Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition

Abstract

Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced. Ultimately, this segregation process produced NZB-129 heteroplasmic mice and their NZB or 129 mtDNA homoplasmic counterparts. Phenotypic comparison of these three mtDNA lines demonstrated that the NZB-129 heteroplasmic mice, but neither homoplasmic counterpart, had reduced activity, food intake, respiratory exchange ratio; accentuated stress response; and cognitive impairment. Therefore, admixture of two normal but different mouse mtDNAs can be genetically unstable and can produce adverse physiological effects, factors that may explain the advantage of uniparental inheritance of mtDNA.

Figure 1. Change in Progeny 129-NZB mtDNA Heteroplasmy in Offspring from Mothers with Varying Levels of Tail Heteroplasmy

(A) Frequency histogram of the segregation of mtDNA variants in the germline (n = 864). (B) The average change in 129-NZB mtDNA heteroplasmy of all the progeny of a particular female (n = 78) relative to her tail heteroplasmy. (C) The average change in 129-NZB mtDNA heteroplasmy in pups of females grouped according to their proportions of the different mtDNA variants, 0%–20% group, n = 341; 20%–40%, group n = 140; 40%–60% group, n = 84; 60%–80% group, n = 168; and 80%–100% group, n = 158. The proportion of NZB mtDNA in the tail of a pup was subtracted from the proportion of NZB in the tail of the mother (both at P10). Positive values represent segregation toward 129 mtDNA and negative values toward NZB mtDNA. Error bars represent ± SEM. See also Figure S6 and Table S1.

Figure 2. Change in mtDNA Heteroplasmy in Organs of Adult Mice Compared to P10 Tail Tip Heteroplasmy, Determined by Fragment Analysis

Positive values represent increased 129 mtDNA content negative values increased NZB mtDNA. More than 50 mice were examined for each tissue except for the ovaries (n = 26), and seminal vesicles (n = 21). Error bars represent ± SEM. See also Figure S1.

Figure 3. Increased 129 mtDNA in the Ovary Compared with Tail at Different Levels of Tail Heteroplasmy Determined by Fragment Analysis

(A) Frequency histogram of the segregation of mtDNA variants in the ovaries compared to tail tissue of individual mice (n = 26). (B) The change in the mtDNA heteroplasmy of ovaries of female mice relative to their tail tissue at P10. (C) The average change in mtDNA variants in the ovaries of mice within groups with different proportions of mtDNA variants, 20%–40% group, n = 6; 40%–60% group, n = 13; and 60%–80% group, n = 5. Positive values indicate segregation toward 129 mtDNA. Error bars represent ± SEM. See also Figures S2, S3, S4, and S5.

Figure 4. Effect of mtDNA Genotype on Fertility and Fecundity of Mice

(A) The average litter size of mice containing different proportions of the 129 and NZB mtDNAs. (B) The average number of pups that were born and survived to weaning. For heteroplasmic mice the mother's percentage of NZB mtDNA is shown, and mice were grouped according to percentage heteroplasmy. For the 0%–20% group 46 litters were examined, 20%–40% group, n = 48; 40%– 60% group, n = 29; 60%–80% group, n = 28; and 80%–100% group, n = 18. 129, segregated to homoplasmic 129 mtDNA (n = 98); Het, mean for all heteroplasmic mice (n = 159); NZB, NZB mice from the backcross (n = 88); NZB N5-10, backcross from generation N5-N10 (n = 67); NZB Seg, mice segregated to homoplasmic NZB from heteroplasmic mice (n = 10). Error bars represent ± SEM.

Figure 5. Behavior and Metabolism of Heteroplasmic Mice and Their Homoplasmic Segregants during the Circadian Cycle

(A) Quantification of activity bouts in light and dark cycles. Left panel: homoplasmic 129 mtDNA animals. Middle panel: heteroplasmic NZB-129 animals. Right panel: homoplasmic NZB mtDNA animals. (B) Average activity bouts during the continuous dark period of the three mtDNA genotype animals. (C) Average food intake of the mice with the three mtDNA genotypes in the dark and light periods. (D) Average differences in respiratory exchange ratio (RER) during the light and dark period of the heteroplasmic and homoplasmic mice. Error bars represent ± SEM.

Figure 6. Anxiety-Related Behavior is Elevated in Heteroplasmic Mice in Response to Stress

(A) Decreased latency to immobility in the forced swim test for 129-NZB heteroplasmic mtDNA versus homoplasmic 129 and homoplasmic NZB mtDNA male mice (n = 9–20; *p % 0.0006). (B) Decreased aversion to anxiogenic center compartment of heteroplasmic mice in the open field (*p % 0.026). (C) Increased time spent in the brightly-lit center compartment in novelty suppression of feeding conflict-based test of heteroplasmic versus homoplasmic mice (*p % 0.006). (D) Increased frequency of NSOF anxiolytic behavior, as determined by body contour (elongation versus contraction) and rearing (*p % 0.001). (E) Decreased latency to feeding in NSOF in heteroplasmic mice (*p % 0.006). Unless otherwise stated, n = 10 and all statistical values were determined by one-way ANOVA, followed by the Student-Newman-Keuls post hoc test. All p values represent significant differences between NZB-129 Hetero. versus homoplasmic 129 and NZB mice. p > 0.05 in all measures for 129 versus NZB. Error bars represent ± SEM.

Figure 7. Heteroplasmic Mice Have Impaired Spatial Learning and Memory

(A) The latency to the first encounter with the target hole for heteroplasmic and homoplasmic mice in the Barnes maze test during the learning period. On the first day the data are from one test trial. On days 2, 3, and 4 each mouse had three trials of 2 min. (B) Representative paths of the mice from day 4 of the acquisition trials. Homoplasmic mice typically used a direct, spatial strategy:. The heteroplasmic mice typically adopted a less direct nonspatial strategy. Het., 129-NZB heteroplasmic mice; 129, 129 homoplasmic mice; NZB, NZB homo-plasmic mice. (C) The effect of heteroplasmy on spatial memory retention. 24 hr after the last acquisition trial the mice were tracked during a 3 min probe trial using the same target hole placement as in the acquisition trials. For all studies, n = 18 for the heteroplasmic mice, n = 17 for the 129 homo-plasmic mice, and n = 9 for the NZB homoplasmic mice. (D) The latency to escape box in cued acquisition trials. Performance of heteroplasmic mice is indistinguishable from both homoplasmic groups in the cued, nonspatial version of the task. For all studies, n = 18 for the 129-NZB heteroplasmic mice (Het.), n = 17 for the 129 homoplasmic mice (129), and n = 9 for the NZB homoplasmic mice (NZB). Error bars represent ± SEM.