The regeneration of nerves throughout the human body has been a subject of much interest in biology recently. The unique characteristics of both the central nervous system (CNS) and the peripheral nervous system (PNS) provide insights into the mechanisms occurring after once damage occurs. A good understanding of tThe differences between these mechanisms also may lead to new therapies for nerve-damaged patients, including stroke patients. Following injury, the nerves in the CNS of an adult mammal will not regenerate, while the PNS will. These differences appear in every stage of axon regeneration.
Within hours of axotomy, the PNS begins regeneration, ultimately growing neurons towards their targets (Bradke et al, 2012). After a crushing injury to a peripheral nerve, axons successfully form new growth cones and begin directed regrowth. However, the opposite can be said for CNS axons. Instead, Aafter injury, the CNS axons retract. They and also develop swollen endings that are called retraction cones, or “frustrated growth cones,”, due to their inability to form proper growth cones and continue with regeneration (Bradke et al, 2012). The differences in the responses between CNS and PNS nerves are due to many factors. The iIntrinsic properties of the CNS axons, a growthn inhibitory environment, and a lack of neurotropic factors all play a role in inhibiting the growth of the axon.
Within Research advances in recent experimentsyears focused in, three areas have worked to further the field of axon regeneration. Studying the effects of the environment on the growth cone, axon growth, and the small GTPases involved in inhibiting regrowth have significantly aeffected the knowledge of axon regeneration.
Growth Cone and Environmental Factors
The first area that has been examined to in effect axon regeneration is the formation of a growth cone. Growth cones regulate neurite extension and sense guidance cues during development. After injury, the formation of a growth cone at the tip of a transected axon is a crucial step during subsequent axonal regeneration. The tips of lesioned axonal stumps located in the PNS transform into growth cones of stereotypic spiky morphology capable of sustained growth. On the contrary, lesioned CNS axons form swellings termed “retraction bulbs” at the tip of their proximal stumps, which are oval structures and lack a regenerative response (Ertürk, 2007).
One of the biggest most intense areas of research is the formation of the new growth cone after axotomy. Initially Immediately after an injury, calcium provides is a big key factor in promoting growth cone formation. An influx of calcium into the axon has been shown to promote sealing of the membrane and transformation of the cytoskeleton to form a growth cone or retraction bulb (Bradke et al, 2012). When a neuron is sliced, the inactive voltage gated calcium channels that are spread throughout the axon become activated in response to the environment. These channels then work to pump calcium that initiates mechanisms to seal and rebuild the membrane around the damaged ends. In the experiments by Spira et. al, the kinetics of the regeneration of proximal and distal axon segments were observed and were shown to correlate to the architectural changes in the axon (Spira et al, 1993). Through inserting microelectrodes into the soma and axon of the neuron of metacerebral neurons, they were able to manipulate the transmembrane potential. Theis changes in potential correlateds to the influx of calcium into the system after axotomy of a neuron. They discovered that this electrophysiological component of the axon’s regeneration is important for the physical changes in the axon. Rapid repolarization was is associated with the formation of a membrane seal over the cut ends by the constriction and subsequent fusion of the axolema. Prior to the formation of a membraneous barrier, electron-dense deposits aggregate at the tip of the cut axon and appear to form an axoplasmic "plug." Electrophysiological analysis revealed that this "plug" does not provide resistance for flow of current flow and that the axoplasmic resistance is homogenously distributed (Spira et al, 1993). These results show that calcium has a direct effect on the regeneration potential of an axon. The voltage-gated channel distribution in the axon directly aeffects the flow of calcium across the membrane and therefore the electric potential of the membrane (Schwab, 2010). Without this change in potential, the axon would not be signaled to change conformation or seal the membrane, integral parts in axon regeneration. This is one key difference between the CNS and PNS nerves discussed before. With the absence of this calcium influx in CNS, the axon fails to grow a new growth cone and instead forms the retraction bulb that will not allow the axons to regenerate.
