A Mouse Model for Manipulating Double-strand DNA Breaks in Aging
2005 senior Scholar Award in agingAmong biological macromolecules the DNA of the genome has a unique position. Unlike other building blocks of macromolecular structures, such as proteins and lipids, DNA molecules cannot be easily replaced by natural turnover. Hence, the need for genomes to rely on advanced maintenance and repair systems to deal with the chemical damage occurring in DNA at tens of thousands of places in each cell on a normal day. Interestingly, defects in genome maintenance leads to the premature appearance of symptoms of aging. For example, the human adult-onset progeroid Werner's syndrome is due to a defect in maintaining parts of the genome, most notably the chromosome ends, called telomeres. Likewise, the childhood Hutchinson Gilford Progeroid Syndrome is caused by a defect in genome maintenance, in this case affecting the genome's 3-dimensional structure through rapid degeneration of the nuclear lamina. In mice, engineered defects in genome maintenance cause the premature appearance of aging phenotypes, providing additional evidence that genome instability is a major cause of aging.
While a causal relationship between DNA damage and aging is now no longer disputed, there is still very limited insight into the proximal causes of the complex degenerative phenotype of old age that gradually unfolds after maturity. It is well established that aging of both humans and rodents is associated with increased numbers of chromosomal aberrations, i.e., large genome alterations, in white blood cells, essentially the only cell type that can be readily assessed for this kind of event. In my laboratory, since the late 1980s, we have developed a series of transgenic mouse models harboring a marker gene that can be recovered from DNA of every tissue or organ and further characterized in the bacterium Escherichia coli. Using this system we demonstrated that during aging all sorts of mutations accumulate. The rate of accumulation as well as the type of events appeared to be organ and tissue-specific. Most notably, similar to the situation in white blood cells, we observed the accumulation of large genome alterations in several organs. Such age-dependent genome alterations, which are also observed in abnormally high numbers in white blood cells from Werner syndrome patients, are likely to result from the erroneous repair of DNA double-strand breaks. We have obtained evidence that these large genomic mutations adversely affect normal patterns of gene expression, thereby greatly increasing the risk of age-related diseases, including cancer.
It would be important to test the possibility that the rate of aging is controlled by the rate at which large genome alterations accumulate in different organs and tissues of the organism. This cannot simply be done by inactivating genes that control the repair of double-strand breaks. Indeed, genome maintenance is very complicated and we are unable to modulate its genes with the necessary precision so as to control the rate of damage accumulation accurately enough. Defects in DNA double-strand break repair have been found to accelerate a number of signs and symptoms of aging. The picture obtained, however, is still far from the phenotype that is obtained after the natural process of aging. In this project we will attempt to gradually increase the rate of double-strand break accumulation in the expectation that this will lead to an accelerated accumulation of large genome alterations. If this is a key molecular determinant of aging, this should lead to the premature appearance of all the common phenotypes of aging.
The system that we will develop is based on introducing restriction enzyme activities in mice in vivo. A restriction enzyme generates DNA double-strand breaks by recognizing and cutting DNA at a particular sequence of nucleotides. Introduction of restriction enzymes into mammalian cells in culture has been shown to result in large genome alterations. The problem is how to create a situation in which such an enzyme can be transferred into cells of a living mouse and then manipulated in a way that double-strand breaks occur, but not at such a high rate that too many cells will die. The idea is to raise the level of such breaks only slightly and then see if this results in a somewhat higher level of genome alterations and an acceleration of all possible symptoms of aging. We will attempt to accomplish this in two ways. First, we will use viruses to bring bacterial genes encoding restriction enzymes into the mouse tissues and cells through repeated infections. This has the advantage that it is relatively easy, but it is difficult to regulate. Second, we will generate mice harboring the restriction enzyme genes in their germline. By using advanced control systems, this should allow us to regulate the production and activity of the restriction enzymes in the animal.
Naturally, the success of this project is uncertain, both for technical and conceptual reasons. However, the potential gain is substantial. Paradoxically, model systems in which the aging phenotype can be accelerated in a controlled manner are much more valuable than models of increased longevity. Indeed, such models will not only give us a major tool for unraveling the mechanistic basis of aging, but will also provide us with the means to test a host of novel interventions in a short-term, highly specific and predictive pre-clinical model for aging-related ailments and functional decline.