Abstract
Mitochondrial DNA deletions affect energy metabolism at tissue-specific and cell-specific threshold levels, but the pathophysiological mechanisms determining cell fate remain poorly understood. Chronic progressive external ophthalmoplegia (CPEO) is caused by mtDNA deletions and characterized by a mosaic distribution of muscle fibers with defective cytochrome oxidase (COX) activity, interspersed among fibers with retained functional respiratory chain. We used diagnostic histochemistry to distinguish COX-negative from COX-positive fibers in nine muscle biopsies from CPEO patients and performed laser capture microdissection (LCM) coupled to genome-wide gene expression analysis. To gain molecular insight into the pathogenesis, we applied network and pathway analysis to highlight molecular differences of the COX-positive and COX-negative fiber transcriptome. We then integrated our results with proteomics data that we previously obtained comparing COX-positive and COX-negative fiber sections from three other patients. By virtue of the combination of LCM and a multi-omics approach, we here provide a comprehensive resource to tackle the pathogenic changes leading to progressive respiratory chain deficiency and disease in mitochondrial deletion syndromes. Our data show that COX-negative fibers upregulate transcripts involved in translational elongation and protein synthesis. Furthermore, based on functional annotation analysis, we find that mitochondrial transcripts are the most enriched among those with significantly different expression between COX-positive and COX-negative fibers, indicating that our unbiased large-scale approach resolves the core of the pathogenic changes. Further enrichments include transcripts encoding LIM domain proteins, ubiquitin ligases, proteins involved in RNA turnover, and, interestingly, cell cycle arrest and cell death. These pathways may thus have a functional association to the molecular pathogenesis of the disease. Overall, the transcriptome and proteome show a low degree of correlation in CPEO patients, suggesting a relevant contribution of post-transcriptional mechanisms in shaping this disease phenotype.
Highlights
The clinical spectrum of mitochondrial DNA deletion syndromes is highly variable, ranging from mild myopathy to the severe syndromic Kearns-Sayre Syndrome 4.0/).(KSS) [1]
We carried out a cytochrome oxidase (COX)/SDH staining in cross sections of muscle biopsies from nine
Chronic progressive external ophthalmoplegia (CPEO) patients (Table S1) Using the images acquired in stained sections as a reference, we identified the corresponding COX+ and COX− unstained fibers in serial sections and dissected them by laser capture microdissection (LCM) using a laser beam with a wavelength of 349 nm
Summary
The clinical spectrum of mitochondrial DNA (mtDNA) deletion syndromes is highly variable, ranging from mild myopathy to the severe syndromic Kearns-Sayre Syndrome 4.0/).(KSS) [1]. The clinical spectrum of mitochondrial DNA (mtDNA) deletion syndromes is highly variable, ranging from mild myopathy to the severe syndromic Kearns-Sayre Syndrome 4.0/). Single size mtDNA deletions generally occur sporadically and are in most instances not transmitted to offspring. Multiple deletions are caused by mutations of nuclear genes coding for the mitochondrial replication machinery and show Mendelian transmission [3,4]. Within a single cell or tissue, mutated mtDNA can coexist with wild type molecules, a situation referred to as heteroplasmy The clinical heterogeneity is considered to depend on the inter- and intraindividual degree of heteroplasmy and size and location of the deletions, and nuclear disease modifiers and environmental factors [5,6,7]. MtDNA deletions occur in muscle fibers and neurons as a function of age [8]
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