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Altered mitochondrial function in fibroblasts containing MELAS or MERRF mitochondrial DNA mutations
1Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Abstract:
A number of human diseases are caused by inherited mitochondrial DNA mutations. Two of these diseases, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres), are commonly caused by point mutations to tRNA genes encoded by mitochondrial DNA. Here we report on how these mutations affect mitochondrial function in primary fibroblast cultures established from a MELAS patient containing an A to G mutation at nucleotide 3243 in the tRNA(Leu(UUR) gene and a MERRF patient containing an A to G mutation at nucleotide 8344 in the tRNA(Lys) gene. Both mitochondrial membrane potential and respiration rate were significantly decreased in digitonin-permeabilized MELAS and MERRF fibroblasts respiring on glutamate/malate. A similar decrease in mitochondrial membrane potential was found in intact MELAS and MERRF fibroblasts. The mitochondrial content of these cells, estimated by stereological analysis of electron micrographs and from measurement of mitochondrial marker enzymes, was similar in control, MELAS and MERRF cells. Therefore, in cultured fibroblasts, mutation of mitochondrial tRNA genes leads to assembly of bioenergetically incompetent mitochondria, not to an alteration in their amount. However, the cell volume occupied by secondary lysosomes and residual bodies in the MELAS and MERRF cells was greater than in control cells, suggesting increased mitochondrial degradation in these cells. In addition, fibroblasts containing mitochondrial DNA mutations were 3-4-fold larger than control fibroblasts. The implications of these findings for the pathology of mitochondrial diseases are discussed.
Insights
Inherited mitochondrial DNA mutations in tRNA genes cause mitochondrial diseases like MELAS and MERRF. These mutations impair mitochondrial function and increase cell size, rather than altering mitochondrial quantity.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Inherited mitochondrial DNA (mtDNA) mutations are linked to human diseases.
- MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) are common mtDNA diseases caused by tRNA gene point mutations.
Purpose of the Study:
- To investigate the impact of specific mtDNA tRNA mutations on mitochondrial function in patient-derived fibroblasts.
- To determine if these mutations affect mitochondrial quantity or bioenergetic capacity.
Main Methods:
- Primary fibroblast cultures from MELAS (3243 A>G tRNA(Leu(UUR))) and MERRF (8344 A>G tRNA(Lys)) patients were used.
- Mitochondrial membrane potential and respiration were measured in permeabilized and intact cells.
- Mitochondrial content was assessed using stereological analysis and marker enzyme assays.
- Cell volume and lysosomal/residual body content were quantified.
Main Results:
- Both MELAS and MERRF fibroblasts exhibited significantly decreased mitochondrial membrane potential and respiration rates.
- Mitochondrial content was comparable between control, MELAS, and MERRF fibroblasts.
- Cell volume was 3-4 fold larger in MELAS and MERRF fibroblasts compared to controls.
- Increased lysosomal and residual body volume suggested enhanced mitochondrial degradation.
Conclusions:
- mtDNA tRNA gene mutations lead to bioenergetically incompetent mitochondria, not changes in mitochondrial amount, in cultured fibroblasts.
- Increased cell size and evidence of mitochondrial degradation are associated with these mutations.
- Findings provide insights into the cellular pathology of MELAS and MERRF diseases.