Genes, ageing and longevity in humans: Problems, advantages andperspectives

S. SALVIOLI1,2,3, F. OLIVIERI4, F. MARCHEGIANI4, M. CARDELLI4, A. SANTORO1,2,

E. BELLAVISTA1, M. MISHTO1,2, L. INVIDIA1, M. CAPRI1,2, S. VALENSIN1,2, F. SEVINI1,2,

E. CEVENINI1,2, L. CELANI1,2, F. LESCAI1,2,3, E. GONOS5, C. CARUSO6, G. PAOLISSO7,

G. DE BENEDICTIS8, D. MONTI9, & C. FRANCESCHI1,2,3,4

1Department of Experimental Pathology, University of Bologna, via S. Giacomo 12, 40126 Bologna, Italy, 2Centro

Interdipartimentale “L. Galvani”, via S. Giacomo 12, 40126 Bologna, Italy, 3ER-GenTech laboratory, via Saragat 1, 44100

Ferrara, Italy, 4Department of Gerontological Sciences, I.N.R.C.A., via Birarelli 8, 60121 Ancona, Italy, 5National Hellenic

Research Foundation, Institute of Biological Research and Biotechnology, 48 Vas. Constantinou Avenue, Athens 11635, Greece,6Immunosenescence Unit, Department of Pathobiology, University of Palermo, Palermo, Italy, 7Department of Geriatric

Medicine and Metabolic Diseases, University of Naples, Naples, Italy, 8Department of Cell Biology, University of Calabria,

Rende, Italy, and 9Department of Experimental Pathology and Oncology, University of Florence, viale Morgagni 50, 50134

Florence, Italy

Accepted by Dr T. Grune

(Received 15 June 2006; in revised form 19 July 2006)

AbstractMany epidemiological data indicate the presence of a strong familial component of longevity that is largely determined bygenetics, and a number of possible associations between longevity and allelic variants of genes have been described. Abreakthrough strategy to get insight into the genetics of longevity is the study of centenarians, the best example of successfulageing. We review the main results regarding nuclear genes as well as the mitochondrial genome, focusing on the investigationsperformed on Italian centenarians, compared to those from other countries. These studies produced interesting results onmany putative “longevity genes”. Nevertheless, many discrepancies are reported, likely due to the population-specificinteractions between gene pools and environment. New approaches, including large-scale studies using high-throughputtechniques, are urgently needed to overcome the limits of traditional association studies performed on a limited number ofpolymorphisms in order to make substantial progress to disentangle the genetics of a trait as complex as human longevity.

Keywords: Genetic polymorphisms, ageing, longevity, centenarians, association studies, mitochondrial DNA

Introduction

Several years ago, Harman proposed the free radical

theory of ageing [1] that has been last revalued and

esteemed to be one of the candidates to explain the

biology of ageing. This theory purports that ageing is

mainly caused by oxidative stress that develops when

the well-regulated balance between pro-oxidants and

antioxidants gets out of control in favour of the pro-

oxidants. Oxidants are mainly produced as by-

products of normal aerobic metabolism, but also by

phagocytic cells, and during lipid peroxidation. They

induce oxidative damage on several substrates (nucleic

acids, lipids and proteins). The cell is equipped with a

series of antioxidant systems in order to cope with the

ISSN 1071-5762 print/ISSN 1029-2470 online q 2006 Informa UK Ltd.

DOI: 10.1080/10715760600917136

Correspondence: C. Franceschi, Department of Experimental Pathology, University of Bologna, via S. Giacomo 12, 40126 Bologna, Italy.Tel: 39 051 2094743. Fax: 39 051 2094747. E-mail: claudio.franceschi@unibo.it

Free Radical Research, December 2006; 40(12): 1303–1323

inescapable and long lasting production of radical

oxygen species (ROS). According to the Harman’s

theory, it is postulated that genes whose product are

part of such antioxidant systems deeply impact on

ageing and longevity. In this review, we will focus on

genes involved in antioxidant systems as well as in

biochemical pathways affected by or sensitive to

oxidative damage. We will review the available data

and hypotheses mainly regarding association studies

on polymorphisms of such genes and human ageing

and/or longevity, focusing in particular on studies on

exceptionally long-living subjects such as centenar-

ians, who are expected to maximize the possible

contribution of genetics to longevity.

Paraoxonase1

In western countries, the major cause of morbidity and

premature death is atherosclerotic related disease [2].

ROS have been demonstrated to play a role in

inflammatory responses and lipidic homeostasis that

lead eventually to the development of atherosclerosis.

In particular, the peroxidation of low density

lipoproteins (LDL) and the capability of oxidized

lipoprotein-associated proteins to modulate local

inflammatory response are recognized to be a major

cause of atherosclerosis [3,4]. Human serum Para-

oxonase1 (PON1) is an A-esterase with peroxidase-

like activity present on the surface of high density

lipoprotein (HDL) and it is highly involved in the

prevention of LDL oxidation [5,6]. It is to note that,

under oxidative stress conditions, also the HDL

become a substrate for lipidic peroxidation. The

following reduction of HDL constitutes an indepen-

dent risk factor for atherosclerosis and furthermore

their oxidation remarkably increases the risk. It has

been observed that PON1 is able to maintain the anti-

atherogenic activity of HDL. In vitro assays have shown

that PON1 can inhibit LDL lipid peroxidation and

inactivate LDL-derived oxidized phospholipids. This

could potentially reduce the amount of the oxidized

lipids involved in the initiation of atherosclerosis [7].

HDL obtained from PON1-knock-out mice cannot

prevent oxidation of LDL in a co-culture model

simulating the artery wall, and their macrophages

contain more oxidized lipids and have an increased

capability to oxidize LDL. These mice are more prone

to atherosclerosis than their wild-type counterparts,

probably as a consequence of increased oxidative stress

[8]. The apoE knock-out (apoE2/2) mouse is a well

known model of atherosclerotic lesion development.

The double knock-out apoE2/2 /PON12/2 mouse has

a susceptibility to develop atherosclerosis which is

higher than that of the single knock-out apoE2/2

mouse suggesting a role for PON1 in the prevention of

the disease [9]. On the contrary, mice overexpressing

PON1 are protected against atherosclerosis in both a

wild-type and an apoE2/2 background [10].

Because of its involvement in antioxidant defence,

PON1 has been taken into consideration as a

candidate longevity gene in a series of association

studies [11–14]. The paraoxonase gene family

contains at least three members, named PON1,

PON2 and PON3, which are located on chromosome

7q21.3–22.1 [15]. The most studied PON1 gene

polymorphisms are due to an amino acid substitution

at position 192 (Gln-Arg) and at position 55 (Leu-

Met) in the coding region of the gene. Alleles at

Codon 192 (Q and R alleles) and 55 (L and M alleles)

in PON1 locus have been associated with enzymatic

activity and concentration, respectively [16–18]. In

recent years, PON1 gene variants have been widely

investigated, especially for their possible role in the

development or severity of coronary artery disease

(CAD). Since cardiovascular diseases are common

age-related diseases, it is possible that exceptionally

long-living subjects such as centenarians, who escaped

the major age-related diseases including CAD, are

enriched in particular polymorphic variants of PON1

gene. Thus, we analysed the genetic variability at

PON1 locus in such individuals, and performed an

analysis of PON1 192 and 55 polymorphisms in a

sample of 579 young Italian individuals and 308

Italian centenarians. We found that the frequency of R

allele, and consequently of R þ carriers (QR þ RR

individuals), significantly increased from young

people to centenarians, thus conferring a small

survival advantage for subjects carrying R allele

compared to subjects carrying Q allele (OR 1.3, CI

1.04–1.6; p ¼ 0.02). Furthermore, when the PON1

192 polymorphism was analyzed together with the

polymorphism at position 55, we found that, among

R þ subjects, the phenomenon was due to an increase

of people carrying M allele at codon 55 locus [11].

