High-frequency inter-parental recombination between mitochondrial genomes of rice cybrids

Analyzing more than 100 independent rice cybrids, we found evidence for inter-molecular recombination between parental mitochondrial genomes occurring at high frequency soon after protoplast fusion. The structure of the region around the atp6 gene showed extensive polymorphism among Indica (MTC-5A), Japonica (Nipponbare), and wild abortive (IR58024A) mitochondrial genomes. Recombination between the mitochondrial genomes of IR58024A and MTC-5A around the atp6 gene was detected by Southern-blot analysis of cybrid plants. Such recombinant mitochondrial molecules were also cloned from IR58024A/Nipponbare cybrid callus. PCR analysis around the atp6 gene demonstrated that inter-parental recombination occurs in practically all cybrid calli within 2 weeks after protoplast fusion. At this point, parental and recombinant mitochondrial genomes coexisted within the callus. Over the course of further cultivation, however, mitochondrial genome diversity decreased as parental and/or recombinant genomes segregated out.     

Bioenergetic dysfunction in Huntington's disease human cybrids

In this work we studied the mitochondrial-associated metabolic pathways in Huntington's disease (HD) versus control (CTR) cybrids, a cell model in which the contribution of mitochondrial defects from patients is isolated. HD cybrids exhibited an interesting increase in ATP levels, when compared to CTR cybrids. Concomitantly, we observed increased glycolytic rate in HD cybrids, as revealed by increased lactate/pyruvate ratio, which was reverted after inhibition of glycolysis. A decrease in glucose-6-phosphate dehydrogenase activity in HD cybrids further indicated decreased rate of the pentose-phosphate pathway. ATP levels of HD cybrids were significantly decreased under glycolysis inhibition, which was accompanied by a decrease in phosphocreatine. Nevertheless, pyruvate supplementation could not recover HD cybrids' ATP or phosphocreatine levels, suggesting a dysfunction in mitochondrial use of that substrate. Oligomycin also caused a decrease in ATP levels, suggesting a partial support of ATP generation by the mitochondria. Nevertheless, mitochondrial NADH/NAD_t levels were decreased in HD cybrids, which was correlated with a decrease in pyruvate dehydrogenase activity and protein expression, suggesting decreased tricarboxylic acid cycle (TCA) input from glycolysis. Interestingly, the activity of alpha-ketoglutarate dehydrogenase, a critical enzyme complex that links the TCA to amino acid synthesis and degradation, was increased in HD cybrids. In accordance, mitochondrial levels of glutamate were increased and alanine was decreased, whereas aspartate and glutamine levels were unchanged in HD cybrids. Conversely, malate dehydrogenase activity from total cell extracts was unchanged in HD cybrids. Our results suggest that inherent dysfunction of mitochondria from HD patients affects cellular bioenergetics in an otherwise functional nuclear background.     

Interspecies transplacement of mitochondria in yeasts

Protoplasts of a respiration-deficient rho⁰ strain of Saccharomyces cerevisiae were incubated with mitochondria isolated from various respiration-competent yeast species under conditions enabling transplacement of mitochondria. Respiration-competent cybrids were selected by plating the protoplasts on agar media containing a non-fermentable energy source. The resulting cybrids contained nuclear DNA of the acceptor S. cerevisiae and mitochondrial DNA of the donor species, as detected by pulsed-field gel electrophoresis of chromosomes and restriction analysis of mitochondrial DNA, respectively. Successful restoration of respiration in the S. cerevisiae mutant was achieved by transplacement of mitochondria isolated from the following Saccharomyces species: S. bayanus, S. capensis, S. delbrueckii, S. exiguus, S. italicus and S. oviformis. Received: 25 February 1997     

Mitochondrial DNA from platelets of sporadic ALS patients restores normal respiratory functions in rho(0) cells

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, which affects the anterior horn cells of the spinal cord and cortical motor neurons. A pathophysiological role for mtDNA mutations was postulated based on the finding that cybrids obtained from mitochondria of sporadic ALS patients exhibited impaired respiratory chain activities, increased free radical scavenging enzymes, and altered calcium homeostasis. To date, however, no distinct mtDNA alterations associated with ALS have been reported. Therefore, we reexamined the hypotheses that mtDNA mutations accumulate in ALS and that cybrids generated from ALS patients' blood have impaired mitochondrial respiration. Cybrid cell lines were generated from 143B osteosarcoma rho(0) cells and platelet mitochondria of sporadic ALS patients or age-matched controls. We found no statistically significant differences in mitochondrial respiration between ALS and control cybrids, even when the electron transport chain was stressed with low concentrations of respiratory chain inhibitors. Mitochondrial respiratory chain enzyme activities were also normal in ALS cybrids, and there was no increase in free radical production. Therefore, we showed that mtDNA from platelets of ALS patients was able to restore normal respiratory function in rho(0) cells, suggesting that the presence of mtDNA mutations capable of affecting mitochondrial respiration was unlikely.     

