Short telomeres are sufficient to cause the degenerative defects associated with aging

Abstract

Telomerase function is critical for telomere maintenance. Mutations in telomerase components lead to telomere shortening and progressive bone marrow failure in the premature aging syndrome dyskeratosis congenita. Short telomeres are also acquired with aging, yet the role that they play in mediating age-related disease is not fully known. We generated wild-type mice that have short telomeres. In these mice, we identified hematopoietic and immune defects that resembled those present in dyskeratosis congenita patients. When mice with short telomeres were interbred, telomere length was only incrementally restored, and even several generations later, wild-type mice with short telomeres still displayed degenerative defects. Our findings implicate telomere length as a unique heritable trait that, when short, is sufficient to mediate the degenerative defects of aging, even when telomerase is wild-type.

Figure 1

Hematopoiesis Is Ineffective in Wild-Type Mice with Short Telomeres (A) Scheme and nomenclature of mTR^+/− breeding. HG7 refers to the seventh heterozygous generation, and wt8^∗1 refers to the first generation born from HG7 crosses. (B–F) Frequency distribution of telomere lengths as measured by qFISH shows that wt^∗ mice have shorter telomeres than wild-type mice (average wild-type = 50,000 telomere fluorescence units [TFU], wt^∗ = 39,000 TFU; p < 0.0001; n = 3 per group). wt^∗ mice have low peripheral-blood absolute neutrophil (C) and platelet counts (D). These cytopenias are associated with a hypocellular bone marrow (D) and atrophic spleen (F). (E) Peripheral white blood cell count after 5-fluorouracil injection on day 0 shows that both wt^∗ and mTR^+/− mice with short telomeres have lower nadir and lower day 12 white counts as compared with wild-type mice. (G) Percentage of bone marrow cells identified by SLAM markers (CD150 and CD48) that are c-kit⁺. Young mice with short telomeres (2 mo), including wt^∗ mice, have increased frequency of SLAM-c-kit⁺ cells as compared with age-matched wild-type mice. This increase is similar to the expansion of this compartment in older wild-type mice (12 mo). Mice were examined at 2 mo of age, and each experiment included at least five mice per genotype. p values < 0.05 are indicated by ^∗, < 0.01 by ^∗∗. Error bars represent standard error of the mean.

Figure 2

wt^∗ Mice Have Lymphocyte Defects that Mirror Immunosenescence (A and B) Absolute B cell lymphopenia in the spleen (A) is associated with low serum IgM levels (B). (C) Fold increase over baseline in antigen-specific IgM 1 wk after immunization. Baseline IgM levels were measured in each mouse 3 days prior to injection. (D and E) Absolute T cell lymphopenia in splenocytes is skewed and favors loss of CD4 cells (D), as shown by the decrease in CD4/CD8 ratio (E). (F–J) T cells from wt^∗ and mTR^+/− mice with short telomeres fail to expand after CD3-CD28 stimulation, as quantitated by the MTT assay (F). This failure to expand is associated with an increase in the proportion of Annexin-V-positive apoptotic cells (G), as well as a decrease in the proportion of live cells in S-phase (I). Representative flow cytometry plots for the quantitation shown in (G) and (I) are show in (H) and (J), respectively. Mice were examined at 3–6 mo of age.

Figure 3

The Short Telotype Predicts Survival (A) Kaplan-Meier survival plot of mTR^−/− mice that died prematurely. Median survival for wild-type, KO_G2, and KO_G10 mice was 476, 212, and 51 days, respectively (p < 0.0001 for both compared to wild-type; log-rank test). (B) Median survival of mTR^−/− mice is predicted by the generation number, a surrogate for telomere length with R² as shown (p < 0.0001, Pearson's test). (C) Survival plot of heterozygous mice that died prematurely. Median survival was 464, 292, 175, and 108 days for HG7–HG10 mice, respectively (p = 0.6, 0.006, and 0.003 for HG8, HG9, and HG10 compared with HG7, respectively; log-rank test). (D) Mendelian ratio of mTR^−/− pups from mTR^+/− parents in successive generations (total n = 3091 pups examined). By the tenth generation of breeding, 9% of pups had the mTR^−/− genotype, in comparison to an expected 25% (p = 0.004; chi-square test).

Figure 4

Histopathology Identifies Mucosal Defects and Bone Marrow Failure as the Cause of Death in Mice with Short Telomeres (A) Photomicrographs of spleen histology in wild-type and mTR^−/− mice showing effacement of the normal white-red pulp architecture and replacement with myelo-erythropoiesis as evidenced by the presence of megakaryocytes (arrows) in the inlet at 200X. (B) Representative photomicrograph of typhlocolitis lesion seen in mTR^−/− mice. Compared to wild-type mice (left panel), the lamina propria and submucosa is infiltrated by neutrophils in the cecum, indicating an ongoing infection (right). There was evidence of colitis in 100% of mTR^−/− mice examined (8 of 8). (C) Micrographs from mid-intestine showing gastrointestinal mucosal defects in wt^∗ mice. Compared with wild-type mice, in which no abnormalities were ever seen (0 of 9), wt^∗ mice had evidence of villous blunting (3 of 6). In mTR^+/− and mTR^−/− mice with short telomeres, more-severe defects were noted, including severe villar blunting adjacent to areas of crypt hyperplasia, as well as regions of complete villous atrophy and crypt loss, which were present in all mTR^−/− mice examined, as illustrated.

Figure 5

Wild-Type Telomerase Elongates Telomeres Incrementally across Generations; Example of mTR^+/− HG4 Family Is Shown (A) Breeding scheme of wt^∗ mice with nomenclature. (B–D) Frequency distribution of telomere length as examined by qFISH on metaphase splenocytes for each wt^∗ generation, compared with true wild-types, are shown in each panel (n = 2 mice per group). Degenerative phenotypes resolve with successive telomere elongation, as shown by an increase in testes weight (C) and the decreasing frequency of aberrant tubules in (D). The number of examined mice is shown below each column, and testes weight was corrected for body weight. Mice were examined at 12 mo of age.