These results were subsequently confirmed in a large

combined group of Italian centenarians and Northern

Ireland octo/nonagenarians and their respective con-

trols for a total of 1479 subjects. In particular, a

significant increase in the frequency of R þ carriers in

Italian centenarians and Northern Ireland octo/nona-

genarian subjects was found in comparison with their

younger control subjects (53.6 vs. 46.1%, p ¼ 0.004)

[12,13] and a logistic regression analysis on the whole

sample highlighted that, when comparing R and Q

alleles, there was a survival advantage for octo/nona-

genarian/centenarian subjects who carried the R allele

(OR 1.3, CI 1.1–1.5; p ¼ 0.007) [12]. In a recent

study, we have found that in people with high risk to

develop CVD such as hypertensive patients, the

frequency of PON1 192 RR genotype is increased in

comparison to non-hypertensive subjects (14.3 vs.

5%, respectively) [19]. This result is apparently in

contrast with our previous studies in which we

reported that subjects with PON1 192 QR genotype

have a higher probability to attain longevity and that

the frequency of PON1 192 RR genotype was around

S. Salvioli et al.1304

9.4% in long-living people [13]. These conflicting

findings can be explained by the hypothesis that

PON1 192 RR genotype has a biphasic, antagonistic

behaviour: it could be considered a risk factor for

hypertension and CVD in the elderly, but a protective

factor for nonagenarians and centenarians. Only few

other studies have taken into consideration the impact

of PON1 gene variation on survival at extremely

advanced age [20–22]. Heijmans and colleagues

performed a population-based study of PON1 192

and 55 polymorphisms among subjects aged 85 years

and over in a cross-sectional and prospective design

[20]. The study revealed that no difference in

genotype distributions between elderly and young

subjects was present and that the risk of all-cause and

cardiovascular mortality was not increased in elderly

subjects with the PON1 LL or RR genotype.

A following paper published by Christiansen et al.

[21] investigated the impact of PON1 gene variability

on mortality using a sample of 1932 Danish

individuals aged 47–93 years. In this study, no

difference in the genotype and haplotype distributions

between different age groups was found.

More recently, Tan et al. [22] presented a novel

survival analysis model that combined population

survival with individual genotype and phenotype

information in assessing the genetic association with

human longevity in cohort studies. In this study, the

Authors considered three SNPs in the human PON1

gene (the two in the coding region, amino acids M55L

and Q192R, and one additional in the promoter

region, C-107T) and they measured the association of

PON1 gene polymorphisms with human survival at

advanced ages in the Danish female 1905 birth cohort

followed from 1998 to 2005. The application of this

novel model to PON1 genotype data detected a

haplotype (T-Q-L) that significantly reduces the risk

of death and thus reveals and stresses the important

role of PON1 genetic variation in promoting human

survival at advanced ages.

Several authors so far have suggested that the

antiatherogenic role of PON1 enzyme is to reduce

oxidative stress during ageing and counteract the

deleterious effects of atherogenesis. Accordingly, the

measure of paraoxonase and arylesterase activities was

investigated in subjects prone to development of

atherosclerosis such as subjects affected by Type 1 or

Type 2 diabetes, familial hypercholesterolaemia, renal

disease and CAD. For all of these age-related

pathologies, a significant decrease of paraoxonase

activity was observed (reviewed in [23]). The increase

of atherosclerosis with age could be due to the

increased susceptibility of LDL and HDL to oxidation

[24,25]. Because of the tight relationship between

PON1 and atherogenesis, some studies have investi-

gated the involvement of PON1 in the ageing process.

In a sample of 129 healthy subjects aged between 22

and 89 years, Seres et al. found that serum PON1

activity significantly decreased with age, while its

arylesterase activity, as well as its concentration and

HDL concentration, remained unchanged with age.

Moreover, the HDL from elderly subjects resulted

more susceptible to oxidative stress than HDL from

young subjects [24]. The authors concluded that the

development of oxidative stress conditions with ageing

could explain, in part, the reduction in PON1 activity.

They also proposed that factors which could

compensate the atherosclerosis could be altered.

Recently, we have investigated the possible role of

PON1 genotypes and phenotypes in aged people free

of overt diseases, focusing our attention especially on

nonagenarians/centenarians. In a sample of 229

participants from 22 to 104 years of age, divided

into young, elderly and nonagenarians/centenarians,

we have evaluated the role played by PON1 genotypes,

paraoxonase actvity, arylesterase activity and paraox-

onase mass concentration in the chance for young and

elderly people to become extremely old. We have

found that paraoxonase activity, arylesterase activity

and paraoxonase specific activity were significantly

lower in nonagenarians/centenarians compared to

elderly and young individuals. On the contrary,

paraoxonase mass concentration was found decreased

in the elderly group and again increased in the

nonagenarian/centenarian group. These parameters

were also analyzed in relation to the PON1 192 and

PON1 55 polymorphisms. This analysis demonstrated

a genetic control for paraoxonase activity that was

maintained throughout life, also in the oldest old

group. This activity showed different mean values

among R and M carriers, having R þ and M 2

carriers the highest paraoxonase activity levels. Using

a multinomial regression logistic model we also

demonstrated that both paraoxonase activity and

R þ and M 2 carriers contributed significantly to the

explanation of the longevity phenotype [14]. On the

whole, we concluded that PON1 activity and genetics

are deeply interconnected throughout life and that

both play a role in human longevity.

Genes for apolipoproteins and proteins related

to lipid metabolism

Beside proteins associated with HDL particles such as

PON1, a large number of studies have considered the

involvement in ageing and longevity of allelic variants

in genes encoding apolipoproteins (APOE, APOB,

APOC1, APOC2, APOC3, APOA1 and APOa),

transfer proteins [microsomal transfer protein

(MTP), cholesteryl ester transfer protein (CETP)]

and transcription factors involved in lipid metabolism

[peroxisome proliferator-activated receptor g

(PPARg)]. One of the most explored loci is that of

the apolipoprotein E (APOE) gene. This locus

appears to be one of the few for which a worldwide

consensus has been reached in terms of negative

Genes, ageing and longevity in humans 1305

association with longevity. Indeed, a higher frequency

of the APOE4 allele in young subjects compared with

old subjects (octogenarians, nonagenarians and

centenarians) has been observed, concluding that the

presence of the APOE4 allele is associated with

decreased lifespan, likely because of an increased

incidence of cardiovascular as well as neurodegenera-

tive diseases [26,27]. Much less is known about other

APO genes and ageing and longevity (reviewed by

Ordovas and Mooser) [28]. In particular, we

investigated common APOB polymorphisms in a

sample of 143 centenarians from southern Italy and a

control sample of 158 individuals [29]. We found that

the frequency of 30APOB-VNTR alleles with fewer

than 35 repeats (small, S) was significantly lower in

centenarians than in controls. A further analysis of

seven different age cohorts (697 individuals from 10 to

109 years old) revealed that there is an age-related

change in the 30APOB-VNTR SS genotype. In

particular, a convex trajectory of the frequency of SS

homozygotes was found. The frequency of SS in the

genotype pool increased from the group aged 10–19

years (3.06 ^ 1.74%) to that aged 40–49 years

(8.51 ^ 4.07%), then it declined reaching the

minimum value in centenarians (1.58 ^ 0.90%)

[30]. This study was repeated in a sample of Danish

people, and neither genotype nor allele frequencies

differed between centenarians and 20–64-year-old

subjects [31]. Furthermore, the demographic-genetic

approach revealed in Danes a significant gene-sex

interaction relevant to Long alleles (more than 37

repeats). The different findings in Denmark and Italy

suggest that these associations between APOB

polymorphism and longevity are population-specific,

and heavily affected by the population-specific genetic

and environmental history. In further studies, we

found that the S alleles lower the average values of

serum Total Cholesterol and LDL-Cholesterol, while

the alleles M and L have no significant effect on the

lipidemic phenotype [32]. Thus, the S alleles would be

advantageous in adults by protecting from coronary

artheriosclerosis and related diseases, while dangerous

in the elderly, probably by lowering serum cholesterol

below a critical threshold. This could explain the

convex frequency trajectory of SS genotypes pre-

viously observed.

