Anticonvulsants Delay Worm Ageing

[Guest guest]

Hi All,

Does the below suggest a new longevity treatment using drugs already

used by people?

Science, Vol 307, Issue 5707, 193 , 14 January 2005

As the Worm Ages: Epilepsy Drugs Lengthen Nematode Life Span

Ingrid Wickelgren

Although pharmacists have proven medications for ailments as

varied as migraines and bacterial infections, they have little to

offer in the fight against aging other than unproven remedies. But

new evidence suggests that the right prescription for longevity may

already be hidden behind the pharmacy counter.

Geneticist Kerry Kornfeld and his colleagues at Washington

University in St. Louis, Missouri, report on page 258 of this issue

that a class of antiseizure drugs markedly extends the life span of

the roundworm Caenorhabditis elegans. The scientists screened 19

classes of medications prescribed for other uses for potential

longevity effects. " These compounds are approved for human use, so

they have [molecular] targets in humans, " says Kornfeld, although he

cautions that there is no evidence yet that the anticonvulsants he

tested slow aging in people.

Because these drugs act on the neuromuscular systems of both

humans and worms, the finding also hints at a direct link between the

neuromuscular system and the aging process, says geneticist

Wolkow of the National Institute on Aging in Baltimore, land.

Furthermore, the data indicate that although the drugs' mechanisms of

action partly involve molecular pathways already known to govern

aging, those pathways tell less than the whole story. " The work opens

up the possibility that there may be new targets not yet explored

that affect aging and neuromuscular function, " says Wolkow. " That's a

pretty important finding. "

With a life span of a few weeks in the lab, C. elegans is a

favorite subject for longevity studies. Since the early 1990s,

researchers have linked mutations in dozens of worm genes to

extensions of the creature's lives. Given all the drugs on the

market, Kornfeld speculated that at least one of them was likely to

retard aging or promote longevity by affecting those gene targets.

Staying alive. Anticonvulsant drugs promote longevity in roundworms

like this one.

CREDIT: THE NATIONAL HUMAN GENOME RESEARCH INSTITUTE

So about 4 years ago, Kornfeld's graduate student Kimberley

Evason began exposing separate groups of 50 worms to various drugs,

from diuretics to steroids, at three different dosages. Most of the

compounds the worms ate off their petri dishes had toxic effects.

After 8 months of negative results, Evason tested the anticonvulsant

ethosuximide (Zarontin). A moderate dose, she found, extended the

worm's median life span from 16.7 days to 19.6 days, a 17% increase.

Lower doses had a lesser effect, and higher doses were toxic.

Evason then discovered that two related anticonvulsants also

lengthened worms' lives, one of them by as much as 47%. By contrast,

a chemically related compound that does not have antiseizure activity

had no similar effect. That is " nice evidence " that the compounds'

ability to extend life span is related to their effectiveness as

anticonvulsants, says geneticist Apfeld of Elixir

Pharmaceuticals in Cambridge, Massachusetts.

The drugs are thought to control seizures in people by acting on

certain neuronal calcium channels. Exactly how the drugs extend life

span in worms is unknown, although they seem to stimulate the

nematode neuromuscular system. Kornfeld's team discovered that the

drugs affect two types of neurons: those that govern egg laying,

leading to earlier release of eggs, and those that control body

movement, making the worms hyperactive.

Unlike many of the genetic mutations that affect worm longevity,

the drugs don't act primarily through the worm's insulin-like

signaling system, the St. Louis group revealed. For example,

treatment with two of the anticonvulsants still lengthened the lives

of worms with life-curbing mutations in an insulin-pathway gene. " We

think the nervous system effects are more complicated than simply

regulating insulin signaling, " Kornfeld says.

The next step is to test whether the drugs have any antiaging

effects on higher organisms, such as flies and mice. " The nervous

system might have a central function in coordinating the progress of

an animal through its life stages, leading ultimately to

degeneration, " Kornfeld speculates. Still, he adds, " it's very early

days for understanding the connection between neural function and

aging. "

Anticonvulsant Medications Extend Worm Life-Span

Kimberley Evason, Cheng Huang, Idella Yamben, F. Covey, and

Kerry Kornfeld

Science 14 January 2005: 258-262.

