To clone genes linked with longevity.
To clone genes linked with in vitro ageing.
To make a comparative analysis of polymorphic markers of the cloned genes in samples of centenarians.
To examine the role of ageing-accelerating and ageing-retarding agents in normal fibroblast cultures.
To establish the biological role of the cloned genes by transfections and antisense micro-injections.
The aim of this proposal is the identification and the study of the function of genes involved in the regulation of ageing and longevity. The four participating laboratories have complementary expertises in the field of molecular and cellular biology of ageing. One laboratory has already identified eight age-associated genes while the others have wide experience in gene and protein characterization, studies on centenarian cells and stress-induced accelerated ageing. The source material for the study will be human diploid fibroblasts either undergoing normal ageing or stress-induced accelerated ageing, and fibroblasts derived from centenarians.
These cellular resources will be used for the isolation of eight specific and longevity-specific genes by three complementary methods:
1) Suppression subtractive hybridisation will be applied to fibroblasts from centenarian versus in vitro normally aged fibroblasts from young subjects in order to identify genes associated with longevity;
2) Differential display will be used to clone genes from fibroblasts undergoing stress-induced accelerated ageing in comparison with unstressed in vitro aged fibroblasts at the same passage number;
3) Phage display technology for cloning genes on the basis of differential protein expression in human diploid fibroblasts undergoing in vitro ageing. In control experiments the specific expression of the isolated genes will be checked in primary and centenarian cells of different origins, as well as in stress induced aged cells.
To discover whether the isolated genes are related to ageing and/or longevity they will be studied by a comparative analysis of possible polymorphic markers in these candidate loci in aged and young individuals as well as in subjects affected by a syndrome of precocious ageing such as Down's syndrome. The analysis will be performed by PCR amplification of the VNTR (variable number of tandem repeats) within or near candidate gene coding regions. Since the research cooperation possesses a unique biological bank of samples of over 700 centenarians from all over Italy, a particular emphasis will be given to studying candidate longevity genes in people of extreme old age, as well as from different ethnical and regional homogeneous groups. Lastly, isolated genes related to ageing or longevity will be examined for their ability to induce or retard senescence respectively, when overexpressed in proliferating cells. In addition, their antisense oligonucleotides will be micro-injected into primary cells and tested for their influence on the in vitro lifespan and stress-induced accelerated ageing. This proposal requires sophisticated cellular and molecular biological methodologies (mentioned above) and rare biological material, which can be contributed by the participating laboratories in this highly complementary constellation. 04 04
Funding SchemeCSC - Cost-sharing contracts
5000 Namur / Namen