Catalase for Longer Life

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Hi All,

The title for the " This Week in Science " description seems to fit the message of

the

below, which is for a pdf-available paper that implicates catalase in longevity.

CR

appears to involve catalase in its anti-oxidase mechanisms:

Agarwal S, Sharma S, Agrawal V, Roy N.

Caloric restriction augments ROS defense in S. cerevisiae, by a Sir2p

independent

mechanism.

Free Radic Res. 2005 Jan;39(1):55-62.

PMID: 15875812

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve & db=pubmed & dopt=Abstra\

ct & list_uids=15875812 & query_hl=22

First, here is the This Week in Science text.

This Week in Science

Science 24 June 2005: 1837 [Full Text]

Catalase for Longer Life

Cell and tissue damage caused by free radical oxygen molecules have been linked

to

aging pathologies, yet the idea that antioxidant defenses can prolong life has

been

controversial. Schriner et al. (p. 1909, published online 5 May 2005; see the

Perspective by ) generated transgenic mice that overexpress catalase in

mitochondria, a major source within the cell of oxygen free radicals. Catalase

removes damaging hydrogen peroxide that can generate reactive oxygen species. In

the

transgenic mice, cellular oxidative damage and age-related decline in heart

function

were reduced and cataract formation was delayed. In addition, life span

increased by

nearly 20%. Thus, antioxidant enzymes can promote mammalian longevity.

Next is the perspective from .

Perspectives BIOMEDICINE:

Enhanced: The Anti-Aging Sweepstakes: Catalase Runs for the ROSes

A.

Science 24 June 2005: 1875-1876. [summary] [Full Text] [PDF]

Organism envy is the unavoidable fate of all who study the physiological

genetics of

aging in mice. If only our favorite rodents had the grace to die in a few weeks

or

months, like worms and flies, or better yet to do so after a round or two of

hermaphroditic self-fertilization! Clinging desperately to the consoling mantra

that

mice are more or less just like people, we hold our breath waiting for a

discovery

in this diminutive mammal worth bragging about. Ergo the exhalations of relief

attending the report of Schriner et. al. [HN1] (1), on page 1909 of this issue,

that

overexpression of human catalase in the mitochondria of mice extends median and

maximal lifespan by about 20% [HN2]. Catalase [HN3] prevents the formation of

reactive oxygen species [HN4] (ROS) that can damage cellular constituents.

Although

20% may not seem like much compared to the 50% life span extension seen in dwarf

mice with hormone-altering mutations (2), it is roughly 5 times that predicted

from

complete abolition of human cancer or heart attack (3), and thus no small

potatoes.

The central mystery for biological gerontology [HN5] is variable-rate synchrony:

If

everything must go to pot all at once as organisms approach emeritus status, why

does it take 2 years to do so in mice, 10 years in dogs, 20 years in horses, 70

years in people, and longer still in whales and some seabirds? What process, or

set

of synchronous processes, sets the tempo of aging, and how does aging lead to

its

unwelcome symptoms? Schriner et al. view their new data as support for the

notion

that oxidative damage is the key villain and, moreover, that mitochondria [HN6]

are

a major source of toxic oxygen radicals. There are still a few gaps in their

story--most laboratory-bred mice die of tumors rather than of the cardiomyopathy

on

which Schriner et al. focus (what are these mice dying of, then?)--but the

report is

the first strong evidence that mouse aging can be delayed by antioxidant

prophylaxis.

Is it safe to conclude that oxygen molecules are the true culprits in causing

aging?

