One way to identify genes that directly regulate aging is to increase or decrease their expression experimentally and assay for effects on the life span. A decreased life span is problematic, as it is likely to result from novel pathologies that do not normally limit the life span. In contrast, an increased life span should result from alterations in limiting processes, and is therefore more likely to identify genes directly related to aging. A strength of the Drosophila model system is that there are a variety of transgenic methods for upregulating or downregulating the expression of specific genes under well-controlled conditions (39).

Extensive correlative evidence suggests that for most organisms, oxidative damage may be a primary cause of aging and functional decline (40-42). Reactive oxygen species (ROS) are generated as a by-product of normal metabolism, and oxidatively damaged molecules and organelles have been found to accumulate in all aging organisms examined, including Drosophila. Not surprisingly, the transgenes tested for effects on the life span in Drosophila have been ones linked to oxidative stress resistance or response. The hsp70 gene was originally identified as one induced in response to heat and oxidative stress (43). The hsp70 family proteins can prevent protein aggregation, facilitate protein refolding, and facilitate entry of damaged proteins into proteolytic pathways (44). The enzymes superoxide dismutase (SOD) and catalase are primary defenses against ROS in all cells. SOD exists in two forms: a primarily cytoplasmic form (Cu/ZnSOD) and a mitochondrial form (MnSOD). SOD converts superoxide to H2O2, and catalase converts H2O2 to H20 and O2. Another important defense against ROS involves the enzyme glutathione reductase. This enzyme generates reduced glutathione, which is an abundant low molecular weight antioxidant.

If overexpression of a gene increases the life span, that gene is by definition a positive regulator of the life span. Transgenic Drosophila containing an extra copy of the native catalase, CuZnSOD, MnSOD, hsp70, or glutathione reductase genes generally exhibited increased gene expression, but were not found to exhibit any consistent increase in the life span under normal culture conditions (30,45-49,54). However, extra copies of hsp70 produced small increases in the life span following mild heat stress, and glutathione reductase overexpression increased survival in hyperoxic atmosphere, a condition known to increase oxida-tive stress.

Relatively large increases in the life span have recently been achieved by driving transgenic overexpression with more complex binary transgenic systems. The GAL4/UAS system (55) was used to produce overexpression of human Cu/ZnSOD in a tissue-specific pattern during Drosophila development and aging, with expression in the adult occurring primarily in motor neurons (56). This expression pattern caused increases in the mean life span of up to 40%. Importantly, O2 consumption was not altered, indicating that metabolic potential was in creased. In other studies, a conditional gene expression system called "FLP-out" (57) was used to drive overexpression of transgenes specifically in the adult (58). Using the FLP-out system, catalase enzyme was overexpressed up to 2.5-fold. Catalase overexpression significantly increased resistance to H2O2 toxicity, but had neutral or slight negative effects on the mean life span. In contrast, overexpression of Cu/ZnSOD using FLP-out extended the mean life span up to 48%. Simultaneous overexpression of catalase with Cu/ZnSOD had no added benefit, apparently due to a preexisting excess of catalase. FLP-out recently has been used to demonstrate that overexpression of the mitochondrial MnSOD also can increase the life span (59). Moreover, the increased mean and maximum life span caused by either Cu/ZnSOD or MnSOD overexpression was not correlated with decreased metabolic activity, suggesting that metabolic potential is increased in each case.

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