Study of molecular mechanisms causing aging. Use of model systems Saccharomyces cerevisiae, C. elegans, and mice, and relevance to aging in humans. Generality of changes in the rDNA, changes in chromatin structure, and DNA damage as molecular causes of aging. Study of mechanism by which calorie restriction extends life span.
Aging in yeast: Budding yeast divide asymmetrically, giving rise to a large mother cell, and a small daughter cell. Mother cells divide a fixed number of times (their life span) then die. Further, as mother cells age, they assume a characteristic morphology, including an increase in size. The yeast Sir complex, which mediates transcriptional silencing at telomeres, regulates the pace of aging. Mutations that knock out SIR genes accelerate aging, and the dominant SIR4-42 mutation slows aging. The nucleolus contains tandemly repeated copies of ribosomal DNA (rDNA). A key event in aging is the generation of an rDNA circle by homologous recombination. Circles replicate each subsequent cell cycle and segregate exclusively to mother cells. SIR2 plays the key role in silencing and repression of recombination in the rDNA thereby extending life span.
Biochemical studies on Sir2p: Sir2p is a simple enzyme with a novel activity – it is an NAD-dependent histone deacetylase (see figure). In vitro the purified recombinant Sir2p will deacetylate the amino-terminal tails of histones H3 and H4, removing acetyls from Lys 9 and 14 of H3, and Lys 16 of H4. These are the Lys residues most important in silencing in vivo. Deacetylation of these lysines by Sir2p likely triggers silenced chromatin in vivo, which is known to be hypoacetylated. Mutations in SIR2 that inactivate the deacetylase fail to promote silencing and longevity in vivo. Since a mouse SIR2 homolog has this same enzymatic activity, the Sir2 deacetylase is likely a general trigger of silencing in eukaryotes.
Importantly, the NAD requirement means that silencing is likely coupled to the metabolic rate of cells. Thus, when metabolic rate is slowed NAD is more available thereby increasing Sir2 activity and triggering more silencing and a longer life span. Consistent with this model, overexpression of Sir2p increases life span in yeast.
Calorie restriction: Limiting calories in the diet extends the life span of rodents by an unknown mechanism. We found that calorie restriction (CR) also extended life span of yeast mother cells, by limiting their concentration of glucose. This extension required SIR2 and sufficient levels of NAD; it was prevented by deleting SIR2 or genes involved in NAD synthesis. CR extends life span in yeast because it increases the rate of respiration. Mutations that block respiration prevent the extension and a genetic trick to increase respiration extends life span in high glucose. CR increases the silencing activity of Sir2p, perhaps because NAD levels rise when respiration increases. We are presently developing a method to calorie restrict C. elegans. We will test possible involvement of each C. elegans SIR2 gene in any extension in life span.
Aging in mice: Several mammalian SIR2 homologs are under study. Knock out mice for five SIR2 genes are being generated. The SIRT2 KO is viable and under study. This particular SIR2 protein appears to be expressed most highly in the brain and possible neorological phenotypes of aging homozygous mice are under investigation. Recent findings also show a possible link between this SIR2 gene and cancer. Mammalian SIR2 substrates other than histones are also being explored. Recent studies show that the tumor suppressor p53 is a substrate for SIRT1, the ortholog of SIR2. Deacetylation of p53 by SIRT1 reduces the activity of the tumor suppressor and blunts apoptosis and senescence in response to radiation or oxidative stress.
Aging in C. elegans: C.elegans has four SIR2 homologs. Strains with a free duplication of the homolog most highly related to yeast SIR2, sir-2.1, show a significantly extended life span. The gene was cloned and inserted into C. elegans to generate transgenic animals with elevated levels of Sir2. These transgenic animals live longer than wild type. sir-2.1 appears to function in the insulin signaling pathway shown to regulate aging and dauer formation in C. elegans. A deletion of sir-2.1 has the opposite effect. Life span is shortened. The roles of yeast SIR2, C. elegans sir-2.1, and mammalian SIRT1 imply that SIR2 genes promote survival in response to nutritional stress in a wide spectrum of organisms.
In another project, the possible role of apoptosis in limiting organismal life span is under study. Mutations that prevent apoptosis are studied for their effects on the soma and the germ line in adult animals. Possible effects on life span are under scrutiny.
Su-Ju Lin, Kaeberlein, M., Andalis, A., Sturtz, L. A., Defossez, P., Culotta, V. C., Fink, G. R. and Guarente, L. Calorie restriction extends life span by increasing respiration. Nature, Jul 18;418(6895):287-8. (2002)
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Luo, J., Nikolaev, A. Y., Imai, S., Chen, D., Shiloh, A., Guarente, L., and Gu, W. Negative control of p53 by Sir2a-mediated deacetylation promotes cell survival under stress. Cell 107, 137-148 (2001).
Tissenbaum, H., and Guarente, L., Increased dosage of a C. elegans SIR2 gene extends life span in C. elegans. Nature, 410, 227-230 (2001).
Johnson, F. B., Marciniak, R., McVey, M., Stewert, S., Hahn, W., and Guarente, L. The S. cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase. EMBO J. 20, 905-913 (2001).