Other studies performed on apolipoprotein A1

(APOA1), apolipoprotein C3 (APOC3) and apolipo-

protein A4 (APOA4) that are tandemly organized

within a short region on chromosome 11q23–q24,

have revealed a MspI-RFLP in APOA1 (275 nt from

the transcription starting site) that modulates the

serum level of LDL. In particular, the A allele reduces

the levels of serum LDL-cholesterol, while the allele P

increases them. Surprisingly, the P allele is more

represented in male centenarians than in 46–80 year

old males [33]. This unexpected finding has been

explained by suggesting that this allele can be risky in

middle-age but protective at very advanced age, and

that the effect of a mutation may depend on the age-

related physiological scenario in which the mutation

acts.

In the case of CETP gene, two different studies have

highlighted the importance of the populations

considered. This gene is characterized by the presence

of a polymorphism at codon 405 (I405V), and the VV

genotype has been reported to be associated with

longevity in Ashkenazi Jewish [34], but not in Italian

population [35]. This seems to indicate that longevity

is not linked to a specific polymorphism of CETP, but

rather that this polymorphism likely interacts with

other factors (gene pool, environment, chance) which

differ from population to population, to determine or

not the “longevity” phenotype.

ApoJ

ApoJ was firstly identified in ram rete testis fluid in

1983 as a secreted glycoprotein enhancing cell

aggregation in vitro (named thus as clusterin) [36].

In humans, it was firstly purified from serum and the

cloned gene was named as CLI (complement cytolysis

inhibitor) [37], SP-40,40 (secreted protein 40,40)

[38] or Apolipoprotein J (ApoJ) [39] due to

similarities with other known apolipoproteins. A

comparison of the ApoJ protein sequences among

mammalian species reveals a high degree of conserva-

tion of ,70–85%, while attempts to clone its

homologues in the worm or the fly by using specific

primers spanning the conserved regions of the gene

appeared negative (our unpublished results). These

two observations along with its wide distribution in

animal tissues and the absence of studies highlighting

functional ApoJ polymorphisms in humans [40,41]

suggest that the protein has evolved in vertebrates to

accomplish a function of fundamental biological

importance.

ApoJ protein is constitutively secreted by a number

of cell types including epithelial and neuronal cells,

while in cells featuring a regulated exocytotic pathway

its secretion depends on the appropriate exogenous

stimulus (reviewed in Trougakos and Gonos 2002)

[42]. However, despite the systematic and combined

effort of several groups, ApoJ function has remained

elusive, the main cause being the intriguingly distinct

and usually opposing functions proposed in an array of

various cell types and tissues. We have developed a

clonal system of rat embryo fibroblast conditionally

immortalized cell lines which undergo senescence

upon SV40 Tantigen inactivation [43,44] and we have

cloned Clusterin/Apolipoprotein J as a senescence

associated gene [45]. We have shown that CLU/ApoJ

is up-regulated during replicative senescence and

stress-induced-premature senescence in various mam-

malian tissues [45,46,47]. Moreover, we have found

that CLU/ApoJ accumulates in human serum during

S. Salvioli et al.1306

in vivo ageing as well as in several age-related diseases,

such diabetes type II, myocardial infarction or

coronary heart [48]. We have also investigated the

effects of CLU/ApoJ on cellular growth and survival

by taking advantage of three human osteosarcoma

(OS) cell lines, since these cells express distinct

endogenous CLU/ApoJ levels and, moreover, they

have distinct and characterized genetic backgrounds.

Following CLU/ApoJ forced over-expression by

means of an artificial transgene we found that

extracellular CLU/ApoJ inhibits cell death in all

three OS cell lines assayed. Interestingly, intracellular

CLU/ApoJ has different effects on cellular prolifer-

ation and survival in these cell lines. Transgenic

KHOS cell lines adapted to moderate intracellular

CLU/ApoJ levels and became resistant to both

genotoxic and oxidative stress, whereas transgenic

SaOS and U2OS cell lines adapted to high intracellu-

lar CLU/ApoJ amounts and were sensitive to the same

cytotoxic agents [49]. To conclude about the primary

CLU/ApoJ function we used small interfering RNAs

(siRNAs) to silence CLU/ApoJ gene expression. Our

data demonstrate that CLU/ApoJ knock down

induces significant reduction of cellular growth,

higher rates of spontaneous endogenous apoptosis

and reduced plating efficiency. These effects are

enhanced in those cell lines expressing high endogen-

ous CLU/ApoJ levels. Moreover, CLU/ApoJ knock

down OS and prostate cancer cells were dramatically

sensitized to various apoptosis-inducing agents. By

assaying the expression levels of several proteins

involved in regulating apoptosis we found that

CLU/ApoJ silencing results in the down-regulation

of the anti-apoptotic molecule bcl-2. In U2OS cells,

which have functional p53, sCLU/ApoJ knock down

apart from bcl-2 down-regulation is also accompanied

by p53 accumulation and up-regulation of its down-

stream pro-apoptotic effector bax and the cyclin-

dependent kinase inhibitor, p21 [50]. Overall, our

results reveal that CLU/ApoJ is a central molecule in

cell homeostasis that exerts a potent cytoprotective

function.

As far as MTP is regarded, it recently attracted great

attention after the finding that a region of chromo-

some 4 appeared to be linked to exceptional longevity

in US Caucasians [51]. In this region (4q25),

containing about 50 genes, a SNPs analysis was

performed and indicated a two-SNPs haplotype of

microsomal transfer protein (MTP) as associated to

longevity [52]. However, further studies were unable

to reproduce these findings on 4q25 and MTP, and a

meta-analysis confirmed this lack of association [53].

IGF-1 and IGF-1 pathway

Hyperglycemia can increase oxidative stress through

a mechanism that promotes the formation of

advanced glycosylation end-products (AGEs) and

PKC activation [54]. Once formed, AGE-protein

adducts are stable and virtually irreversible. More-

over, during oxidative stress, AGEs formation

increases substantially [55]. The glycosilation of

LDL represents the cause leading for the modifi-

cation of the putative LDL receptor binding domain.

These glycated LDL, that are no more recognized by

LDL receptors, become the substrate for macro-

phages that stimulate foam cells formation and

promote atherosclerosis. Monocyte-macrophage

interaction with AGEs also results in the production

of mediators such as interleukin-1, tumor necrosis

factor-a, platelet-derived growth factor, and insulin

growth factor-I [56,57,58], which have a pivotal role

in the pathogenesis of atherosclerosis [59].

As IGF-1 is an important regulator of the insulin

action by stimulating the glucose transport, Paolisso

et al. have investigated this molecule in centenarians

and they have found a decrease in IGF-1 plasma levels

[60,61] together with a well preserved insulin action,

thus indicating that insulin responsiveness is funda-

mental to reach the extreme limits of human life span

[62,63]. In contrast, several papers reported low levels

of IGF-1 to be associated with cardiovascular

pathologies [64,65,66,67]. Also, we have found that

in aged people high levels of this hormone are

beneficial especially for the physical performance and

the maintenance of muscle strength [68]. Apparently,

these last results do not fit with those obtained from

centenarians showing that reduced IGF-1 is associ-

ated with longevity [61]. Indeed, this apparent

paradox could be explained with the evolutionary

theory of ageing which assumes that expression of

particular genes could be beneficial early in life, but

could become detrimental with age [69]. In this case,

high levels of IGF-1 can be helpful to avoid disability

and frailty in the elderly, but later in life (after the age

85), low levels of IGF-1 could promote survival by

avoiding cancer development and growth.

