Mitochondrial Inverted Repeats Strongly Correlate with Lifespan: mtDNA Inversions and Aging

<div><p>Mitochondrial defects are implicated in aging and in a multitude of age-related diseases, such as cancer, heart failure, Parkinson’s disease, and Huntington’s disease. However, it is still unclear how mitochondrial defects arise under normal physiological conditions. Mitochondrial DNA (mtDNA) deletions caused by direct repeats (DRs) are implicated in the formation of mitochondrial defects, however, mitochondrial DRs show relatively weak (Pearson’s <i>r</i> = −0.22, <i>p</i><0.002; Spearman’s <i>ρ</i> = −0.12, <i>p = </i>0.1) correlation with maximum lifespan (MLS). Here we report a stronger correlation (Pearson’s <i>r</i> = −0.55, <i>p</i><10^–16; Spearman’s <i>ρ</i> = −0.52, <i>p</i><10^–14) between mitochondrial inverted repeats (IRs) and lifespan across 202 species of mammals. We show that, in wild type mice under normal conditions, IRs cause inversions, which arise by replication-dependent mechanism. The inversions accumulate with age in the brain and heart. Our data suggest that IR-mediated inversions are more mutagenic than DR-mediated deletions in mtDNA, and impose stronger constraint on lifespan. Our study identifies IR-induced mitochondrial genome instability during mtDNA replication as a potential cause for mitochondrial defects.</p></div

The majority of inversions are caused by mtDNA replication.

<p>(<b>A</b>) Diagram showing inversion products generated by recombination or replication mechanisms. IRs are indicated by thick arrows. Thin half-arrows indicate LR PCR primers (p_LR1 and p_LR2) and qPCR primers (p_q1 and p_q2) used to quantify the inversion. (<b>B</b>) Quantification of the recombination and replication inversion products using qPCR. Total DNA from a 24-months-old mouse brain was first amplified with 8 or 12 cycles of LR PCR with the primers p_LR1 and p_LR2 (B), p_LR1 alone (S), or no primer control (N). Inversions resulting from replication errors can be amplified with p_LR1 alone, while inversions resulting from homologous recombination require both p_LR1 and p_LR2 primers._. The LR PCR was followed by qPCR with primers p_q1 and p_q2 to specifically quantify the inversion. The Ct values, inversely proportional to log of template concentration, are plotted for each PCR reaction. Error bars indicate s.e.m. (<i>n = </i>6 for N; <i>n</i> = 12 for groups B and S). (<b>C</b>) A replicate of (<b>B</b>) using the total DNA from a brain of a different mouse (30-months-old).</p

Correlations of DRs and IRs with MLS in different clades.

<p><b>Bold</b> font, <i>p</i><0.05; <b>*</b>, <i>p</i><0.001. Two-tailed. CAIC, comparative analysis by phylogenetically independent contrasts. –Mammalia, excluding Mammalia. NCBI taxonomic information was used to construct the phylogenetic tree and assuming equal branch lengths. The correlations of IRs and MLS decreased from –0.55 to –0.34 for mammals but were still significant (<i>p</i> = 7.2×10⁻⁷). So, the strong correlation between IRs and MLS is not a false correlation caused by phylogeny.</p

Inverted repeats (IRs) show stronger negative correlation with maximum lifespan (MLS) than direct repeats (DRs).

<p>(<b>A</b>) The mutagenic score calculated as where <i>i</i> is the number of identical matches in <i>l</i> bps for each DR length, has a good fit with the number of experimentally reported human mtDNA deletions. The human mtDNA deletions are from MITOMAP database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073318#pone.0073318-MITOMAP1" target="_blank">[30]</a>. (<b>B, C</b>) The correlation between total mutagenic score, calculated as a sum of mutagenic scores of all DR (<b>B</b>) and IR (<b>C</b>) lengths for each species, and MLS within mammals. (<b>D, E</b>) The correlation between total mutagenic score and MLS within Chordata. (<b>F, G</b>) The correlation within Rodentia. <i>R.</i>, <i>Rattus</i>.</p

A schematic model showing the role of p21-PARP-1 in acute cigarette smoke (CS)-induced DNA damage and cellular senescence.

<p>CS exposure causes DNA damage including double-strand break (DSB). CS exposure also increases the level of p21, and p21 gene deletion augments PARP-1 activity and the levels of non-homologous end joining (NHEJ) proteins to repair damaged DNA. Consequently, CS-induced cellular senescence is attenuated by p21 deletion.</p

p21 deletion increased the levels of PAR, which was reduced by 3-AB treatment.

