Researchers at the National Brain Research Centre, Gurgaon, have experimentally demonstrated how neurons that are injured or damaged can be functionally restored by fusion of the severed axons. Neurons can break during accidental injury and day-to-day stress-induced injury. Carpel tunnel syndrome is a typical case of peripheral nerve damage arising from several severed axons.
Neuronal damage arises when the axons are severed into distal and proximal fragments. Experiments carried out on Caenorhabditis elegans by a team led by Anindya Ghosh-Roy from NBRC has now shown how the touch sensation of the worms is restored when the two severed axon fragments fuse. The results were published in the Proceedings of the National Academy of Sciences.
“This is the first detailed cell biological study to show the basis of functional regeneration of damaged neurons in C. elegans, ” says Dr. Sandhya Padmanabhan Koushika from the Department of Biological Sciences at the Tata Institute of Fundamental Research (TIFR), Mumbai, and one of the authors of the paper.
They also found that functional recovery takes place in an age-dependent manner, with better recovery in the late larval stage and less recovery in adult worms.
The researchers used two femtosecond lasers to locate and cut the axons involved in touch sensation. During regeneration, only the axons that successfully fuse with their distal counterparts contribute to functional recovery. “There is no fusion, there is no functional recovery,” Dr. Ghosh-Roy says.
The researchers genetically proved that three molecules — ced-7, psr-1 and eff-1 — are essential for fusion. “In this study we showed that mutants lacking any of these three molecules show normal axon growth but fusion of the axons is perturbed. As a result, the touch sensation is not restored,” says Atrayee Basu, a graduate student from NBRC and the first author of the paper.
Dr. Ghosh-Roy’s team has experimentally shown that let-7 mutants (worms that do have let-7 microRNA that is present in neurons) have higher amounts of ced-7 molecule. The ced-7 molecule is important for the recognition of the proximal and distal ends of the injured neurons. “We have molecularly shown that the level of ced-7 is increased in the mutants. This leads to enhanced functional restoration,” he says.
Effect of age
In humans, the ability to regenerate neurons gets reduced with age; the researchers have made similar observations in worms. In older worms, even when fusion takes place the functional restoration does not happen. It could be because the fusion is not complete.
The researchers used synaptic vesicles that travel from one end of the axon to the other to see if incomplete fusion prevents the vesicle movement or reduces the amount of vesicles travelling across the point of fusion to the distal axon.
“In larval stage we could see the synaptic vesicle movement across the point of fusion. But in adults, due to aging, only very few axons show vesicles moving from one end to the other. So the cytoplasmic continuity is compromised in the adult stage,” says Dr. Koushika.
Complete fusion needed
The age-related decline in functional restoration is overcome in let-7 mutants. “In mutants, the vesicle transport is maintained in adults indicating that the fusion is complete; the mutants show functional restoration unlike in wild-type worms of the same age,” says Dr. Ghosh-Roy.
The researchers carried out experiments to find out why despite fusion the cytoplasmic continuity is compromised in adults stage. They found that the amount of eff-1 protein at the tip of the growing axon is high in the larval stage. But in adult worms, the eff-1 enrichment is reduced.
In let-7 mutants, the amount of eff-1 protein at the tip of the axons in adult worms is high and this improves the cytoplasmic continuity.
Functional recovery after injury might come into play after spontaneous breakage of axonal process during day-to-day stress induced injury.
The finding that the fusion of the severed axons can be genetically improved raises hopes for treating nerve injuries in human in future.
