Re: Gene removal makes flies live up to six times longer, study finds

November 19, 2005

--- " nebula331@... " <nebula331@...> wrote:

> Gene removal makes flies live up to six times longer, study finds

> Experiments have produced one of the longest recorded life-span extensions in

any

> organism and opened doors for anti-aging research in humans, researchers say.

> (Story in World Science)

> http://www.world-science.net/othernews/051117_lifespanfrm.htm

Hi All,

The story is:

This week in Science 2004

Science 310 (5751) 1087-1088

edited by Stella Hurtley and Phil Szuromi

Kinases Involved in Promoting Longevity in Yeast

In many organisms, nutrient-sensing and caloric intake regulate aging and

longevity,

and in the budding yeast Saccharomyces cerevisiae, calorie restriction can

increase

replicative life span. Kaeberlein et al. (p. 1193; see the Perspective by Rine)

analyzed 564 single-gene yeast deletion strains and identified 10 gene deletions

that significantly increase replicative life span. Six of these encoded

components

of the highly conserved, nutrient-responsive TOR and Sch9 pathways. Calorie

restriction of cells lacking TOR1 or Sch9 failed to increase life span further.

Thus, it appears that TOR and Sch9 kinases are involved in a pathway through

which

excess caloric intake limits life span in yeast and, perhaps, higher eukaryotes.

Rine J.

CELL BIOLOGY: Twists in the Tale of the Aging Yeast.

Science. 2005 Nov 18;310(5751):1124-1125.

PMID: 16293743

The study by Kaeberlein et al. on page 1193 of this issue (1), a recent Science

paper by Lamming et al. (2), and a report by Fabrizio et al. (3) present the

three

latest chapters of the fascinating saga of how the life span of the budding

yeast,

Saccahromyces cerevisiae, is regulated (see the figure). To put this work into

context, yeast mortality was a minor concern of humans until a study revealed

that

yeast have an ortholog of the human gene in which mutations cause Werner

syndrome.

This condition has characteristics that resemble premature aging. Mutations in

the

yeast version of this gene results in substantial shortening in one measure of

yeast

life span: the number of times a cell can divide, which is defined as its

replicative life-span (4). This conserved feature of life span regulation in

yeast

and metazoans was thus of particular interest to all scientists, especially

those of

us past a certain age.

Subsequent work built upon this lead and found that SIR2, a gene originally

famous

for its role in nicotinamide adenine dinucleotide (NAD)-dependent deacetylation

of

histones in gene silencing, also promotes longevity of yeast by suppressing

recombination in the repeated array of ribosomal DNA (rDNA) genes. This

suppression

blocks the accumulation of extrachromosomal circles of rDNA, whose abundance

normally limits longevity and leads to the death of yeast mother cells. This

result

was fabulously interesting in the yeast field but lacked resonance in other

organisms where there was no known link between aging and rDNA recombination.

Caloric restriction has long been recognized as the most common contributor to

longevity in a range of organisms. Interest in yeast aging was reignited by the

discovery that it too is extended by caloric restriction. The first paper to

look at

the involvement of SIR2 in the link between caloric restriction and yeast aging

(5)

encountered some complexity: rDNA recombination in yeast is repressed by SIR2

but is

enhanced by FOB1, a gene necessary for a replication fork barrier between rDNA

repeats. In sir2 mutant cells with normal Fob1 function, extrachromosomal rDNA

circles accumulate and limit longevity. Nonetheless, in a fob1 mutant, caloric

restriction was reported to extend life span in a SIR2-dependent way. Because

caloric restriction would lead to more NAD and less of its reduced form, NADH,

this

result suggested that Sir2 protein and its NAD cofactor may be a universal

mediator

of the effect of caloric restriction on longevity. The excitement was all the

more

enjoyable because resveratol, an agonist of Sir2 enzymatic activity, is present

in

red wine, giving a new excuse for the pleasure of self-medication.

Figure: Effects of SIR2 and SCH9 on replicative and chronological life span.

Caloric restriction operates independently of SIR2 and its cousin HST2 to

promote

replicative life span, as long as extrachromosomal rDNA circles are prevented by

the

action of SIR2 and HST2. In contrast, SIR2 prevents the extension of

chronological

life span by caloric restriction.

