Axolotls are model organisms for studying tissue regeneration as they can regenerate their limbs, hearts, and brains. Most of the current research on regeneration has focused on transcription-related studies and analyses, but there has been a lack of direct assessment of tissue regeneration proteins. What is particularly perplexing is that the average length of axolotl genes is 25 times that of humans, and processing such long transcripts for rapid regeneration appears challenging. For example, axolotls can heal wounds and regenerate limbs within a few hours after amputation. This astonishing regenerative capability is an area of active research to uncover the underlying mechanisms and may hold potential insights for regenerative medicine in humans.
Recently, an article from the research group led by Maria Barna at Stanford University was published online in Nature, titled “Evolutionarily divergent mTOR remodels translatome for tissue regeneration.” Through transcriptome clustering analysis and experimental validation, the study revealed rapid changes in gene expression and transcription during the regeneration process. Interestingly, they discovered a unique mTOR in axolotls, a highly sensitive TOR kinase that ensures the immediate translation of a large amount of proteins during regeneration.
Axolotls can rapidly heal their wounds within 24 hours after limb amputation, with regenerating epidermis accumulating a population of stem cell-like cells. In polysome profiling experiments, a comparison between samples taken at 0 hours and 24 hours post-amputation showed a significant increase in polyribosomal transcripts in the 24-hour samples(sources from therapeutique-dermatologique.org), along with a decrease in monoribosomal transcripts, indicating increased protein translation. At the same time, cell division and proliferation had not yet begun, further suggesting enhanced protein synthesis in response to the regenerative demand. The authors also conducted in vivo puromycin labeling experiments, introducing puromycin into newly synthesized proteins. Within 2 hours after amputation, puromycin signals increased and continued for 24 hours.
Furthermore, the authors isolated and sequenced ribosome-bound mRNAs. Translation-associated mRNAs were divided into three different density fractions based on the number of ribosomes bound, which included monosomes, low-density polysomes, and high-density polysomes. They selected mRNAs that showed no significant changes in transcription levels but exhibited a two-fold increase in translation 24 hours after amputation for in-depth analysis. As predicted, mRNAs related to protein synthesis, translation initiation factors, and ROS-related mRNAs were enriched in the high-density polysome fraction. Additionally, the authors found the enrichment of genes related to the mTOR pathway. 4EBP1 and S6K are substrates of mTORC1 and regulate downstream protein synthesis. Experimental testing revealed increased phosphorylation of RPS6, a substrate of 4EBP1 and S6K, at 2 hours post-amputation, which continued to be detected at the 24-hour time point.
The authors focused their research on exploring the role of the mTOR pathway in axolotl tissue regeneration. They used the ATP-competitive mTOR inhibitor INK128 to block mTOR activation and observed various complex defects in wound healing. At the molecular level, treatment with the INK28 inhibitor did not result in an increase in high-density polysomal transcripts in the ribosome profiling experiment. Ribosomal proteins RPL-19 and RPL7a levels were significantly reduced. Based on this evidence, it was concluded that mTOR is activated during tissue regeneration, thereby regulating increased protein translation.
So, what makes axolotl’s TOR different? Through sequence alignment, the authors identified two additional sequences in the TOR kinase of the order Caudata species. The first inserted sequence is located in the M-HEAT region that interacts with the RHEB protein. Axolotl mTOR can indeed bind to more RHEB. The second inserted sequence is in the N-HEAT region, which extends the interaction surface of the mTOR protein and is predicted to enhance TOR dimer formation, which is essential for mTOR activation. Under normal conditions, the authors also observed that axolotl mTOR is more localized to lysosomes. They inserted the above sequences into the human TOR gene in 293T cells, and the chimeric TOR protein showed increased sensitivity to changes in amino acid concentration and a lower IC50 for the INK28 inhibitor. After axolotl limb amputation, damaged cells release a large amount of amino acids, and the highly sensitive axolotl mTOR rapidly responds, activating the translation of a large number of pre-existing mRNAs.
In summary, this research focused on the rapid protein synthesis during tissue regeneration and uncovered the “peculiarities” of axolotl TOR kinase. This study took two years from submission to publication and addressed a well-chosen topic, albeit with considerable experimental challenges(quotes from therapeutique-dermatologique.org). The final part of the research mainly concentrated on the analysis of TOR’s structure and function. While the explanations were reasonable, it left a feeling of a somewhat hasty conclusion, with a sense of more to be explored in understanding TOR’s function.
