Stress/longevity

[Guest guest]

Hi All,

That the worms were isogenic and that stress caused differential effects on

longevity in the pdf-available below may be of note.

http://edition.cnn.com/2005/TECH/science/07/25/stress.worms.reut/index.html

Nat Genet. 2005, 5 pages, in press.

A stress-sensitive reporter predicts longevity in isogenic populations of

Caenorhabditis elegans

Shane L Rea, Deqing Wu, R Cypser, W Vaupel & E

Published online: 24 July 2005 | doi:10.1038/ng1608

Abstract | Full text | PDF (317K) | Supplementary Information

Abstract: When both genotype and environment are held constant, 'chance'

variation

in the lifespan of individuals in a population is still quite large. Using

isogenic

populations of the nematode Caenorhabditis elegans, we show that, on the first

day

of adult life, chance variation in the level of induction of a green fluorescent

protein (GFP) reporter coupled to a promoter from the gene hsp-16.2 predicts as

much

as a fourfold variation in subsequent survival. The same reporter is also a

predictor of ability to withstand a subsequent lethal thermal stress. The level

of

induction of GFP is not heritable, and GFP expression levels in other reporter

constructs are not associated with differences in longevity. HSP-16.2 itself is

probably not responsible for the observed differences in survival but instead

probably reflects a hidden, heterogeneous, but now quantifiable, physiological

state

that dictates the ability of an organism to deal with the rigors of living.

Chance has a large and probably ineradicable role in determining variation among

individuals in age at death1, 2. In humans, as well as populations of laboratory

animals, 60 & #8722;90% of the variation in age at death is independent of

genotype3.

In isogenic populations (where genetic variance is essentially zero), in a

uniform

environment, some individuals die early in life and others live for a long

time1, 4.

Differences in individual lifespan in Caenorhabditis elegans populations can be

as

great as 50-fold4, 5 and still have almost as much variation in time of death as

do

the human population of the US1, 2, 6. Such observations make suspect the

popular

notion of a " genetic program that regulates longevity " 7. Instead, geriatric,

demographic and evolutionary evidence suggests an alternative paradigm of aging

that

encompasses a variety of often highly plastic processes, influenced by genetic,

environmental and stochastic phenomena1, 2, 6. Here we show that the ability of

individual isogenic worms to respond to stress on the first day of adult life

has a

large stochastic component and is a good predictor of their subsequent

longevity.

The optical transparency of C. elegans allows for noninvasive visual assessment

of

living worms without compromising subsequent measurement of longevity. We used a

chromosomally integrated transgenic strain (TJ375) containing the 400-bp

hsp-16.2

promoter coupled to the gene encoding green fluorescent protein (GFP) but

encoding

no HSP-16.2 product (Fig. 1a). This reporter provides an accurate assessment of

the

total amount of native HSP-16.2 protein8. We observed no detectable GFP in

uninduced

worms (Supplementary Fig. 1 online), but GFP became readily apparent after

exposure

to a 1- to 2-h heat shock at 35 °C (Fig. 1b) and peaked 15 & #8722;18 h later

(Fig.

1c,d).

Fig. 1 ... There were no significant differences between progeny derived from

the

original subpopulations expressing high or low levels of GFP for any of the

three

parameters (P >0.3, t-test).

Heat-shocked populations had a wide and essentially normally distributed

variation

in individual GFP fluorescence (Fig. 1c & #8722;g), even though individuals were

isogenic and grown in an environment designed to minimize environmental

heterogeneity. This variation was observed as soon as GFP expression was

detectable

and continued until GFP had completely dissipated (several days, data not

shown).

The degree of heterogeneity increased markedly with time (Fig. 1c,d) and was

replicable, quantifiable and heritable (Fig. 1g & #8722;i).

