Telomeric Aging Markers

All vertebrate telomeres contain a characteristic 6-base repeated sequence, TTAGGG (Meyne et al., 1989). According to the telomere hypothesis, DNA replication in somatic cells leads to telomere shortening, which forms the basis of a cellular mitotic clock (Sherr and DePinho, 2000). Moreover, several premature aging syndromes lead to accelerated loss of telomeric DNA, which eventually results in accelerated replicative senescence of the cells (Kipling et al., 2004; Metcalfe et al., 1996; Pandita, 2002; von Zglinicki et al., 2005). Cumulatively, these results suggest that it is also possible to ascertain aging and senescence phenotypes in zebrafish by qualitative and quantitative measurements of telomere metabolism and maintenance.

20 Gy

Figure 28.2. Whole-mount SA-J-gal detection of genotoxic stress-induced embryonic senescence. Wild-type zebrafish embryos were treated with IR (0, 10, and 20 Gy) at 6 hpf. Subjected embryos were raised by 6 dpf. The 6-day larval fish were fixed and stained for SA-S-gal. Both 10 and 20 Gy of IR obviously induced SA-J-gal in larval zebrafish. While 10 Gy-treated larval fish did not exhibit a particularly abnormal phenotype with more than 90% survival rate, 20 Gy-treated larval fish showed severe malformation and did not subsequently survive at all.

To detect and measure telomeric DNA in zebrafish, we have adopted a previously established procedure utilizing quantitative measurement of in situ hybridization (Q-FISH) with a fluorescence-labeled telomere-specific probe (Miller et al., 1992). Importantly, telomeric repeat sequences of TTAGGG are conserved in all vertebrates, from fish to humans. Thus, zebrafish telomeric sequences can be detected by the same procedure used for human telomeres, utilizing a fluorescein- or Cy3-conjugated peptide nucleic acid (PNA) probe as confirmed previously (Baerlocher and Lansdorp, 2004; Sola and Gornung, 2001). Since zebrafish embryos are transparent, we can easily detect the telomere signals through fluorescence

Figure 28.2. Whole-mount SA-J-gal detection of genotoxic stress-induced embryonic senescence. Wild-type zebrafish embryos were treated with IR (0, 10, and 20 Gy) at 6 hpf. Subjected embryos were raised by 6 dpf. The 6-day larval fish were fixed and stained for SA-S-gal. Both 10 and 20 Gy of IR obviously induced SA-J-gal in larval zebrafish. While 10 Gy-treated larval fish did not exhibit a particularly abnormal phenotype with more than 90% survival rate, 20 Gy-treated larval fish showed severe malformation and did not subsequently survive at all.

microscopy by staining with the PNA probe. We could quantify the fluorescence intensity of zebrafish telomeres by the telomere analysis program, TEL-TELO V1.0a, developed and provided by Peter Lansdorp at the Terry Fox Laboratory, as previously performed for human and mouse telomeres (Baerlocher and Lansdorp, 2004).

We have also performed Southern blot analysis to confirm telomere lengths in muscle from 6, 18, 30, and 42-month-old fish for 10 fish in each age group. Southern analysis can demonstrate length of terminal restriction fragments (TRF), containing DNA with uniform telo-meric TTAGGG repeats, obtained by digestion of genomic DNA using Hinf I and Rsa I restriction enzymes in zebrafish, as well as in humans and mice. Through Southern analysis in adult fish muscle samples, we found that the telomere lengths were stochastically shortened late in the life of the fish (e.g., at more than 30 months), irrespective of their constitutive telomerase activity throughout their lives.

Telomerase is a ribonucleoprotein complex that consists of two main subunits: TR, the telomerase RNA template for addition of TTAGGG repeat sequence (telomerase RNA complex, TERC); and TERT, telomer-ase reverse transcriptase catalytic subunit of telomerase and other associated proteins. While TR is expressed ubiquitously and constitutively in various types of cells, TERT is not expressed in most normal somatic cells. However, stem cells and germ cells as well as approximately 80-85% of cancer cells express TERT in humans.

Zebrafish telomerase activity has been examined by Telomeric Repeat Amplification Protocol (TRAP) assay, which is a sensitive and efficient PCR-based telomerase detection method (Wright et al., 1995). There is a causal relationship between telomerase expression, telomere maintenance and extension of cellular lifespan in mammalian cells. Due to tightly regulated expression of TERT as described above among normal tissues of adult humans, telomerase activity is almost undetectable except in germ-line cells, although very small amounts of activity are detectable in bone marrow, peripheral blood lymphocytes, and skin epidermis. In addition, mice normally exhibit telomerase activity in colon, liver, ovary, and testis but not in brain, heart, stomach, and muscle. However, strikingly in zebrafish, the results showed the constitutive activation of telomerase in nearly all the tissues, including muscle, heart, and brain, throughout their lives (Kishi et al., 2003) (Figure 28.3).

Once the unlimited growth potential and remarkable regeneration ability of zebrafish is appreciated, the above results may not seem surprising. On the other hand, strong link between cell proliferation and telomerase activity is biologically and evolutionarily very intriguing.

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