The preeminence of SIR2 took a surprising hit when a study showed that extension

of

yeast life span by caloric restriction was SIR2-independent. Specifically,

caloric

restriction could, in fact, extend the life span of yeast lacking both FOB1 and

SIR2. The discrepancy was due to reliance in the earlier work on a uniquely odd

yeast strain (6). The view in the field was that the high level of recombination

in

rDNA in sir2 mutants created a potentially yeast-specific mechanism of aging

that

masked the ability of caloric restriction to extend life span. Only when rDNA

recombination was vastly reduced in a fob1 sir2 double mutant could caloric

restriction extend life span. In essence, the study predicted a gene or pathway

for

controlling life span in yeast in response to caloric restriction that was both

SIR2-independent and independent of recombination in the rDNA.

Just as we were beginning to suspect that all that red wine consumption was in

vain,

faith was restored in SIR2 and in one of its molecular cousins (2). In this

study,

the authors used a petri-plate assay for the " recombinogenic state " of rDNA

chromatin to screen the yeast genome for genes that, like SIR2, could extend

life

span through effects on rDNA. Remarkably, HST2, one of the four paralogous

cousins

of SIR2 in the yeast genome, can repress rDNA recombination and extend yeast

life

span when it is overproduced. Although caloric restriction can still extend the

life

span of hst2 mutants, just as it did for sir2 mutants, it cannot extend the life

span of a hst2 sir2 double mutant. Thus, although SIR2 could not explain all the

effects of caloric restriction on aging, the combination of SIR2 and its HST2

cousin

comes close to doing so.

However, as clear and interesting as this work is, there are two pesky

complications. First, if caloric restriction has effects on yeast longevity

independent of rDNA stability, they would likely have been missed by this study

because of the rDNA bias in the screen used to uncover the role of HST2. Second,

the

ability of fob1 to suppress the aging phenotype of sir2mutants was crucial to

the

argument for a caloric restriction effect on longevity independent of SIR2.

Recall

that the elevated recombination of rDNA and creation of extrachromosomal rDNA

circles in yeast cells expressing FOB1 creates a form of aging that masks the

impact

of caloric restriction. Alas, it appears that fob1 mutants cannot suppress rDNA

recombination in the combined absence of both SIR2 and HST2 (2). Hence, there

could

still be a caloric restriction effect on yeast aging, independent of both SIR2

and

HST2, that would be masked by the extrachromosomal rDNA circles resulting from

the

heightened recombination in the rDNA.

The work of Kaeberlein et al. (1) breaks through the historical bias that

influenced

key previous studies of yeast aging by screening a collection of mutant yeast,

each

with a single gene deletion, for the influence of genes on longevity. The

elegance

of this screen lies not in its genetic sophistication, but rather in the

intersection of pragmatism and dedication to measure directly the replicative

life

span of multiple cells from hundreds of mutants. The results so far are

fascinating.

Mutant forms of TOR1 and SCH9 (genes encoding two protein kinases involved in

nutrient sensing), as well as mutations in other genes of these nutrient-sensing

pathways, extend the life span of yeast. Tor and Sch9 mutants also extend the

life

span of Drosophila melanogaster and Caenorhabditis elegans, encouraging

extrapolation of these results more broadly. Indeed, SCH9 is an ortholog of AKT,

a

gene that encodes a protein kinase in the insulin- insulin growth factor

pathway,

that figures prominently in life span studies of mice and worms. The increased

life

span of these Tor1 and Sch9 yeast mutants is SIR2-independent, but at present

there

are no experimental data on whether HST2, or even another SIR2 cousin, might be

necessary. On one hand, the contribution of rDNA recombination to aging seems to

be

yeast-specific, and hence so are the roles of SIR2 and HST2 in rDNA

recombination.

On the other hand, the impact of TOR and SCH9/AKT on aging appears to be

conserved,

and hence their SIR2-independent effects on aging in yeast may well be

rDNA-independent. Owing to the difficulty of the assay, the work reported by

Kaeberlein et al. surveys only ~10% of yeast genes for their contributions to

longevity. Hence, further advances are virtually certain as this work progreses.

These studies on replicative life span ignore chronological life span, which is

of

equal interest given that most somatic cells are not replicating for the major

portion of their life span. In the third new twist to this story, Fabrizio et

al.

(6) report that rather than promoting chronological life span, SIR2 limits

chronological life span. Moreover, in sir2 mutants, caloric restriction is

needed to

extend chronological life span, providing another example of a SIR2-independent

response to caloric restriction. As with the recent work on SCH9 and TOR1 (1),

there

are no experimental data on whether HST2, or even another SIR2 cousin, might be

also

involved. Clearly, studies of chronological life span are also likely to benefit

from an unbiased screen of the yeast knockout mutant collection. Because of the

nature of the chronological aging assay, it may be possible to exploit the

molecular

bar codes unique to each mutant to screen all viable mutants in parallel.