Recent evidences from evolutionary biology show

that, in a variety of model systems from invertebrates

to mammals (yeast, worms, fruit flies and rodents),

mutations in genes that share similarities with the

human genes involved in the insulin/IGF-1 signal

response pathway are responsible for an extension of

life span [70–74]. On the basis of this consideration

and on the basis of the findings that insulin

responsiveness impacts on human longevity, we tested

the hypothesis that human loci which share similarities

with genes that regulate the insulin pathway in

invertebrates could affect human longevity [61]. In

particular, we investigated polymorphisms at IGF-1R

(insulin-like growth factor type 1 receptor), PI3KCB

(phosphoinositide 3-kinase), IRS-1 (insulin receptor

substrate-1) and FOXO1A genes as well as their

possible effects on IGF-1 plasma levels. These genes

represent key molecules of the insulin/IGF-1 pathway

and their sequential activation determines a cascade

Genes, ageing and longevity in humans 1307

activation signal. This study was performed on 496

Caucasian healthy subjects subcategorized into two

groups by splitting the sample at the age of 85 years.

The major findings emerging from the study were that

free IGF-1 plasma levels displayed an age-related

decrease and that these levels were affected by the

polymorphisms at IGF-1R and PI3KCB. The poly-

morphisms considered in the study were the following:

a G to A transition at nucleotide 3174 in exon 16

of the IGF-1R and a T–C transition located

359 bp upstream from the starting codon of

PI3KCB. Again, subjects carrying at least one A allele

at the IGF-1R locus have lower plasma IGF-1 levels

than the rest of the population and these subjects are

more represented in the long-lived individuals in

respect to younger subjects. Accordingly, Holzenber-

ger and colleagues have demonstrated that the

inactivation of IGF-1R prolongs the life span and

increases the resistance to oxidative stress in mice [75].

When the polymorphisms of IGF-1R and PI3KCB

were considered together, another interesting result

emerged, i.e. the proportion of IGF-1R/PI3KCB-

Aþ/Tþ carriers is significantly increased among long-

lived individuals. Intriguingly, in animal models like C.

elegans and M. musculus the down-regulation of IGF-1

pathway is associated with an extension of life span

[76–78], whereas high levels of IGF-1 are associated

with a shortened life span [78]. Moreover, caloric

restriction reduces plasma free IGF-1 levels [80,81]

and cannot be excluded that the peculiar nutritional

regimen of centenarians could resemble that of

subjects who have undergone a moderate caloric

restriction. To date, this study represents the first

indication that also in humans, polymorphisms of

genes regulating the IGF-1/insulin pathway affect

longevity, contributing to the hypothesis that the

impact of the IGF-1/insulin pathway on longevity is a

property that has been evolutionarily conserved

throughout the animal kingdom. Further studies

performed in European and Japanese populations

give results that are in agreement with those previously

reported and add further indications that IGF-1

pathway is involved in human longevity [82,83].

Taking into account that high levels of circulating

IGF-1 are positively associated with muscle strength

in the elderly [68], we can hypothesize that a

phenomenon of antagonist pleiotropy occurs in ageing

and longevity regarding IGF-1. According to such an

interpretation, high level of IGF-1 could be beneficial

in the elderly as a protective factor toward sarcopenia

but the price to pay would be a higher risk of cancer

incidence. Conversely, in extreme ages and in

centenarians, low levels of IGF-1 would give a survival

advantage (low cancer incidence) but in this case the

price to pay would be physical frailty and loss of

muscle mass and strength.

Furthermore, it is interesting to note that YTHDF2,

a gene recently found to be associated with longevity

in a study conducted on 412 Italian participants,

including 137 centenarians, and expressed in a wide

variety of organs but mainly in testis, placenta and

pancreas [84], has been reported to be regulated by

high glucose concentration (see GenBank accession

no. AF192968 [85]). Thus, it could be worthwhile to

test the hypothesis of a possible role of YTHDF2 in

carbohydrate homeostasis and in the insulin/IGF-1

signaling pathway.

Genes for cytokines

ROS are signaling molecules for a wide variety of

inflammatory stimuli and serve as signaling messen-

gers for the evolution and perpetuation of the

inflammatory process, but relatively little is known

about the relationship between oxidative stress and

production of inflammatory cytokines [86,87]. Oxi-

dative stress and pro-inflammatory cytokines trigger

common signal transduction pathways that lead to

amplification of the inflammatory cascade, mainly

through activation of mitogen-activated protein

kinases (MAPK) and nuclear factor kappaB (NF-

kB). ROS could activate ubiquitous transcription

factors, such as NF-kB and AP-1, up-regulating

monocyte chemotactic molecules expression, respon-

sible for the initial inflammatory events and up-

regulating the cytokines gene expression [88,89].

Cytokines, in turn, could cause the formation of nitric

oxide (NO) that combines with superoxide to form the

potent oxidant peroxynitrite, thus generating a vicious

circle [90]. As an example, it was reported that

oxidative stress increases the production of tumor

necrosis factor alpha (TNF-a), a potent pro-inflam-

matory cytokine; TNF-a impairs the production of

glutathione (GSH), an intracellular antioxidant,

leading to a pathogenic “loop” [89]. Moreover, ROS

can modulate apoptosis in a variety of cell types,

including human inflammatory cells [91]. From

invertebrate to humans the cellular response to a

variety of stressors involves the up-regulation of

mediators such as ROS and pro-inflammatory

cytokines, such as interleukin-1 (IL-1), interleukin-6

(IL-6) and TNF-a and this type of cellular response

appears to be highly maintained during the evolution

[69]. On this basis, the stress response and inflam-

mation could be considered an integrated evolutionary

conserved defence network, and oxidative stress could

modulate immune response and inflammation, balan-

cing the pro-inflammatory/anti-inflammatory cyto-

kines production [92,13]. In particular, IL-6, TNF-a

and IL-1 are pro-inflammatory pleiotropic cytokines

capable of regulating proliferation, differentiation and

activity of a variety of cell types [93,94]. TGF-b1 is a

multifunctional cytokine that regulates cell prolifer-

ation, differentiation and migration, and it was

considered an anti-inflammatory molecule [95].

Interleukin-10 (IL-10) is a powerful cytokine that

S. Salvioli et al.1308

inhibits lymphocyte replication and secretion of

inflammatory cytokines [96]. Ageing seems to be

associated with a pro-inflammatory shift in cytokine

expression profile, as suggested by the increased

circulating levels of pro-inflammatory cytokines in

healthy elderly humans, as a consequence of age-

related alterations in the endocrine system, cellular

metabolism, and redox regulation [94,97]. In order to

test whether the model of inflamm-ageing does apply

not only to human ageing but also to human longevity,

we performed a series of studies on such cytokines in

centenarians. Data obtained in these studies suggested

that longevity is associated with the capability of cells

to cope with a variety of stressors, including oxidative

stress, modulating the inflammatory response [13].

Moreover, it has been demonstrated that centenarians

have lower oxidative stress than subjects 70–99 years

old, suggesting that a reduction of protective mechan-

isms against ROS during ageing could induce a greater

toll of oxidative damage, deleterious for longevity

[98,99]. In summary, the capability to maintain a

lower production of pro-inflammatory cytokines and

the capability to be well protected from oxidative stress

appears to be favourable for reaching the extreme limit

of human life span in good health conditions and could

be genetically controlled [69,99].

Moreover, recent data suggest that increased levels

of pro-inflammatory cytokines, such as IL-6, IL-1 and

TNF-alpha and a decreased production of anti-

inflammatory cytokines such as TGF-b1 and IL-10

during ageing, represents the biological background

favouring the susceptibility to the major age-related

diseases, such as cancer, cardiovascular disease,

insulin resistance, type 2 diabetes and Alzheimer’s

disease and can also be strong short-term predictors of

mortality associated with age-related chronic diseases

[100–104,69,13].