Can we now turn our attention to the secondary questions of how they cause

physiological decline in the superannuated? There are still some grounds for

skepticism here. The search for antioxidant drugs that slow aging and extend

life

span in mammals has produced much frustration and a lamentable absence of

authentic

anti-aging pills. Mice heterozygous for the mitochondrial form of superoxide

dismutase, an enzyme that destroys a highly reactive derivative of oxygen called

superoxide [HN7], show high levels of DNA oxidation in multiple organs. In spite

of

their abnormally oxidized DNA, these animals show no decline in lifespan and no

acceleration in certain hallmarks of aging: cataracts, immune dysfunction, and

protein modifications (4). Thus, mice can live reasonably long and healthy lives

despite unusually high levels of oxidative damage. Furthermore, skin-derived

fibroblast cells from three different kinds of long-lived dwarf mice are

resistant

to multiple forms of stress, including oxidants, ultraviolet light, heat, the

heavy

metal cadmium, and a DNA alkylating agent (5). Mutations that extend worm

longevity

also typically lead to, and perhaps act through, increased resistance to

multiple

forms of stress (6). Thus, it seems plausible that many age-retarding mutations

may

work by inducing cellular signaling pathways, still poorly defined, that augment

defenses against a multitude of insults, including the oxidative ones.

[Figure] Maximum life-span estimates in various populations. Mutations (or

calorie

restriction) increase maximal survival in mice by ~50% at most. In contrast,

porcupine longevity is greater by about 500%. The value for whales is

speculative,

based on a small sample number. This juxtaposition suggests that further

progress in

understanding the pathways that control aging rates in mammals may benefit from

greater emphasis on interspecies comparisons.

The Schriner et al. paper increases to nine the number of mouse genes whose

mutation

extends maximal longevity. It is also the second gene whose overexpression in

mice

has this effect. Five of these loci modulate signals mediated by insulin-like

growth

factor-I (IGF-I) [HN8], with lower levels of IGF-I associated with longer life

span

and slower aging. In dogs, too, genetic polymorphisms that diminish body size by

altering IGF-I levels increase life span (7). It remains to be seen whether

mammalian longevity induced by any of these genetic effects, or by diets low in

calories or nutrients [HN9], are accompanied by changes in cellular resistance

to

oxidants, nonoxidant injuries, or both.

So far, the best one can accomplish by combinations of genetic and dietary

intervention is to increase median and maximal life span of a given mammalian

species by about 80% (8). Natural selection, however, routinely engenders new

species that outlast their progenitor species by a factor of10 or more. Among

the

rodents, species specific maximal longevities range from 2 to 4 years in shrews,

mice, and rats, to at least 27.3 years in naked mole rats, porcupines, and

perhaps

beavers (see the figure). How does nature do this? Does she rely solely on

antioxidant mechanisms or on pathways that control multiple forms of cellular

stress

resistance? The recent demonstration (9) [HN10] that the replicative potential

of

mouse cells in culture can be increased to levels seen in human cells by

reducing

environmental oxygen suggests that resistance to oxidative damage may contribute

to

the remarkable longevity of our own species. Similarly, resistance of cultured

fibroblasts to oxidative stress correlates with maximal life span across a range

of

mammalian species (10). Thus, oxidation defenses have become the heavy favorite

among players wishing to bet on a single horse in the anti-aging sweepstakes.

But

attractive dark horses still lurk within the pack of remaining hypotheses.

Perhaps

the winning bet will prove to be a trifecta: oxidative damage, protein

malformation,

and DNA breakage [HN11](order to be determined).

Research in basic biogerontology may lead to a pill that slows aging and, as a

pleasant side effect, delays all age-related diseases. Deceleration of aging and

associated late-life illnesses is now readily achievable in mice, and thus is a

conceivable goal for preventive medicine. The value of studies like that of

Schriner

et al. is not that they serve as blueprints for genetic surgeons--gene therapy

for

aging is still as distant a dream as other sci-fi perennials--but as pointers

toward

the currently less sexy discipline of comparative physiology. It is time to

exploit

the modest but growing collection of anti-aging mutations, together with the

large

and potent collection of slow-aging organisms donated to us by 100 million years

of

natural selection, to map out the common pathways by which aging can best be

modulated.

Here, are the major text excerpts for the article of interest.

Schriner SE, Linford NJ, GM, Treuting P, Ogburn CE, Emond M, Coskun PE,

Ladiges W, Wolf N, Van Remmen H, Wallace DC, Rabinovitch PS.

Extension of murine life span by overexpression of catalase targeted to

mitochondria.

Science. 2005 Jun 24;308(5730):1909-11. Epub 2005 May 5.