As we have seen already in the roller coaster ride of yeast aging research, even

these fine new additions are unlikely to be the last surprises in the story.

Although the analysis of single and double mutants has proven useful, there is

nothing quite as revealing as the phenotypes of the right triple mutant, unless

of

course it is the critical quadruple mutant, genotypes that may provide even more

twists to this story.

Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO,

Kirkland

KT, Fields S, Kennedy BK.

Regulation of Yeast Replicative Life Span by TOR and Sch9 in Response to

Nutrients.

Science. 2005 Nov 18;310(5751):1193-1196.

PMID: 16293764

Calorie restriction increases life span in many organisms, including the budding

yeast Saccharomyces cerevisiae. From a large-scale analysis of 564

single-gene–deletion strains of yeast, we identified 10 gene deletions that

increase

replicative life span. Six of these correspond to genes encoding components of

the

nutrient-responsive TOR and Sch9 pathways. Calorie restriction of tor1D or sch9D

cells failed to further increase life span and, like calorie restriction,

deletion

of either SCH9 or TOR1 increased life span independent of the Sir2 histone

deacetylase. We propose that the TOR and Sch9 kinases define a primary conduit

through which excess nutrient intake limits longevity in yeast.

Calorie restriction (CR) is the only intervention known to increase life span in

yeast, worms, flies, and mammals, but the molecular mechanism for this

phenomenon

has not been clear. In yeast, CR due to reduced glucose concentration of the

culture

medium increases replicative life span (the number of daughter cells produced by

a

given mother cell before senescence) by 20 to 40% (1–3). This increased life

span

has been attributed to activation of Sir2 (1), a histone deacetylase that is

dependent on NAD (the oxidized form of nicotinamide adenine dinucleotide) (4)

and

that promotes longevity by inhibiting the formation of extrachromosomal

ribosomal

DNA (rDNA) circles (ERCs) in the nucleolus (5). Recently, however, the link

between

Sir2 and CR has been called into question with the discovery that Sir2 is not

required for life-span extension by CR (3).

To identify genes that regulate longevity in the budding yeast, a large-scale

analysis of replicative life span was conducted with the MATa haploid open

reading

frame (ORF) deletion collection, a set of 4800 single-gene–deletion strains (6).

Because replicative life-span analysis requires labor-intensive

micromanipulation of

daughter cells from mother cells, fewer than 80 different genes have been

previously

examined for their effect on replicative life span (7). Here we examined the

replicative aging properties of 564 single-gene–deletion strains (Fig. 1A; table

S1).

Fig. 1. TOR activity is an important modifier of yeast longevity.

(A) The distribution of observed strain mean life spans for 564

single-gene–deletion mutants (broken line) shows an overrepresentation of

short-lived (dark arrow) and long-lived (light arrow) mutants relative to

expected

mean life-span distribution (solid lines) for wild-type cells of the same sample

size (n = 5).

( Deletion of TOR1 increases life span.

regulated by TOR, increases life span. Mean life spans are shown in parentheses.

[View Larger Version of this Image (10K GIF file)]

An iterative method was designed to identify 95% of strains with mean

replicative

life span at least 30% longer than wild type (8). For each single-gene–deletion

strain, replicative life span was initially determined for five individual

mother

cells. If the mean life span was less than 26 generations, the strain was

classified

as not-long-lived (NLL). This lower cutoff value is predicted to result in

misclassification of a long-lived strain less than 5% of the time (fig. S1). If

the

mean life span was less than 20, the strain was classified as short-lived (SL).

If

the mean life span was greater than 36, the strain was putatively classified as

long-lived (LL), and an additional 10 cells were examined. This upper cutoff

value

is predicted to result in misclassification of a strain with wild-type life span

less than 2% of the time. For the remaining strains with a five-cell mean life

span

between 26 and 36 generations, an additional five cells were analyzed (one

iteration), and the same classification scheme was applied. This process was

repeated until every strain was either classified as SL, NLL, or LL or until

replicative life span had been determined for a minimum of 15 cells for each

unclassified strain. The replicative life-span data for strains from which at

least

15 mother cells had been assayed were compared with cell life-span data from

wild-type mothers, matched by experiment, by using a Wilcoxon rank-sum test to

generate a P value. Strains with P 0.1 were classified as LL, and strains with

P >

0.1 were classified as having a life span not significantly extended (NSE). Of

the

564 strains analyzed, 114 were classified as SL, 254 as NLL, 152 as NSE, and 44

as

LL. Although nearly 20% of the gene deletions resulted in a significantly

shortened

life span, relatively few of these are likely to represent a true premature

aging

phenotype, because dysregulation of many different cellular processes will

decrease

fitness and longevity (9). For this reason, we focused on genes that, when

deleted,

resulted in increased replicative life span, reasoning that the proteins encoded

by

these genes must impede the normal aging process.

