Dataset Information

Differential analysis of dystrophic dog muscle after MuStem cell therapy

ABSTRACT: Duchenne Muscular Dystrophy (DMD), the most common form of inherited neuromuscular disorder, is caused by mutations in the dystrophin gene leading to progressive muscle loss. Secondary changes in signaling pathways and energy metabolism are aggravating factors. Thus, for the evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscle. MuStem cells constitute a new progenitor cell population proposed as a source of cells for a therapeutic strategy. When injected in Golden Retriever Muscular Dystrophy (GRMD) dogs, MuStem cells contribute to myofiber regeneration, satellite cell replenishment, and dystrophin expression. This cell therapy leads to muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. In this study, we used isotope-coded protein labeling (ICPL) to compare the protein expression profiles of muscles coming from three different types of dogs: healthy, GRMD and GRMD after injection of MuStem cells. Based on the comparison of three biological replicates, these triplex ICPL experiments led to the identification of proteins differentially expressed after treatment. This study contributes to a better understanding of the mechanisms of action of MuStem cells.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Canis Familiaris (dog) (canis Lupus Familiaris)

TISSUE(S): Muscle Cell, Femoral Muscle

DISEASE(S): Duchenne Muscular Dystrophy

SUBMITTER: Melanie Lagarrigue

LAB HEAD: Charles Pineau

PROVIDER: PXD001768 | Pride | 2019-05-24

REPOSITORIES: Pride

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Publications

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

Lardenois Aurélie A Jagot Sabrina S Lagarrigue Mélanie M Guével Blandine B Ledevin Mireille M Larcher Thibaut T Dubreil Laurence L Pineau Charles C Rouger Karl K Guével Laëtitia L

Proteomics 20160701 14

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-cod ...[more]

PMID: 27246553

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Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on human skeletal muscle transcripts from a patient with Becker muscular dystrophy. Methods: Tissue homogenates were prepared from frozen sections of a muscle biopsy obtained from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control. Ribosome-protected fragments and total RNA were prepared from a single homogenate, so starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. RPF-Seq libraries were built using the TruSeq Small RNA Sample Preparation Kit (Illumina) and RNA-Seq libraries were built using the TruSeq RNA Sample Preparation v2 Kit (Illumina). RPF-Seq and RNA-Seq libraries were subjected to 50 cycles of single-end sequencing on an Illumina HiSeq 2000 instrument. Trimmed and filtered RPF- and RNA-Seq reads were mapped to RefSeq fasta sequences downloaded from the UCSC genome browser (hg19 assembly). Results: Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that results in the expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosomal profiling. Conclusions: Our results provide a molecular explanation for the rescue of 5’ truncating mutations via a heretofore undescribed mechanism of post-transcriptional regulation of dystrophin expression. The presence of a glucocorticoid-inducible IRES within a highly conserved region of the DMD sequence strongly suggests a programmed role for alternate translation initiation, and ongoing efforts to understand the relevant cell lineage-specific and/or conditional activation signals will shed light on underlying mechanisms of IRES control and elucidate potentially novel functions of dystrophin. Skeletal muscle ribosome-protected fragment and RNA-Seq profiles from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control were generated by deep sequencing using the Illumina HiSeq 2000.

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