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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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