Of the 44 single-gene–deletion strains initially classified as LL, 13 result in

a

significant increase in replicative life span (Table 1). Verification was

accomplished by determining the replicative life span for the corresponding gene

deletion strain from the haploid MATa deletion collection and, in select cases,

by

generating a new deletion allele in the parental BY4742 strain. Of the 13 genes,

FOB1 served as a proof of principle that our method can identify a true-positive

aging gene, because deletion of FOB1 is known to increase life span by reducing

the

formation of ERCs (10). In two cases, gene deletions conferring increased life

span

occurred in overlapping ORFs encoded on opposite strands (REI1 contains YBR266C;

IDH2 overlaps YOR135C), and longevity was comparable for overlapping deletion

pairs

(table S2). The identification of two different overlapping gene pairs from this

screen suggests that a high fraction of true-positive genes were successfully

identified.

Table 1. Long-lived deletion strains. From a screen of 564 single-gene–deletion

strains, 13 genes were found to increase replicative life span when deleted.

GDP,

guanosine diphosphate; GTP, guanosine 5'-triphosphate; PI3-like kinase, a kinase

like phosphatidylinositol 3-kinase.

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

Deletion strain Protein function

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

bre5 Ubiquitin protease

fob1 rDNA replication fork barrier protein

idh2 Isocitrate dehydrogenase

rei1 Protein of unknown function with similarity to human ZPR9

rom2 GDP-GTP exchange factor for Rho 1p

rpl31a Ribosomal protein L31

rpl6b Ribosomal protein L6

tor1 PI3-like kinase involved in regulation of cell growth

ure2 Regulator of nitrogen catabolite repression

ybr238c Protein of unknown function

ybr255w Protein of unknown function

ybr266c Hypothetical ORF overlapping REI1

yor135c Hypothetical ORF overlapping IDH2

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

The most striking feature of the 10 (excluding the overlapping dubious ORFs and

FOB1) newly identified aging genes is that 6 are implicated in the TOR signaling

pathway. TOR proteins are highly conserved from yeast to humans and regulate

multiple cellular processes in response to nutrients, including cell size,

autophagy, ribosome biogenesis and translation, carbohydrate and amino acid

metabolism, stress response, and actin organization (11). Yeast has two TOR

proteins, Tor1 and Tor2. Tor2 is essential and, therefore, not represented in

the

deletion collection. Deletion of TOR1 was identified from this screen and found

to

increase both mean and maximum life span by 20% (Fig. 1B). Two downstream

targets of

Tor1 and Tor2 were also identified: Ure2, which regulates activity of the

nitrogen-responsive transcription factor Gln3, and Rom2, a proposed activator of

protein kinase C (12, 13). Deletion of three genes that are transcriptionally

up-regulated by TOR increased life span: YBR238C, a gene of unknown function

(14),

and RPL31A and RPL6B, encoding two components of the large ribosomal subunit

(Fig.

1C). Not all TOR-regulated ribosomal protein gene deletions examined conferred

increased life span. Unlike the case in most multicellular eukaryotes, many of

the

ribosomal protein genes are duplicated in yeast (e.g., RPL31A and RPL31B), which

allows for viable deletion of either paralog (but not both simultaneously). The

relative importance of each paralog for ribosomal function, perhaps reflecting

differential expression levels, may determine the longevity phenotype on

deletion,

with the gene coding for the more abundant member of the pair more likely to

influence life span. Consistent with this idea, rpl31aD mother cells are

long-lived

and slow growing, whereas rpl31bD mother cells are not (fig. S2).