On the whole, data obtained from genetic analysis

of candidate genes in centenarians and in sample of

patients suffering by major age-related diseases,

confirmed the hypothesis that genes related to

inflammation are implicated in human longevity and

in the susceptibility to many age-related diseases. A

great amount of papers reported a positive association

between some polymorphic markers of IL-6 gene and

longevity, and the capability of producing low levels of

IL-6 throughout life span appears to be beneficial for

longevity [13,94,105,106]. Nevertheless, it appears

that IL-6 polymorphism does not affect life expect-

ancy neither in the Sardinian population, nor in

people from Southern Italy, suggesting that the effect

of IL-6 polymorphism on longevity might be

population-specific and dependent on gene-environ-

ment interactions [107,108].

Particular attention was devoted to two anti-

inflammatory cytokines, i.e. TGF-b1 and IL-10. We

reported an association between TGF-b1 poly-

morphic markers and human longevity [109]. In this

study, the plasma levels of biologically active TGF-b1

were significantly increased in the elderly group,

independently from TGF-b1 genotypes.

The relation of some IL-10 polymorphisms, such as

T-819C, A-592C and A-1082G promoter SNPs, with

age was evaluated in different population samples,

suggesting that IL-10 may be a promising candidate

ageing-related gene [107,110–114]. In particular, IL-

10 and TNF-a have complex and opposing roles, and

an autoregulatory loop appears to exist [115].

Furthermore, their synergistic role in the control of

immune-inflammatory responses has been hypoth-

esised, and the interaction between TNF-a -308

and -1082 IL-10 polymorphisms promoter was

observed [112].

Instead, no particular polymorphism in the IL-1

gene cluster was reported to confer a survival advantage

in the last decades of life, and the observed age-related

increase in IL-1Ra plasma levels seems not to be

genetically controlled [93]. However, the studies of IL-

6 and TNF-a suggest that the levels of these pro-

inflammatory molecules are, to a large extent,

genetically determined, even in elderly individuals,

and therefore, it is important to elucidate the genetic

factors involved in the regulation of these proteins,

especially in age-related diseases [116]. Recently, many

associations between a great number of polymorphic

markers of cytokine genes and the incidence and/or the

prognosis of age-related diseases, such as cardiovas-

cular disease, type 2 diabetes, cancer and Alzheimer’s

disease have been reported [117–126].

An additive effect of variants of genes of cytokines

(IL-6 G174C) and of other genes involved in

metabolic pathways such as PPARg (Pro/Ala) on the

total variation of obesity-related factors (BMI, insulin

resistance, triglyceride levels and so on) has been

recently reported [126].

In summary, a great number of genetic markers

related to a pro-inflammatory phenotype are associ-

ated with the major age-related diseases, and have

been found to be under-represented in centenarians,

while genetic variants associated with an anti-

inflammatory activity have been found to be more

represented in centenarians, confirming that the

balancing during ageing between pro and anti-

inflammatory mechanisms is largely under genetic

control. Cytokines could be thus an important trait

that links mitochondria and oxidative stress on one

side and inflammation and ageing on the other side

[127].

SOD and PARP genes

Studies performed on animal models [128] and on

octo/nonagenarian subjects [129] evidenced that the

level of nonenzymatic antioxidants declines during

senescence, whereas systemic oxidant load tends to

increase in older adults [129]. For these reasons, the

Genes, ageing and longevity in humans 1309

enzymes involved in the inactivation of ROS

(“enzymatic antioxidants”), such as superoxide dis-

mutase (SOD), and enzymes involved in the repair of

the DNA injuries caused by oxidative damage or other

genotoxic stress, such as PARP (poly (ADP-ribose)

polymerase), are thought to play a key role in

controlling the rate of the ageing process, and have

been extensively studied in relation to longevity.

SOD enzymes catalyze the breakdown of super-

oxide into hydrogen peroxide and water and are

therefore central regulators of ROS levels [130].

Different SODs have evolved to inactivate both

intracellular and extracellular superoxide radicals

produced as by-products of metabolic oxidation.

Mammalian cells, in particular, contain a manganese

superoxide dismutase (Mn SOD/SOD2) localized

within the mitochondrial matrix [131], a copper- and

zinc-containing superoxide dismutase (Cu/Zn SOD/

SOD1) localized predominantly in cytoplasmic and

nuclear compartments [132], and a copper- and zinc-

containing SOD predominantly found in extracellular

compartments (EC SOD/SOD3) [133].

The Cu/ZnSOD and MnSOD overexpression has

been found to increase life-span in Drosophila

[134,135], while null mutations in these genes greatly

decrease viability and life-span in the same species

[136,137], and only MnSOD mutation has a severe

effect in mice [138].

The role of enzymatic antioxidants, SOD in

particular, in human ageing has been addressed by

different studies. The enzyme activities of SOD

(Cu/Zn-SOD), glutathione peroxidase, catalase and

glutathione reductase (GR) in erythrocytes was

analysed in a cross-sectional study [139] conducted

on 41 Danish centenarians and on 52 control subjects

(aged between 60 and 79 years). Cu/Zn-SOD activity

was found to be decreased in centenarians (particu-

larly in centenarians with the lowest cognitive and

physical functional capacity), and the result was

interpreted as a reduced demand for the enzyme at

lower metabolic rate and oxygen consumption

(whereas subjects with high GR activity were more

frequent than expected in centenarians).

In a different study [140], plasma levels of non-

enzymatic antioxidants (vitamin C, uric acid, vitamin

E, vitamin A, carotenoids, total thiol groups), and the

activity of enzymatic antioxidants were assayed in 32

healthy centenarians, 17 elderly subjects aged 80–99

years, 34 elderly subjects aged 60–79 years, and 24

adults aged less than 60 years. In particular, plasma

SOD, plasma glutathione peroxidase (GPX), and red

blood cell (RBC) SOD activities were measured.

A constant decrease with age of the nonenzymatic

antioxidants and a concomitant increase of the

enzymatic antioxidant activities were observed in the

three younger age classes, but not in centenarians,

who on the contrary showed the highest levels of

vitamins A and E, and activities of both RBC and

plasma SOD which were not increased or decreased

with respect to each other age class. From the results

of the above cited studies, it can be hypothesized that

normal ageing is probably characterized by a pro-

oxidant status, accompanied by a decrease of non-

enzymatic antioxidants and by a compensatory

increased activity of enzymatic antioxidants, whereas

healthy centenarians show a completely different

profile, in which high levels of vitamin A and vitamin

E are associated with normal or reduced activities of

enzymatic antyoxidants. A reduced cellular suscepti-

bility to free radicals attack in centenarians was also

suggested in studies [141–143] which analysed the

modifications of the plasma membrane composition in

centenarians. Altogether, these studies suggested that

the erythrocyte membranes from centenarians have

some distinct features in comparison with elderly

subjects, that might act in a protective way against

injuries [144].

Other genes whose products have an antioxidant

action include GSTT1. GSTT1 gene is a member of

the Glutathione-S-Transferase superfamily, involved

not only in protection against ROS, but also against

xenobiotics and exogenous carcinogens [145]. We

studied polymorphisms in CYP1A1, GSTT1 and

GSTM1 genes and found that centenarians have an

increased proportion of a deletion of GSTT1 gene

with respect to younger controls [146]. Accordingly,

the presence of one or two alleles of such a gene has

been observed to be correlated with an increase in

mortality [147,148].