<p>The levels of PAR and PARP-1 were determined by Western blot in lungs of C57BL/6J (<b>A</b>), and p21^-/- mice as well as WT littermates (<b>B</b>) in response to CS. PAR levels were increased in lungs of p21^-/- mice as compared to WT mice. 3-AB treatment reduced PAR level in lungs of both p21^-/- and WT mice. Intact PARP-1 level was not altered by either p21 deficiency or 3-AB treatment. Gel pictures shown are representative of at least 3 separate mice. Fold change is indicative of the alteration of PAR and PARP-1 compared with air-exposed and vehicle (Veh)-treated WT mice after normalizing to corresponding GAPDH or β-actin. Data are shown as mean ± SEM (n = 3-13 per group).^*<i>P</i><0.05 <i>vs</i> air group; ⁺<i>P</i><0.05, ⁺⁺<i>P</i><0.01, ⁺⁺⁺<i>P</i><0.001 <i>vs</i> Veh group; ^†††<i>P</i><0.001 <i>vs</i> WT mice.</p

3-AB increases CS-induced cellular senescence, but does not affect neutrophil influx in mouse lungs.

<p>3-AB augmented CS-induced increase in SA-β-gal activity in WT, but not p21^-/- mice (<b>A</b>). p21 deletion increased the expression of PCNA in lungs as compared to WT mice exposed to CS, which was reduced by 3-AB treatment (<b>B</b>). p21 deletion attenuated CS-induced neutrophil influx in BAL fluid, which was not affected by 3-AB (<b>C</b>). Original magnification, ×200. Gel pictures shown are representative of at least 3 separate mice. Relative density ratio is indicative of results after normalizing to corresponding GAPDH. Data are shown as mean ± SEM (n = 3-4 per group). ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs</i> air group; ⁺<i>P</i><0.05, ⁺⁺⁺<i>P</i><0.001 <i>vs</i> Veh group; ^††<i>P</i><0.01, ^†††<i>P</i><0.001 <i>vs</i> WT mice.</p

NMR tissue-specific changes in IGF system components with age.

<p>Changes in IGF-1 and -2, IGFBP-1 through -6, PAPP-A, and IGF-1 and -2 receptor mRNA levels were analyzed by real-time PCR in lung, liver and kidney of young (8–12 months), middle-age (4.5–6 years) and old (17–22 years) NMR (n = 4–5). Only data showing a significant up- or down-regulation with age are displayed. Groups not connected by the same letters are significantly different at <i>P</i> < 0.05.</p

Generation of R26NHEJ knock-in mouse model.

<p>(<b>A</b>) The pROSA26PA-NHEJ vector containing NHEJ reporter construct targeted to the ROSA26 genomic sequence. The construct consists of GFP exons separated by the Pem1 intron, interrupted by the killer exon Ad2. Flanking Ad2 are unique I-SceI recognition sites for inducing DSB. Successful NHEJ repair leads to the reconstitution of GFP. Two <i>loxP</i> sites in direct orientation flanking Neomycin/Kanamycin genes (Neo/Kana) and Bacterial origin of Replication (OriC) are located downstream. This vector was targeted by homologous recombination into the endogenous ROSA26 locus of C57BL/6 mouse ES cells. (<b>B</b>) NHEJ construct integrated into ROSA26 locus in the mouse genome. (<b>C</b>) DNA from G418-resistant ES cell clones was digested with <i>Bam</i>HI and hybridized to a GFP probe (indicated in B). (<b>D</b>) Founder mice were genotyped using PCR primers indicated in (B). The positive control lanes contain PCR reactions with genomic DNA from ES cell clones shown in (C) as a template. Negative control lane contains PCR reactions with DNA from a wild-type C57BL/6 mouse as template and is followed by a No template control.</p

Analysis of deletions and insertions at NHEJ junctions in cells from young and old mice.

<p>A total of 300 independent junctions, 30 young and 30 old, for each cell type, were analyzed. The complete list of junction sequences is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004511#pgen.1004511.s004" target="_blank">Table S1</a>. (<b>A</b>) Average deletion size decreases with age in heart fibroblasts and increases in lung and skin fibroblasts. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test). (<b>B</b>) Large deletions are more frequently found in old lung and skin fibroblasts and in young heart fibroblasts. The graph shows percentage of NHEJ clones containing deletions larger than 500 bp. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test). (<b>C</b>) Average size of insertions increases in astrocytes and decreases in kidney and lung fibroblasts. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test). (<b>D</b>) The frequency of insertions decreases with age in kidney and lung fibroblasts. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test).</p