PMID: 15879174

Published online 5 May 2005 [DOI: 10.1126/science.1106653] (in Science Express

Reports ) [Abstract] [Full Text] [PDF] [supporting Online Material]

To determine the role of reactive oxygen species in mammalian longevity, we

generated transgenic mice that overexpress human catalase localized to the

peroxisome, the nucleus, or mitochondria (MCAT). Median and maximum life spans

were

maximally increased (averages of 5 months and 5.5 months, respectively) in MCAT

animals. Cardiac pathology and cataract development were delayed, oxidative

damage

was reduced, H2O2 production and H2O2-induced aconitase inactivation were

attenuated, and the development of mitochondrial deletions was reduced. These

results support the free radical theory of aging and reinforce the importance of

mitochondria as a source of these radicals.

A causative role for reactive oxygen species (ROS) in aging processes, referred

to

as the free radical theory of aging (1), proposes that ROS in biological systems

attack molecules and cause the functional decline of organ systems that

eventually

leads to death. Accumulation of this damage over time is thought to result in

pathologies associated with aging, including arteriosclerosis, neoplasia, and

cataracts (2). ROS are generated, in large part, from single electrons escaping

the

mitochondrial respiratory chain and reducing molecular oxygen to form the

superoxide

anion (). Superoxide dismutase (SOD) converts into hydrogen peroxide (H2O2)

that

then produces a highly reactive hydroxyl radical (·OH) in the presence of

reduced

metal atoms unless H2O2 is removed by the action of glutathione peroxidase or

catalase.

The hypothesis that longevity can be enhanced by increasing antioxidant defenses

has

been controversial because of contradictory findings in invertebrate models of

aging. These include whether or not the overexpression of SOD or catalase

enhances

the life span of the fruit fly Drosophila melanogaster (3–6) and whether

synthetic

antioxidants extend the life span of the nematode Caenorhabditis elegans (7–10).

Although there is an increasing number of long-lived mutant mouse models, most

of

them do not directly test the free radical theory of aging. Overexpression of

the

antioxidant protein thioredoxin was reported to increase mean and maximum life

span

in a short-lived strain, although the identities of the specific agents that

limited

life span were not determined (11).

To determine the role of H2O2 in limiting mammalian life span, we targeted human

catalase, normally localized in the peroxisome (PCAT), to the nucleus (NCAT) and

mitochondria (MCAT). Catalase activities in MCAT animals were elevated in heart

and

skeletal muscle of both founder lines (Fig. 1, A and and in brain (Fig. 1C)

of

the 4033 founder line. Furthermore, catalase activity in the cardiac

mitochondrial

fraction of MCAT animals was 50 times higher than that in their wild-type

littermates (Fig. 1D). Quantitative reverse transcription polymerase chain

reaction

(RT-PCR) confirmed transgene expression in these tissues (fig. S1) (12).

Endogenous

catalase expression was similar between MCAT and wild-type animals, with the

highest

expression in liver, kidney, and lung (fig. S1). Confocal immunolocalization

revealed that about 10 to 50% of cells in the MCAT heart expressed detectable

levels

of human catalase, co-localized with a mitochondrial marker in heart and

fibroblast

cultures from MCAT transgenic animals (Fig. 2). Human catalase was not detected

in

heart or fibroblasts from wild-type littermates. PCAT and NCAT gene products

localized to peroxisomes and nuclei, respectively, as previously described (13).

To determine whether the expression of PCAT, NCAT, or MCAT could modulate life

span,

we maintained transgenic animals and wild-type littermates until death. PCAT

animals

showed a slight extension of median life span of 3 months (10%) and 3.5 months

(13%)

in the two founder lines compared with controls (Fig. 3A); this was significant

only

for the 2088 line (P = 0.02). Differences in maximal life span were not

statistically significant. NCAT mice showed only 1-month (4%) and 3-month (11%)

increases in median life span in the two founders; neither was significant (Fig.

