Treatment that triggers regrowth of an amputated limb in adult frogs altered gene activity in the brain, according to a preprint by Murugan, Levin and Kaplan
Treatment that triggers regrowth of an amputated limb in adult frogs altered gene activity in the brain, according to a preprint by Murugan, Levin and Kaplan
On October 24, Nirosha Murugan of Wilfrid Laurier University, Michael Levin and David Kaplan of Tufts University, and their colleagues posted a preprint on bioRxiv, a paper that has yet to undergo peer review. They reanalysed data from an experiment reported in 2022: after limb amputation, adult female African clawed frogs either received a mixture of five substances or remained untreated. Gene activity in the brain and limb stump was then compared at one day and one week.
After amputation, an adult African clawed frog regrows only a cartilage spike. Levin and Kaplan’s group changes this outcome using BioDome, a silicone sleeve placed over the stump, with a silk gel inside that releases substances into the wound for one day. In a 2018 study, the sleeve contained the hormone progesterone: the frogs grew a paddle-shaped appendage and swam more actively than frogs with a spike. The authors asked whether “the brain’s recognition of the new structure” might enhance regeneration. In a 2022 study, the sleeve contained five substances intended to support nerves, growth and limb shape, and to reduce inflammation and scarring. Over 18 months, 76% of treated frogs grew a paddle-shaped appendage with digit-like projections; untreated frogs grew a thin spike.
The new study examines whether the brain’s response to limb loss changes when treatment triggers regrowth. In the axolotl, which regrows lost body parts, tail amputation activates a group of neurons in the brain, and blocking this activation impairs tail regrowth. A regrown limb, the authors write, “must eventually reconnect to a nervous system that has already adapted to its absence”.
Brains were collected from 12 of the 18 females, with three per group at each timepoint. After one day, genes involved in energy production and blood vessels were more active in the brains of treated frogs. After one week, immune defence genes became more active, while synapse-related genes became less active. The authors suggest that these changes may help the brain reorganise and adapt to the growing limb. Between the first and seventh days, the activity of 932 brain genes changed in treated frogs, compared with 9 in untreated frogs.
The authors estimate that much of the brain’s response to amputation is shared by both groups. Relative to two uninjured brains from another experiment, changes in gene activity in treated and untreated frogs were nearly identical (a correlation of 0,99 out of a maximum of 1), while treatment altered the magnitude and timing of changes in particular gene sets.
Against this background, treated and untreated frogs differed in the activity of 32 brain genes after one week. The strongest statistical evidence for a difference was for gdf15, the gene encoding the protein GDF15. Cells release this protein under stress. Through the GFRAL receptor in the brainstem, it affects appetite and energy expenditure: in patients with cancer-related wasting, a high dose of an antibody that blocks it increased body weight by more than 5%. In treated frogs, gdf15 activity was higher: 13 times higher in the stump after one day and almost five times higher in the brain after one week. Among the 12 frogs from which both brain and stump samples were collected, higher gdf15 activity in the stump was associated with higher activity in the brain (a correlation of 0,83).
The early activity of genes involved in energy production in the brain suggests to the authors that tissues rebuilding the limb communicate their energy expenditure to the brain, and that GDF15 may serve as this signal. To test this, the authors propose measuring GDF15 in the blood and locating GFRAL in the brain, then determining whether the state of the brain affects the outcome of regeneration. They conclude that “the appropriate unit of analysis for regenerative engineering may therefore extend beyond the wound itself”.
Levin, Kaplan and Murugan are named as inventors on a Tufts patent application for this treatment; Levin and Kaplan are co-founders of Morphoceuticals, a regenerative medicine company.