II. Measuring age-related changes using C. elegans.
Caenorhabditis elegans is a free-living, hermaphroditic nematode that displays extensive conservation of fundamental biological processes with other animals. Based on the pioneering studies of Sydney Brenner and colleagues beginning in the 1960s, C. elegans has become a powerful experimental model system. C. elegans is extremely well understood at the cellular anatomical level, since the entire cell linage of the 959 somatic nuclei has been determined. The C. elegans genome is fully sequenced, and many genes have highly conserved human counterparts. C. elegans is excellent for studies of aging because the adults display the progressive, degenerative changes that are typical of aging in larger animals but the adult lifespan is only about 15 days. The powerful methods for genetic analysis of C. elegans have resulted in the discovery of genes and pathways that modulate the rate of aging.
The aims of our initial project were to develop methods to measure age-related changes quantitatively and determine the relationships among age-related changes using longitudinal studies. There were several rationales for studying markers of aging. Compared to vertebrate systems where many age-related changes have been described, relatively little is known about the degenerative changes that occur during C. elegans aging. In addition, the short life span of C. elegans should make it feasible to conduct longitudinal studies to define the relationships among these changes. We analyzed the declines of three physiological processes: reproduction, body movement and pharyngeal pumping. We measured the span of time required for partial or complete declines in these processes, and then conducted longitudinal studies of wild-type and mutant worms. As C. elegans hermaphrodites grow old, they undergo a stereotyped degeneration of function. Reproductive function fails first, followed by a loss of neuromuscular activity such as body movement and pharyngeal pumping, ending in death (Figure 2). We carefully measured these changes, and displayed them in ways that facilitate comparing different factors that can extend longevity (Figure 3). The declines of body movement and pharyngeal pumping were positively correlated, and both were positively correlated with life span. The positive correlations indicate that these declines in physiological processes either cause a reduced survival probability or there is a common regulatory system that mediates all these declines. These studies were led by Cheng Huang, a graduate student (Huang et al., 2004).
A critical question in aging research is why does the reproductive system age so rapidly? Human females undergo menopause at about age 50, and C. elegans hermaphrodites cease reproduction about day 10 of their 15-day lifespan. To address this important issue, we have focused on measuring the development and aging of the reproductive system. We have identified four factors that can delay reproductive aging of mated hermaphrodites that have abundant sperm: cold temperature, caloric restriction, reducing the activity of the insulin/insulin-like growth factor pathway, and the anticonvulsant drug ethosuximide. Surprisingly, using the reproductive tract to generate progeny early in life neither accelerates nor delays reproductive aging (Figure 4). This observation suggests that reproductive aging is not controlled by use-dependent factors. These studies were led by Stacie Hughes, a graduate student, and lay the foundation for a further mechanistic understanding of this process (Hughes et al., 2007). We published a review of approaches for measuring and analyzing age-related changes in WormBook (Collins et al., 2007).
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Figure 2. Age-related declines of physiological processes.
III. Identification and characterization of drugs that extend lifespan.
We initiated a project to identify drugs that can delay aging. One reason this is important is because there are currently no well-documented pharmacological treatments that delay normal human aging and extend human lifespan. We chose to screen drugs used to treat a variety of human diseases, reasoning that these compounds might have effects on aging that had not been previously identified. After screening about 20 compounds, we identified two drugs that significantly extended C. elegans life span, the anticonvulsant medication ethosuximide and the neuroactive drug valproic acid.
Ethosuximide is a small, heterocyclic ring compound that prevents absence seizures in humans and has been a preferred drug for treating this disorder since its introduction in the 1950s. An important question is whether the anticonvulsant activity in humans and the lifespan extension activity in worms have a similar mechanism. If this is the case, then other drugs with similar structures and anticonvulsant activity might also affect lifespan. Trimethadione and 3,3-diethyl-2-pyrrolidinone (DEABL) have anticonvulsant activity and structures similar to that of ethosuximide (Figure 5). Trimethadione is approved for human use and the treatment of absence seizures. DEABL is not used to treat humans. Both compounds caused significant extensions of mean and maximum C. elegans lifespan (Figure 6).
For the treatment of seizures, the therapeutic range of ethosuximide in humans is 40-100 microgram /mL. Worms cultured with the optimal dose of ethosuximide for lifespan extension had an internal concentration of about 30 microgram/mL. This value is near the therapeutic range, suggesting that the anticonvulsants may have similar targets in worms and humans. Time of administration experiments were used to determine the developmental stage when the drugs function to extend lifespan. Exposure to trimethadione only during embryonic and larval development had no effect on lifespan. In contrast, exposure to trimethadione only during adulthood caused a significant extension of mean lifespan.
To determine if these drugs delay age-related declines of physiological processes, we analyzed body movement and pharyngeal pumping. Treatments with ethosuximide and/or trimethadione significantly extended the span of time that animals displayed fast body movement, fast pharyngeal pumping, and any pharyngeal pumping. These findings indicate that ethosuximide and trimethadione delay the aging process. Together our findings suggest that the life-span-extending activity of these compounds is related to the anticonvulsant activity and implicate neural activity in the regulation of aging. These studies were led by Kim Evason, a MD/PhD student, and published in Science (Evason et al., 2005). In addition, we wrote a review of this topic for Archives of Neurology (Evason and Kornfeld 2006).
