Dmd Gene Mutations Cause Cell-autonomous Alterations in Macrophages

Abstract

Background
DMD is the largest human gene known spanning ~2.3 mega bases or 0.08% of the human genome. It encodes the dystrophin protein and its expression is driven by eight promoters giving rise to multiple isoforms. These specific dystrophin isoforms interact with unique associated proteins creating intricate scaffolds and signalling complexes, compositions of which can vary in different cells. Mutations in the DMD gene that lead to the loss of dystrophin production cause Duchenne muscular dystrophy. This highly debilitating and lethal disease presents in early childhood with progressive muscle loss with sterile inflammation, accompanied by brain and bone impairments. This diversity of symptoms illustrates the importance of dystrophin in various non-muscle cells. In this respect, the immune cells, and particularly macrophages, are known to be involved in the initiation and progression of the DMD pathology. Therefore, this project investigated the impact of DMD gene mutations on macrophages hypothesizing that DMD gene mutations cause cell-autonomous alterations in macrophages.

Methods and findings
Using combinations of proteomics and functional assays, this study demonstrates for the first time that mutations affecting specific dystrophin isoforms can result in cell-autonomous abnormalities in mouse macrophages. Two DMD mouse models have been used in this investigation: The mdx lacks the full-length isoforms and the mdxβgeo has all dystrophin expression ablated. Firstly, we characterised the Dmd gene expression in differentiating myeloblasts and demonstrated the specific Dp71 transcript (Dp71b) expression there. The Dp71b expression was bi-phasic, with mRNA levels gradually reduced as cells differentiated into macrophages and its expression increasing again in peritoneal macrophages. Peritoneal macrophages from mdxβgeo but not mdx mice showed significant adhesion and migration deficits, measured using live cell microscopy. Moreover, proteomic studies of naïve mdxβgeo bone marrow cells differentiated for 7 days in vitro into mature macrophages revealed upregulated expression of 139 proteins and downregulation of further 188 proteins.
Bioinformatics analyses performed using MetaCore showed alterations of specific pathways: adhesion, phagocytosis, cell cycle and cytoskeleton organisation. These global protein changes extended into protein phosphorylation, methylation and acetylation. Functional analyses confirmed that mdxϐgeo bone marrow-derived macrophages exhibited an abnormal flatter morphology, larger surface area and different cytoskeletal organisation when compared with C57BL/6 controls. These cells were also less adherent. Following LPS stimulation, these bone-marrow derived mdxϐgeo macrophages demonstrated reduced migration (p=0.009) and invasion in Boyden chamber assay (p = 0.058). Their capability to internalize Dextran (MW 40 000) was also reduced after 2 h (p = 0.022) and 4 h (p = 0.050) incubation. The cell cycle was one of the top pathways found to be altered in the proteomics analysis. Interestingly, while preliminary functional assays in mature bone marrow derived macrophages at day 7 did not reveal clear directional alterations, at day 10 dystrophic macrophages showed increased ki67 staining (p=0.01) suggesting higher proliferation rate. Additionally, this study provides for the first time, an indication that kainate receptors might be expressed in macrophages and that Dmd mutations affect their expression and function.

Conclusions
In this study, a new role of the Dmd gene in the specific cells of the immune system has been identified and this data suggest that the expression of specific Dp71 dystrophin may have a role in the development and/or differentiation of macrophages. Further studies are clearly needed, and this should include analysis of human macrophages, as somatic mutations or epigenetic changes affecting Dmd expression in myeloid progenitors and macrophages may affect their functions. Moreover, findings described here demonstrate that the DMD pathology may still not be understood fully as it extends beyond muscle fibre damage due to sarcolemma instability. As this disease leads to progressive, severe disability and death of young men and no treatment improves the long-term outcome, our findings raise the possibility that by exploring these alternative targets, effective therapies could be developed.

Date of Award	12 Jun 2018
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	Darek Gorecki (Supervisor)