In C. elegans, the functional recovery of the damaged neurons happens only when proximal axons successfully fuse with their distal counterparts. A team led by Dr. Anindya Ghosh-Roy from the National Brain Research Centre, Gurgaon, found that the ability to regenerate neurons gets reduced with age as the fusion is not complete. The work raises hopes for treating nerve injuries in humans by genetically silencing a specific microRNA.
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. While we might not realise the damage to a few neurons in the skin, neuronal injury on a larger scale can lead to loss of senses or in the case of the spinal cord injury even locomotion.
Neuronal damage arises when the axons are severed into distal and proximal fragments. While fusion of these fragments has been observed, the functional significance has not been clear till now. Experiments carried out on Caenorhabditis elegans by a team led by Dr. Anindya Ghosh-Roy from NBRC has now shown how the touch sensation of the worms is restored when the proximal part of the axon fuses with the distal part of the axon. 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 functional recovery of the damaged axons drops sharply on day four in the case of C. elegans. The adult worms live for about three weeks,” says Dr. Ghosh-Roy. Dr. Ghosh-Roy is an Intermediate Fellow of the Wellcome Trust DBT India Alliance.
The researchers used two femtosecond lasers to locate and cut the axons involved in touch sensation. There are two touch sensation axons in the right and left side of the worm. While the touch sensation persists when one of the two axons is injured, it strongly reduces when both the axons are cut.
During regeneration, only the axons that successfully fuse with their distal counterparts contribute to functional recovery. Regrowth of the proximal axons without fusing with the distal axon will not result in functional recovery, they found. “There is no fusion, there is no functional recovery,” Dr. Ghosh-Roy says.
While it is already known that three molecules — ced-7, psr-1 and eff-1 — are essential for fusion, the researchers genetically proved it. “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.
let-7 mutants show enhanced functional restoration
A microRNA molecule let-7, whose is present in neurons, helps in the transition from the larval stage to the adult stage in C. elegans. It promotes the maturation process in the worms. In the absence of let-7, the worms remain as juveniles and the neurons remain at a younger stage.
Dr. Ghosh-Roy’s team has experimentally shown that let-7 mutants (worms that do have let-7 microRNA) 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, which is one of the three molecules that regulates the axon fusion, is increased in let-7 mutants. This leads to enhanced functional restoration,” he says.
Effect of age on functional recovery
In humans, the ability to regenerate neurons gets reduced with age. Now, these researchers have made similar observations in worms. They found that in older worm, even when fusion takes place the functional restoration does not happen. It could be because the fusion is not complete and the cytoplasmic continuity is lacking.
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 into the distal portion from the proximal [after travelling across the point of fusion] confirming cytoplasmic continuity. But in adults, due to aging, there are fewer vesicles moving, very few axons show connection between the distal and proximal injured parts and there is less vesicle movement in the axons that appear to be fused. 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. Due to complete fusion, 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. The elevated levels of eff-1 protein at the axon tips in adults improve the cytoplasmic continuity.
While axon fusion might not help recover the lost function after large injury, as several axons get severed, this phenomenon might come into action after spontaneous breakage of axonal process during day-to-day stress induced injury. According to Dr. Ghosh Roy axon fusion process might be very crucial in the maintenance of neuronal integrity throughout the life span of an organism. The finding that functional recovery can be genetically improved by silencing the let-7 microRNA raises hopes for treating nerve injuries in humans.