towards wild-type levels in Fiona animals. This work also identified potential mdx markers specific to increased utrophin (DUS3, TPI1) and highlights novel mdx biomarkers (GITR, MYBPC1, HSP60, SIRT2, SMAD3, CNTN1). We define a panel of putative protein mdx biomarkers to evaluate utrophin based strategies which may help to accelerate their translation to the clinic. http://dx.doi.org/10.1016/j.nmd.2017.06.269
P.240 Proteomic identification of novel brain and serum biomarkers linked to the pathophysiology of Duchenne muscular dystrophy S. Murphy 1, M. Zweyer 2, P. Dowling 1, M. Henry 3, P. Meleady 3, D. Swandulla 2, K. Ohlendieck 1 1 Maynooth University, National University of Ireland, Maynooth, Ireland; 2 University of Bonn, Bonn, Germany; 3 Dublin City University, Dublin, Ireland Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood, and is characterised by primary abnormalities in the Dmd gene encoding the membrane cytoskeletal protein dystrophin. Aside from the well-established skeletal muscle degeneration and cardio-respiratory complications, extra-muscular manifestations are also evident. Comparative label-free liquid chromatography mass spectrometric analyses of brain tissue and serum derived from the mdx-4cv animal model of the disease have identified novel proteomic markers of astrogliosis and inflammation respectively. The elucidation of proteome-wide changes in the mdx-4cv dystrophin-deficient brain revealed alterations in proteins involved in cytoskeletal stabilisation, calcium homeostasis, metabolism and reactive gliosis. Elevated abundance of some brain-associated proteins were also detected in serum samples, suggesting that the presence of brain-derived proteins in serum could potentially reflect the extent of dystrophinopathy in the brain. A large number of differentially abundant proteins in immunodepleted mdx-4cv serum were also identified by label-free mass spectrometry. In particular these proteins were associated with chronic tissue damage, necrosis and sterile inflammation. A variety of these potential circulatory biomarkers linked to muscular dystrophy were validated independently by comparative immunoblot analysis and enzyme-linked immunosorbent assays. Further research will be required to investigate whether such protein biomarkers are passively leaked or actively secreted into the circulatory system. These novel protein marker candidates identified by proteomic profiling of brain tissue and serum offer insights into the pathophysiology of dystrophinopathy and may be further investigated for their potential use in the diagnosis, prognosis and therapeutic monitoring of Duchenne muscular dystrophy. http://dx.doi.org/10.1016/j.nmd.2017.06.270
P.241 Comparative high resolution proteomic analysis of dystrophic mouse models reveals a core dystrophic proteome and the impact of aging T. van Westering 1, H. Johansson 2, A. Coenen-Stass 1, S. Miyatake 3, J. Tanihata 3, S. Takeda 4, T. Yokota 5, J. Lehtiö 2, M. Wood 1, S. El Andaloussi 6, T. Roberts 7, Y. Aoki 4 1 University of Oxford, Oxford, UK; 2 Karolinska Institutet, Stockholm, Sweden; 3 National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; 4 National Institute of Neuroscience, National Center of Neurology and
P.238 Dystrophin Dp71 is expressed in skeletal muscle T. Kawaguchi 1, E. Niba 2, A. Rani 2, S. Yoshida 1, S. Sakakibara 3, N. Maeda 3, O. Sato 3, M. Matsuo 2 1 Daiichi Sankyo RD Novare co., Ltd., Tokyo, Japan; 2 Kobe Gakuin University, Kobe, Japan; 3 Daiichi Sankyo co., Ltd., Tokyo, Japan Duchenne muscular dystrophy (DMD) is a common inherited neuromuscular disorder characterized by progressive muscular degeneration. DMD is caused by a mutation in the DMD gene, which encodes dystrophin protein generated from a 14 kb transcript containing 79 exons. The DMD gene has at least four internal promoters that give rise to shorter dystrophin transcripts. One of them is Dp71, which is a 71 kDa protein transcribed from the promoter located in intron 62 of the DMD gene. Dp71 is known to be expressed ubiquitously in various tissues, except for skeletal muscle. However, our PCR analysis indicated that Dp71 mRNA was expressed in human skeletal muscle. Simple Western® is a fully automated capillary electrophoresis based immunodetection technology with high quantitativeness and reproducibility. Using this method, the expression of Dp71 protein in skeletal muscle was quantified. Both signal peaks of full-length dystrophin (Dp427) and Dp71 were identified in human skeletal muscle. In C57BL/6 mice, both Dp427 and Dp71 were shown to be expressed. On the other hand, in mdx mice (a mouse model of DMD), Dp427 was not detected, and the expression level of Dp71 protein was higher than that of the C57BL/6 mice. As we had confirmed the expression of Dp71 in skeletal muscle, these results raised the possibility that the expression of the Dp71 protein, which is controlled by a different promoter on the DMD gene, may be activated in mdx mice because of the Dp427 deficiency in skeletal muscle. These findings can aid in accelerating the understanding of the physiological role of Dp71. We considered that in future exon skipping treatment for DMD patients, the state of skeletal muscle could be evaluated by quantifying the expression of Dp71 in addition to that of Dp427. http://dx.doi.org/10.1016/j.nmd.2017.06.268