We considered whether hsp-16.2::GFP expression might predict longevity. Because

our

initial findings (on individual, isogenic worms measured manually; Supplementary

Fig. 2 online) suggested that there was a significant correlation between GFP

expression and subsequent longevity (r = 0.48; P = 0.002), we extended our

studies

to large populations. We sorted worms into classes with high, median or low GFP

expression at various times after heat induction (Fig. 1b,f) and then tested

their

resistance to a lethal thermal stress or kept them for longevity analysis. We

routinely observed significant differences in longevity and thermotolerance

among

worms that expressed GFP at high, median or low levels (Figs. 2 and 3). When

sorted

after a 2-h induction at 35 °C, worms expressing different levels of GFP showed

large differences in mean remaining lifespan and thermotolerance. In a typical

experiment, we found mean remaining lifespans of 16.4 d in worms expressing the

highest levels of GFP after heat shock but only 3.2 d in worms expressing the

lowest

levels of GFP after heat shock (Fig. 2a and Supplementary Table 1 online). Worms

expressing the highest GFP levels also had greater thermotolerance (9.5 h) than

the

average (6.7 h) or than worms expressing the lowest levels of GFP (4.0 h; P <

0.001;

Fig. 2d).

Figure 2. (a) A representative longevity assessment showing mean remaining

adult

lifespan after heat shock (mean s.e.m.; high = 16.4 1.5 d, median = 11.3 0.7

d,

low = 3.2 0.4 d; N = 30; P < 0.001). ( ... Thermotolerance (survival at 35

°C) of

worms derived from the same populations that were sampled to generate the

longevity

data in a (mean s.e.m.; high = 9.5 0.5 h, median = 6.7 0.4 h, low = 4.0 0.4

h; N

= 31 & #8722;33; P < 0.00001). (e) ... (f) Combined data for all thermotolerance

experiments (high = 10.07 0.20 h, N = 394; median = 8.27 0.15 h, N = 401; low

=

6.68 0.18 h, N = 404; P < 10-10).

Sorting and other conditions were as described for Figure 2. (a) Data from a

typical

longevity analysis shown (mean s.e.m.; high = 24.4 1.1 d, median = 18.7 1.1

d,

low = 15.35 1.0 d; N = 40; P < 0.025). ( The difference in average longevity

of

the subpopulations of worms expressing high and low levels of GFP for each of

nine

experiments is shown by a point. Details are given in Supplementary Table 2

online.

© Combined data for all longevity experiments (mean s.e.m.; high: 20.86 0.93

d,

median = 18.20 0.80 d, low = 14.03 0.85 d; N = 149 & #8722;150; P = 0.03 for

high

versus median and P < 0.001 for other comparisons). (d) Survival trajectories of

worms used to generate the data in a (see also Supplementary Tables 1 and 4

online).

(e) Survival trajectories of subpopulations of worms expressing high and low

levels

of GFP (as in d) plotted from 5 d onward. The curves are significantly different

(P

< 0.05). (f) Not all GFP reporter constructs are biomarkers for longevity. Worms

expressing the oxidative stress reporter gst-4::GFP were sorted into populations

constitutively expressing high, median or low levels of GFP, and their survival

was

then assessed. Shown is the combined data (N = 291 for each subpopulation) from

five

independent experiments. Controls include an unsorted population (Presort) and a

sorted but unselected population (Random). The curves are not significantly

different (P > 0.1; see also Supplementary Table 1 and Supplementary Fig. 3

online).

We next determined whether sorting at different time intervals after the heat

shock

affected differences in survival. We sorted worms at various times after

induction

and found difference in mean remaining lifespan to be as much as 10 & #8722;15 d

(Fig.

2b and Supplementary Table 2 online), averaging 8.0 d over all 19 experiments

(Fig.

2c). After 2 h of induction, lifespan averaged 15.1 d in worms expressing the

highest levels of GFP and only 7.1 d in worms expressing the lowest levels of

GFP,

more than a twofold difference (P = 8.0 10-29; Fig. 2c and Supplementary Table

1

online). Sorting earlier than 9 h after induction gave nonsignificant results.

