Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

Gellerich, Frank N.; Deschauer, Marcus; Chen, Ying; Müller, Tobias; Neudecker, Stephan; Zierz, Stephan

doi:10.1016/s0005-2728(02)00305-5

Cited by 78 publications

(53 citation statements)

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“…All patients with single, large‐scale mtDNA deletions assessed in this study showed a combined complex I and IV deficiency, consistent with the removal of several mt‐encoded structural subunits and mt‐tRNAs, as previously reported 18, 20, 22, 33. However, 3 distinct respiratory chain profiles were observed, suggesting differential rates of COX‐I and NDUFB8 decline.…”

Section: Discussionsupporting

confidence: 89%

“…Larger mtDNA deletions have been associated with more severe disease and earlier disease onset 15, 16, 17, 18, 19. Whereas a highly significant,20 significant,15, 16, 21, 22 weak,18, 23 or no24 correlation has been reported between the percentage of COX‐deficient fibers and the degree of mtDNA heteroplasmy, most studies found no correlation between the biochemical defects and the nature of the deletion (the number of protein‐encoding genes and the complexes affected by the deletion) or deletion size alone 4, 15, 20, 22, 25. Interestingly, a higher proportion of COX‐deficient ragged‐red fibers was found in patients with deletions encompassing the 3 mtDNA‐encoded COX genes,26, 27 whereas an isolated complex I deficiency was reported in patients with smaller mtDNA deletions removing only complex I genes 26…”

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confidence: 99%

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Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

Rocha

Rosa

Grady

et al. 2018

Annals of Neurology

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ObjectiveSingle, large‐scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large‐scale mtDNA deletions in skeletal muscle.MethodsWe investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large‐scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction.ResultsWe have defined 3 “classes” of single, large‐scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class.InterpretationCombining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex‐specific protein‐encoding genes. Furthermore, removal of mt‐tRNA genes impacts specific complexes only at high deletion levels, when complex‐specific protein‐encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115–130

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Section: Discussionsupporting

confidence: 89%

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confidence: 99%

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

Rocha

Rosa

Grady

et al. 2018

Annals of Neurology

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“…Single mtDNA deletions cause KSS, CPEO, or PS, whose affected tissues show impaired electron transport activity, ATP production, and mitochondrial protein synthesis [26,56,57]. Muscle biopsies from patients with KSS or CPEO show ragged-red fibers and cytochrome oxidase-negative fibers [58,59].…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial DNA deletions inhibit proteasomal activity and stimulate an autophagic transcript

Alemi

Prigione

Wong

et al. 2007

Free Radical Biology and Medicine

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Deletions within the mitochondrial DNA (mtDNA) cause Kearns Sayre Syndrome (KSS) and Chronic Progressive External Opthalmoplegia (CPEO). The clinical signs of KSS include muscle weakness, heart block, pigmentary retinopathy, ataxia, deafness, short stature, and dementia. The identical deletions occur and rise exponentially as humans age, particularly in substantia nigra. Deletions at >30% concentration cause deficits in basic bioenergetic parameters, including membrane potential and ATP synthesis, but it is poorly understood how these alterations cause the pathologies observed in patients. To better understand the consequences of mtDNA deletions, we microarrayed 6 cell types containing mtDNA deletions from KSS and CPEO patients. There was a prominent inhibition of transcripts encoding ubiquitin-mediated proteasome activity, and a prominent induction of transcripts involved in the AMP kinase pathway, macroautophagy, and amino acid degradation. In mutant cells, we confirmed a decrease in proteasome biochemical activity, significantly lower concentration of several amino acids, and induction of an autophagic transcript. An interpretation consistent with the data is that mtDNA deletions increase protein damage, inhibit the ubiquitin-proteasome system, decrease amino acid salvage, and activate autophagy. This provides a novel pathophysiological mechanism for these diseases, and suggests potential therapeutic strategies.

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“…The combined COX/SDH histochemical method enables the visualization of cells with mitochondrial dysfunction. This technique, with early studies dating back to 1968, remains popular, with many considering it the "gold standard" for identifying mitochondrial diseases in patients 14,19,26,27 . It is now frequently used to investigate mtDNA mutation-driven aging and aging-related disorders 12,13,18,20,21,24 .…”

Section: Figure 1 Mitochondrial Respiratory Complexes I-vmentioning

confidence: 99%

Visualization of Mitochondrial Respiratory Function using Cytochrome <em>C</em> Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Ross

2011

JoVE

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Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2 . The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3 . A wealth of evidence has been compiled in support of this theory 1,4 , an example being the mtDNA mutator mouse 5 ; however, the precise role of mtDNA damage in aging is not entirely understood 6,7 .Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2 . Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9 . Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis [10][11][12] . The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14 . Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21 .Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23 . 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22 . In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22 . Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24 . Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9 . Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22 . Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25 . Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented. Video LinkThe video component of this article can be found at

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Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

Cited by 78 publications

References 58 publications

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

Mitochondrial DNA deletions inhibit proteasomal activity and stimulate an autophagic transcript

Visualization of Mitochondrial Respiratory Function using Cytochrome <em>C</em> Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

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