Oxidative stress results in many damaging cellular

effects; among them, the effects on DNA are the most

harmful to the cell function. Oxidative stress induces

DNA single strand breaks and leads to the activation

of the DNA repair enzyme poly (ADP-ribose)

polymerase (PARP). Poly (ADP-ribosyl)ation is a

post-translational modification of proteins which is

catalyzed mostly by PARP-1 [149,150], an abundant

nuclear protein that binds to a DNA single-strand

break (SSB) and catalyses the formation of poly

(ADP-ribose) polymers on itself and other acceptor

proteins (Lindahl et al. 1995). Poly (ADP-ribose)

polymers formation is suggested to be important to

protect DNA from breaks and to attract DNA repair

proteins to the site of damage [151,152]. PARP-1 is

also involved in base excision repair (BER) [153]. Poly

(ADP-ribosyl)ation capacity in mononuclear blood

cells positively correlates with the species-specific

longevity of 13 mammalian species [154], and the

observed differences in the catalytic capacity may be

explained by sequence divergence in the PARP-1

protein [155]. Similarly, poly (ADP-ribosyl)ation

capacity of human limphoblastoid cells correlates

with longevity of the donors [156]. PARP-1 activity

appears to finely regulate the rate of genomic

instability events, caused by DNA-damaging agents

[157,150]. However, excessive activation of PARP-1

S. Salvioli et al.1310

can lead to cell death (suicide hypothesis), and it is

probably implicated in a variety of insults, including

cerebral and cardiac ischemia, 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine-induced Parkinsonism,

traumatic spinal cord injury and streptozotocin-

induced diabetes [158], and has been hypothesised

to be the central mediator in all the mechanisms by

which hyperglycaemia induces diabetic vascular

dysfunction [159]. It has been hypothesised that

PARPs might regulate cell fate as essential modulators

of death and survival transcriptional programs [158].

Recent results suggest that PARP-1 activity might

be impaired in the elderly. In particular, the results of

a recent study [160] suggest that increasing age is

associated with an impaired capacity of PARP-1 in

base excision DNA-repair, which in turn is suggested

to be due to the reduced bioavailability of zinc ions

observed in the elderly. Interestingly, the PARP-1

impairment seems to be enhanced in old infected

patients (acute and remission phases of broncho-

pneumonia infection), but reversed in centenarians,

where good zinc ion bioavailability and higher

capacity of PARP-1 in base excision DNA-repair

were observed [160].

The influence of the genetic variability of PARP and

SOD2 genes on the inter-individual variability of

human longevity has been tested in an association

study [161] in which a (gt)n repeat polymorphism in

exon 1 of the PARP-1 gene, and a T/C substitution

(which changes Valine16 to Alanine16) in the

mitochondrial targeting domain of the SOD2 gene

were analysed in two age groups respectively

composed of centenarians (109 from northern Italy

and 87 from southern Italy) and of younger control

subjects (from 10 to 85 years, 119 from northern Italy

and 239 from southern Italy) matched for sex and

geographic area. The results of this study suggested

that these polymorphisms do not affect inter-

individual variability in life expectancy.

Two other studies obtained negative results about

the possibile association of PARP-1 gene polymorph-

isms and human longevity. In the first [156], 437

DNA samples (239 centenarians and 198 controls)

were analysed for a polymorphic dinucleotide repeat

located in the promoter region of PARP-1, but no

significant enrichment of any of the alleles or

genotypes identified among centenarians or controls

was observed. In the same study, maximal oligonu-

cleotide-stimulated PARP-1 activity in lymphoblas-

toid cell lines was found to be significantly higher in

centenarians (n ¼ 49) than in younger controls

(n ¼ 51). The second study [162] was performed by

analysing two single nucleotide polymorphisms in the

PARP-1 gene in 648 DNA samples from a French

population (324 centenarians and 324 controls) and

even in this case no significant difference in genotype

frequency was found. Furthermore, none of the

genotype combinations at any polymorphic site

studied could be related to a high or low level of

poly(ADP-ribosyl)ation capacity, suggesting that the

longevity-related differences in the poly(ADP-ribosy-

l)ation capacity of human lymphoblastoid cell lines

cannot be explained by the genetic polymorphisms in

the PARP-1 coding sequence studied until now.

Nevertheless, experimental evidence seems to indicate

a link between poly(ADP ribosyl)ation and longevity

in mammals [163].

p53

The p53 tumor suppressor gene (TP53, Trp53) is a

pivotal gene controlling DNA repair, cell cycle,

apoptosis and cell senescence. Due to its extensive

involvement in biological phenomena so crucial for

cell life and death, it is conceivable that p53 gene can

be involved also in ageing and longevity. Accordingly,

it has been described that mice with increased p53

activity display an increased resistance to cancer, a

premature ageing phenotype and a reduced longevity

[164]. The complete deficiency of p53 leads to a

dramatic shortage of life expentancy due in this case

to increased cancer incidence in both mice [165] and

humans (Li-Fraumeni patients, [166]). Thus p53 has

to be considered a longevity-assurance gene in the

sense that it avoids premature death by protecting

from cancers in young age, and at the same time it is

a gene involved in ageing, since it appears that its

expression, while protecting from cancer, induces

ageing of the organism. On the basis of these results,

we looked at possible involvement of p53 gene

polymorphisms in ageing and longevity. In fact, it is

known that p53 gene harbours a number of

polymorphisms, the most studied of which is the

G-to-C transversion at codon 72 in the hexon 4. This

mutation leads to an aminoacidic change at position

72 in the protein from Arginine to Proline [167].

This mutation lies in the proline-rich domain and

confers a number of biochemical and biological

differences to the two resulting p53 isoforms [168]

and in particular as far as apoptosis, the Arg allele is

endowed with a greater apoptotic potential with

respect to Pro one [169–172]. By contrast, the Pro

allele appears to be much more efficient in cell cycle

arrest and cell senescence [173,174]. Interestingly,

these differences appeared more overt when cells

were obtained from aged subjects and centenarians

[173,174]. In the Caucasian population, the fre-

quency of the Arg allele is roughly 70%, while that of

the Pro allele is around 30% [175]. We wondered if

this polymorphism could impinge upon longevity,

and to test this hypothesis, we studied a total of 1086

Italian subjects of different ages (from young to

elderly people, including 307 centenarians). Besides

this G-to-C transversion, we also studied two

additional polymorphisms of p53 gene, that is the

16 bp Insertion/Deletion (Ins/Del) in Intron 3 and

Genes, ageing and longevity in humans 1311

the C to T transition in Intron 6, and found no

difference in the frequency distribution of these

polymorphisms in the age classes considered. We

concluded that p53 polymorphisms do not have an

effect on longevity [176–178]. In contrast, van

Heemst et al. reported that in a study of 1226 people

aged 85 years and over, Pro/Pro homozygous

subjects appear to have an increased survival despite

a 2.54 fold increased proportional mortality from

cancer [179]. The conclusion of this study was that

the survival advantage brought by Pro allele over-

whelms the increased risk of cancer. The discrepancy

with other data [176–178] nevertheless casts some

doubts on this general conclusion. The authors

themselves acknowledge that the study could be

underpowered and should be repeated in other

populations. In fact, Pro/Pro genotype is the more

rare and thus the number of subjects needed to have

a strong statistical power is likely not affordable

by a single study. Moreover, it is possible or even

likely that the effects observed by all these studies

[176–179] are population-specific. In our opinion

this is the reason why these results are apparently

in contrast.

On the basis of our preliminary results, we are

tempted to speculate that p53 codon 72 polymorph-

ism impinges upon not only cancer, but also other

common age-related diseases, and the algebraic sum

of the positive and negative effects on these different

pathologies gets to zero, at least on the Italian

population, so that in studies performed on Italian

subjects, the frequency distribution of p53 codon 72

genotypes does not change over age, but on the other

side it may vary in other (ethnically different)

populations in which the algebraic sum of these

effects is different. To conclude, p53 codon 72

polymorphism appears to impinge upon longevity,

and this effect might be population-specific, likely

depending on the interactions with both gene pools

and environmental conditions.

mtDNA in human longevity

Mitochondria are semi-autonomously functioning

organelles, harboring some life important cellular

process, such as the lipidic metabolism, the citric acid

cycle and the respiratory chain coupled to oxidative

phosphorylation (OXPHOS), that generates approxi-

mately 90% of cellular adenosine triphosphate (ATP)

[180]. Mitochondria are also provided with a small

genome, the mitochondrial DNA (mtDNA), that is

the only repository of genetic information outside the

nucleus. Human mtDNA is a 16,569 bp double-

stranded circular genome containing 37 genes encod-

ing 13 proteins (all of which are essential components

of the respiratory chain), 22 tRNAs and 2 rRNAs; it is

present in hundreds to thousands of copies per cell,

and transmitted as a non-recombinant unit only

through maternal inheritance [181,182]. The

mutation rate of mtDNA is about ten times higher

than nuclear genome since mtDNA is much more

exposed to ROS-induced damage than the nuclear

genome, because of the high vicinity to the oxidative

environment present in mitochondria [183]. More-

over, mtDNA has no histone-like protection, and less

efficient DNA repair systems, thus causing mtDNA to

accumulate various mutations in mitotic [184] and

post-mitotic tissues [185,186]. Nonetheless, it is

emerging that mtDNA is packaged into protein-

DNA complexes, called mitochondrial nucleoids (mt

nucleoids) that look like globular foci in human cells

[187]. Among the proteins discovered to interact with

mtDNA in mt nucleoids, some have known functions

related to mtDNA transaction, while others have not.

