miRNAs are small non-coding RNA molecules that play important functions in transcriptional and post-transcriptional regulation of gene expression. miRNAs have been shown to regulate many important biological processes and be linked to several diseases, cancer and neurogenesis, among others. Moreover, miRNAs have been related to liver and heart regeneration. Now a recent paper from the laboratory of Karen Echeverri reports on the function of miR-125b in spinal cord regeneration in axolotls (http://www.ncbi.nlm.nih.gov/pubmed/24719025).
Whereas in mammals the glial scar inhibits axonal regeneration after spinal cord injury, axolotls have no problems in regenerating their spinal cord. Importantly, no glial scar is detected in axolotls after injury. The rationale behind this study was to concentrate on the environment of the injury site in order to analyze whether intrinsic differences could help to explain the differences between the regenerative capacities shown by axolotls and mammals. That is, the authors tried to identify molecules that promote a regeneration-permissive environment in axolotls.
Previous works by the same research group had focused on analyzing the role of miRNAs in axolotl tail regeneration. Here, they carried out a comparative miRNA profiling analysis at different time points after spinal cord injuries in rats and axolotls. The authors chose 1 and 7 days after complete transection of the spinal cord. Of the approximately 4,000 miRNAs of several vertebrate species included in their array, they found 14 showing significant differences after injury in axolotl versus rat. One of the identified miRNAs was miR-125b, previously related to cancer and stem cell differentiation. Before injury, miR-125b expression in axolotl was concentrated mainly in radial glial cells. In rats, also in an uninjured animal, astrocytes had the highest level of expression; however, in this uninjured situation miR-125b showed 8-fold higher expression in axolotls than in rats. One day after amputation the levels of miR-125b in axolotls decreased about 40%, whereas in rats this decrease was less than 1%. The authors hypothesized that this strong and early downregulation of miR-125b in axolotls could be contributing to creating a permissive environment for regeneration. To test it they analyzed how regeneration proceeded after modulating the levels of miR-125b. The overexpression of miR-125b resulted in an aberrant sprouting of the axons on both sides of the injury together with a reduced growth of the axons through the lesion site by 7 days post-injury, when controls showed normal full regeneration. On the other side, the inhibition of miR-125b in axolotls (up to levels similar to those observed in rats) strongly inhibited also axonal regeneration. Interestingly, this inhibition resulted also in a significant deposition of fibrin that reminded the glial scar found in rats. These results suggested that the levels of miR-125b must to be tightly regulated to allow regeneration in axolotls.
Next, they carried out bioinformatics analyses to try to identify putative target genes regulated by miR-125b. Among them, they identified a homologue to Sema4D, a gene that is upregulated in mice at the wound site after spinal cord injury. In uninjured axolotls Sema4D protein was not detected in glial cells, although it was transiently upregulated at 3 days after injury. On the other side, the inhibition of miR-125b resulted in the upregulation of Sema4D at the injury site; conversely, the overexpression of miR-125b decreased the levels of Sema4D. These results suggested that the regulation of Sema4D levels by miR-125b might play an important role during regeneration. In fact, the overexpression of Sema4D at the injury site inhibited axonal regeneration in axolotl. Then, the authors analyzed the relationship between miR-125b, Sema4D and regeneration in rats. To do that, they modulated in vivo the levels of miR-125b after spinal cord injury. By 7 days post-amputation the levels of miR-125b significantly decreased whereas Sema4D levels increased. The overexpression of miR-125b resulted in a reduced expression of Sema4D, further supporting the results obtained in axolotls. Remarkably, these animals showed improved locomotive abilities compared to controls, reduced glial scar formation and increased number of axons projecting into the scar tissue.
Finally, because the overexpression of miR-125b probably affects the expression of many genes other than Sema4D, they performed microarrays analyses to compare control versus treated animals overexpressing miR-125b. Remarkably, they found that many genes involved in glial-scar formation were downregulated, whereas genes related to neurite outgrowth were upregulated after overexpressing miR-125b.
Taking all the results together, the authors propose a model in which miR-125b would promote a regeneration-permissive environment by downregulating the expression of glial-scar genes and other genes such as Sema4D and, at the same time, inducing positive factor for axonal regrowth. The further characterization of this miRNA-regulated pathway in axolotls and rat can uncover fundamental differences between these two species and help us to understand their different regenerative capabilities.