Rejuvenation Research, in press (SENS 3 meeting issue)
Metallothionein down-regulation in very old age: a phenomenon associated with cellular
senescence?
M. Malavolta1, C. Cipriano1, L. Costarelli1, R. Giacconi1, A. Larbi2, G. Pawelec2, G. Dedoussis3, G.
Herbein4, D. Monti5, J. Jajte6, L. Rink7, E. Mocchegiani1
1
Immunology Ctr. (Section: Nutrition, Immunity and Ageing), Res. Dept. INRCA, Ancona, Italy;
2
Center for Medical Research, ZMF, Univ. of Tubingen, Medical School, Tubingen, Germany
3
Dept. of Nutrition Science and Dietetics, Harokopio University of Athens, Greece
4
Dept. of Virology, Franche-Comte University, Besancon, France.
5
Experimental Pathology and Oncology, University of Florence, Florence, Italy.
6
Dept. of Toxicology, Faculty of Pharmacy, Medical University, Lodz, Poland.
7
Institute of Immunology, RWTH-Aachen University Hospital, Aachen, Germany.
Word count: 1498
Address for correspondence and reprints: Marco Malavolta, Res. Dept. INRCA, via Birarelli 8,
Ancona, Italy. Tel. +39-071-8004116. Fax +39-071-206791. email: [email protected]
Key Words: Metallothioneins, Ageing, Cell Senescence, Zinc, T cell clones
Abbreviated Title: Metallothioneins in very old age
Abstract
It is known that metallothionein (MT) mRNA expression first increases with age, but then decreases
again in the very elderly. Here, we report that MT protein levels also decrease in very old age, and
that this is independent of dietary zinc intake. Age-related changes of MT as well as alterations of
zinc homeostasis (intracellular labile zinc and NO-induced zinc release) occur both in human
PBMC ex vivo and also in CD4+ T cell clones progressing through their finite lifespan in vitro.
These results suggest that phenomena observed in very old people can be at least partially attributed
to diminished cell proliferation.
Introduction
Metallothioneins (MTs) are ubiquitous proteins that bind multiple Zn atoms under physiological
conditions. They are thought to play a role in zinc and copper homeostasis, to regulate synthesis,
assembly, or activity of Zn metalloproteins and to protect against damage caused by reactive
oxygen species.1,2 MTs transduce signals via Zn that activate intracellular antioxidant stress
responses2 and are also involved in modulating cellular respiration and energy metabolism.3
Therefore, these proteins are being intensively studied in the context of ageing and longevity
mechanisms. In nematodes, basal levels of MTs in daf-2 long-lived mutants were found to be higher
than in wild-type.4 Prolonged life span associated with reduction of age-related stress biomarkers
was observed in cardiac-specific MT transgenic mice.5 An involvement of MTs in human longevity
is suggested by the association of a polymorphism in the MT1a gene coding region with longevity
in an Italian population6. However, the precise function of these proteins in aging is still debated
because their protective role could also be deleterious in the case of excessive sequestration of Zn.7
The increased expression of MT mRNA during ageing but decreased levels found in healthy
nonagenarians/centenarians suggests a possible selection for survival of low expressors.8 Most
studies have been at transcriptional level, however, and due to a possible lack of correlation or timeshift delay between MT mRNA and protein production, this could be a confounding factor.9,10
Additionally, as dietary intake of zinc influences MT levels11 and zinc deficiency increases with
age,12 age-dependent MT expression may reflect changes in dietary habits rather than intracellular
or physiological age-related modification. Employing culture models can overcome some of these
problems.13 Here, we assess age-related patterns of MT expression at the protein level in blood
mononuclear cells in relation to Zn intake compared to changes in MT profile observed in long term
clonal cultures of CD4+ T cells (TCC). Intracellular labile Zn (iZnL) and NO-induced release of Zn
(iZnR) were monitored in order to assess functional relationships with Zn homeostasis.
