Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA^Leu(UUR) 3243A>G mutation and harbor ∼10–30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50–90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90–100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20–30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50–90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling. Significance Mitochondria generate signals that regulate nuclear gene expression via retrograde signaling, but this phenomenon is rendered more complex by the quantitative differences in the percentage of mutant and normal mtDNAs that can exist within patient cells. This study demonstrates that depending upon its relative cytoplasmic levels, a single mtDNA point mutation can cause a discrete set of cellular transcriptional responses within cells of the same nuclear background. This qualitative regulation of nuclear gene expression by quantitative changes in mtDNA mutant levels challenges the traditional “single mutation–single disease” concept and provides an alternative perspective on the molecular basis of complex metabolic and degenerative diseases, cancer, and aging.

In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-β (Aβ) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Aβ peptide (Aβ_1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Aβ_1-42specifically inhibited the terminal complex cytochromecoxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu²⁺and specific “aging” of the Aβ_1-42solution. Maximal COX inhibition occurred when using Aβ_1-42solutions aged for 3-6 h at 30°C. The level of Aβ_1-42-mediated COX inhibition increased with aging time up to ∼6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Aβ as the only Aβ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant Aβ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Aβ is associated with brain mitochondria and that Aβ_1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu²⁺. We conclude that Cu²⁺-dependent Aβ-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimer's disease.