Genetic studies have elucidated mechanisms that regulate aging,

but there has been little progress in identifying drugs that delay

aging. Here, we report that ethosuximide, trimethadione, and 3,3-

diethyl-2-pyrrolidinone increase mean and maximum life-span of

Caenorhabditis elegans and delay age-related declines of

physiological processes, indicating that these compounds retard the

aging process. These compounds, two of which are approved for human

use, are anticonvulsants that modulate neural activity. These

compounds also regulated neuromuscular activity in nematodes. These

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.

Aging is characterized by widespread degenerative changes.

Although treatments for aging would be desirable, the development of

such treatments is challenging. Approaches based on rational design

require information about the aging process, but little information

is currently available. Approaches based on random screens of

potential treatments require relevant and feasible assays of aging,

but the time and effort necessary to measure aging are substantial

obstacles.

To address these challenges, we exploited the C. elegans model

system. These animals age rapidly, and many processes are conserved

between nematodes and vertebrates, including aspects of the aging

process (1). To identify compounds that delay aging, we assayed 19

drugs from a variety of functional or structural classes that have

known effects on human physiology (2). We reasoned that such

compounds might have an undiscovered effect on aging. For each drug,

hermaphrodites were cultured with three different concentrations from

before fertilization until death, and the adult life-span [fourth

larval (L4) stage to death] of about 50 animals was measured. To

focus on aging, we excluded dead worms that displayed internally

hatched progeny, an extruded gonad, or desiccation due to crawling

off the agar.

Ethosuximide had the greatest effect on adult life-span, extending

mean adult life-span from 16.7 to 19.6 days (17% increase) (Fig. 1B

and Table 1). A dose-response analysis revealed that worms cultured

with external concentrations of 2 and 4 mg/ml ethosuximide displayed

the largest extensions of mean life-span; lower concentrations caused

smaller extensions, whereas higher concentrations caused toxicity and

reduced life-span (3). This effect was temperature sensitive;

ethosuximide extended mean life-span by 35% at 15°C, 17% at 20°C, and

insignificantly at 25°C (3).

Table 1. Mean and maximum life-spans. Most strains were fed live

E. coli OP50 and cultured at 20°C. Exceptions were wild-type strain

N2 cultured at 15°C (WT, 15°C), N2 fed live B. subtilis (WT, B.

subtilis), and N2 fed UV-killed OP50 (WT, UV/E. coli). External drug

concentrations are shown in milligrams per milliliter for

ethosuximide (ETH), trimethadione (TRI), and succinimide (SUC). (4/0)

and (0/4) indicate culture with drug from fertilization to L4 and L4

to death, respectively. Genotypes with no drug treatment are compared

with line 1, and differences were not analyzed for statistical

significance. Otherwise, comparisons are to the same genotype with no

drug treatment. For these comparisons in the columns showing life-

spans, numbers with no asterisks are not significant (P > 0.05); *, P

< 0.05; **, P < 0.005; ***, P < 0.0001. Maximum adult life-span is

the mean life-span of the 10% of the population that had the longest

life-spans. N, number of hermaphrodites analyzed, with number of

independent experiments in parentheses. N.D., not determined.

---------------------------------------------------------------------

-----------

Genotype Drug Mean life-span ± SD (days) % Change in mean life-

span Maximum life-span ± SD (days) % Change in maximum life-

span N

----------------------------------------------------------------------

----------

WT None 16.7 ± 3.7 23.3 ± 1.7 976(19)

ETH(2) 18.9 ± 6.0*** +13 28.9 ± 1.7*** +24 479(10)

ETH(4) 19.6 ± 5.3*** +17 28.5 ± 1.8*** +22 458(10)

TRI(4) 24.6 ± 8.4*** +47 36.5 ± 2.2*** +57 482(9)

TRI (0/4) 20.7 ± 6.7*** +24 30.1 ± 2.3*** +29 124(2)

TRI (4/0) 15.5 ± 3.4 -7 21.0 ± 1.0 -10 110(2)

