Makoto Yoneda

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.

Accumulation of Deletions and Point Mutations in Mitochondrial Genome in Degenerative Diseases

Accumulation of various mutations in the mitochondrial genome is proposed as an important contributor to aging and degenerative diseases. Extensive fragmentation of mtDNA was detected in association with increased 8-hydroxydeoxyguanosine content in the heart mitochondrial DNA (mtDNA) from a patient with premature aging and mitochondrial cardiomyopathy, who carried a mutation within the mitochondrial tRNA(Asp) gene. This result suggests that damage to mtDNA by hydroxyl radical and accumulation of deleted mtDNA can be accelerated by a specific mitochondrial genotype. Similarly, extensive fragmentation of mtDNA was also detected in cultured cells exposed to a high oxygen concentration atmosphere, implying that mtDNA is vulnerable to reactive oxygen species. To clarify the role of point mutations accumulated in mtDNA, we examined the sequence heterogeneity of mtDNA in the skeletal muscle of a MELAS patient who carried a mutation within the mitochondrial tRNA(leu)(UUR) gene. The analysis revealed that the frequency of mutant clones in the MELAS muscle was significantly higher than those in an age-matched control muscle and a control placenta. Some of these nucleotide substitutions were missense and nonsense mutations, which potentially have deleterious effects on the mitochondrial function. The frequency of nucleotide substitutions in the striatum of three patients with Parkinsons disease was also significantly higher than that in control tissues. We also observed increased protein modification by 4-hydroxy-2-nonenal, a lipid peroxidation by-product, in Parkinsons disease. These results suggests that a vicious cycle contributes to the progression of degenerative process. In this cycle, first a primary mitochondrial mutation(s) induces a mitochondrial respiratory defect, which increases the leakage of reactive oxygen species (ROS) from the respiratory chain. Then the ROS would trigger accumulation of secondary mtDNA mutations in postmitotic cells, leading to further aggravation of mitochondrial respiratory defects and increased production of ROS and lipid peroxides from mitochondria, and thus resulting in degeneration of cellular components.

Increased mitochondrial damage by lipid peroxidation in trophoblast cells of preeclamptic placentas

Lipid peroxides and their related free radicals have been implicated in the pathogenesis of placental dysfunction in preeclampsia. Recent studies suggest that the placenta is a source of the increased lipid peroxides in the maternal circulation of women with preeclampsia. We examined intracellular localization of 4‐hydroxy‐2‐nonenal (HNE: a major aldehydic product of lipid peroxidation)‐modified proteins in human placentas by immunohistochemistry, and immunoblotting. The trophoblast layer of the chorionic villi showed intense immunoreactivity for HNE‐modified proteins in 4 of 12 preeclamptic placentas, whereas no staining was observed in 12 normal placentas. Immunoblotting revealed that three immunoreactive proteins with apparent molecular mass of 110 kDa, 75 kDa, and 70 kDa were localized in the mitochondrial fraction. The present results indicate that the damage to mitochondrial proteins by lipid peroxidation byproducts and subsequent dysfunction of trophoblasts contribute to the pathophysiology of preeclampsia.

Quantitation of mitochondrial DNA carrying tRNALys mutation in MERRF patients

An A to G transition at nucleotide position 8,344 in tRNALys of mitochondrial DNA has been recently identified as a causative mutation of myoclonus epilepsy associated with ragged-red fibers (MERRF). To investigate if the degree of heteroplasmy of mitochondrial DNA is correlated with the severity of MERRF, we have developed a novel method for quantitation of the mutant mitochondrial DNA by polymerase chain reaction using a mismatched primer. With the method, populations of mutant mtDNAs from 5 cases of MERRF carrying the tRNALys mutation were analyzed. The tight linkage of the severity of symptoms and the degree of heteroplasmies is not necessarily observed for all cases, though there is a tendency that patients with less wild type mtDNAs show severer clinical symptoms and earlier onset.

Pleiotropic molecular defects in energy-transducing complexes in mitochondrial encephalomyopathy (MELAS)

The extent of molecular defects in the mitochondrial energy-transducing system was examined in autopsied tissues of a 14-year-old male with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) in order to elucidate the underlying molecular and genetic abnormalities. The patient also had other multiorganic disorders: hypertrophic cardiomyopathy, nephrotic syndrome, and pseudohypoparathyroidism. Enzymic activities of complex I and IV were severely decreased, and those of complex III and V were mildly decreased in the mitochondria isolated from various tissues, but the severity of the deficiencies varied from tissue to tissue. In contrast, complex II and citrate synthase activities were normal or were decreased to a lesser extent than the enzymic activities of other complexes in all the tissues examined. These results suggest that the energy-transducing complexes, namely complexes, I, III, IV, and V, that contain mitochondrially synthesized subunits, were selectively affected. Immunoblot analysis demonstrated that the decreased enzymic activities were based on decreased contents of subunits in these complexes. The multiorganic manifestation of the disorder may result from wide and uneven distribution of abnormal mitochondria that have pleiotropic molecular defects in the energy-transducing complexes among the organs of the patient.

