Overview
Study of molecular mechanisms regulating aging. Use of model systems Saccharomyces cerevisiae, C. elegans, mice, mammalian cells, and relevance to aging in humans. Study mechanisms by which SIR2-related genes regulate life span and calorie restriction (CR).

Research Summary
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. The SIR2 gene is the key determinant of life span and impacts the nucleolus, which contains tandemly repeated copies of ribosomal DNA (rDNA). A key event in aging is the generation of rDNA circles by homologous recombination.

Biochemical studies on Sir2p: Sir2p is a simple enzyme with a novel activity – it is an NAD-dependent protein 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. The mammalian SIR2 homolog Sirt1 has this same enzymatic activity, but can also deacetylate other substrates, including p53, forkhead, NF-kB, p300, and other transcription factors. Importantly, the NAD requirement means that silencing is likely coupled to the metabolic strategy of cells. When metabolic strategy is altered, for example during CR, the NAD/NADH ratio changes thereby altering Sir2 activity.

Calorie restriction in yeast: 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, drives down the levels of NADH, and increases the silencing activity of Sir2p.

Aging in mice: The mammalian ortholog Sirt1 has several functions that trigger physiological changes seen during CR. First, Sirt1 makes cells more resistant to oxidative or radiation-induced stress, which is a phenotype of rodent cells from CR animals. Second, Sirt1 promotes mobilization of fat from white adipose tissues, a change triggered by CR that is sufficient to extend the life span. It does this by modulating the activity of the key regulator of white fat, PPARg. Third, Sirt1 mediates the metabolism of energy sources in metabolically active tissues. CR animals are efficient in metabolism rendering them insulin-sensitive. Fourth, Sirt1 regulates the induction of insulin in pancreatic beta cells, an obvious component of energy utilization during CR. In several cases, Sirt1 is activated by starvation or stress to carry out these functions. Molecular mechanisms of Sirt1 activation and its functions in triggering the physiological changes elicited by CR are under study.

There are six other mammalian SIR2-related genes besides Sirt1. Functional studies of Sirt2, 3, 4, and 7 are being carried out. At least some of them also appear to play a role in CR. Sirt2 appears to regulate insulin sensitivity in mice. Recent findings also show a possible link between Sirt2 and cancer. Sirt4, like Sirt1, plays a role in regulating insulin production in beta cells. This regulation is mediated from the mitochondria, the cellular compartment where Sirt4 resides. Knock out mice for Sirt1-4 are available for these studies.

Aging in C. elegans: C. elegans has four SIR2 homologs. Strains with a free duplication of the SIR2 ortholog, sir-2.1, show a significantly extended life span. Transgenic animals with elevated levels of sir-2.1 also live longer than wild type. A deletion of sir-2.1 has the opposite effect, life span is shortened. sir-2.1 appears to function in the insulin signaling pathway shown to regulate aging and dauer formation in C. elegans. This gene may also function in a second pathway to regulate life span. In another project, the possible role of cell death genes in limiting organismal life span is under study. In a third project, mutants that show premature aging have been identified and are being characterized.

Selected Publications
Kubova, J., and Guarente, L. How does calorie restrriction work? Genes and Dev. 17, 313- 321, (2003).

Hekimi, S. and Guarente, L. Genetics and the specificity of the aging process. Science, 299, 1351-1354, (2003).

Lin, S. J., Ford, E., Haigis, M., Liszt, G., and Guarente, L. Calorie restriction extends life span by lowering the level of NADH. Genes Dev. 18, 12-16, (2004).

Motta, M. C., Divecha, N., Lemieux, M., Kamel, C., Chen, D., Gu, W., Bultsma, Y., McBurney, M., and Guarente, L. Mammalian SIRT1 represses forkhead transcription factors. Cell 116, 551-563, (2004).

Picard, F., Kurtev, M., Chung, N., Topark-Ngarmm, A., Senawong, T, Machado de Oliveira, R., Leid, M, McBurney, M. and Guarente, L. SIRT1 regulates white fat in mice:a mechanistic link between calorie restriction and aging. Nature, 429, 771-776. published online June 2, (2004).

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