DEABL(2) 21.8 ± 7.6*** +31 34.7 ± 1.6*** +49 92(2)

SUC(2) 16.0 ± 4.0 -4 23.5 ± 1.9 +1 94(2)

WT, 15°C None 23.6 ± 5.3 +41 32.9 ± 2.7 +41 116(2)

ETH(4) 31.9 ± 8.2*** +35 45.2 ± 2.4*** +37 93(2)

WT, B. subtilis None 19.5 ± 5.4 +17 28.8 ± 1.5 +24 108

(2)

TRI(4) 27.1 ± 6.6*** +39 38.2 ± 1.8*** +33 115(2)

WT, UV/E. coli None 20.0 ± 6.3 +20 30.4 ± 2.3 +30 51(2)

ETH(4) 22.8 ± 4.8* +14 29.8 ± 1.4 -2 45(2)

TRI(4) 28.1 ± 6.0*** +41 37.3 ± 0.7** +23 49(2)

daf-16 (m26) None 14.4 ± 3.3 -14 20.0 ± 1.0 -14 123(3)

ETH(2) 16.7 ± 3.0*** +16 21.3 ± 0.8** +7 119(3)

TRI(2) 16.7 ± 3.2*** +16 22.0 ± 0.0 (N.D.) +10 58(1)

daf-16 (mu86) None 14.4 ± 3.2 -14 19.6 ± 0.9 -16 113(2)

ETH (0.5) 16.0 ± 3.5** +11 21.1 ± 0.5** +8 105(2)

TRI(4) 17.4 ± 4.5** +21 24.2 ± 1.2** +23 53(1)

daf-2 (e1370) None 34.6 ± 10.9 +107 52.6 ± 5.0 +126 142

(3)

ETH(4) 39.3 ± 11.5** +14 56.8 ± 2.9* +8 118(3)

TRI(4) 37.6 ± 8.5* +9 50.0 ± 3.5 -5 50(1)

unc-31 (e928) None 22.8 ± 8.4 +37 36.2 ± 3.3 +55 56(3)

ETH(2) 27.4 ± 11.1** +20 44.4 ± 2.4*** +23 95(3)

TRI(4) 28.3 ± 5.4*** +24 35.8 ± 2.4 -1 55(1)

unc-64 (e246) None 22.6 ± 10.7 +35 42.8 ± 3.3 +84 227

(4)

ETH(2) 24.4 ± 10.7* +8 43.7 ± 2.6* +2 160(3)

TRI(4) 28.1 ± 10.2*** +24 43.3 ± 2.9 +1 245(2)

aex-3 (ad418) None 19.3 ± 5.3 +16 29.1 ± 2.5 +25 209(4)

ETH(4) 21.8 ± 6.8*** +13 34.4 ± 2.1*** +18 236(4)

TRI(4) 26.0 ± 6.0*** +35 34.3 ± 1.0*** +18 113(2)

tax-4 (p678) None 20.1 ± 6.6 +20 31.4 ± 2.4 +35 144(3)

TRI(4) 27.4 ± 4.9*** +36 35.2 ± 1.2* +12 52(1)

osm-3 (p802) None 20.2 ± 6.6 +21 32.1 ± 4.1 +38 161(4)

ETH(2) 22.1 ± 7.1 +9 34.0 ± 3.6 +6 131(3)

TRI(4) 23.6 ± 5.4** +17 32.7 ± 3.0 +2 30(1)

eat-2 (ad465) None 20.1 ± 6.3 +20 31.4 ± 3.1 +35 192(4)

----------------------------------------------------------------------

----------

TRI(4)

----------------------------------------------------------------------

----------

28.6 ± 10.0***

----------------------------------------------------------------------

----------

+42

----------------------------------------------------------------------

----------

47.2 ± 7.3**

----------------------------------------------------------------------

----------

+50

----------------------------------------------------------------------

----------

68(1)

----------------------------------------------------------------------

----------

Concentrations of 0.5, 5, or 10 mg/ml succinimide also did not

significantly increase mean life-span.

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 (4, 5) (Fig. 1A).

An important question is whether the anticonvulsant activity in

humans and the life-span 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 life-span.

