Cellular senescence is a state in which cells stop dividing but remain metabolically active. Moreover, they usually express a pro-inflammatory secretome, upregulate immune ligands and are positive for specific activities such as senescence-associated b-galactosidase (SA-bgal). Cells can enter senescence after DNA damage and activation of oncogenes, for example, to prevent cell proliferation. Recently, senescence has been linked to aging and aged-related pathologies, as in many species senescent cells accumulate with aging. In mammals, our limited regenerative abilities are further compromised with age. A recent report indicates that the decline in muscle regeneration with age could be related to an increase of cellular senescence. But what happens in those other vertebrates such as amphibians capable of regenerating repetitively over most part of their lifespan? Very few studies have addressed the regulation of senescence and its relationship with regeneration in those regeneration-competent species. Now, a recent paper from the laboratory of Maximina Yun (http://www.ncbi.nlm.nih.gov/pubmed/25942455) reports on cellular senescence during amphibian limb regeneration.
First, the authors set up a system to identify senescent cells in cell culture and tissue sections in salamanders. As senescence stops the proliferation of damaged or dysfunctional cells they use UV irradiation to induce DNA damage leading to senescence. Twelve days after irradiation about 80% of newt A1 cells entered a senescent state characterized, among others, by high levels of SA-bgal activity, sustained production of reactive oxygen species (ROS) and extended mitochondrial and lysosomal networks. Also, they acquired a secretory phenotype. In contrast to quiescent cells, these senescence ones did not re-enter the cell cycle upon serum stimulation. All these traits observed in salamander senescent cells were comparable to those previously observed in mammalian cells.
Next the authors analysed cellular senescence in vivo in normal and regenerating newts. During regeneration they observed a significant induction of cellular senescence during the intermediate stages of this process. However, the number of senescent cells decreased at later stages. Senescent cells were found at the amputation plane and within the blastema and included different cell types such as cartilage, muscle, fibroblasts and epidermal glands. Similar results were observed in axolotls, another amphibian model for regeneration. Remarkably, this induction and posterior disappearance of senescent cells was specific of regeneration, as it was not observed during normal limb development.
Salamanders can go through multiple consecutive rounds of regeneration so the authors checked the senescence response over repetitive events of amputation and regeneration. Interestingly, no accumulation of senescent cells was observed after five regeneration cycles over a period of 1.5 years. These results indicate that senescent cells were effectively eliminated during each round of regeneration. Moreover, whereas in other species, including mammals, senescent cells accumulate with age the authors found that senescent cells in heart, spleen and liver did not accumulate in older salamanders. These results suggest that some mechanism of senescent cell clearance functions in both normal and regenerating salamanders. In order to determine whether such mechanism really exists, the authors implanted either senescent cells or normal cells labelled with GFP within newt limbs and followed them over time. Implanted normal cells persisted for at least 40 days and contributed to different structures. However, 80% of the implanted senescent cells were cleared after 2 weeks. These results suggest that salamanders have an active mechanism to get rid of senescent cells. A remarkable observation done by the authors was that when a mixture of 1:1 senescent and normal cells was implanted both cell populations were cleared with time. The reason for that is that these salamander senescent cells were capable to induce senescence in the neighbour cells (a property of the senescence cells seen in other systems). The authors showed here that this effect was mediated by a paracrine factor.
Finally, the authors tried to better characterize this clearance mechanism. Based on previous results on the role of the immune system in both the clearance of senescent cells in other systems as well as in amphibian regeneration, they analysed the role of macrophages in their system. First, they saw that, in vivo, macrophages and senescent cells are in close proximity during limb regeneration. The implantation of senescent cells triggers the recruitment of macrophages to their vicinity, which was not observed when normal cells were implanted. Then, after macrophage depletion, these implanted senescent cells remained over time and were not cleared. Therefore, these results indicate that macrophages were actively involved in the clearance of senescent cells during limb regeneration.
Overall, this study shows for the first time that salamanders possess a senescence surveillance mechanism that operates during regeneration. Remarkably, senescence is strongly induced in the regenerating blastema at mid stages of regeneration. Future experiments should try to determine the biological significance of this senescence upregulation for a successful regeneration.