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Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion

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Abstract

Duchenne muscular dystrophy results from loss of the protein dystrophin, which links the intracellular cytoskeletal network with the extracellular matrix, but deficiency in this function does not fully explain the onset or progression of the disease. While some intracellular events involved in the degeneration of dystrophin-deficient muscle fibers have been well characterized, changes in their secretory profile are undescribed. To analyze the secretome profile of mdx myotubes independently of myonecrosis, we labeled the proteins of mdx and wild-type myotubes with stable isotope-labeled amino acids (SILAC), finding marked enrichment of vesicular markers in the mdx secretome. These included the lysosomal-associated membrane protein, LAMP1, that co-localized in vesicles with an over-secreted cytoskeletal protein, myosin light chain 1. These LAMP1/MLC1-3-positive vesicles accumulated in the cytosol of mdx myotubes and were secreted into the culture medium in a range of abnormal densities. Restitution of dystrophin expression, by exon skipping, to some 30 % of the control value, partially normalized the secretome profile and the excess LAMP1 accumulation. Together, our results suggest that a lack of dystrophin leads to a general dysregulation of vesicle trafficking. We hypothesize that disturbance of the export of proteins through vesicles occurs before, and then concurrently with, the myonecrotic cascade and contributes chronically to the pathophysiology of DMD, thereby presenting us with a range of new potential therapeutic targets.

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Abbreviations

LAMP1:

Lysosome-associated membrane protein 1

MLC1:

Myosin light chain 1

MLC1-3:

Myosin light chain 1 and 3

PARP1:

Poly ADP-ribose polymerase 1

SILAC:

Stable isotope labeling with amino acids in cell culture

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Acknowledgments

We would like to thank Douglas Johnson for his help with preliminary SILAC experiments, Dr. Toshifumi Yokota, Dr. Qi Lu, Dr. Jyoti Jaiswal and Dr. Kanneboyina Nagaraju for useful discussion and technical advice. This work was supported by IDDRC 1P30HD40677 and NCMRR 2R24HD050846 NIH grants and by funding from the Foundation to Eradicate Duchenne and from Wellstone Center, U54HD053177, W81XWH-05-1-0616, and by the ANR Genopath IN-A-FIB and the AFM (Association Française contre les Myopathies). The monoclonal antibodies F310 and S21 developed by Dr. Stockdale F.E. [Developmental Studies Hybridoma Bank (DSHB)] were developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Center for Genetic Medicine, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue, Washington, DC, USA

    Stephanie Duguez, William Duddy, Helen Johnston, Kristy J. Brown, Yetrib Hathout & Terence Partridge

  2. Institut de Myologie, UM76, Inserm U974, CNRS, UMR7215, Université Pierre et Marie Curie-Paris 6, Paris, 75013, France

    Stephanie Duguez, William Duddy, Marie Catherine Le Bihan, Anne Bigot & Gillian Butler-Browne

  3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Jeanne Lainé & Marie Catherine Le Bihan

  4. Département de Physiologie, Université Pierre Et Marie Curie-Paris 06, Site Pitié-Salpêtrière, Paris, 75013, France

    Jeanne Lainé

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  1. Stephanie Duguez
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Correspondence to Terence Partridge.

Electronic supplementary material

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18_2012_1248_MOESM1_ESM.tif

Supplemental Figure 1: Cell death is not greater in mdx myotubes. (A) Caspase 9 activity in mdx and wild-type myotubes cultured without (-) serum. (B) Cleaved PARP1 protein level in mdx and wild-type myotubes. Immunoblots of cleaved PARP1 with an image of a coomassie-stained gel showing MHC. Lanes 1 to 5 are wild-type or mdx myotube samples cultured without serum, lanes 6 and 7 cultured with serum. (C) Measurements of myotube cell death when cultured for 24 h without serum. Values are mean ± SD, n = 5 per group. ***p < 0.001, mdx vs. wild-type H-2K myotubes. WT: wild-type; PARP: ADP-ribose polymerase; MHC: myosin heavy chain

18_2012_1248_MOESM2_ESM.tif

Supplementary material 2 (TIFF 24292 kb) Evans Blue and propidium iodide do not diffuse into mdx and wild-type myotubes. (A) Representative images of Evans Blue staining in mdx and wild-type myotubes cultured in the absence of serum. Diffusion into the myotubes was not observed in the presence of serum either (data not shown). (× 20 objective). (B) Representative images of propidium iodide staining in mdx and wild-type myotubes after 12 h of incubation. Myotubes incubated for 1 h were also negative (data not shown). (× 20 objective)

