tp53 mutation rescues the premature infertility of tert-/- zebrafish
Loss of male fertility is one of the early phenotypes of zebrafish aging in both wild type and terthu3430/hu3430 mutants (tert-/- in this study) [13]. tp53M214K/M214K homozygous mutants (tp53-/- in this study) rescues the proliferative decline of testis in tert-/- zebrafish, resulting in partial restoration of tissue integrity [22]. To test whether tp53 mutation had a functional effect on tert-/- zebrafish fertility, we have conducted fertility assays over their lifespan. We selected 6 months-old as the earliest time point, outcrossed mutant males with wild type females and evaluated the percentage of fertilized eggs. Consistent with our previous reports, by the age of 6 months, tert-/- males were almost completely infertile (Fig. 1A, N = 7, wt vs tert-/- p < 0.0001, tp53-/- vs tert-/- p < 0.0001, tert-/- vs tert-/- tp53-/- p < 0.0001). In the tert-/- tp53-/- background, fertility at the age of 6 months was slightly lower than that of wild type siblings (Fig. 1A, N = 7, wt vs tert-/- tp53-/- p = 0.0149, tert-/- tp53-/- ns p = 0.1635). However, approximately 60% of the eggs laid by wild-type females were still fertilized, indicating a clear functional rescue of the male infertility observed in tert-/- males (Fig. 1A, N = 7, tert-/- vs tert-/- tp53-/- p < 0.0001).
At 9 months of age, less than 3% of eggs laid by wild type females were fertilized by the tert-/- males, indicating a severe loss of fertility (Fig. 1B, N = 4–7, wt vs tert-/- p < 0.0001, tp53-/- vs tert-/- p < 0.0001). In contrast, 9 month-old tert-/- tp53-/- males maintained their fertility, with ca. 70% of eggs successfully fertilized (Fig. 1B, N = 4–7, wt vs tert-/- tp53-/- ns p = 0.2834, tp53-/- vs tert-/- tp53-/- ns p = 0.5225, tert-/- vs tert-/- tp53-/- p < 0.0001). We also examined the fertility of 12 month-old males and tert-/- tp53-/- now showed a significantly reduced fertility when compared to wild type and tp53-/- siblings. (Fig. 1C, N = 5–7, wt vs tert-/- tp53-/- p = 0.0013, tp53-/- vs tert-/- tp53-/- p = 0.0002). Approximately 20% of the eggs laid by wild type females were fertilized by tert-/- tp53-/-, although the difference was not statistically significant from tert-/- siblings (Fig. 1C, N = 5–7, tert-/- vs tert-/- tp53-/- p = 0.4266).
tp53 mutation reduces tert-/- premature aging phenotypes
The rescue of male fertility prompted us to investigate whether tp53 mutation improved aging phenotypes, such as kyphosis and cachexia, both related to muscle tissue atrophy. Starting from 12 months of age until time of dead, we monitored the incidence of these phenotypes. At 12 months of age, ca. 52% of tert-/- zebrafish developed cachexia and/or kyphosis (Fig. 2A,B N = 12–28, wt vs tert-/- p < 0.0001, tp53-/- vs tert-/- p < 0.0001, tert-/- vs tert-/- tp53-/- p < 0.0001). This increase in incidence of aging phenotypes was accompanied by weight loss of tert-/- zebrafish by a mean weight of ca. 0.4 grams compared to ca. 0.55 grams in wild type and tp53-/- siblings (Fig. 2C N = 12–28, wt vs tert-/- p = 0.0039, tp53-/- vs tert-/- p = 0.0162). In the tert-/- tp53-/- background, cachexia and kyphosis incidence was ca. 13% and significantly lower than the tert-/- siblings (Fig. 2B N = 12–28, tert-/- vs tert-/- tp53-/- p < 0.0001). Furthermore, tert-/- tp53-/- zebrafish exhibited higher body weights than the tert-/- siblings, even though it did not reach the statistical significance.
tp53 mutation does not reduce tert-/- chronic inflammation.
