Evolutionary conservation of the clk-1-dependent mechanism of longevity: loss of mclk1 increases cellular fitness and lifespan in mice

Abstract

Inactivation of the Caenorhabditis elegans gene clk-1, which is required for ubiquinone biosynthesis, increases lifespan by an insulin signaling-independent mechanism. We find that homozygous inactivation of mclk1, the mouse ortholog of clk-1, yields ES cells that are protected from oxidative stress and damage to DNA. Moreover, in the livers of old mclk1(+/-) mice, hepatocytes that have lost mclk1 expression by loss of heterozygosity undergo clonal expansion, suggesting that their resistance to stress allows them to outcompete cells that still express the gene. mclk1(+/-) mice, whose growth and fertility are normal, also display a substantial increase in lifespan in each of three different genetic backgrounds. These observations indicate that the distinct mechanism by which clk-1/mclk1 affects lifespan is evolutionarily conserved from nematodes to mammals and is not tied to a particular anatomy or physiology.

Figure 1.

Reduction in the level of DNA damage in mclk1^-/- ES cells and in mclk1^+/- mice. (A) DNA damage measured by the comet assay. Staining is for DNA and the presence of a tail associated with a nucleus signals the presence of fast-migrating damaged DNA. Two independent fields of view are shown for each genotype. Much fewer mclk1^-/- ES cells show nuclei with tails, and the tails are smaller. (B) The number of nuclei with tails, without consideration of the size of the tails, was determined for mclk1^-/- and mclk1^+/+ ES cells (three samples of 100 cells for each genotype) as well as for liver cells of mclk1^+/- and mclk1^+/+ mice (n = 7 mice for each genotype; three samples of 100 cells for each mouse). Error bars represent the standard deviation of the means.

Figure 2.

Increased lifespan of mclk1^+/- mice. Kaplan-Meier survival curves are shown with p values calculated by the Mantel-Haenszel log rank test. Green indicates the mclk1^+/- mice and red the wild-type mice in all three panels. (A) Lifespan extension in the 129Sv/J genetic background. mclk1^+/- mice (n = 10) lived, on average, 15% longer than their wild-type (n = 12) littermates (824.8 ± 103.8 vs. 720.2 ± 96.1 d; p = 0.00045). All animals were female. (B) Lifespan extension in the 129Sv/J × Balb/c background. mclk1^+/- mice (n = 9) live, on average, 31% longer than their wild-type littermates (n = 5) (980.4 ± 105.9 vs. 749.8 ± 57.2 d; p = 0.00025). (C) Lifespan extension in the C57BL/6J background. There are both males and females in the C57BL/6J study, and both sexes behave similarly. Although the study in the C57BL/6J background is not finished, the available data show a median survival of 686 d for mclk1^+/+ (n = 5) and of 821.5 d for mclk1^+/- (n = 8), a difference that is already significant at p = 0.00345. Currently, the median lifespan of the males is 726 d for mclk1^+/+ (n = 3) and 837 d for mclk1^+/- (n = 5) (p = 0.026).

Figure 3.

Groups of cells lacking mCLK1 expression can be observed in the livers of mclk1^+/- mice with extended longevity. Immunohistochemical analyses with anti-mCLK1 antibody revealed that groups of cells lacked mCLK1 expression in the livers of old mclk1^+/- mice only. Uniform staining is seen in young (5-mo-old) mclk1^+/+ (A) and mclk1^+/- (D) mice. However, while there is uniform staining in 25-mo-old mclk1^+/+ mice (B), the staining is patchy in similar mclk1^+/- mice (E). (E) Large groups of cells without staining surround the central veins (upper arrow) and appear to expand throughout the whole classical hepatic lobule, which is the region drained by a central vein. (E) Other central veins appear surrounded by mCLK1-positive cells only (e.g., lower arrow). RISH with antisense DIG-labeled probe for mclk1 similarly showed uniformly positive cells in 25-mo-old mclk1^+/+ mice (C), but in similar mclk1^+/- mice (F) there were groups of cells that either lacked (e.g., left arrow) or expressed (e.g., right arrow) the signal for the mclk1 transcripts (blue). The nuclei (pink) were counterstained by nuclear fast red.

Figure 4.

LOH at the mclk1 locus. LCM of groups of 20–30 cells were obtained from mCLK1-negative (-) or mCLK1-positive (+) regions of sections from livers of old mclk1^+/- animals stained for the mCLK1 protein by immunocytochemistry. DNA isolated from these cells was then amplified by whole-genome MDA. Amplified DNA was used for PCR amplification with mclk1-specfic primers. This yields two products from mclk1 heterozygous DNA: one corresponding to the wild-type gene (300 bp) and a larger one corresponding to the disrupted allele (600 bp). (A) DNA specifically corresponding to the wild-type mclk1 allele is lost from cells that do not express mCLK1. (Lane 1) Negative control provided by LCM buffer, without any captured cells, but which subsequently underwent all procedures (DNA extraction, MDA, and PCR). (Lane 2) PCR from captured cells expressing mCLK1. (Lane 3) PCR from captured cells, not expressing mCLK1. (Lane 4) PCR production from DNA of a wild-type mouse tail (positive control). (B) Control for extracted DNA quality. Wild-type DNA from the igf1r locus (on chromosome 11) and p53 locus (on chromosome 7) can be unfailingly PCR amplified from both mCLK1-negative and mCLK1-positive cells. The same sample obtained by LCM and whole-genome MDA is being used for PCR in lanes 1, 3, and 5 from a mCLK1-positive group of cells, and in lanes 2, 4, and 6 from a mCLK1-negative group of cells.

Figure 5.

Quinones in mclk1^+/- mice. (A) Reverse-phase HPLC chromatograms show the elution of UQ6, DMQ9, and UQ9 standards, and the elution of quinones from representative livers of mclk1^+/- and mclk1^+/+ mice. UQ6 is added in the liver samples as an internal standard. No DMQ9 peak was detected in any of the liver samples from mclk1^+/- animals (n = 7; age range: 14–22 mo). (B) Ubiquinone levels in livers and kidneys of mclk1^+/- mice. In the livers, but not in the kidneys, ubiquinone levels were significantly decreased compared with that in wild-type littermates (n = 7 for each genotype; three measurements were taken for each liver; p = 0.0024). The error bars represent the 95% confidence interval (approximately two times the standard error of the mean).