3B). Targeting catalase to the mitochondria, however, afforded 4.5-month (17%, P

<

0.0001) and 5.5-month (21%, P = 0.0002) increases in median life spans of

founders

4403 and 4033, respectively (Fig. 3C). There was a similar extension of maximal

life

span: The 10% longest-lived MCAT animals showed a 4.5-month longer median life

span

than wild-type littermates (both founders combined, P = 0.001). Increased life

span

was evident in both males (P < 0.0001) and females (P = 0.0003) without any

statistically significant sex differences (fig. S2). The MCAT longevity data fit

a

Gompertz distribution (exponential increase in mortality rate with age) with

parallel log mortality rates for MCAT and wild-type littermates (fig. S3), a

result

often interpreted as a delay in onset of aging. None of the transgenic lines

showed

a difference in weight or food consumption when compared to littermate controls

(table S1), and there were no gross physical abnormalities.

Table S1. Food consumption data, four day observation. N= average of 9 for each

of

the 16 cohorts. There was no difference in food consumption between MCAT

transgenic

and wild type mice.

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

Line Sex Genotype Age* Body weight (gm) Total gm food consumed per gm bw Daily

gm

food consumed per gm bw

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

4033

female MCAT old 29±3 0.65±0.08 0.163±0.020

WT old 30±5 0.58±0.15 0.145±0.037

MCAT young 27±4 0.48±0.10 0.120±0.025

WT young 26±3 0.54±0.12 0.135±0.029

male

MCAT Old 36±3 0.44±o.11 0.110±0.027

WT Old 37±4 0.38±0.04 0.095±0.010

MCAT Young 37±3 0.49±0.80 0.123±0.020

WT Young 39±3 0.47±0.14 0.118±0.035

4403

female MCAT Old 28±2 0.68±0.17 0.170±0.043

WT Old 29±2 0.63±0.10 0.158±0.025

MCAT Young 33±6 0.48±0.06 0.120±0.016

WT Young 34±4 0.48±0.07 0.120±0.035

male MCAT Old 37±7 0.48±0.07 0.120±0.015

WT Old 32±4 0.50±0.05 0.125±0.014

MCAT Young 41±4 0.45±0.08 0.113±0.021

WT Young 44±2 0.38±0.01 0.095±0.003

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

*Old = 24 to 30 months, young = 9 to 12 months.

Young (9 to 11 months) and older (20 to 25 months) MCAT and wild-type

littermates

were examined by histopathology. Little abnormality was seen in either group at

9 to

11 months of age. In older animals, there was a trend toward reduced

splenomegally

and splenic lymphoid neoplasia in MCAT (1 of 21) compared with wild-type (4 of

24)

mice, but this effect was not statistically significant. Cardiac pathology

(subendocardial interstitial fibrosis, hyaline cytoplasmic change, vacuolization

of

cytoplasm, variable myocyte fiber size, hypercellularity, collapse of

sarcomeres,

mineralization, and arteriolosclerosis) was the most consistent difference

between

20- to 25-month MCAT and wild-type mice. These changes are also commonly

observed in

elderly human hearts, often in association with congestive heart failure (14);

the

latter has also been associated with functional abnormalities of mitochondria

(15).

The severity of pathology was graded on a score of 0 to 4 for a cross-sectional

cohort of 21 MCAT and 20 wild-type mice age 20 to 25 months from both founder

lines.

The severity of arteriosclerosis was 1.29 on average for MCAT and 1.85 for

wild-type

(P = 0.04). The severity of cardiomyopathy was 1.19 for MCAT and 2.00 for

wild-type

(P = 0.004; P = 0.002 when combined with arteriosclerosis). This demonstrates

the

potential of the MCAT protein to protect the heart and suggests that these mice

experience a prolonged health span as well as life span. The severity of

cataracts,

quantitated on a four-point scale by slit-lamp examination, was reduced in

17-month-old founder 4033 MCAT mice compared with age-matched wild-type mice

(1.5 ±

0.13 and 1.95 ± 0.13, respectively, P = 0.003) but not in founder 4403 compared

with

wild-type. However, this trend became of borderline significance at 27 months (P

=

0.06), and by the age of 30 months both groups had similar cataract scores of

2.5.

