Restorative Neurology and Neuroscience

Adult fish, in contrast to mammals, are capable of regenerating axonal tracts as well as cells and even entire tissues in the central nervous system (CNS). The zebrafish is a powerful genetic model for studies on the developing CNS and is now emerging as a CNS regeneration model. Here we review recent progress in adult regeneration paradigms in zebrafish ranging from axonal re-growth models to those of tissue regeneration. Moreover, we review the tools that have become available in zebrafish to elucidate the factors contributing to successful CNS regeneration. Since many molecular pathways are shared between zebrafish and mammals, it is hoped that insights from zebrafish may contribute to future therapeutic approaches in CNS injury and disease.

Cell adhesion molecules (CAMs) play important roles in cell-cell and cell-extracellular matrix interactions in both mature and developing nervous system. During development, they are involved in cell migration, axon guidance, target recognition, and synapse formation; while in the mature nervous system, they maintain synaptic connections, cell-cell contacts, and neuron-glial interactions. Injuries to the nervous systems break the stable state of the tissues and the repair of damaged tissues and regeneration of axons require the participation of CAMs both as adhesion molecules and as signal transduction molecules. One group of the well-studied CAMs in the nervous system is the immunoglobulin superfamily including L1 and neural cell adhesion molecule (NCAM). This review will be focussed on the involvement of L1, NCAM, and polysialylated NCAM in neural repair and axon regeneration after nerve injury and their potential applications in the treatment of CNS injury.

Following injury to the adult mammalian central nervous system, regenerative growth of severed axons is very limited. The lack of neuronal repair is often associated with significant functional deficits, and depending on the severity of injury, may result in permanent paralysis distal to the site of injury. A detailed understanding of the molecular mechanisms that limit neuronal growth in the injured spinal cord is an important step toward the development of specific strategies aimed at restoring functional connectivity lost as a consequence of injury. While rapid progress is being made in defining the molecular identity of CNS growth inhibitory constituents, comparatively little is known about their receptors and downstream signaling mechanisms. Emerging new evidence suggests that the mechanisms for myelin inhibition are likely to be complex, involving multiple and distinct receptor systems that may operate in a redundant manner. Furthermore, the relative contribution of a specific ligand-receptor system to bring about growth inhibition may greatly vary among different neuronal cell types. Myelin-associated glycoprotein (MAG), for example, employs different mechanisms to inhibit neurite outgrowth of cerebellar, sensory, and retinal ganglion neurons in vitro. Nogo-A harbors distinct growth inhibitory regions, which employ different signaling mechanisms. The Nogo-66 receptor 1 (NgR1), a shared ligand binding component in a receptor complex for Nogo-66, MAG, and OMgp, participates in neuronal growth cone collapse to acutely presented myelin inhibitors, but is dispensable for longitudinal neurite outgrowth inhibition on substrate-bound Nogo-66, MAG, OMgp, or crude CNS myelin in vitro. Consistent with the idea of cell-type specific mechanisms for myelin inhibition, different types of CNS neurons possess very different regenerative capacities and respond differently to experimental treatment strategies in vivo. We speculate that differences in regenerative axonal growth among different fiber systems are a reflection of their intrinsic ability to elongate axons and their distinct cell surface receptor profiles to respond to the growth inhibitory extracellular milieu. The existence of cell type specific mechanisms to impair regenerative axonal growth in the CNS may have important implications for the development of treatment strategies. Depending on the fiber tract injured, different ligand-receptor systems may need to be targeted in order to elicit robust and long-distance regenerative axonal growth.

A wide variety of molecules are involved as attractive or repulsive guidance cues in the developing nervous system. Some of these molecules are also expressed in the CNS of adult mammals where, following injury, they may repel regenerating axons, inhibit axonal regrowth, or control the behaviour of other cells important for the development of the meningeal and glial scars or the immune response to injury. Ephrins, semaphorins, Slits, Netrins, bone morphogenetic proteins (BMPs) and Wnts are among the most likely molecules to be involved in limiting axonal regeneration in the injured spinal cord. The receptors for these molecules are not universally expressed by neurons but there is evidence that ephrins and semaphorins limit regeneration of particular classes of axon into spinal cord lesion sites. It is likely that other repulsive guidance cues will also differentially affect the regeneration of specific tracts within the spinal cord. In addition to direct effects on axonal regeneration, many axonal guidance molecules have effects on glial, meningeal or immune system cells which also modulate the responses of CNS tissue to injury.

