Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc-/- mice with short telomeres

Abstract

Reconstitution of telomerase activity is proposed as a potential gene therapy to prevent, or rescue, age-related diseases produced by critical telomere shortening. However, it is not known whether or not short telomeres are irreversibly damaged. We addressed this by re-introducing telomerase in late generation telomerase-deficient mice, Terc-/-, which have short telomeres and show severe proliferative defects. For this, we have crossed these mice with Terc+/- mice and analyzed telomere length, chromosomal instability and premature aging of the progeny. The Terc-/- progeny had one set of chromosomes with normal telomeres, whereas the other set remained with critically short telomeres; these mice presented chromosomal instability and premature aging. In contrast, Terc+/- progeny showed all chromosomes with detectable telomeres, and did not show chromosomal instability or premature aging. These results prove that critically short telomeres can be rescued by telomerase, and become fully functional, thus rescuing premature aging. This has important implications for the future design of telomerase-based gene therapy of age-related diseases.

Fig. 1. Generation of G4 Terc^+/– and G4 Terc^–/– littermates via a G3 Terc^–/– × Terc^+/– cross. Wild-type and null Terc alleles were identified by PCR (see Methods). The positions of the PCR bands corresponding to the wild-type and null alleles, as well as that of an internal control (IC) for PCR efficiency, are indicated by arrows.

Fig. 2. (A) Telomere length of both p- and q-arms, as well as the average of both arms (average p+q) from chromosome Y, chromosome 2, as well as all chromosomes, are represented. Standard errors are also included; however, some of the errors are so small (see Table I) that the error bars are not visible. (B) Illustrative images of combined Q-FISH and chromosome 2 painting. For each case represented, both chromosome 2 are shown (mice numbers 1, 2, 4 and 6). Notice undetectable chr2-telomeres in the G3 Terc^–/– father (number 1), as well as in the G4 Terc^–/– sibling (number 4). The G4 Terc^+/– sibling (number 6) and the Terc^+/– mother (number 2) show detectable chr2-telomeres.

Fig. 2. (A) Telomere length of both p- and q-arms, as well as the average of both arms (average p+q) from chromosome Y, chromosome 2, as well as all chromosomes, are represented. Standard errors are also included; however, some of the errors are so small (see Table I) that the error bars are not visible. (B) Illustrative images of combined Q-FISH and chromosome 2 painting. For each case represented, both chromosome 2 are shown (mice numbers 1, 2, 4 and 6). Notice undetectable chr2-telomeres in the G3 Terc^–/– father (number 1), as well as in the G4 Terc^–/– sibling (number 4). The G4 Terc^+/– sibling (number 6) and the Terc^+/– mother (number 2) show detectable chr2-telomeres.

Fig. 3. (A) Telomere length distribution of chr2-telomeres in primary splenocytes from the parents and the G4 progeny as determined by Q-FISH. The histogram depicts a population of critically short chr2-telomeres in the G3 father and in the G4 Terc^–/– littermates. In contrast, the G4 Terc^+/– littermates show disappearance of the peak corresponding to short telomeres. (B) Telomere length distribution of all chromosome telomeres in primary splenocytes from the parents and the G4 progeny as determined by Q-FISH. The histogram depicts a population of critically short telomeres (≤1 kb) in the G3 father and in the G4 Terc^–/– littermates. The G4 Terc^+/– littermates show disappearance of ≤1 kb telomeres.

Fig. 3. (A) Telomere length distribution of chr2-telomeres in primary splenocytes from the parents and the G4 progeny as determined by Q-FISH. The histogram depicts a population of critically short chr2-telomeres in the G3 father and in the G4 Terc^–/– littermates. In contrast, the G4 Terc^+/– littermates show disappearance of the peak corresponding to short telomeres. (B) Telomere length distribution of all chromosome telomeres in primary splenocytes from the parents and the G4 progeny as determined by Q-FISH. The histogram depicts a population of critically short telomeres (≤1 kb) in the G3 father and in the G4 Terc^–/– littermates. The G4 Terc^+/– littermates show disappearance of ≤1 kb telomeres.

Fig. 4. Measurement of terminal restriction fragments (TRFs) in bone marrow cells from both parents and the G4 progeny. Decrease in telomere length is visualized by the appearance of TRF bands below the 6.5 kb marker in the G4 Terc^–/– mice that are not present in the G4 Terc^+/– littermates (indicated by open circles). +/–, Terc^+/– (numbers 2, 3, 6, 7 and 9); –/–, Terc^–/– (numbers 1, 4, 5 and 8).

Fig. 5. Histology of testis, small intestine and bone marrow from littermate 21-day-old G4 Terc^–/– and G4 Terc^+/– siblings (numbers 4 and 6, respectively). Magnifications were 20 and 40×, as indicated.