Trimethadione and 3,3-diethyl-2-pyrrolidinone (DEABL) have

anticonvulsant activity and structures similar to that of

ethosuximide (4, 6) (Fig. 1A). 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 life-span (Fig. 1, C and D, and Table 1). Trimethadione

caused the largest extension of mean (47%) and maximum (57%) life-

span of the three compounds. Succinimide, similar in structure but

lacking in anticonvulsant activity in vertebrates, did not extend

life-span (Fig. 1A and Table 1). These findings suggest that

ethosuximide, trimethadione, and DEABL may extend life-span by a

similar mechanism that may be related to the mechanism of

anticonvulsant activity.

For the treatment of seizures, the therapeutic range of

ethosuximide in humans is 40 to 100 µg/ml (5). Worms cultured with an

external concentration of 2 mg/ml ethosuximide had an internal

concentration (±SD) of 30.5 ± 22.2 µg/ml. This value is near the

therapeutic range, suggesting that the anti-convulsants may have

similar targets in worms and humans.

To determine the developmental stage at which the drugs function

to extend life-span, trimethadione was administered from

fertilization until the L4 stage or from the L4 stage until death.

Exposure to trimethadione only during embryonic and larval

development had no effect on life-span. In contrast, exposure to

trimethadione only during adulthood caused a significant extension of

mean life-span (24%) (Fig. 1D and Table 1).

To determine whether these drugs delay age-related declines of

physiological processes, we analyzed self-fertile reproduction, body

movement, and pharyngeal pumping. The declines of pharyngeal pumping

and body movement are positively correlated with each other and with

life-span (7). The decline of self-fertile reproduction is not

correlated with life-span, suggesting that this age-related change is

regulated independently (7). 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 (Fig. 2, B to D and F). Neither compound

significantly extended the span of time that animals displayed self-

fertile reproduction (Fig. 2, A and F). These measurements can be

used to define stages of aging (7). Both compounds extended Stage II,

the postreproductive period characterized by vigorous activity (Fig.

2E). Trimethadione also extended Stage IV, the terminal phase

characterized by minimal activity. These findings indicate that

ethosuximide and trimethadione delay the aging process.

Several genetic and environmental manipulations can extend C.

elegans life-span. To investigate the relationships between the anti-

convulsants and these regulators of aging, we examined the effect of

combining two treatments. Worms cultured on nonpathogenic Bacillus

subtilis or ultraviolet (UV)–irradiated E. coli display an extended

life-span (8, 9). Trimethadione extended the life-span of worms

cultured on B. subtilis and UV-irradiated E. coli (Table 1),

indicating that the primary mechanism of the anticonvulsant life-span

extension is not a reduction of bacterial pathogenicity.

Nutrient limitation extends life-span and can be caused by a

mutation of the eat-2 gene that is important for pharyngeal pumping

(1, 10, 11). Trimethadione significantly extended the life-span of

eat-2 mutants (42%) (Fig. 1E and Table 1), indicating that the

primary mechanism of life-span extension is not nutrient limitation.

Furthermore, wild-type animals treated with ethosuximide or

trimethadione were not nutrient limited, because they displayed

normal pharyngeal pumping, food ingestion, and body morphology (they

did not appear thin or starved), and they produced an approximately

normal number of progeny (3).

An insulin-like signaling pathway regulates C. elegans life-span.

This pathway requires the function of sensory neurons that may

mediate the release of an insulin-like ligand, the daf-2 insulin-like

growth factor (IGF) receptor gene, and a signal transduction cascade

that regulates the daf-16 forkhead transcription factor gene. Loss-of-

function daf-16 mutations reduce life-span and suppress the life-span

extensions caused by mutations in upstream signaling pathway genes

such as daf-2 (12). Treatment with ethosuximide or trimethadione

significantly extended the life-span of two loss-of-function mutants,

daf-16(m26) (16%) and daf-16(mu86) (11 to 21%) (Fig. 1, F and G, and

Table 1), although the percentage change caused by trimethadione was

less than that in wild-type animals (47%). These results indicate

that part of the anticonvulsant action is independent of daf-16. Part

of the anti-convulsant action may require daf-16. However, the

reduced effect of trimethadione is consistent with other

possibilities, such as deleterious consequences of combining a

mutation and a drug that both cause pleiotropic effects (13).