A major goal is to elucidate the mechanism of action of the drug in extending lifespan. To identify genes that are necessary for the activity of ethosuximide, we conducted a genetic screen for mutations that cause resistance to the lethal effects of high doses of ethosuximide. We identified about 50 mutations that cause resistance to ethosuximide, and we mapped many of these mutations to positions in the C. elegans genome (Figure 7). We molecularly identified two genes that can be mutated to cause resistance: osm-3 and che-3. Both genes are necessary for the function of a small number of ciliated neurons in the head of the animal (Figure 8). We showed that mutations in several other genes that affect these neurons cause resistance to ethosuximide, indicating that these ciliated neurons mediate the effects of the drug. Control experiments indicate that the mutants are not abnormal in uptake of ethosuximide or other drugs, indicating that the neurons are the target of the drugs. Consistent with this conclusion, we showed that ethosuximide treatment causes many phenotypes associated with diminished function of these neurons, including defects in chemotaxis, dauer larvae formation, and L1 arrest. These findings indicate that ethosuximide extends lifespan by decreasing the activity of ciliated neurons in the head of the worm. These neurons are important for sensing chemical cues in the environment, including food, suggesting that ethosuximide may extend lifespan by decreasing the perception of food (Figure 9). It is interesting that ethosuximide treatment does not cause caloric restriction, since treated animals have normal numbers of progeny. Therefore, animals that have impaired food perception and normal food intake may experience a lifespan extension. These studies were led by Jim Collins, a graduate student (Collins et al., 2008).
Valproic acid is an important human pharmaceutical used to treat seizure disorders and migraine headaches. It has not previously been reported to extend lifespan or delay aging. Dose response studies showed valproic acid causes substantial extensions of mean (29-35%) and maximum (42-43%) adult lifespan. In addition, valproic acid delays the age-related decline of body movement that is characteristic of worm aging. Administration of valproic acid only during adulthood extended mean lifespan significantly, indicating that valproic acid functions in adults to delay age-related degenerative changes. Valproic acid is a small, carboxylic acid. A group of compounds that are structurally related to valproic acid have been used to conduct elegant structure-activity studies. We have discovered that one of these related compounds, valproimide, could also extend the lifespan of C. elegans.
To characterize the mechanism of action of valproic acid, we tested combinations of valproic acid and heterocyclic ring anticonvulsants. Worms cultured with 4 mg/mL trimethadione and 1 mg/mL valproic acid displayed the largest lifespan extension (61% increase). These findings suggest that valproic acid and trimethadione have different mechanisms of action and demonstrate the potential of combining drugs to produce a longer extension of lifespan. Valproic acid promoted dauer larvae formation and nuclear location of DAF-16. Both findings are suggestive that valproic acid may extend lifespan by influencing the insulin/IGF signaling pathway. The studies of valproic acid were led by Kim Evason and Jim Collins (Evason et al., 2008). We published a review article in Experimental Gerontology describing drugs that affect aging in C. elegans (Collins et al., 2006).
Collins, J.J., K. Evason and K. Kornfeld. 2006. Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Experimental Gerontology, 41: 1032-1039.
Collins, J.J., C. Huang, S. Hughes and K. Kornfeld. 2007. The measurement and analysis of age-related changes in Caenorhabditis elegans. WormBook, ed. The C. elegans Research Community, WormBook, http://www.wormbook.org.
Collins, J.J., Evason, K., Pickett, C.L., Schneider, D.L., and K. Kornfeld. 2008. The Anticonvulsant Ethosuximide Disrupts Sensory Function to Extend C. elegans Lifespan. PLoS Genetics, 4(10): e1000230. doi:10.1371/journal.pgen.1000230 PMID: 18949032
Evason, K., C. Huang, I. Yamben, D.F. Covey and K. Kornfeld. 2005. Anticonvulsant medications extend worm life-span. Science, 307: 258-262.
Evason, K., and K. Kornfeld. 2006. Effects of anticonvulsant drugs on lifespan. Archives of Neurology, 63: 491-496.
Evason, K., Collins, J.J., Huang, C., Hughes, S., and K. Kornfeld. 2008. Valproic acid extends C. elegans lifespan. Aging Cell, 7: 305-317. PMID: 18248662,
Huang, C., C. Xiong and K. Kornfeld. 2004. Measurements of age-related changes of physiological processes that predict life span of Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA., 101: 8084-8089.
Hughes, S., K. Evason, C. Xiong and K. Kornfeld. 2007. Genetic and Pharmacological Factors That Influence Reproductive Aging in Nematodes. PLoS Genet, 3(2): e25. doi:10.1371/journal.pgen.0030025.
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Figure 5.Anticonvulsant medications.