In mammals, among the proteins present in mtDNA

nucleoids there are factors involved in mtDNA

replication and transcription, such as TFAM, Twin-

kle, mtSSB, polymerase g, BRCA1, PRSS15 (LON),

and others involved in TCA cycle, such as aconitase

(for a review, see [188] and [189]). A possible role in

the protection of mtDNA from ROS damage can be

hypothesised for such proteins. The accumulation of

mutations in mtDNA, together with strong evidence

of a decline in the OXPHOS capacity of mitochondria

occurring with age in a variety of tissues such as

skeletal muscles, heart, brain, liver and other organs

[190,191] has given support to the “mitochondrial

theory of ageing” [192], an extension of the original

oxygen free radical theory of ageing proposed by

Harman [1]. It poses that accumulation of mutations

in mtDNA and consequent mitochondrial dysfunc-

tion are the major contributors to ageing and age-

related neurodegenerative diseases. In 2002, Lin and

his group found that mtDNA obtained from brain of

elderly subjects had a higher aggregate volume of

mutations than that obtained from brain of younger

subjects [193]. However, according to some authors,

specific mtDNA point mutations responsible for the

defects seen in ageing and age-related diseases have

yet to be identified [194].

Recently, somatic mutations in the control region

(CR) of the mtDNA have been associated with ageing

[195]. The A189G and T408A CR mutations

accumulate with age in skeletal muscle [196], while

a C150T mutation is more represented in white blood

cells and dermal fibroblasts of centenarians with

respect to young and old people in the Italian

population [197]. In any case, the correlation between

accumulation of somatic mutations, ageing and

longevity is still debated [198]. Recently, a murine

model has been set up in order to understand whether

the accumulation of mtDNA mutations is a cause or

rather an effect of ageing. In such a model, animals

expressing a proofreading-deficient version of mtDNA

polymerase g accumulate mtDNA mutations and

display features of accelerated ageing [199,200].

S. Salvioli et al.1312

Surprisingly, no increased oxidative stress occurs in

such mtDNA mutator mice [201]. On the basis of

these results, it can be assumed that the capability to

maintain mtDNA integrity and to avoid the accumu-

lation of mtDNA mutations is likely a feature of

longevity. However, this model does not really mimic

natural ageing, but it is rather to be considered as a

sort of genetic “disease”, since the mutator mice

accumulate mutations mainly in rapidly renewing

tissues rather than in post-mitotic ones, as it could be

expected, and the authors suggest that the resulting

phenotype could be due to the exhaustion of stem

cells, a phenomenon still not proven in physiological

ageing, where instead “old” microenvironment plays a

critical role for the function of cells (for a discussion

see Santoro et al. [198]).

Interestingly, recent findings by Sato et al. [202]

propose that mitochondrial complementation

phenomena do exist. They suggest that the mitochon-

dria can share mtDNA molecules and their products

as well, in order to avoid the expression of pathogenic

mutated forms of mtDNA. It is supposed that free

mixing of mitochondrial genetic components through-

out the mitochondrial network would protect mam-

malian mitochondria from direct expression of

respiratory defects caused by accumulated mtDNA

mutations. This innovative finding, if confirmed,

would challenge the mitochondrial theory of ageing

and lead to a re-consideration of the role of

mitochondria in ageing and longevity.

Beside somatic mutations, mtDNA is characterized

also by an inherited or inter-individual sequence

variability. Studies on mtDNA variations in human

populations have identified peculiar mutations that

were assumed to be neutral, thus avoiding elimination

by selection and becoming prevalent through genetic

drift. Such an assumption explains how mutations

which occurred thousands of years ago are nowadays

present at high frequency, and create groups of related

mtDNA haplotypes (called haplogroups), sharing a

specific set of stable polymorphic restriction sites

[203–205].

However, recent findings regarding the population-

and continent-specific distribution of such hap-

logroups, have suggested that such variants may not

be as neutral as previously supposed, but rather

influence and modulate mitochondrial metabolism, so

that factors such as climate and food or oxygen

availability can select them [205,206]. If this

hypothesis would come true, it is conceivable that

inherited mtDNA variability can impinge upon a

variety of phenotypes, including ageing, longevity and

age-related diseases. Accordingly, the analysis of

mtDNA haplogroups is currently providing new

insights into the association of mtDNA-inherited

variants in several neurodegenerative diseases such as

Parkinson’s and Alzheimer’s diseases [207–209].

As far as ageing is regarded, it has been observed

that in male centenarians from northern Italy mtDNA

haplogroup J is over-represented [210], suggesting a

protective role for this mtDNA variant against ageing.

However, when a large population from southern Italy

was studied, no association was found between

haplogroups J and longevity. On the whole, these

results once again support the idea that the effect of

mtDNA inherited variants on longevity is population-

specific and strongly depends on the interactions with

the environment.

This observation has been confirmed in two other

European studies [211,212] and in Japanese cente-

narians, where a sublineage of haplogroup D is more

frequent [213,214]. Furthermore, it has been found

that cytochrome b gene in mtDNA from centenarians

is characterised by inherited variants different from

those found in Parkinson’s patients [215]. Thus, the

available data support the hypothesis that mtDNA

lineages are qualitatively different from each other,

bearing mutations able to modify mitochondrial

functions, and consequentely affecting the probability

to reach longevity and avoid pathologies. To this

regard, it has been reported that mitochondria bearing

mtDNA belonging to haplogroups H and T displayed

a significant difference in the activity of complex I and

IV of respiratory chain in sperm cells [206]. We have

recently obtained data in vitro on a cybrid cell lines

model suggesting that mtDNA haplogroups can affect

the expression of cytokine and cytokine receptor

genes, thus suggesting that there is a cross-talk

between mitochondrial and nuclear genome, and

that mtDNA haplogroups modulate differently the

expression of nuclear genes [216].