Another important factor in the formation of the a new growth cone is the microtubule integrity and regrowth. Microtubules form the foundation for axonal shafts and growth cones. They allow for transport of important organelles and stability of the structure. Most importantly, dynamic microtubules protrude through growth cones allowing for axon elongation (Ertürk, 2007). Therefore, stable and active microtubules promote growth in regenerating axons. Again, a difference exists in between the microtubules of CNS nerves compared and to PNS nerves. As expected, the PNS nerves show more dynamic microtubule interaction and more stability of the microtubules after injury. In their experiments, by Ali Ertürk (et al.), they coimmunostained GFP-labeled axonal shafts, growth cones, and retraction bulbs for detyrosinated tubulin using an anti-Glu-tubulin antibody and tyrosinated tubulin using an anti-Tyr antibody as markers for stable and dynamic microtubules, respectively. It was foundThe results showed that in growth cones, the stable mictotubules were aligned in parallel bundles, which added stability to the whole structure. Also, the dynamic tubules used for growth were found in organized bundles that reached to the edges of the growth cone. In the retraction bulbs, howeverin contrast, no organization appeared. There were no parallel bundles in the retraction bulb and even moreover, the presence of the microtubules was not consistent (Ertürk, 2007). Especially These results establish that dynamic mictrotubles were not present in the CNS axon, and were disorganized when present. This means that the misorganization and mislocalization of microtubules is a important contributing factor to the lack of CNS axon regeneration.
AOnce again, another difference is observed between the CNS and PNS axons centers in the next step of regeneration. Following the generation of a growth cone, the cone must migrate through the substrate towards the target site of the neuron.
The experiments by Chierzi (et al.), examines this aspect of axon regeneration. Theiry examinationed included the dorsal root ganglia and the retinal ganglion cells of, PNS and CNS nerves, respectively. The allowed the axons to grow over a permissive surface, then preformed an axotomy and evaluated the ability of the axons to regenerate (Chierzi et al, 2005). Both neurons were cultured in serum-free medium while and nuerites were allowed to grow. After 2-5 days in vitro, the axons were axotomised at least 100 micrometers from their cell bodies. A scratch on the plastic substrate marked the beginning of the axon and the axons were observed at two time points (Chierzi et al, 2005). Also, immunohistochemistry was preformed for tubulin and actin to discover determine where these elements were at different times in the regeneration. After four hours, the dorsal root ganglia had a three times higher growth rate than the retinal cells (Chierzi et al, 2005). Though this may seem like the retinal cells were only delayed in growing, the actin was still present in the axon and even after 20 hours, minimal growth had occurred. This shows that the regeneration was dependent on the ability of the axon to grow, not the latency of the growth (Chierzi et al, 2005).
HoweverFurthermore, the abovese results also speak to the intrinsic nature of the axons. CNS nerves, like such as the retinal nerve of in the experiments, show less regeneration potential as a whole. This inability to regenerate could be the result of the lack of new growth cones or a miscommunication between the growth cone and the environment. Yet, Ooverall, there is a direct correlation between the ability to re-initiate growth cones and complete axonal elongation with in the successful regeneration of the axon.
The role of the ERK and PKA pathways was also examined within these experiments. The dorsal root ganglia were pretreated with inhibitors to the cascades prior to the in vitro axotomy. The ERK pathway was inhibited with U0126, which blocks MEK1 without affecting PKA catalytic activity. This inhibition was shown to counteracted axonal elongation and formation of a new growth cone. Theseis results was were then confirmed with a second ERK inhibitor PD98059, which gave similar results (Chierzi et al, 2005). When the cAMP binding site of PKA was inhibited by rp-cAMPS, similar results were formedobtained. The amount of regenerated axons were was significantly reduced (Chierzi et al, 2005). Since the inhibitors were administered applied right before axotomy, it is clear that the inhibition of these pathways affects axonal elongation. Both the ERK and PKA pathways could be key to communicating with the cytosol at the occurrence of axotomy in PNS nerves to promote regeneration.
GTPases and Regeneration Inhibitors
Finally, the third last major research field in axon regeneration is the study of the glia-derived inhibitory factors in the mammalian CNS. These inhibitory factors are present in the membranes of the oligodendrocytes and 3 bind directly to one of two Nogo receptor complexes on in the membrane of the neurons. The three factors that bind directly are Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). The receptors can be either composed of either Nogo receptor (NgR)·p75 neurotrophin receptor LINGO-1 or NgR·TROY·LINGO-1.The receptor complex then activates the small RhoA GTPase to induce growth cone collapse and exert inhibition of axonal regrowth as well as compensatory sprouting of remaining axons (Lu et al, 2013).