Material and Methods
Subjects
A total of 460 healthy elderly volunteers aged 61-102 years, recruited in five European countries
(Zincage project, www.zincage .org) according to the SENIEUR protocol14 , were grouped into 4
age classes: 61-70 years (95 F, 79 M), 71-80 years (96 F, 79 M), 81-90 years (47 F, 29 M) and > 90
years (26 F, 9 M).
Zinc score
A food frequency questionnaire was used to assess dietary zinc intake. Individual food consumption
was calculated as frequency (1 for never or less than 1 time/month, 2 for occasionally, 3 for
sometimes and 4 for every day) multiplied by quantity (0 for no consumption, 1 for small, 2 for
medium and 3 for abundant). To provide a continuous variable representative of zinc intake, a “zinc
score” was calculated for each volunteer as the sum of all estimated zinc intakes derived from listed
food items. The general formula used for zinc score calculation was: Zinc score = Consumption x
Zinc content. European National and USDA food composition tables were used to define zinc
content for all food items.
PBMC recovery and storage
PBMCs were obtained by ficoll-hypaque (density d = 1,077) (Biochrom AG, Berlin, Germany)
gradient centrifugation of heparinzed blood and frozen in FCS containing 10% of DMSO under
liquid nitrogen until use.
T cell clones
CD4+ TCC from 8 healthy old people (61-89 years) and 5 nonagenarians/centenarians (90-102
years) were obtained by limiting dilution in the presence of IL-2 as previously described.13 Because
different T cell clones reach senescence after different numbers of population doublings, age is
expressed as % of completed lifespan. TCC were considered senescent and age set to 100% when
they stopped growing. Four time points representating clone ages were taken: 38-54%, 54-69%, 7094% and 95-100% (n = 10 per time point).
Flow Cytometric (FC) analysis of iZnL, iZnR and MT
iZnL and iZnR were assessed by single color flow cytometry of PBMC stained with Zinpyr-1, as
previously reported.15 MT were assessed by flow cytometry using mouse anti-horse Metallothionein
clone E9 (Dakocytomation, Denmark), as previously reported.15 MT data are reported as MFI or
Log(MFI).
Statistical Analysis
Significant differences were calculated by univariate analysis using the Zn Score as a covariate and
gender as a fixed factor. Due to skewed distribution, log transformed values of MT and Zn Score
were used for the “ex-vivo” data. Regression analysis and curve estimation was performed by SPSS
11.0. Significant differences among time points of TCC were detected by ANOVA.
Results
A) MT, iZnL and iZnR in PBMC “ex-vivo”: MTs showed an increasing trend up to 71-80 years,
with a subsequent rapid decline in nonagenarians. A quadratic model best approximated MT data in
dependence of age (R2 = 0.020, p = 0.011; y = -0.032 x2 + 4.67 x - 98.06). Mean values of MT,
iZnL and iZnR after adjusting for Zn score are reported in Fig.1 (Panels: A1-A3). MT values in the
60-70 and 71-80 yr-olds were significantly higher than the 81-90 (p = 0.010 and p = 0.001
respectively) and > 90 yr-olds (p = 0.012 and p = 0.003, respectively) with no gender-related
differences. The iZnL was also higher in the two younger age groups compared to both 81-90 (p =
0.010 and p = 0.049, respectively) and > 90 yr-olds (p = 0.012 and p = 0.033, respectively), again
without gender-related differences. The best fit was found with a quadratic model (R2 = 0.055, p <
0.0001; y = -0.0002 x2 + 0.021 x + 0.586). The iZnR was higher in the 60-70 and 71-80 yr-olds
compared to both the 81-90 (p = 0.002 and p = 0.020, respectively) and > 90 groups (p < 0.001 and
p = 0.002, respectively) with no gender-related differences. Both an exponential decay and a linear
regression (R2 = 0.085, p < 0.0001) described the age-related decline of iZnR.