Several different mitochondrial DNA regions are involved in intergenomic recombination in Brassica napus cybrid plants

Summary An important mitochondrial (mt) DNA polymorphism was detected by SalI restriction enzyme analysis in five Brassica napus cybrids plants which combine B. napus chloroplasts and a cytoplasmic male sterility (cms) trait from Raphanus sativus. Novel restriction fragments observed in these cybrids were analysed. One of them was found to be constituted by fragments of both parent mt genomes. Sites involved in rut recombination in cybrids were compared by molecular hybridization to sites supposedly implicated in intragenomic mt recombination in B. oleracea The results indicate that mt recombination events arising through protoplast fusion involve several different rut DNA regions. Some of these regions appeared homologous to regions presumably involved in intragenomic mt recombination in B. oleracea.     

MELAS mitochondrial DNA mutation A3243G reduces glutamate transport in cybrids cell lines

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) is commonly associated with the A3243G mitochondrial DNA (mtDNA) mutation encoding the transfer RNA of leucine (UUR) (tRNA ^Leu(UUR)). The pathogenetic mechanisms of this mutation are not completely understood. Neuronal functions are particularly vulnerable to alterations in oxidative phosphorylation, which may affect the function of the neurotransmitter glutamate, leading to excitotoxicity. In order to investigate the possible effects of A3243G upon glutamate homeostasis, we assessed glutamate uptake in osteosarcoma-derived cytoplasmic hybrids (cybrids) expressing high levels of this mutation. High-affinity Na⁺-dependent glutamate uptake was assessed as radioactive [³H]-glutamate influx mediated by specific excitatory amino acid transporters (EAATs). The maximal rate (V_max) of Na⁺-dependent glutamate uptake was significantly reduced in all the mutant clones. Although the defect did not relate to either the mutant load or magnitude of oxidative phosphorylation defect, we found an inverse relationship between A3243G mutation load and mitochondrial ATP synthesis, without any evidence of increased cellular or mitochondrial free radical production in these A3243G clones. These data suggest that a defect of glutamate transport in MELAS neurons may be due to decreased energy production and might be involved in mediating the pathogenic effects of the A3243G mtDNA mutation.     

Mitochondrial DNA depletion analysis by pseudogene ratioing

The mitochondrial DNA (mtDNA) depletion status of ρ⁰ cell lines is typically assessed by hybridization or polymerase chain reaction (PCR) experiments, in which the failure to hybridize mtDNA or amplify mtDNA using mtDNA-directed primers suggests thorough mitochondrial genome removal. Here, we report the use of an mtDNA pseudogene ratioing technique for the additional confirmation of ρ⁰ status. Total genomic DNA from a U251 human glioma cell line treated with ethidium bromide was amplified using primers designed to anneal either mtDNA or a previously described nuclear DNA-embedded mtDNA pseudogene (mtDNAψ). The resultant PCR product was used to generate plasmid clones. Sixty-two plasmid clones were genotyped, and all arose from mtDNAψ template. These data allowed us to determine with 95% confidence that the resultant mtDNA-depleted cell line contains less than one copy of mtDNA per 10 cells. Unlike previous hybridization or PCR-based analyses of mtDNA depletion, this mtDNAψ ratioing technique does not rely on interpretation of a negative result, and may prove useful as an adjunct for the determination of ρ⁰ status or mtDNA copy number.     

Puerarin protects Alzheimer's disease neuronal cybrids from oxidant-stress induced apoptosis by inhibiting pro-death signaling pathways

Mitochondrial oxidative stress induced by reactive oxygen species (ROS) has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease (AD). We used mitochondrial transgenic neuronal cell cybrid models of sporadic AD (SAD), which overproduce ROS compared to control cybrids, to investigate the protective effects of puerarin, an isoflavone purified from Chinese herb radix of Pueraria lobata, on viability, endogenous ROS and intracellular signaling pathways. SAD cybrids had increased apoptosis and increased accumulation of ROS that was inhibited by puerarin. Western blotting demonstrated that SAD cybrids had increased basal activation of the caspase-3, p38 and c-Jun N-terminal kinase (JNK) that were inhibited by puerarin. Puerarin was also found to decrease Bax/Bcl-2 ratio. These results suggest that expression of SAD mitochondrial genes in cybrids activates oxidative-stress-related signaling pathways and reduces viability, and that the protective effects of puerarin inhibit oxidative-stress-induced apoptosis through down-regulation of Bax/Bcl-2 ratio, which blocks the activation of JNK, p38 and caspase-3. Therefore, puerarin may act as an intracellular ROS scavenger, and protect neurons against oxidative-stress-induced apoptosis.