Worms

expressing different levels of GFP also had significantly different

thermotolerance

9 & #8722;36 h after induction (Fig. 2d,e and Supplementary Tables 1 and 3 online)

in

14 of 16 replicates. This difference in thermotolerance was very robust,

averaging

3.4 h (P = 1.7 10-28; Fig. 2f and Supplementary Table 1 online). Differences in

survival and thermotolerance were highest 18 h after induction, when variance

was

maximal.

In previous studies, incubation of the TJ375 reporter strain for 2 h at 35 °C

resulted in subsequent thermotolerance and increased longevity in a process

called

hormesis8. In the experiments reported here, the 2-h heat induction also

resulted in

hormesis for both longevity and thermotolerance, but only in a subpopulation of

worms. Using a discrete two-population frailty model, we found that 27% of the

worms

were damaged by this treatment (D.W., S.L.R., T.E.J. & J.W.V., unpublished

data).

These results are different because we used new induction conditions to maintain

a

more uniform environment (abrupt versus slow temperature shift). When we

decreased

induction time to 1 h, we observed significant differences in survival between

worms

expressing the highest and lowest levels of GFP in seven of nine experiments

(Fig.

3a & #8722;e and Supplementary Tables 1, 2 and 4 online). Average lifespan

differed by

as much as 14 d in one study (Fig. 3b). Furthermore, after 1 h of heat shock, we

observed a robust hormesis effect, consistent with previous observations9, 10.

To correct for possible inter-relationships between the effect of heat on

survival

and its effect on fertility11, we crossed two temperature-sensitive fertility

mutations, fer-15(b26) and spe-9(hc88), into the TJ375 reporter strain to form a

new

strain, TJ550. At the nonpermissive temperature, the combination of both

mutations

completely blocked reproduction but not germ-cell formation or proliferation5.

We

still observed significant differences in survival between worms on this

background

expressing the highest and lowest levels of GFP in all six replicates (Fig. 2b

and

Supplementary Table 2 online).

Individual differences in hsp-16.2::GFP reporter expression may result from

genetic

variation: epigenetic changes may occur in isogenic individuals during

propagation

that lead to differential inactivation or expression of one or more of the large

number of repeats in the transgenic array present in the reporter strains12. To

address this possibility, we determined whether differences in levels of GFP

expression were heritable. We sorted a population 11 h after heat shock into

subpopulations containing a few hundred of the initial population of 60,000

worms

(Fig. 1i). We collected progeny, allowed them to grow to maturity, induced them

by

heat shock and assessed their levels of GFP expression. Progeny of worms

expressing

the highest and lowest levels of GFP had almost identical average levels (298.0

versus 288.6 GFP units) and variance in GFP expression (P = 0.5, 2 test for

distributional difference; Fig. 1i), essentially recapitulating observations

from

the parental population. Therefore, precise level of GFP expression is not

heritable. Although further experimentation may identify discrete causal factors

that determine variance in GFP expression, our results were obtained from an

isogenic population, maintained in a uniform environment during their

propagation.

Nonheritability of GFP expression level suggests that there is a large

underlying

stochastic component specifying level of GFP expression in individual worms,

similar

to that observed in bacteria13, 14.

Finally, we also asked whether level of GFP fluorescence was a predictor of

longevity when GFP was tagged to promoters of non-stress-inducible genes (myo-2

and

mtl-2). It was not. Because GFP fluorescence depends on redox activation, we

also

tagged the promoter of a gene normally activated in response to oxidative stress

(gst-4) and again found no relationship between GFP levels and subsequent

longevity

(Fig. 3f and Supplementary Fig. 3 and Supplementary Table 1 online). We conclude

that HSP-16.2 expression level in young adults is a robust predictor of

remaining

lifespan and that variation in this reporter is not heritable.