B) MT, iZnL and iZnR in TCC: Because no significant differences were observed between TCC
from old and nonagenarian/centenarian donors, all clones were grouped together according to % of
lifespan accomplished. Similar results to those obtained “ex-vivo” were observed with “in-vitro”
ageing of TCC (Fig.1, Panels: B1-B3). MTs were higher (p < 0.05 for each comparison) in the
early passage TCC (38-54% and 55-69% of lifespan) than during the last passages, approaching
senescence (95-100% of lifespan). iZnL was higher (p < 0.05 for each comparison) in the “middleaged” clones (55-69% and 70-94% of lifespan) than at late passage (95-100% of lifespan). iZnR
was significantly lower (p < 0.05 for each comparison) in very late passage TCC than the rest of
clone lifespan.
Discussion
The present work establishes that MT protein as well as mRNA levels are down-regulated at very
old age, accompanied by a concomitant decrease of iZnL and iZnR, starting from 70-80 years.
Reduced dietary zinc intake may cause these changes, but this is unlikely due our using the Zn score
as a covariate to abrogate the confounding effect of Zn intake. Exactly the same was found in late
passage/senescent TCC despite a constant concentration of Zn in the culture medium, consistent
with the decrease of MT at very old age not being simply due to dietary Zn deficiency. An
explanation for this age-related pattern may reside in the increased pro-inflammatory condition of
the elderly (which is known to up-regulate MT).16 The same could apply to the TCC model, in
which increasing autocrine secretion of cytokines such as TNF- reflect an enhanced proinflammatory environment17.
Alternatively, as MTs are involved in cell proliferation, which is. associated with increased net
retention of extracellular zinc, through a coordinated modulation of zinc transporters and upregulation of MTs.18 the, concomitant decrease of MT, iZnL and iZnR in late passage TCC as well
as in people older than 80 years could be related to accumulation of senescent cells. Loss of
telomere repeats occurs during the life span of most T cell clones19 and telomere length in
lymphocytes from healthy people declines with age.20 Even though cellular senescence can be
triggered by mechanisms other than telomere shortening, including epigenetic modulation of the
INK4a/ARF locus, and DNA damage, the final result is limited cellular proliferation.20 Subsequent
changes in zinc homeostasis could be the consequence of this phenomenon. It is also interesting to
observe that MT, iZnL and iZnR strongly decrease between the 70’s and 90 years but more slowly
thereafter, due to the presence of nonagenarians/centenarians with functional characteristics similar
to 10-20 years younger individuals. Although these results were derived from a cross-sectional
study and could therefore result from population selection, the exact same findings in the in vitro
longitudinal TCC model reinforce the conclusion that dynamic changes in these factors are indeed
occurring within the individual as he or she ages. Accordingly, interventions aimed at modulating
MT may be beneficial for restoring immune function at old age.
Acknowledgements: This work was supported by EU (ZINCAGE FOOD-CT-2003-506850,
LIFESPAN LSHG-CT-2007-036894), Deutsche Forschungsgemeinschaft SFB685-B04 and PA
361/11-1; SFB 685 B04 and INRCA. We are grateful to Mss A Rehbein and K. Hähnel for their
dedicated work in generating and propagating TCC.
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Figures Legends
Fig.1. Panel A: Mean values ± SEM of MT (A1), iZnL (A2) and iZnR (A3) adjusted for Zn score
in PBMC from different age groups (a = “61-70 years”, b = “71-80 years”, c = “81-90 years”, d =
“> 90 years”). Panel B: Mean values ± SEM of MT (B1), iZnL (B2) and iZnR (B3) in TCC at
different time points of their lifespan (a = 38-54%, b = 55-69%, c =70-94%, d = 95-100%).
Superscript indicates a significant difference (p < 0.05 at least) with respect to the respective age
group.
Abbreviations: MT = Metallothioneins, iZnL = intracellular labile zinc, iZnR = intracellular NOinduced release of zinc.