Low grade sterile inflammation is a recognized feature of aging and another premature phenotype of tert-/- zebrafish. To test whether tp53 mutation could attenuate inflammation in tert-/- zebrafish, we examined the expression of genes related to type I interferon response and senescence associated inflammatory phenotype (SASP) in 12-month-old zebrafish. We chose the head kidney because it hosts the adult hematopoietic system and give rise to blood cells that regulate the communication between organs. In tert-/- zebrafish, expression levels of isg15 (Fig. 3A, N = 5–6, wt vs tert-/- p = 0.0062, tp53-/- vs tert-/- p = 0.0116) and ifn-i (Fig. 3B, N = 5–6, wt vs tert-/- p = 0.0493, tp53-/- vs tert-/- p = 0.0312) were significantly higher compared wild type and tp53-/- siblings. Similarly, mmp15a expression levels were elevated in tert-/- zebrafish (Fig. 3C, N = 5–6, wt vs tert-/- ns p = 0.0772, tp53-/- vs tert-/- ns p = 0.0896). In contrast to previous results, tert-/- tp53-/- zebrafish expression levels of isg15 (Fig. 3A, N = 5–6, wt vs tert-/- tp53-/- ns p = 0.0571, tp53-/- vs tert-/- tp53-/- ns p = 0.0988), ifn-i (Fig. 3B, N = 5–6, wt vs tert-/- tp53-/- ns p = 0.0558, tp53-/- vs tert-/- tp53-/- p = 0.0356) and mmp15a (Fig. 3C, N = 5–6, wt vs tert-/- tp53-/- ns p = 0.1301, tp53-/- vs tert-/- tp53-/- p = 0.1494) were higher compared wild type and tp53-/- siblings.
tert-/- reduces tumor incidence of tp53-/- zebrafish
During our observation of aging zebrafish, we noticed that tp53-/- zebrafish started developing macroscopically visible tumors from the age of 11 months. By the time they reached 18 months of age, ca. 65% of tp53-/- fish had developed visible tumors (Fig. 4A, N = 36–44, wild type vs tp53-/- p < 0.0001). The incidence of cancer was 30% in tert-/- tp53-/- (Fig. 4A N = 36–44, wild type vs tert-/- tp53-/- p = 0.0005) and 15% in tert-/- zebrafish (Fig. 4A, N = 36–44, wild type vs tert-/- ns p = 0.0720). However, histological assessment revealed that macroscopic assessment of tumors underestimated the true tumor incidence in terms of incidence and onset. Nevertheless, all four genotypes exhibited a similar trend in susceptibility to tumor incidence. Approximately 10% of tert-/- zebrafish developed tumors, exclusively carcinomas, (Fig. 4B, N = 59), while tumor incidence was ca. 20% in tert-/- tp53-/- zebrafish (Fig. 4B, N = 21). In tert-/- tp53-/- zebrafish, the majority of tumors were sarcomas (Fig. 4B, N = 3/4). Consistent with the macroscopic assessment, tp53-/- fish had the highest tumor incidence, approximately 30%, exclusively sarcomas (Fig. 4B, N = 22).
tert-/- rescues tp53-/- reduced lifespan
Telomerase deficiency and dysfunctional telomeres result in reduced lifespan [11, 12, 13, 15]. Inhibition of p53 via pifithrin-alpha (PFTa) and morpholinos against tp53 were shown to increase median life span of G2 tert-/- zebrafish larvae [15]. Consitent with previous work, tert-/- zebrafish ehxibited a shorter lifespan with a median of 15.5 months (Fig. 5, N = 28–34, wt vs tert-/- p = 0.0004). Unexpectedly, in contrast to G2 tert-/- larvae experiments, tp53-/- fish had the shortest life span, with a median of 14 months (Fig. 5, N = 28–34, wt vs tp53-/- p < 0.0001), primarily due to malignant tumors. Strikingly. after the wild type fish, tert-/- tp53-/- zebrafish had the longest lifespan, with the median of 20.5 months (Fig. 5, N = 28–34, wt vs tert-/- tp53-/- ns p = 0.1175, tert-/- vs tert-/- tp53-/- p = 0.0441, tp53-/- vs tert-/- tp53-/- p = 0.0004). Overall, our data suggest that the molecular phenotypes associated with telomerase deficiency, which are rescued by the loss of p53, contribute to the rescue of fertility, cachexia and kyphosis. However, the loss of p53 increases tumor incidence in tert-/- zebrafish, leading to earlier onset of cancer. Interestingly, telomerase deficiency improves the short lifespan of tp53 mutants, and vice versa, indicating a more complex mechanism of action for than initially thought.