The ability of the MCAT protein to enhance protection of mitochondria from ROS

was

investigated by measuring aconitase activity in isolated heart mitochondria from

5-,

19-, and 30- to 33-month-old animals (Fig. 4, A, B, and C). Aconitase is rapidly

inactivated in H2O2-treated mitochondria isolated from wild-type hearts of all

ages.

This inactivation was significantly attenuated in MCAT heart mitochondria at all

ages compared to controls, suggesting that these mitochondria eliminate H2O2

more

effectively and are thereby better protected from oxidative damage. The MCAT

protein

also decreased mean H2O2 production from heart mitochondria 25% compared with

wild-type animals (Fig. 4D), a significant difference (P = 0.004).

To determine whether MCAT overexpression could reduce oxidative damage to total

DNA,

we measured 8-hydroxydeoxyguanosine (8-OHdG) in DNA from skeletal muscle and

heart.

An age-related increase in 8-OHdG in skeletal muscle, but not heart, was

observed in

control animals, and MCAT mice were protected from this change (Fig. 4E) (P =

0.03).

Mitochondrial deletions associated with oxidative damage were measured as low

molecular weight products by long-extension polymerase chain reaction (LX-PCR).

These increased with age in both wild-type heart and skeletal muscle (16);

however,

a statistically significant decrease in the number of deletion products was

noted in

21-month-old MCAT skeletal muscle (Fig. 4F). A decrease was also detected in

30+-month MCAT skeletal muscle and 21-month-old MCAT heart, but neither reached

statistical significance.

To examine the possibility that combined enhanced antioxidant defenses might

provide

further extension of life span in mammals, we bred hemizygous

PCAT-overexpressing

animals to hemizygous SOD1-overexpressing animals (17). The double transgenic

mice

had an 18.5% extension of median life span compared with wild-type (P < 0.0001)

and

a 7% extension compared with PCAT littermates (P = 0.036), but without extension

of

maximum life span (Fig. 3D). There were no apparent deleterious phenotypic

changes

in these animals. It seems likely that SOD1 x MCAT or SOD2 x MCAT mice might

exhibit

an even greater extension of longevity, because both the combination of

antioxidant

enzymes chosen for enhancement and the subcellular localization appear to have

profound effects on the life span extension phenotype.

The life span extension of MCAT mice (Fig. 3) was similar in magnitude to that

resulting from knockout of the fat-specific insulin receptor (18) but less than

that

achieved by caloric restriction or dwarfism or that observed in other genetic

models

of delayed and decelerated aging (19). However, the effect of MCAT on life span

is

accomplished without apparent deleterious side effects and without disabling a

major

transduction pathway. Although the MCAT longevity phenotype likely results from

the

direct beneficial effects of reduced oxidative stress in aging, it is also

possible

that indirect effects, such as a stress response secondary to reduced

intracellular

H2O2-dependent signaling, may also contribute to the longevity phenotype. The

mosaic

pattern of catalase expression might also play a role in modulating the life

span

extension. Mosaicism may result from selection against cells expressing high

catalase activities during early development because ROS may be an important

mitogen

(20). In addition, silencing of the CAG promoter-enhancer and/or the progressive

loss of transgene expression as the founder C3H alleles from the B6 (B6C3F1)

hybrid

embryos were diluted out through successive B6 back crosses may have reduced or

modified MCAT expression (21). As a result, the observed MCAT protection against

mitochondrial H2O2 toxicity, oxidative DNA damage, and mitochondrial DNA

deletion

accumulation might have been much higher in the aging cohort mice than in the

mice

that were subsequently tested in biochemical assays. Aging cohort and cardiac

pathology studies were performed on mice two to four generations after

establishing

the transgenic lines. Biochemical tests were done at generation 9 or later, when

the

genetic background was >99% B6 and the mice had been moved to a new facility,

and

the life span extension phenotype appears to be diminished. Nonetheless, these

results support the conclusion that mitochondrial ROS can be an important

limiting

factor in determining mammalian longevity and provide impetus for studies of new

and

combined antioxidant mouse models.

Supporting Online Material

Methods

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

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