18_2012_1248_MOESM3_ESM.jpg

Supplementary material 3 (JPEG 678 kb) Proteins identified by SILAC are not passively released into the culture medium. Myotubes were incubated with 3, 10 and 40 kDa dextran for 5 min. (x20 objective). (A). Representative images of mdx myotubes incubated with 3, 10 and 40 kDa dextran. Positive myotubes were observed with a dextran size of 3 kDa but, in general, myotubes were not permeable to sizes of 10 and 40 kDa. Similar results were observed for wild-type myotubes (images not shown; data presented in Table II). (B) Positive control of dextran leakage into wild-type myotubes. Live myotubes were permeabalized by adding 0.2 % triton × 100 into the culture medium.

18_2012_1248_MOESM4_ESM.tif

Supplementary material 4 (TIFF 6256 kb) Myosin light chain is consistently detected in the culture medium and in the plasma blood samples. (a) Western blots of myosin light chain 1-3 on myotube samples. anti-myosin light chain 1-3 (F310), give satisfactory results by western blot on myotube samples. (b) Immunoblots of MLC1-3 after 24 h of culture in medium containing serum. 10 μl of culture medium were loaded from each flask. Lanes 1-5: culture medium containing serum from mdx or wild-type myotubes (n = 5/group). MLC1-3 is observed in all five mdx culture medium samples and is absent in all five wild-type culture medium samples, commending its potential as a biomarker. Bands at 100 kDa on the gels show the consistency of the sample loading. (c) Immunoblots showing MLC1-3 in the blood plasma from two wild-type and two mdx mice aged 15-20 days. Albumin levels on the gels show the consistency of the sample loading. WT: wild-type; MLC1-3: myosin light chain 1-3

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Supplementary material 5 (XLS 414 kb) Table 1: Total list of proteins that are detected in the culture medium of mdx and wild-type myotubes. A total of 267 proteins that were consistently observed in both forward and reverse SILAC experiments. We found 234 proteins released in excess by the mdx myotubes, and 13 proteins by the wild-type. Eighty-two proteins were found at a lower band then their expected molecular weight, and 40 proteins were found at a higher molecular weight than expected. Proteins were sorted based on their localization in the cells (according to the DAVID database). The ratio between labeled and unlabeled (i.e., mdx labeled vs. wild-type unlabeled or vice versa) of the two experiments are shown, as well as the theoretical and observed molecular weights of the proteins detected on the gels. We report the accession number given by UniProtKB. We report the proteins that are classically described to be secreted or involved in secretion pathways according to the UniProtKB database: 43 of these proteins are classically secreted, and 11 proteins are involved in secretion pathways. Table 3: Cluster-based protein function analysis of the secretome of mdx myotubes 107 protein clusters have been identified. The table gives enrichment score for each cluster, as well as the annotation terms belonging to each cluster. The number of protein involved, and their Uniprot accession number are also given for each pathways (annotation terms). The Fisher exact p value of each annotation term inside the cluster is given and reflects the relevance of the enrichment. Table 4: Total list of proteins secreted by PMO Ex23-treated and untreated mdx myotubes309 proteins were observed in both the forward and reverse SILAC experiments, with 235 over-secreted by untreated mdx myotubes and 74 by PMO Ex23-treated. Proteins were sorted based on their localization in the cells (according to DAVID database). The ratio between labeled and unlabeled (i.e., untreated labeled vs. PMO Ex23-treated unlabeled or vice versa) of the two experiments are shown. We report the accession number given by UniProtKB. The table report the proteins that were also identified the analysis of the secretome of mdx vs. wild-type myotubes

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Supplementary material 6 (DOCX 14 kb) Table 2: Mdx and wild-type myotube intracellular accumulation of dextran. To assay passive leakage across the cell membrane, mdx and wild-type myotubes were incubated with 3, 10, or 40 kDa dextran for 5 or 20 min, at day 6 of differentiation. Myotubes were cultured for the preceding 24 h in medium with or without serum (see materials and methods for details). The table summarizes the number of myotubes positive for dextran versus the total number of myotubes analyzed. Mdx: mdx myotubes, WT: wild-type myotubes

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Duguez, S., Duddy, W., Johnston, H. et al. Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion. Cell. Mol. Life Sci. 70, 2159–2174 (2013). https://doi.org/10.1007/s00018-012-1248-2

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  • DOI: https://doi.org/10.1007/s00018-012-1248-2

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