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Supplemental Discussion Text Biomarkers of C. elegans ageing In ageing populations, we defined three behavioral classes that have distinct life expectancies (Suppl. Fig. 1c) and that correlate with the extent of bodywall muscle decline (Suppl. Fig. 2b). We also identified a vacuolated body phenotype (possibly due to yolk and lipid inclusions in the pseudoceolomic space) (Suppl. Fig. 1a) and GFP reporters for muscle nuclear changes (Figs. 3a-3e), sarcomere integrity (Figs. 3g, 3h), hypodermal nuclear changes (Suppl. Figs. 4a, 4b), and yolk distribution (Figs. 4a,4b) that, although reporting progressive changes rather than clear "on-off" switches, should enable more precise evaluation of the ageing process than a longevity endpoint can reflect. Likewise, such biomarkers might permit the distinction of mutations that confer accelerated ageing from those that cause general unhealthiness (Garigan et al., Genetics, v. 161(3), pp. 1101-1112, 2002). Evaluation of biomarkers of C. elegans ageing in longlived mutants should facilitate more detailed understanding of genetic effects on healthspan and longevity. Cell death in C. elegans ageing Our results make it clear that extensive neuronal cell loss is not a major contributor to senescent decline. In addition, although muscle nuclei, sarcomeres and cytoplasm undergo progressive changes with age, we do not see much evidence of muscle cell loss per se. We did not detect extensive apoptosis in any tissue we examined at the cell or ultrastructural level, even in Class C animals that had significant deficits in locomotion and cellular integrity. Thus it appears that apoptosis is not a major factor in the senescent decline of C. elegans. Consistent with this conclusion, mutants defective in apoptosis do not exhibit lifespan changes (Hengartner, Exp. Geront., v.32, pp.363-374, 1997), and apoptotic gene expression changes do not occur with age (Lund et al., Current Biology, in press). We have observed occasional necrotic cells in aged animals (especially in intestine and hypodermis)(Suppl. Figs. 5b-5d), but the stochastic and mostly end-stage occurrence of these suggests that broad-based necrotic cell death is also unlikely to be a major factor in the process of senescence per se. We have noted an increase in structures that resemble autophagic vacuoles in intestine of many aged animals (Suppl. Fig. 5a), raising the possibility that autophagy contributes to the end stages of decline in C. elegans. Why do worms die? How do C. elegans die in old age? Nematodes have been suggested to die of bacterial infection (Gems and Riddle, Genetics, v. 154(4), pp. 1597-1610, 2000; Garigan et al., Genetics, v. 161(3), pp. 1101-1112, 2002). Although we observed 5 possible cases of bacterial invasion of body tissue during our ultrastructural analysis (into vulva, pharynx, sensilla, gut; data not shown), this was not universal. We also noted catastrophic failures such as membrane disruption of the intestine or hypodermis increase in Class C animals, and these appeared to initiate necrotic tissue failure (Suppl. Figs. 5b5d). These stochastic breakdowns appear likely to contribute to death of the animal, and if so, lead us to predict multiple "causes" of cell death and tissue failure among elderly C. elegans. However, defining exactly how nematodes die in old age awaits systematic EM autopsy of the recently dead. Questions on the integrity of the neuromuscular junction during ageing The profound decline in C. elegans bodywall muscle occurs in dramatic contrast to the apparent cellular integrity of the nervous system, and immediately raises the question of whether nerve/muscle connections deteriorate with age, a significant issue in human ageing. When we examined the expression of a GFP-tagged UNC-49B GABA receptor that is present on both nerve and muscle at the neuromuscular junction (Bamber et al., J. Neurosci. v. 19, pp. 5348-5359, 1999), we failed to detect any major differences in overall fluorescence patterns over time (data not shown). Thus, large-scale elimination of this receptor from the NMJ does not occur late in life. Definition of how the neuromuscular junction changes with age in C. elegans, however, awaits extensive serial section EM coupled with quantitative studies of multiple proteins localized to either side of the NMJ. Catastrophic events with stochastic origins characterize other ageing C. elegans tissues EM data indicates that many additional cell types (but not all) exhibit age-related deterioration. This general decline occurs stochastically, much like we observed for muscle decline. In the hypodermis, GFP reporters indicate mid-life nuclear changes. With onset around day 10, we observe a transition from uniform labeling of hypodermal nuclei expressing pcol-12GFP/NLS to a more mottled appearance (similar to that in muscle nuclei) that features prominent nucleoli (Suppl. Fig. 4a,4b). EM revealed cytoplasmic thinning in the hypodermal syncytium as well as frequent signs of plasma membrane weakness that lead to degradation, loss of organelles and local necrotic events (Suppl. Fig. 5c, 5d). Intestine initially accumulates abundant lipid inclusions including yolk and lipofuscin (Suppl. Fig. 4c, 4d; Suppl. Fig. 5b). Microvillar structures decline later in life, but lumenal integrity is always maintained due to continued reinforcement by the terminal web (Suppl. Fig. 5a, 5b). Autophagic and necrotic events appear to increase in ageing intestine, resulting in organelle losses and cytoplasmic changes (Suppl. Figs. 5a, 5b)-- the resulting local catastrophes can cause dramatic regional defects within the body. By contrast, we found that the excretory canal (an excretory cell that functions as the "kidney") appears intact, even in aged Class C animals (Suppl. Fig. 5c). However, since the excretory canal is essential for nematode viability, we cannot rule out an experimental bias for aged animals in which this cell is maintained. Changes within the gonad are complex with different structures undergoing various degrees of decline, but one unexpected finding was that in some day Class A animals, a small number of healthy-looking oocytes were maintained within the markedly deteriorating gonad sheath (data not shown).