P.239 Identification of serum protein biomarkers for utrophin based DMD therapy S. Guiraud, B. Edwards, S. Squire, A. Babbs, N. Shah, A. Berg, H. Chen, K. Davies Medical Research Council Functional Genomics Unit, University of Oxford, Oxford, UK Despite promising therapeutic avenues, there is currently no effective treatment for Duchenne muscular dystrophy (DMD), a lethal, X-linked recessive disorder caused by the loss of the cytoskeletal protein dystrophin. A highly promising approach to therapy, applicable to all DMD patients irrespective to their genetic defect, is to modulate utrophin, a functional paralogue of dystrophin, able to compensate for the primary defects of DMD restoring sarcolemmal stability. One of the major difficulties in assessing the effectiveness of therapeutic strategies is to define appropriate outcome
S167
measures. The identification of non-invasive biomarkers will accelerate evaluation of utrophin modulators and translation to patients. In the present study, we utilised an aptamer based proteomics approach to profile 1,310 proteins in plasma of wild-type, mdx and Fiona (mdx overexpressing utrophin) mice. Comparison of the C57 and mdx sera revealed 83 proteins with statistically significant >2 fold changes in dystrophic serum abundance. A large majority of previously described biomarkers (ANP32B, THBS4, CAMK2A/ B/D, CYCS, CAPNI) were normalised towards wild-type levels in Fiona animals. This work also identified potential mdx markers specific to increased utrophin (DUS3, TPI1) and highlights novel mdx biomarkers (GITR, MYBPC1, HSP60, SIRT2, SMAD3, CNTN1). We define a panel of putative protein mdx biomarkers to evaluate utrophin based strategies which may help to accelerate their translation to the clinic. http://dx.doi.org/10.1016/j.nmd.2017.06.269
P.240 Proteomic identification of novel brain and serum biomarkers linked to the pathophysiology of Duchenne muscular dystrophy S. Murphy 1, M. Zweyer 2, P. Dowling 1, M. Henry 3, P. Meleady 3, D. Swandulla 2, K. Ohlendieck 1 1 Maynooth University, National University of Ireland, Maynooth, Ireland; 2 University of Bonn, Bonn, Germany; 3 Dublin City University, Dublin, Ireland Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood, and is characterised by primary abnormalities in the Dmd gene encoding the membrane cytoskeletal protein dystrophin. Aside from the well-established skeletal muscle degeneration and cardio-respiratory complications, extra-muscular manifestations are also evident. Comparative label-free liquid chromatography mass spectrometric analyses of brain tissue and serum derived from the mdx-4cv animal model of the disease have identified novel proteomic markers of astrogliosis and inflammation respectively. The elucidation of proteome-wide changes in the mdx-4cv dystrophin-deficient brain revealed alterations in proteins involved in cytoskeletal stabilisation, calcium homeostasis, metabolism and reactive gliosis. Elevated abundance of some brain-associated proteins were also detected in serum samples, suggesting that the presence of brain-derived proteins in serum could potentially reflect the extent of dystrophinopathy in the brain. A large number of differentially abundant proteins in immunodepleted mdx-4cv serum were also identified by label-free mass spectrometry. In particular these proteins were associated with chronic tissue damage, necrosis and sterile inflammation. A variety of these potential circulatory biomarkers linked to muscular dystrophy were validated independently by comparative immunoblot analysis and enzyme-linked immunosorbent assays. Further research will be required to investigate whether such protein biomarkers are passively leaked or actively secreted into the circulatory system. These novel protein marker candidates identified by proteomic profiling of brain tissue and serum offer insights into the pathophysiology of dystrophinopathy and may be further investigated for their potential use in the diagnosis, prognosis and therapeutic monitoring of Duchenne muscular dystrophy. http://dx.doi.org/10.1016/j.nmd.2017.06.270
P.241 Comparative high resolution proteomic analysis of dystrophic mouse models reveals a core dystrophic proteome and the impact of aging T. van Westering 1, H. Johansson 2, A. Coenen-Stass 1, S. Miyatake 3, J. Tanihata 3, S. Takeda 4, T. Yokota 5, J. Lehtiö 2, M. Wood 1, S. El Andaloussi 6, T. Roberts 7, Y. Aoki 4 1 University of Oxford, Oxford, UK; 2 Karolinska Institutet, Stockholm, Sweden; 3 National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; 4 National Institute of Neuroscience, National Center of Neurology and