Protein kinase A (PKA) and Sch9 are nutrient-responsive protein kinases that

modulate replicative aging in yeast (1, 15). Mutations that decrease PKA

activity

increase replicative life span and have been suggested as genetic models of CR

(1,

3). TOR is thought to act both upstream and parallel to PKA, whereas Sch9 is

thought

to act in a pathway parallel to PKA and TOR (16, 17). TOR, PKA, and Sch9

regulate

the expression of common downstream targets, including ribosomal proteins, such

as

Rpl31a and Rpl6b (18, 19). CR of tor1D or sch9D cells failed to significantly

increase the life span of these long-lived mutants (Fig. 2, A and , which

indicates that, similar to PKA, Sch9 and TOR are targets of CR in yeast. CR by

growth on low glucose, or mutations resulting in decreased PKA activity,

increase

life span additively with deletion of FOB1 (3). Deletion of TOR1 or deletion of

SCH9

also resulted in an additive increase in life span when combined with deletion

of

FOB1 (Fig. 2, C and D). The already long life span of the sch9D fob1D or tor1D

fob1D

mother cells was not further increased by CR (Fig. 2E). Life-span extension by

CR

also occurs independently of Sir2, as long as ERC formation is kept low through

deletion of FOB1 (3). Deletion of either TOR1 or SCH9 also increased the life

span

of sir2D fob1D cells (Fig. 2F). These epistasis experiments suggest that

decreased

activity of the nutrient-responsive kinases Sch9 and TOR in response to CR

results

in increased replicative life span in yeast.

Fig. 2. TOR1 or SCH9 deletion mutants are genetic mimics of CR.

(A) CR fails to further increase the life span of cells lacking TOR1.

( CR fails to further increase the life span of cells lacking SCH9.

(D) Deletion of SCH9 increases life span additively with deletion of FOB1.

(E) Deletion of either TOR1 or SCH9 fails to increase the life span of

calorie-restricted fob1D cells.

(F) Deletion of either TOR1 or SCH9 increases the life span of sir2D fob1D

double-mutant cells.

Mean life spans are shown in parentheses.

Life-span extension by CR in yeast was initially characterized in the

short-lived

strain background PSY316 (1). PSY316 is unique among yeast strains used for

longevity studies in that, although CR increases lifespan by 30 to 40%, deletion

of

FOB1 or overexpression of SIR2 fails to result in increased life span (20). To

determine whether TOR activity is a general or strain-specific determinant of

replicative life span, we examined the effect of TOR1 deletion on life span and

Sir2

activity in the PSY316 background. Deletion of TOR1 significantly increased life

span in PSY316, but had no effect on Sir2-dependent silencing at telomeres,

similar

to the effect of CR by growth on low glucose (Fig. 3, A and . Thus, like CR,

decreased TOR activity is a strain-independent mechanism to achieve enhanced

longevity in yeast.

Fig. 3. Decreased TOR activity, like CR, is a strain-independent modifier of

replicative life span.

(A) Deletion of TOR1 increases life span in the PSY316 background. Mean life

spans

are shown in parentheses.

( Deletion of TOR1 and CR have no effect on Sir2-dependent silencing of a

telomeric URA3 marker gene, as measured by survival in the presence of 5-FOA, in

the

PSY316 background. An extra copy of Sir2 (SIR2-ox) increases silencing of

telomeric

URA3.

TOR activity is a primary determinant of replicative aging in yeast, and genetic

analysis indicates that Sir2-independent life-span extension by CR is mediated

by

reduced signaling through TOR, Sch9, and PKA, resulting in down-regulation of

ribosome biogenesis. Recently, an alternative model has suggested that

Sir2-independent CR is caused by decreased ERC formation, resulting from nuclear

relocalization and activation of the Sir2 homolog Hst2 (21). However, as long as

ERC

formation is maintained at a low level, CR increases life span to a greater

extent

in cells lacking Sir2 than in cells where Sir2 is present, seemingly

inconsistent

with Hst's simply playing a role redundant to Sir2's. CR increases life span

additively with deletion of FOB1, which suggests a mechanism for CR that is

independent of ERCs. ERCs also affect aging only in yeast, whereas the

longevity-promoting role of CR has been evolutionarily conserved.

Decreased activity of TOR and Sch9 orthologs increases life span in

Caenorhabditis

elegans (22, 23) and Drosophila melanogaster (24), as does mutation of the

TOR-regulated S6 kinase (24), which promotes ribosomal protein maturation in

multicellular eukaryotes. Therefore, the data presented here are consistent with

a

model whereby CR increases life span through a highly conserved,

Sir2-independent

signaling network from nutrients to ribosomes.

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

__________________________________

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November 19, 2005