Giuseppe Attardi

Proteolytic Processing of OPA1 Links Mitochondrial Dysfunction to Alterations in Mitochondrial Morphology

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase γ. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.

Isolation of human cell lines lacking mitochondrial DNA.

Publisher Summary This chapter discusses isolation of human cell lines lacking mitochondrial DNA. An understanding of the molecular genetic mechanisms by which these mutations act can provide insights into the etiology, pathogenesis, and ultimately the treatment of these diseases, and may enhance the knowledge of mitochondrial biogenesis. For investigating nucleomitochondrial interactions and the molecular pathogenetic mechanisms of mtDNA mutations, it is useful to manipulate the mtDNA complement of a cell, move mitochondria from one cellular environment to another, or introduce new genes into mitochondria. As an initial step towards these goals, there is an isolation human cell lines that completely lack mtDNA (ρ0 cell lines). The chapter describes the theory and the methods utilized to isolate such cell lines.

Giant-size rapidly labeled nuclear ribonucleic acid and cytoplasmic messenger ribonucleic acid in immature duck erythrocytes

Abstract The pattern of RNA synthesis has been investigated in immature erythrocytes present in the blood from normal and anemic ducks and maintained in vitro in a suitable medium. The majority of the RNA synthesized in vitro by these cells remains associated with the nuclear fraction and consists of RNA species of high sedimentation coefficient. This heavy RNA appears to include two distinct types: (a) ribosomal-type RNA, representing presumably precursors of ribosomal RNA, sedimenting between 30 and 50 s; (b) RNA different from ribosomal RNA in base ratios and with sedimentation coefficients ranging from 30 to 80 s or more. Under the conditions of cell maintenance in vitro used in the present work, the synthesis of presumptive ribosomal RNA precursors appears to be limited in time, ceasing after a few hours: in addition, only a relatively small conversion of such precursors to mature ribosomal RNA takes place in immature erythrocytes. The heavy non-ribosomal type nuclear RNA represents a heterogeneous mixture of molecules characterized by high U and relatively low GC content. All the evidence accumulated in the present work using enzymic tests, changes in the extraction procedure and in the conditions of sedimentation analysis, thermal denaturation tests, reconstruction experiments and base composition studies, supports the conclusion that the high sedimentation coefficients of these molecules are a reflection of their large size, corresponding to molecular weights from 2 × 10 6 to 10 7 or possibly more. These giant-size molecules are metabolically unstable, with a halflife of about 30 min; under the experimental conditions used in the present work, they continue to be synthesized and destroyed for as long as two days. A minor portion of the RNA synthesized in vitro by immature duck erythrocytes during a two-to three-hours incubation is found to be associated with the polysome fraction, and contains, in addition to a variable but in general relatively small amount of ribosomal RNA, a heterogeneous RNA fraction with sedimentation coefficients between 6 and 35 s, and base ratios different from those of ribosomal RNA and reflecting rather the base composition of DNA: this fraction can be identified as messenger RNA. In blood cell populations rich in immature erythrocytes at early stages of development, the labeling of the polysomal messenger RNA increases dramatically, and a large fraction of it sediments as a relatively sharp peak with a sedimentation coefficient of about 9 s: this corresponds to a molecular weight of about 150,000, which would be expected for the messengers of hemoglobin chains. Both base composition and kinetics data strongly suggest that no relationship of precursor-to-product type or, at any rate, not a simple one, exists between the heavy non-ribosomal type nuclear RNA and the cytoplasmic messenger RNA.

Animal Mitochondrial DNA: An Extreme Example of Genetic Economy

Publisher Summary This chapter describes animal mitochondrial DNA. It focuses on the aspects of the structure, function, and evolution of animal mitochondrial DNA (mtDNA). It also explores the questions raised by the recent unravelling of the mitochondrial genetic code and of the primary structure and gene organization of animal mtDNA. The most conspicuous among the features of animal mtDNA from animal cells, yeast, and filamentous fungi, is the use of UGA as a tryptophan codon rather than as a stop codon. There are genetic code differences between the individual mitochondrial genetic systems, even within the same philogenetic group. In mammalian, but not in fungal mitochondria, besides AUG, also AUA, AUU, and AUC can function as initiator codons; in this role, AUU and AUC may indeed be read by N-formyl-methionyl-tRNA and thus code for N-formyl-methionine, like AUG and AUA. There is a case among mammalian mitochondrial tRNAs where a C in the first position of the anticodon, rather than a U, enables it to pair with A and G in third codon position. The mtDNA of all animal cells so far analyzed is in the form of circular molecules of relatively uniform length, with their sizes ranging between 15.7 and 19.5 kb.

In vitro genetic transfer of protein synthesis and respiration defects to mitochondrial DNA-less cells with myopathy-patient mitochondria.

A severe mitochondrial protein synthesis defect in myoblasts from a patient with mitochondrial myopathy was transferred with myoblast mitochondria into two genetically unrelated mitochondrial DNA (mtDNA)-less human cell lines, pointing to an mtDNA alteration as being responsible and sufficient for causing the disease. The transfer of the defect correlated with marked deficiencies in respiration and cytochrome c oxidase activity of the transformants and the presence in their mitochondria of mtDNA carrying a tRNA(Lys) mutation. Furthermore, apparently complete segregation of the defective genotype and phenotype was observed in the transformants derived from the heterogeneous proband myoblast population, suggesting that the mtDNA heteroplasmy in this population was to a large extent intercellular. The present work thus establishes a direct link between mtDNA alteration and a biochemical defect.

Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication

The recently discovered aging-dependent large accumulation of point mutations in the human fibroblast mtDNA control region raised the question of their occurrence in postmitotic tissues. In the present work, analysis of biopsied or autopsied human skeletal muscle revealed the absence or only minimal presence of those mutations. By contrast, surprisingly, most of 26 individuals 53 to 92 years old, without a known history of neuromuscular disease, exhibited at mtDNA replication control sites in muscle an accumulation of two new point mutations, i.e., A189G and T408A, which were absent or marginally present in 19 individuals younger than 34 years. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in three subjects in very low amounts (A189G). Furthermore, in several older individuals exhibiting an accumulation in muscle of one or both of these mutations, they were nearly absent in other tissues, whereas the most frequent fibroblast-specific mutation (T414G) was present in skin, but not in muscle. Among eight additional individuals exhibiting partial denervation of their biopsied muscle, four subjects >80 years old had accumulated the two muscle-specific point mutations, which were, conversely, present at only very low levels in four subjects ≤40 years old. The striking tissue specificity of the muscle mtDNA mutations detected here and their mapping at critical sites for mtDNA replication strongly point to the involvement of a specific mutagenic machinery and to the functional relevance of these mutations.

MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination.

We have investigated the pathogenetic mechanism of the mitochondrial tRNALys gene mutation (position 8344) associated with MERRF encephalomyopathy in several mitochondrial DMA (mtDNA)–less cell transformants carrying the mutation and in control cells. A decrease of 50–60% in the specific tRNALys aminoacylation capacity per cell was found in mutant cells. Furthermore, several lines of evidence reveal that the severe protein synthesis impairment in MERRF mutation–carrying cells is due to premature termination of translation at each or near each lysine codon, with the deficiency of aminoacylated tRNALys being the most likely cause of this phenomenon.

The tRNA genes punctuate the reading of genetic information in human mitochondrial DNA

A detailed transcription map of HeLa cell mitochondrial DNA (mtDNA) has been constructed by using the S1 protection technique to localize precisely the sequences coding for the ribosomal RNA (rRNA) and poly(A)-containing species on the physical map of the DNA. This transcription map has been correlated with the positions of the tRNA genes derived from the mtDNA sequence. It has been shown that, with the exception of the D loop and another small segment near the origin of replication, the mtDNA sequences are completely saturated by the rRNAs, poly(A)-containing RNAs and tRNA coded for by the two strands. No evidence for intervening sequences has been found. The sequences coding for the individual poly(A)-containing RNA and rRNA species appear to be immediately contiguous on one side, and most frequently on both sides, to tRNA coding sequences. Furthermore, the H strand sequences coding for the two rRNAs, the poly(A)-containing RNAs and the tRNAs appear to be adjacent to each other, extending from coordinate 2/100 to coordinate 95/100 of the genome relative to the origin taken as 0/100. The results are consistent with a model of transcription of the H strand in the form of a single molecule which is processed into mature RNA species by precise endonucleolytic cleavages, occurring in almost all cases immediately before and after a tRNA sequence. The tRNA sequences may play an important role as recognition signals in the processing of the primary transcripts.

MtDNA mutations in aging and apoptosis.

There is considerable evidence that the oxidative phosphorylation capacity of human mitochondria declines in various tissues with aging. However, the genetic basis of this phenomenon has not yet been clarified. The occurrence of large deletions in mtDNA from brain, skeletal, and heart muscles and other tissues of old subjects at relatively low levels has been well documented. We discuss their possible functional relevance for the aging processes. On the contrary, until very recently, only inconclusive and often discordant evidence was available for the accumulation of mtDNA point mutations in old individuals. In the past few years, however, an aging-dependent large accumulation of mtDNA point mutations has been demonstrated in the majority of individuals above a certain age. These mutations occur in the mtDNA main control region at critical sites for mtDNA replication in fibroblasts and skeletal muscles. The extraordinary tissue specificity and nucleotide selectivity of these mutations strongly support the idea of their being functionally relevant. Evidence in agreement with this conclusion has been provided by the very recent observation that an mtDNA mutation occurring in blood leukocytes near an origin of replication, which causes a remodeling of this origin, occurs at a strikingly higher frequency in centenarians and monozygotic and dizygotic twins than in the control populations, strongly pointing to its survival value. The present article reviews another area of active research and discussion, namely, the role of pathogenic mtDNA mutations in causing programmed cell death. The available evidence has clearly shown that mtDNA and respiration are not essential for the process of apoptosis. However, the limited and sometimes contradictory data indicate that the absence or impaired function of mtDNA can influence the rate of this process, most probably by regulating the production of reactive oxygen species or the lack thereof.

Low Reserve of Cytochrome c Oxidase Capacity in Vivo in the Respiratory Chain of a Variety of Human Cell Types

The question of whether and to what extent thein vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16–40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O2 consumption rate supported by 0.4 mmtetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7–22% excess) for ADP + glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold.