Defects of mitochondrial respiratory enzymes in cloned cells from MELAS fibroblasts

Abstract

Defects in mitochondrial DNA (mtDNA) are associated with mitochondrial encephalomyopathies (McKusick 251900). The mutations include deletions but also duplications and point mutations. In individuals with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), a common A-to-G substitution at position 3243 in the leucine (UUR) tRNA gene has been elucidated (Goto et al 1990). Furthermore, this mutant mtDNA always coexists with the wild-type mtDNA, and the proportion of the mutated mitochondrial genome is different in each tissue of one individual.This mutation creates an ApaI restriction site, and we can then easily make the molecular diagnosis for this syndrome. Blood samples are also available for molecular genetic analysis (Hammans et al 1991), but a new problem arises: do the individuals with the mutation always develop MELAS or not? It is important to answer this question whether the point mutation affects the functional mitochondrial abnormality. A defect of complex I has been detected in muscles from patients with MELAS. However, activity of this enzyme does not always decrease in fibroblasts from a MELAS patient despite their containing the mutated mitochondrial genome. We therefore studied how the mutant mtDNA coexists with wild-type mtDNA in one cell and affects functional abnormality. MATERIALS A N D M E T H O D SSkin fibroblasts from patients with MELAS were grown in Eagle's minimum essential medium (MEM) supplemented with 10% (v/v) fetal bovine serum, 0.15 % (w/v) sodium bicarbonate, 50#g/ml streptomycin and 100 U/ml penicillin. When cells were 60% confluent in a 25 crn z flask, the medium was replaced with 8 ml of opti-MEMI (Gibco). A 20 #g sample of SV40 DNA lacking its replication origin were diluted in 50 #1 of water. SV40 DNA and lipofectin solutions (50 #1 of each) were gently mixed and incubated for 15 rain at room temperature. The SV40 DNA-lipofectin complex was dropped into the cells in the opti MEM. After incubation for 12-16 h under 5% carbon dioxide at 37°C, the cells were harvested with phosphate-buffered saline containing 0.3% trypsin. About 3000 cells were distributed into each a welt of 96-well culture plate filled with 0.8 ml of the culture medium. The cells were taken out 797

Clinical and biochemical phenotype of the MELAS mutation

“…However, the activity of complex I decreased at lower population of this mutation (Figure 4). These results agree with our previous data (Miyabayashi et al 1992). The reason for this result is that complex I has more component peptides encoding from mtDNA than does complex IV, and mtDNA mutation is more harmful to its assembly.…”

“…The restriction fragments were separated by electrophoresis on a 3% agarose gel and were photographed under UV light in the presence of ethidium bromide. The quantitative analysis of mutated and wild-type mtDNA was done using isotope-labelled primer (Miyabayashi et al 1992).…”

Oxidative phosphorylation defect in the brains of carriers of the tRNAleu(UUR) A3243G mutation in a MELAS pedigree

“…The available data suggest that the most important respiratory chain abnormality associated with the A3243G mutation is a deficiency in the activity of complex I. [11][12][13] Little is known about the rate-limiting steps in the respiratory chain in different tissues, but it is possible that com-plex I has a more important regulatory role in the brain than in tumor cells in vitro. Third, it is possible that mtDNAs are organized differently in CNS cells than in other cells.…”

“…[7][8][9] The nature and extent of the biochemical defect produced by the A3243G mutation has been investigated in immortal tumor cell lines repopulated with high proportions of the A3243G mutant (cybrid cells) 10 -12 and in fibroblasts. 13 These cells exhibit a severe defect in mitochondrial translation and deficiencies in the ac-tivities of the respiratory chain complexes, particularly in complex I when the proportion of mutant mtDNAs exceeds 95%. 10 Similarly high thresholds have been observed for other mtDNA tRNA mutations studied in cybrid cells, in primary myoblast cultures, 14 or in single muscle fibers.…”

Mitochondrial DNA Density Homeostasis Accounts for the Threshold Effect in Human Mitochondrial Disease

“…In particular, cells appear to be able to withstand high levels of heteroplasmy without showing any significant metabolic or physiological defect. For instance, fibroblasts possessing the MELAS mutation were shown to have unaffected respiratory enzyme activity until mutant load exceeded around 60% [10]. Also, Chomyn et al [11] showed that oxygen consumption of cells does not significantly reduce until MELAS heteroplasmy exceeds ∼90%.…”