Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans.

BACKGROUND: We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee) individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens. RESULTS: We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. CONCLUSIONS: Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans.

Inheritance of mitochondrial DNA recombinants in double-heteroplasmic families: potential implications for phylogenetic analysis.

Recently, somatic recombination of human mitochondrial DNA (mtDNA) was discovered in skeletal muscle. To determine whether recombinant mtDNA molecules can be transmitted through the germ line, we investigated two families, each harboring two inherited heteroplasmic mtDNA mutations. Using allele-specific polymerase chain reaction and single-cell and single-molecule mutational analyses, we discovered, in both families, all four possible allelic combinations of the two heteroplasmic mutations (tetraplasmy), the hallmark of mtDNA recombination. We strongly suggest that these recombinant mtDNA molecules were inherited rather than de novo generated somatically, because they (1) are highly abundant and (2) are present in different tissues of maternally related family members, including young individuals. Moreover, the comparison of the complete mtDNA sequence of one of the families with database sequences revealed an irregular, nontreelike pattern of mutations, reminiscent of a reticulation. We therefore propose that certain reticulations of the human mtDNA phylogenetic tree might be explained by recombination of coexisting mtDNA molecules harboring multiple mutations.

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Hippocampal N-acetyl aspartate levels do not mirror neuronal cell densities in creatine-supplemented epileptic rats.

For neuroprotective therapy of neurodegenerative diseases creatine treatment has gained special interest because creatine has been shown to cross the blood-brain barrier, accumulate in the human brain in vivo and cause delayed neuronal cell death in a large number of animal models. Here, we used the pilocarpine model of temporal lobe epilepsy to determine whether creatine administration is able to attenuate the epilepsy-associated decrease in hippocampal N-acetyl aspartate (NAA) concentrations, impairment of mitochondrial function and neuronal cell loss. In vivo1H-NMR spectroscopy showed, in epileptic rats after creatine administration, higher hippocampal NAA concentrations, suggesting improved neuronal survival. However, in vitro observation of hippocampal slices from creatine-treated epileptic rats revealed a more pronounced loss of pyramidal neurons and decrease in activity of mitochondrial enzymes in hippocampal subfields. This indicates that NAA concentrations measured by in vivo1H-NMR spectroscopy reflect alterations of metabolism rather than neuronal cell densities. Our data indicate an adverse effect of creatine on neuronal survival under conditions of enhanced neuronal activity.

Expression pattern of mitochondrial respiratory chain enzymes in skeletal muscle of patients harboring the A3243G point mutation or large-scale deletions of mitochondrial DNA.

To assess the detailed expression pattern of mitochondrial-encoded proteins in skeletal muscle of patients with mitochondrial diseases we performed determinations of cytochrome content and enzyme activities of respiratory chain complexes of 12 patients harboring large-scale deletions and of 10 patients harboring the A3243G mutation. For large-scale deletions we observed a mutation gene dose-dependent linear decline of cytochrome aa3 content, cytochrome c oxidase (COX) activity, and complex I activity. The content of cytochromes b and the complex III activity was either not affected or only weakly affected by the deletion mutation and did not correlate to the degree of heteroplasmy. In contrast, in skeletal muscle harboring the A3243G mutation all investigated enzymes containing mitochondrial-encoded subunits were equally affected by the mutation, but we observed milder enzyme deficiencies at a comparable mutation gene dose. The results of single fiber analysis of selected biopsies supported these findings but revealed differences in the distribution of COX deficiency. Whereas predominantly type I fibers were affected in A3243G and deletion CPEO biopsies, we observed in MELAS and KSS biopsies higher quantities of COX-deficient type 2 fibers. Our findings indicate different pathomechanisms of deletion and A3243G mutations.

Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus.

Mitochondrial function is a key determinant of both excitability and viability of neurons. Here, we demonstrate seizure-dependent changes in mitochondrial oxidative phosphorylation in the epileptic rat hippocampus. The intense pathological neuronal activity in pilocarpine-treated rats exhibiting spontaneous seizures resulted in a selective decline of the activities of NADH-CoQ oxidoreductase (complex I of the respiratory chain) and cytochrome c oxidase (complex IV of respiratory chain) in the CA3 and CA1 hippocampal pyramidal subfields. In line with these findings, high-resolution respirometry revealed an increased flux control of complex I on respiration in the CA1 and CA3 subfields and decreased maximal respiration rates in the more severely affected CA3 subfield. Imaging of mitochondrial membrane potential using rhodamine 123 showed a lowered mitochondrial membrane potential in both pyramidal subfields. In contrast to the CA1 and CA3 subfields, mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and the parahippocampal gyrus. The changes of oxidative phosphorylation in the epileptic rat hippocampus cannot be attributed to oxidative enzyme modifications but are very likely related to a decrease in mitochondrial DNA copy number as shown in the more severely affected CA3 subfield and in cultured PC12 cells partially depleted of mitochondrial DNA. Thus, our results demonstrate that seizure activity downregulates the expression of mitochondrial-encoded enzymes of oxidative phosphorylation. This mechanism could be invoked during diverse forms of pathological neuronal activity and could severely affect both excitability and viability of hippocampal pyramidal neurons.