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The proportions of mutant and wild-type mtDNA are crucial in determining the severity of mitochondrial diseases. It has been generally considered that deletion-mutant mtDNA has replication advantages and accumulates with time. Here, we examine the tissue-by-tissue proportions of mutant mtDNA with a 4696-bp deletion ([DELTA]mtDNA) and wild-type mtDNA in mitochondrial disease model mice (mito-mice). Comparison of the proportions of [DELTA]mtDNA in each tissue at various ages showed that the rate of accumulation of [DELTA]mtDNA differed among tissues. The heart, skeletal muscles, kidney, liver, testis, and ovary showed increases in the proportion of [DELTA]mtDNA with age, but the pancreas, spleen, brain, and blood showed only a slight or no increase in proportion. In contrast to the somatic tissues, however, the germ cells of female mito-mice and resultant offspring showed a strong decrease in [DELTA]mtDNA with maternal age. The decrease was so acute that some offspring showed complete disappearance of [DELTA]mtDNA, even though their elder brothers and sisters had high proportions of [DELTA]mtDNA. Female germ cells have a machinery that prevents the inheritence of defective mtDNA to the following generation since germ cells are kept for a long time until they are ovulated.

Mitochondrial disease model mice, mitomice, were created using zygotes of B6mtspr strain mice carrying mitochondrial DNA (mtDNA) from Mus spretus as recipients of exogenous mitochondria carrying wild-type and a deletion mutant mtDNA ([DELTA]mtDNA) of M. musculus domesticus. In these experiments, mtDNAs from different mouse species were used for identification of exo- and endogenous wild-type mtDNAs in the mitomice. Results showed transmission of exogenous [DELTA]mtDNA, but not exogenous wild-type mtDNA, of M. m. domesticus to following generations through the female germ line. Complete elimination of exogenous wild-type mtDNA would be due to stochastic segregation, whereas transmission of exogenous [DELTA]mtDNA would be due to its smaller size leading to a propagational advantage. Tissues in mitomice of the [F.sub.3] generation carrying exogenous [DELTA]mtDNA showed protection from respiration defects until AmtDNA accumulated predominantly. This protection from expression of mitochondrial dysfunction was attained with the help of endogenous wild-type mtDNA of M. spretus, since mitomice did not possess exogenous wild-type mtDNA of M. m. domesticus. These observations provide unambiguous evidence for the presence of interaction between exogenous mitochondria carrying [DELTA]mtDNA and endogenous mitochondria carrying M. spretus wild-type mtDNA.

Abstract Background Mitochondrial DNA (mtDNA) with pathogenic mutations has been found in patients with cognitive disorders. However, little is known about whether pathogenic mtDNA mutations and the resultant mitochondrial respiration deficiencies contribute to the expression of cognitive alterations, such as impairments of learning and memory. To address this point, we used two groups of trans-mitochondrial mice (mito-mice) with heteroplasmy for wild-type and pathogenically deleted (Δ) mtDNA; the "low" group carried 50% or less ΔmtDNA, and the "high" group carried more than 50% ΔmtDNA. Results Both groups had normal phenotypes for not only spatial learning, but also memory at short retention delays, indicating that ΔmtDNA load did not affect learning and temporal memory. The high group, however, showed severe impairment of memory at long retention delays. In the visual cortex and dentate gyrus of these mice, we observed mitochondrial respiration deficiencies, and reduced Ca2+/calmodulin-dependent kinase II-α (α-CaMKII), a protein important for the establishment of spatial remote memory. Conclusion Our results indicated that normal mitochondrial respiratory function is necessary for retention and consolidation of memory trace; deficiencies in this function due to high loads of pathogenically mutated mtDNA are responsible for the preferential impairment of spatial remote memory.

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-[mt.sup.spr] strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-[mt.sup.spr] mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.

Mito-mice carrying various proportions of deletion mutant mtDNA (ΔmtDNA) were generated by introduction of the ΔmtDNA from cultured cells into fertilized eggs of C57BL/6J (B6) strain mice. Great advantages of mito-mice are that they share exactly the same nuclear-genome background, and that their genetic variations are restricted to proportions of pathogenic ΔmtDNA. Since accumulation of ΔmtDNA to more than 75% induced respiration defects, the disease phenotypes observed exclusively in mito-mice carrying more than 75% ΔmtDNA should be due to accumulated ΔmtDNA. In this study, we focused on the expressions of hearing loss and diabetic phenotypes, since these common age-associated abnormalities have sometimes been reported to be inherited maternally and to be associated with pathogenic mutant mtDNAs. The results showed that accumulation of exogenously introduced ΔmtDNA was responsible for hearing loss, but not for expression of diabetic phenotypes in mito-mice. [Copyright &y& Elsevier]

Patient studies suggested that accumulation of pathogenic mitochondrial (mt) DNAs having large-scale deletion or point mutation and the resultant mitochondrial respiratory abnormalities are associated with a wide variety of disorders, such as mitochondrial diseases, neurodegenerative diseases, diabetes, and aging. Although the pathogenicities of these mtDNA mutations were proved by co-transmission of the mutant mtDNAs and respiration defects to human mtDNA-less cells, there is as yet no convincing reverse genetic evidence to explain whether accumulation of these pathogenic mutant mtDNAs in tissues is responsible for the expressions of various clinical phenotypes. In such situation, we have succeeded in generating mice with pathogenic deletion mutant mtDNA, named "mito-mice", by introduction of mitochondria with mtDNA which is deleted 4,696 bp (nt 7,759-12,454) including 6 tRNA genes and 7 structural genes (del-mtDNA). In the mito-mice, del-mtDNA was transmitted maternally, and its accumulation induced mitochondrial dysfunction in various tissues, resulting in mitochondrial disease phenotypes, such as low body weight, lactic acidosis, ischemia, myopathy, hart block, deafness, male infertility, and renal failure. Thus, mito-mice are the first model animal for mtDNA-based diseases, and the mice should be valuable for screening effective drugs and testing therapies.