Proteasome and immunoproteasome

Ageing is characterised by an accumulation of

oxidised proteins which are normally degraded by

the proteasome. Beside the increased oxidative stress,

this accumulation could be due to an impaired

function of the 20S proteasome in aged cells. To test

the importance of such phenomenon for human

ageing and longevity, we have analysed the expression

and the proteolytic activity of the proteasome in cells

from centenarians. Analysis of several proteasome

subunits RNA expression levels, determination of one

peptidase activity and identification of oxidised

proteins revealed that cells from centenarians have a

preserved proteasome function [217]. In addition, it

was found that cells from centenarians exhibit

characteristics similar to cells from younger rather

than the older control donors in all three assays. We

then moved to the study of possible involvement in

ageing and longevity of genetic variants of immuno-

proteasome subunits. Immunoproteasome is indeed a

crucial complex for the processing of antigens and

MHC-I presentation. We then studied polymorphisms

in LMP-2, LMP-7 and MECL-1 genes, whose

Genes, ageing and longevity in humans 1313

expression is needed for immunoproteasome assem-

bly. A non conservative nucleotide base pair change at

amino acid position 60 (in exon 3) in LMP2 gene,

resulting in two alleles, Arginine (R) or Histidine (H),

resulted to be associated with different autoimmune

diseases, but not with ageing [218,219]. However, it is

worthy to note that a modulation of proteasome

activity depending on the LMP2 R60H polymorphism

was observed in tissues derived from elderly people

(brain and blood) [219,220]. In particular, the

modulation of TNF-a induced-apoptosis in periph-

eral blood nuclear cells (likely mediated by IkB-a

degradation by proteasomes) was detectable only in

elderly subjects, being not present in samples from

young donors. Such results suggest that the effect of

the polymorphism is tissue-dependent and age-

dependent. In a recent study, we suggested that

LMP2 aminoacid 60 lies in a putative binding groove

for a regulatory molecule, able to enhance the 20S

proteasome activity [220]. On the whole, data

suggested how the activity/expression of such

hypothetical protein should be strictly regulated and

could vary on the basis of different factors, such as the

tissue analysed and the age of the subject. Further-

more, the putative regulatory site has been predicted

into the immunoproteasome, and no data about the

proteasome constitutive isoform are available. Hence,

we are tempted to speculate that the increase of

immunoproteasome content during ageing in specific

tissues such as human brain [221], could increase the

modulatory capability of the putative/predicted regu-

latory molecule, and in turn the importance of the

LMP2 codon 60 polymorphism, on the whole

proteasome activity. However, in cells like fibroblasts,

not directly involved in antigen-processing, it has been

recently reported that the levels of immunoprotea-

some subunits do not decrease but their induction by

IFNg is lost in senescent cells [222].

Conclusions

An impressive and coherent series of epidemiological

data in different populations (New England Amer-

icans, Mormons, Ashkenazi Jewish, Islandic, Okina-

wan Japanese, Italians, Irish, Dutch, among others)

indicate the presence of a strong familial component

of longevity. These studies demonstrate that parents,

siblings and offspring of long-lived subjects (but not

the spouses of the long-lived subjects who shared with

them most part of their adult life) have a significant

survival advantage, a higher probability to have been

or to became long-living persons and to have a lower

risk to undergo the most important age-related

diseases, such as cardio- and cerebro-vascular dis-

eases, diabetes and cancer, when compared to the

appropriate controls [223–229]. Thus longevity is

present in many generations of the same families in

spite of the great variations in life style and life

expectancy of the last century, and this strongly

suggests that this familiar trait is largely determined by

genetics. On the other side, many experimental

models have demonstrated that the modification of

even only one gene can deeply alter animal ageing or

longevity. In recent years, a great number of studies

have been performed in order to identify this genetic

component of human longevity, in particular by

seeking genes whose variants were correlated to

increased (or decreased) life-span. Different

approaches have been used, such as association

studies, case-control studies, epidemiological studies,

longitudinal studies on cohorts of old people, in the

attempt to find genes or gene variants that correlate

with longevity (or, at variance, with ageing or age-

associated diseases) in humans. Nevertheless, studies

on humans suffer by a variety of inescapable pitfalls

that render the results from these studies more

“confused” than those from strictly controlled animal

models in which variables such as life-style, environ-

mental conditions, gene pools, etc. are minimized.

Paradoxically this is also the strength of studies in

humans which do not suffer from the intrinsic

limitation consequent to the highly “artificial”

conditions in which animals used in biogerontological

studies to assess the determinants of longevity are

exposed life-long. The most clear example to this

regard is represented by studies on the ageing of the

immune system and the role played by immuno-

senescence in the onset of age-related pathologies.

Studies in humans and particularly on centenarians

clearly anticipated a variety of key observations such as

the progressive establishment of a peculiar chronic

inflammatory status we proposed to call inflamm-

aging, the accumulation of memory cells with age, the

exhaustion of virgin T cells and the role of viruses such

as cytomegalovirus (CMV), among other [230–238].

Such observations would be hampered or highly

biased by the “clean” conditions typical of well

controlled animal houses, quite distant from the real

“wild” conditions where every type of animal is

exposed life-long to a variety of pathogens. These were

indeed the conditions that modelled our genome and

that of other animals during evolution. We surmise

that similar bias may occur in the genetics of longevity,

when studies in humans and other animal models are

compared or extrapolated. Moreover, humans are

clearly different and more complex with respect to all

other animals either biologically or culturally. In

particular, all factors related to environment and

lifestyle are incomparably different from any labora-

tory environment and the effect of culture cannot be

underestimated. For these reasons, the results of these

studies are often “disturbing” in the sense that they are

difficult to compare each other, or even contrast.

Indeed, only in few cases the results have been

replicated in different populations (as in the case for

example of APOE4). On the contrary, as discussed

S. Salvioli et al.1314

all along this review, many associations between gene

variants and longevity have been found only in specific

populations. This should be not unexpected, since

ageing and longevity are complex traits resulting not

only and not exclusively from genetics, but rather from

the interactions between genetics, environment and

chance. In this perspective, it is conceivable that

longevity can be achieved by different combinations of

these three components, and thus, when gene pool

and environment change, the combinations leading to

longevity change as well [239].

In conclusion, the main lesson from these studies is

that is very difficult to identify “longevity genes” in

humans with “classical” association studies, because

wild type species (as humans are) are endowed with

such an intrinsic genetic complexity and environmen-

tal conditions can be so different from Country to

Country that render longevity-assurance combi-

nations, and thus “longevity genes”, almost exclu-

sively peculiar to any specific population. This should

not imply a diminished interest in regard to genes

whose effects on longevity are evident only in one or

few populations, but rather suggests that we urgently

need new approaches to move up one step on our

general understanding of the complex traits such as

human longevity. These could be, for example, studies

on very large population samples containing people of

different ethnic origin, so that internal comparisons

can be made avoiding methodological biases, or

studies on families (siblings or offspring of long-living

people, for example). Furthermore, high-throughput

analyses of many different polymorphisms must be

envisaged in order to obtain more informative data

about a candidate gene. Second, new computational

approaches have to be developed in order to interpret

the data. Third, in silico bioinformatics models

should be considered as a possible useful tool to

further implement benchtop data.

Finally, it has to be taken into account that longevity

is a post-reproductive phenomenon, so that longevity

as such has not been subjected to evolutionary

selection, and thus the genetics of longevity likely

does not conform to the classical genetic rules [239].

This aspect, together with other phenomena such as

antagonistic pleitropy, further complicates the study

of this peculiar aspect of human genetics. The

researches in this field are still in their infancy, and

the studies performed until now should be considered

little more than pioneering works that paved the way

to the discovery of this exciting aspect of science and

human life as well.

Acknowledgements

This work was supported by: EU Projects “T-CIA”

FP5 Contract n QLK6-CT-2002-02283 and

“GEHA—Genetics of Healthy Aging” FP6-503270

Grants; the PRRIITT program of the Emilia-Romagna

Region (and Fondi Strutturali Obiettivo 2); MIUR

(Italian Ministry of University) Fondo per gli

Investimenti della Ricerca di Base (FIRB) 2001,

protocol RBNE018AAP and #RBNE018R89; Italian

Ministry of Health Grant (Ricerche Finalizzate 2002

and 2003); FISM (Federazione Italiana Sclerosi

Multipla) Grant (Finalised Project “Immunoprotea-

some in Multiple Sclerosis: Genetics and Biological

Role in the Pathogenesis of the Disease”) to CF; Italian

Ministry of Health (Ricerca Finalizzata “Markers

genetici di sindrome coronaria acuta e valutazione

della L-arginina nella prevenzione di eventi ischemici”)

Grant to DM and CF; EU Project “ZINCAGE” 6th

FP, Contract n. FOOD-CT-2003-506850, and MIUR

(PRIN 2004) Grant to DM; University of Bologna

Ricerca Fondamentale Orientata (RFO 2005), and

Roberto and Cornelia Pallotti Legacy for Cancer

Research Grants to CF and SS.

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