Life-span extension is caused by loss-of-function mutations of

genes important for the function of sensory neurons (osm-3 and tax-

4), for neurotransmission (unc-31, unc-64, and aex-3), and for

transmission of the insulin-like signal (daf-2) (12, 14, 15) (Table

1). Ethosuximide and/or trimethadione significantly increased the

life-span of osm-3, tax-4, unc-31, unc-64, aex-3, and daf-2 loss-of-

function mutants from 8 to 36% (Fig. 1, H to M, and Table 1). These

results indicate that part of the anticonvulsant action may be

different than the action of these mutations. The effects of

ethosuximide and/or trimethadione were only partially additive with

several mutations, notably daf-2, unc-64, and osm-3. Thus, part of

the activity of the anticonvulsants may be similar to the effects of

these mutations, several of which affect neural function. However, an

absence of full additivity is also consistent with other

possibilities (13).

Anticonvulsants affect the neural activity of vertebrates. To

determine whether these drugs have a similar activity in nematodes,

we analyzed neuromuscular behaviors. C. elegans egg laying is

mediated by HSN neurons that innervate the vulval muscles (16, 17).

Wild-type hermaphrodites lay eggs that have matured to about the 30-

cell stage of development. Trimethadione and ethosuximide caused wild-

type hermaphrodites to lay eggs at much earlier stages of

development, often the 1- to 7-cell stage (Fig. 3C). The control

drug, succinimide, did not stimulate egg laying (Fig. 3C). A delay in

egg laying can result in an egg-laying defective (Egl) phenotype

characterized by progeny that hatch internally. Approximately 8.9% of

wild-type hermaphrodites displayed an Egl phenotype during their

lifetime; ethosuximide and trimethadione reduced this to 2.9 and

1.2%, respectively (Fig. 3A). To investigate whether the

anticonvulsants act presynaptically on the HSN neurons or

postsynaptically on the vulval muscles, we analyzed an egl-1 mutant

that lacks HSNs as a result of a developmental abnormality (17).

Ethosuximide did not cause egl-1 mutants to lay eggs at earlier

stages of development (Fig. 3D), indicating that the vulval muscles

are not sufficient and the HSN neurons are necessary for the

anticonvulsant to stimulate egg laying. This result is consistent

with the model that the drug acts presynaptically.

Treatment with ethosuximide or trimethadione caused wild-type

hermaphrodites to display hyperactive motility, indicating that these

drugs stimulate neuromuscular activity (Fig. 3B). To analyze this

phenotype, we examined sensitivity to the acetylcholinesterase

inhibitor aldicarb. Aldicarb causes paralysis of body movement

resulting from the accumulation of acetylcholine at the neuromuscular

junction (18). Mutations that reduce synaptic transmission cause

resistance to aldicarb (18). In contrast, mutations that stimulate

synaptic transmission cause hypersensitivity to aldicarb-mediated

paralysis (19). Trimethadione treatment of wild-type animals caused

hypersensitivity to aldicarb-mediated paralysis (Fig. 3E). The

control drug, succinimide, did not cause hyperactive motility or

aldicarb hypersensitivity (3). These results indicate the

anticonvulsants stimulate synaptic transmission in the neuromuscular

system that controls body movement.

Ethosuximide and trimethadione effectively treat absence seizures

in humans by regulating neural activity. A likely target of

ethosuximide is T-type calcium channels, although it is possible that

these compounds act on multiple targets (20–22). These anti-

convulsants also affected neural activity in nematodes, and the

anticonvulsant and the life-span extension effects of the compounds

may act through similar mechanisms. The findings presented here are

consistent with the model that the effect on neural activity causes

the life-span extension, although they do not exclude the possibility

that the drugs affect neural activity and aging by different

mechanisms. Furthermore, the interactions with the insulin-signaling

mutants suggest the intriguing possibility that neural activity

regulates aging by both daf-16–dependent and daf-16–independent

mechanisms.