Two components have been examined by Lu (et al.) are the myelin-assoiated glycoprotein (MAG) and Nogo-A (Niederöst et al, 2002). The signaling mechanisms responsible for the transduction of the inhibitory properties of MAG and Nogo-A domains are not well understood. Cytoskeletal components may be required for proper axonal path finding. and Tthe formation of axons and dendrites are is regulated by members of the Rho family, including RhoA, Rac1, and Cdc42. Complex communication between Rho proteins are crucial for this regulation. Rho proteins serve as a molecular switch by cycling between an inactive GDP-bound state and an active GTP-bound state (Lu et al, 2013). In their active state, these GTPases bind characteristic sets of effector proteins. The most important effector of RhoA in the growth cone is probably the serine–threonine kinase Rho-kinase ROCK, a negative regulator of growth cone motility (Lu et al, 2013).
The involvement of the Rho family of small GTPases in the responses of Nig, nogo-66, and MAG, was studied by Miederöst et al., through examining the amount of Rho and Rac present in cells and the growth of neurons exposed to these GTPases. The group found that inactivating endogenous RhoA activity allowed not only more cells to extend neurites, but their outgrowth response was markedly improved on all three inhibitory substrates., Hhowever, the MAG treatment showed the most improvement. To determine whether ROCK was involved in the MAG- or Nogo-A-induced neurite growth inhibition, cerebellar granule cells were treated with various concentrations of a ROCK-selective inhibitor,Y27632. Application of Y27632 negated the inhibitory effect of MAG and significantly improved neurite outgrowth response on NiG and Nogo-66-GST substrates (Niederöst et al, 2002). All of these results show that Rho and Rac play a crucial role in inhibiting neuron regeneration.
This group also studied the effects of C3 transferase had the ability to inhibit the effects of RhoA activity and whether this could protect the neurons. The group found that although the overall organization of microtubules of C3 transferase-treated cells was not significantly changedaffected, they seemed to be more bundled in the central domain of the growth cone. No apparent cytoskeletal reorganization of microtubules was detectable in C3 transferase-treated growth cones that had advanced onto oligodendrocytes, a characteristic which is often not seen in collapsing growth cones (Niederöst et al, 2002) However Tthe effects of thethe differentdifferent Nogo receptor complexes were also examined. The experiments by Yan Lu (et al.) examines the difference between these two receptors. As previously mentioned, the receptors can be either the Nogo receptor (NgR)·p75 neurotrophin receptor LINGO-1 or NgR·TROY·LINGO-1. Using the MASCOT program and a GST precipitation assay, the group determined that RhoGDIα was a cofactor of the TROY receptor, ultimately promoting inhibition of regeneration. It was also found that Nogo-66 enhanced TROY/RhoGDIα interaction. Increasing Nogo-66 increased RhoA activity and neurite outgrowth inhibition (Lu et al, 2013). This might be attributed to the dissociated RhoA from RhoGDIα, which was recruited to TROY. (Lu et al, 2013).
Overall, both the roles of the inhibition factors and receptors have been examined, and. Rho and Rac have been determined to play an important role in inhibiting axonal regeneration, yet the different waysVarious approaches to increase the activity of these GTPases has only not slightly been studieed in detail and deserves future attention.determined.
Future of Axon Regeneration Research
It is clear that more research and experimentation will be necessary to solve the problem of regenerating axons. This solution is necessary to help treat stroke patients and damage to nerves. So far, targeting regenerating axons has focused on three main components: the ability of the growth cone to form, the migration of the axon, and the inhibitory factors present around the nerve. In the future, Hhowever, further and future insights into all three components is necessary.
For growth cone development, the differences between CNS and PNS nerves and the reasons for failure of the growth cone to develop have been determined. However, the process that is defective still needs to be examined in more detail. The development of treatments to improve the probability ofinitiate growth cone regeneration may beis fundamental to induce axonal regeneration in the injured CNS.
Similarly, in the migration and response of the axons, the differences in neurons have been examined. While the stable structure of microtubules and the pathways necessary for regeneration have been examined, further steps advancements are needed to examine how these factors can be manipulated to cause regeneration. A method of for stabilizing microtubules in vivo deserves exploration and study must to be determined and its the ability to induce regrowth must be reexamined. Also, the pathways must be manipulated using various methods, such as gene knockouts. This would not only provide further insights into the exact effects each pathway has, but also would lead to a therapeutic solution to regeneration using these pathways.
Finally, the inhibitory factors around in nerves and the GTPases associated with them require have the greatest necessity attention for in future research and experimentation. While the molecules present on in the membranes and the different interactions have been began to be determinedstudied, the consequences of these interactions have not been fully explored. Multiple GTPases have been shown to be important in the process of nerve regeneration and, however multiple molecules have been shown to affect these GTPases. In the future, it would be necessary to examine the different interactions and how manipulation of each interaction affects nerve regeneration in vivo.
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