Eric A. Shoubridge

The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes

In mammals, mitochondrial DNA (mtDNA) sequence variants are observed to segregate rapidly between generations despite the high mtDNA copy number in the oocyte. This has led to the concept of a genetic bottleneck for the transmission of mtDNA, but the mechanism remains contentious. Several studies have suggested that the bottleneck occurs during embryonic development, as a result of a marked reduction in germline mtDNA copy number. Mitotic segregation of mtDNAs during preimplantation, or during the expansion of primordial germ cells (PGCs) before they colonize the gonad, is thought to account for the increase in genotypic variance observed among mature oocytes from heteroplasmic mothers. This view has, however, been challenged by studies suggesting that the bottleneck occurs without a reduction in germline mtDNA content. To resolve this controversy, we measured mtDNA heteroplasmy and copy number in single germ cells isolated from heteroplasmic mice. By directly tracking the evolution of mtDNA genotypic variance during oogenesis, we show that the genetic bottleneck occurs during postnatal folliculogenesis and not during embryonic oogenesis.

A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy

NADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system.

Mutations in COX15 Produce a Defect in the Mitochondrial Heme Biosynthetic Pathway, Causing Early-Onset Fatal Hypertrophic Cardiomyopathy

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patients heart and fibroblast mitochondria, and levels of heme O were increased in the patients heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%-70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency.

Deletion mutants are functionally dominant over wild-type mitochondrial genomes in skeletal muscle fiber segments in mitochondrial disease

We mapped the distribution and expression of wild-type and deleted mitochondrial DNA (mtDNA) molecules in skeletal muscle fibers of patients with mitochondrial disease. We show that ragged red fiber segments, which are characteristic histological features of these myopathies, represent focal accumulations of mitochondria containing predominantly deleted mtDNAs and that the mutant genomes are absent or extremely rare in normal fiber segments. This suggests that the mtDNA mutations play a direct role in focal mitochondrial accumulation. Although levels of wild-type mtDNAs and mRNAs in ragged red fiber segments are near normal, mitochondrial function, as revealed by cytochrome oxidase cytochemistry, is severely impaired. This suggests that the presence of mutant mtDNAs interferes with the expression of coexisting wild-type mtDNAs in these segments at a posttranscriptional level.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B12 (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Mitochondrial DNA and the Mammalian Oocyte

In mammals, mitochondria and mitochondrial DNA (mtDNA) are transmitted through the female germ line. Mature oocytes contain at least 100,000 copies of mtDNA, organized at 1-2 copies per organelle. Despite the high genome copy number, mtDNA sequence variants are observed to segregate rapidly between generations, and this has led to the concept of a developmental bottleneck for the transmission of mtDNA. Ultrastructural investigations of primordial germ cells show that they contain approximately 10 mitochondria, suggesting that mitochondrial biogenesis is arrested during early embryogenesis, and that the mitochondria contributing to the germ cell precursors are simply apportioned from those present in the zygote. Thus, as few as 0.01% of the mitochondria in the oocyte actually contribute to the offspring of the next generation. Mitochondrial replication restarts in the migrating primordial germ cells, and mitochondrial numbers steadily increase to a few thousand in primordial oocytes. Genetic evidence from both heteroplasmic mice and human pedigrees suggests that segregation of mtDNA sequence variants is largely a stochastic process that occurs during the mitotic divisions of the germ cell precursors. This process is essentially complete by the time the primary oocyte population is differentiated in fetal life. Analysis of the distribution of pathogenic mtDNA mutations in the offspring of carrier mothers shows that risk of inheriting a pathogenic mutation increases with the proportion in the mother, but there is no bias toward transmitting more or less of the mutant mtDNAs. This implies that there is no strong selection against oocytes carrying pathogenic mutations and that atresia is not a filter for oocyte quality based on oxidative phosphorylation capacity. The large number of mitochondria and mtDNAs present in the oocyte may simply represent a genetic mechanism to ensure their distribution to the gametes and somatic cells of the next generation. If true, mtDNA copy number, and by inference mitochondrial number, may be the most important determinant of oocyte quality, not because of the effects on oocyte metabolism, but because too few would result in a maldistribution in the early embryo.

Mutation in TACO1, encoding a translational activator of COX I, results in cytochrome c oxidase deficiency and late-onset Leigh syndrome

Defects in mitochondrial translation are among the most common causes of mitochondrial disease, but the mechanisms that regulate mitochondrial translation remain largely unknown. In the yeast Saccharomyces cerevisiae, all mitochondrial mRNAs require specific translational activators, which recognize sequences in 5′ UTRs and mediate translation. As mammalian mitochondrial mRNAs do not have significant 5′ UTRs, alternate mechanisms must exist to promote translation. We identified a specific defect in the synthesis of the mitochondrial DNA (mtDNA)-encoded COX I subunit in a pedigree segregating late-onset Leigh syndrome and cytochrome c oxidase (COX) deficiency. We mapped the defect to chromosome 17q by functional complementation and identified a homozygous single-base-pair insertion in CCDC44, encoding a member of a large family of hypothetical proteins containing a conserved DUF28 domain. CCDC44, renamed TACO1 for translational activator of COX I, shares a notable degree of structural similarity with bacterial homologs, and our findings suggest that it is one of a family of specific mammalian mitochondrial translational activators.

The Role of Mitochondrial DNA Copy Number in Mammalian Fertility

Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000–50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.

LRPPRC and SLIRP interact in a ribonucleoprotein complex that regulates posttranscriptional gene expression in mitochondria.

The PPR family protein LRPPRC is implicated in the French Canadian variant of Leigh syndrome, a fatal neurodegenerative disease. LRPPRC functions in posttranscriptional mitochondrial gene expression as part of an RNP complex with SLIRP, a stem-loop RNA-binding protein, to regulate the stability and handling of mature mRNAs.

Identification and Characterization of a Common Set of Complex I Assembly Intermediates in Mitochondria from Patients with Complex I Deficiency

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.