The Schiner group set out to answer the following questions with their experiments...
1. How are mice manufactured to test the role of H2O2 in life span?
2. Does MCAT, NCAT, or PCAT affect life span?
3. Can over expression of MCAT prevent disease?
4. Can MCAT overexpression reduce oxidative damage to DNA?
5 Are combined antioxidant defenses responsible for extension of life in mammals?
Question: How are mice manufactured to test the role of H2O2 in life span?
In order for the group of researchers to perform their experiments, they first needed to generate transgenic mice. Numerous lines of founder mice were created, each overexpressing human catalase in select areas of the cell. The transgenes developed by the researchers targeted the expression of human catalase in the mitochondria, the nucleus, or the peroxisome depending on the founder line being developed. The process needed to create these transgenic mice is depicted in the illustration below.
First, a desired transgene (a specific sequence of nucleotides) was created for the expression of human catalase. This sequence was amplified by polymerase chain reaction, creating millions of copies1. Then, the following step were taken: 1) An embryo was harvested from a mouse while it is still in the pronuclear stage. 2) An injection pipette is used to inject a suspension of the transgene into the sperm’s pronucleus in the embryo2.
The transgene combines with the DNA of the sperm cell through recombinant DNA technology. Restriction enzymes locate a particular sequence in the DNA and then cleave the sperm cell ’s DNA at the restriction site. The transgenes are then inserted into the host DNA and is then bound by DNA ligase.1 As the embryo undergoes development the transgene will become part of the mouse’s DNA and will be found in every cell of the host. The embryo is implanted into a pseudopregnant female. After the mice have reached several weeks of age, a small piece of the tail of each newborn mouse will be taken and analyzed through the southern blotting method for the presence of the desired transgene. The transgenic mice expressing human catalase will be will then be bred for multiple generations until they express the desired quantities of the target transgene in the desired part of the cell.
Courtesy of Lawrence Berkely Nat’l Lab
Roy Kaltschmidt – LBL photographer
Answer: Transgenic mice overexpression human catalase localized to mitochondria (MCAT), the nucleus (NCAT), and the peroxisome (PCAT) were generated.
Question: Does MCAT, NCAT, or PCAT affect life span?
Of the three different transgenic mice generated, the mice which overexpressed human catalase localized to the mitochondria (MCAT) showed the most significant extension of median life span. The median life span of MCAT mice was 4.5 to 5.5 months longer than that of the wild type mice. NCAT mice only showed an increase in median life span of 1 to 3 months, while PCAT mice showed an increase of 3 to 3.5 months over the wild type mice. These increases were expressed by both sexes and there were no apparent abnormalities of the transgenic mice when compared to their wild type littermates. The results suggest that the MCAT mice actually delayed the effects of aging that are the result of oxidative damage caused by free radicals.
Answer: Only the MCAT mice showed significant increase of median life span, as compared to the control mice.
Question: Can over expression of MCAT prevent disease?
The two groups of mice (wild type and over expressed MCAT) were examined for disease throughout their lives. When mice from both groups were young (9 to 11 months of age) there was little difference between the two groups. However, the older the mice became, the ability of MCAT expression to prevent disease because clear.
To compare the prevalence of each disease between the groups of mice, a scale of 0 to 4 was established (4 being the most severe).
The combined arteriosclerosis and cardiomyopathy results show the potential for MCAT expression to protect the heart and prevent health deterioration while increasing longevity. These results overwhelmingly prove the correlation between over expression of MCAT and disease prevention.
Question: Can MCAT overexpression reduce oxidative damage to DNA?
To determine if MCAT overexpression can reduce oxidative damage to DNA,
8-hydroxydeoxyguanosine was measured in DNA from skeletal and heart muscle in control and MCAT mice. 8-hydroxydeoxyguanosine is a DNA base oxidative product, which is indicative of oxidative damage to DNA. It is considered a “biomarker” of oxidative stress to DNA. Structurally, 8-OHdG is a hydroxyl guanine nucleotide that has been damaged at the C8 position from a radical. Please refer to Figure 1.1 below. It is subsequently removed from DNA by endonuclease repair enzymes. Because DNA repair is known to normally occur relatively quickly and efficiently, the amount of 8-OHdG in serum directly reflects the amount of damage within the entire body. Species with greater longevity have been found to have lower levels of DNA damage than similar, shorter-lived species. As a result, the degree of DNA damage is thought to be a determinant of longevity. Levels of 8-OHdG can be determined by an ELISA assay3.
Figure 1.1 A Hydroxyl radical (OH · ) is thought to produce dehydroxyguanosine hydroxylation at the C8 position of guanine
Mitochondrial base deletions associated with oxidative change were measured as low-molecular weight products by long-extension polymerase chain reaction (LX-PCR).
In control animals, 8-OHdG was observed to increase with age in skeletal muscle, but not in heart muscle.
MCAT mice did not have an increase in 8-OHdG with age.
In control mice, the number of mitochondrial deletions increased in both skeletal and heart muscle.
In MCAT animals, a statistically significant decrease in deletion products was observed in 21-month-old skeletal muscle. However, the decrease noted in 21-year-old heart muscle and 30+-month-old skeletal muscle was not statisitically significant.
Please refer to Figure 4E below, which represents these results, compiled by Schriner, et al. 8-OHdG in skeletal muscle is represented by the black bar and that in the heart, by the white bars
Answer: With increasing age, oxidative damage to DNA appears to be statistically lower in MCAT mice than in their wild type counterparts, primarily in skeletal muscle tissue.
Question: Are combined antioxidant defenses responsible for extension of life in mammals?
Transgenic mice were produced that overexpress both PCAT and SOD. PCAT animals overexpress human catalase localized to the peroxisome. Superoxide dismutase (SOD) converts the superoxide anion O2- into hydrogen peroxide, which then creates the highly reactive hydroxyl radical when exposed to metal atoms. However, H2O2 can be removed by glutathione peroxidase or catalase. In order to generate the “double” transgenic mice, hemizygous PCAT overexpressing animals were bred with hemizygous SOD-overexpressing animals4.
Figure 1.1 The relation between superoxide dismutase and catalase function. Figure courtesy of Dr. Jennifer McDowall.
The median life span of double transgenic mice increased by 18.5% compared to the control mice.
There was a 7% extension of median life span compared with mice with only PCAT overexpression.
However, there was no apparent increase in maximum life span in the mice that overexpressed SOD and PCAT compared with the control mice.
There were no apparent deleterious phenotypic changes in the double transgenic mice.
Schriner et al hypothesize that mice with overexpression of SOD and MCAT (rather than PCAT) would cause a greater extension of lifespan because the antioxidant enzymes enhanced at the mitochondria appear to have a more significant impact on longevity than do
Please refer to Figure 3D below, which represents the aforementioned results, compiled by Schriner, et al.
Answer: The overexpression of both PCAT and SOD appears to extend the median life span of mice compared to the wild type mice and those only overexpressing catalase localized to the peroxisome.