For C. elegans, mutational analysis has long been the preferred approach for

understanding gene action and biological function15, no less so for aging and

lifespan. Despite the success of the genetic approach in explaining lifespan

extension between distinct genotypes in C. elegans, most lifespan variation is

not

under genetic control. Even under rigidly controlled laboratory conditions, 60%

of

the variation in longevity in F2 intercrosses in nematodes is not genetic16.

Similar

observations have been made in all species that have been studied1, 3; in

humans,

only 25% of the variation in lifespan (even after excluding early deaths due to

childhood disease and accident) is due to measurable genetic effects1, 2, 17,

leaving most variation in lifespan unexplained or 'environmental', some of which

results from chance or stochastic events in individuals1, 2.

Stochastic variation arises from fundamental thermodynamic and statistical

mechanical considerations. A large fraction of individual variation in lifespan

must

stem from the fact that life results from an integrated series of metabolic

reactions that themselves are under physical constraints of the specificity and

rigidity with which they, too, can be regulated18. At the molecular level, two

points are germane to this study. First, when the number of molecules regulating

a

biological process becomes countably small, chance distributions come into play

such

that some regulatory molecules can vary severalfold between individual cells19.

Second, the Maxwell-Boltzmann (M-W) equation specifies the distribution of

kinetic

energies among molecules and requires kinetic energy to be a distributed

function.

This equation was used to develop a general theory explaining mortality

kinetics20.

Several sources of variation at the molecular level could conceivably alter GFP

(HSP-16.2) expression level and simultaneously affect more global processes.

These

include intracellular differences and fluctuations in the rates of molecular

processes such as transcription, ribosome loading and translation (as previously

postulated21). Chance variation in the number of HSF effecter molecules present

in

each cell at the time of heat shock also could have marked phenotypic

consequences.

Variation in the frequency of mitochondrial genomic rearrangements, as

previously

observed in isogenic populations of C. elegans22, 23, 24, could have an effect.

There is a growing body of research describing variation among isogenic

individuals

at the molecular level, typically in microbial or yeast cultures where such

effects

can be visualized13, 14, 25. Substantial variation among genetically identical

individuals is a fact of nature, and inherent molecular variability implies that

biochemical and molecular genetic processes must have inherent variability.

From the earliest studies of aging C. elegans populations26, it has been

apparent

that individual age at death varies greatly in isogenic populations, with

several

weeks separating those dying on the first day of adult life (excluding larval

and

embryonic death) from the last to die. In the case of long-lived Age mutants,

this

span can be several months. Stochastic variation provides a means by which we

can

start to understand this huge variation in lifespan. Genetic regulatory systems

can

be viewed in terms of robustness or sensitivity toward chance environmental

fluctuations, either maintaining expression of a single phenotype or leading to

the

expression of a distributed phenotype. When multiple phenotypes are useful, such

as

for sampling changing environments, genetic systems that have a built-in

capacity to

uncover variance, or indeed amplify it, could be selected27. Such systems might

be

of particular use to self-fertilizing organisms such as C. elegans.

A biomarker of aging is defined as " a biological parameter of an organism that

either alone or in some multivariate composite will, in the absence of disease,

better predict functional capability at some later age than will chronological

age " 28. This study suggests that level of HSP-16.2 production may be such a

biomarker of aging and that it is a robust predictor of individual longevity.

The

hsp-16.2::GFP reporter construct used in this study provided no functional

HSP-16.2

protein. It is unlikely that GFP conferred the longevity effect, because other

GFP

reporters we tested resulted in no differences in longevity. It is also unlikely

that endogenous HSP-16.2 proteins alone were responsible for the differences in

longevity, because overexpression of HSP-16.2 increases longevity by only a few

days29. Instead, the hsp-16.2::GFP reporter is probably conveying information

about

the general physiological state of the cell or organism with respect to its

ability

to withstand stress and its likelihood of subsequent survival. Future studies

will

probably identify additional biomarkers for longevity in C. elegans that also

indicate something about the physiologic state of the organism.

....

Al Pater, PhD; email: old542000@...

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