Muscle histopathology in myoclonus epilepsy with ragged-red fibers (MERRF)

Histopathologic findings were examined in skeletal muscle biopsies from 6 patients with myoclonus epilepsy with ragged-red fibers (MERRF) who had an A to G base substitution at mitochondrial DNA (mtDNA) nucleotide pair 8344. In addition to variation in fiber size and ragged-red fibers, all specimens in cross sections showed focal cytochrome c oxidase (CCO) deficiency, suggesting that this finding is crucial in elucidating the role of the mutant mtDNA in the pathogenesis of this disorder. Along the length of single muscle fibers, defects in CCO activity were distributed segmentally with blurred borders in 5 patients which were in contrast with segmental defects with sharply delineated borders seen in chronic progressive external ophthalmoplegia with deleted mtDNA. These morphologically heterogeneous defects in CCO activity may in part be due to differing populations of and distributions of wild and mutants mtDNAs.

Detection and quantification of point mutations in mitochondrial DNA by PCR.

Publisher Summary This chapter describes three useful methods for detection and quantification of mutant mitochondrial DNA (mtDNA) by the polymerase chain reaction (PCR). The methods include (1) PCR–restriction fragment length polymorphism (PCR–RFLP), (2) PCR–mediated restriction site modification (PCR–RSM), and (3) PCR– single-strand conformation polymorphism (PCR-SSCP). Numerous deleterious point mutations of mtDNA, now amounting to more than 30, are associated with various types of human disorders involving deficiencies in the mitochondrial oxidative phosphorylation apparatus. Most of the mutations occur in heteroplasmic form—that is, the mutant and wild-type mtDNAs coexist in varying proportions in different tissues of an individual, causing the clinical features of the diseases. Therefore, detection and quantification of the mutant mtDNA are essential for the diagnosis of the diseases and for understanding the molecular basis of their pathogenesis.

Primary structure of the smallest (6.4-kDa) subunit of human and bovine ubiquinol-cytochrome c reductase deduced from cDNA seqences

Amino acid sequences of the smallest subunit of human and bovine ubiquinol‐cytochrome c reductase were deduced from nucleotide sequence of recombinant cDNA clones isolated by screening the corresponding cDNA libraries. Both proteins were composed of 56 amino acids. They were 84% homologous to each other in the coding nucleotide sequences and 88% homologous in the amino acid sequences. Southern blot analysis with human DNA suggested the presence of a single gene coding for the protein. Northern blot of human mRNAs from different tissues confirmed the existence of a single species of transcript among the tissues but the human gene is highly expressed in bioenergetically active tissues like heart and skeletal muscle.

Mitochondrial DNA Mutations as an Etiology of Human Degenerative Diseases

Because mitochondrial DNA (mtDNA) is exclusively maternally transmitted, mutations of mtDNA are implicated to be the cause of maternally inherited diseases. Recent extensive studies have clarified three types of mtDNA mutations in several human diseases.

REVERSIBLE CELL CYCLE ARREST WITH CONCOMITANT P21/WAF1 OVEREXPRESSION AND MITOCHONDRIAL DESTRUCTION BY NITRIC OXIDE

When human hepatocellular carcinoma Hep‐G2 cells were treated with the NO‐generating compounds, S‐nitroso‐N‐acetyl‐DL‐penicillamine(SNAP) or (±)‐(E)‐4‐ethyl‐2‐(E)‐hydroxyimino]‐5‐nitroso‐3‐hexenamide, cells stopped growing. Most cells were found to be in either G1 or G2/M phase and the dye‐exclusion test revealed that the cells were alive. Electron microscopic examination confirmed the integrity of cells and nuclei. Nuclear staining with the DNA‐binding dye H33258 revealed that cells did not undergo apoptosis although dramatic changes in mitochondrial morphology were noticed within 6 hr of treatment with SNAP by electron microscopy. Western and northern blot analysesrevealed that cells overexpressed p21/WAF1. The growth arrest was released by withdrawal of the NO‐generating compound and cells started to divide within 24 hr after withdrawal of the compounds.