After injury to the mammalian central nervous system (CNS), neurons are not able to regenerate their axons and recovery is limited by restricted plasticity. Axon regeneration is inhibited by the presence of the various inhibitory molecules, including chondroitin sulfate proteoglycans (CSPGs) which are upregulated around the injury site. Plasticity after the end of critical periods is restricted by extracellular matrix changes, particularly the formation of CSPG-containing perineuronal nets. Enzymatic removal of chondroitin sulfate (CS) chains with chondroitinase ABC promotes axon regeneration and reactivates plasticity. This review details the structures and properties of the different CSPGs in the normal and damaged CNS, the use of the enzyme chondroitinase ABC to promote neural regeneration and plasticity, and discusses mechanisms of action and possible therapeutic uses of this enzyme.

This review will describe the unique advantages that are offered by the visual system of mammals and other vertebrates for studying the regenerative responses of the central nervous system (CNS) to injury, and recent insights provided by such studies. In the mouse and rat visual system a variety of experimental paradigms promote survival of retinal ganglion cells (RGC) and optic nerve regeneration, probably through stimulation by neurotrophic factors (NTF) either directly, or indirectly through retinal astrocyte/Müller cell intermediary activation. NTF induce disinhibition of axon growth through regulated intramembranous proteolysis of p75 $^{NTR}$ , and the inactivation of RhoA and EGFR signalling. The concomitant release of metalloproteinases (MMP) and plasminogen activators from RGC axons, and tissue inhibitors of metalloproteinases from optic nerve glia repress scarring and thereby reduce titres of scar-derived inhibitory ligands expressed in the wound. MMP also degrade myelin-derived inhibitory ligands along regenerating axon trajectories after regulated release from glia at the growing front of regenerating RGC axons. Optic nerve transection induces apoptosis of RGC which is blocked by anti-apoptotic regimes and thus, in combination with blockers of axon-growth inhibitory signalling and promoters of axon growth may be a therapeutic formula for promoting sustained axon regeneration. All these findings in the visual system are translatable to the CNS as a whole and thus strategies that successfully promote visual axon regeneration will be equally effective elsewhere in the CNS. Future developments likely to advance the field of regenerative research include a greater understanding of phylogenetic differences in the response of the CNS to injury, the role of NTF, cAMP, EGFR, glia/neuron interactions in disinhibiting and promoting axon growth, the control of neuron death, and effective drug delivery.

The use of genetically modified mice to study axon regeneration after spinal cord injury has served as a useful in vivo model for both loss-of-function and gain-of-function analysis of candidate proteins. This review discusses the impact of genetically modified mice on axon regeneration after spinal cord injury in the context of axon growth inhibition by myelin, the glial scar, and chemorepellent molecules. We also discuss the use of mice which transgenically express fluorescent proteins in specific axons for increasing our understanding of how spinal cord axons behave after injury.

The olfactory nerve differs from cranial nerves III-XII in that it contains a specialised type of glial cell, called 'olfactory ensheathing cell' (OEC), rather than Schwann cells. In addition, functional neurogenesis persists postnatally in the olfactory system, i.e. the primary olfactory pathway continuously rebuilds itself throughout adult life. The presence of OECs in the olfactory nerve is thought to be critical to this continuous growth process. Because of this intrinsic capacity for self-repair, the mammalian olfactory system has proved as a useful model in neuroregeneration studies. In addition, OECs have been used in transplantation studies to promote pathway regeneration elsewhere in the nervous system. Here, we have reviewed the parameters that allow for repair within the primary olfactory pathway and the role that OECs are thought to play in this process. We conclude that, in addition to intrinsic growth potential, the presence of an aligned substrate to the target structure is a fundamental prerequisite for appropriate restoration of connectivity with the olfactory bulb. Hence, strategies to promote regrowth of injured nerve pathways should incorporate usage of aligned, oriented substrates of OECs or other cellular conduits with additional intervention to boost neuronal cell body responses to injury and/or neutralisation of putative inhibitors.

Astrocytes comprise a heterogeneous cell population that plays a complex role in repair after spinal cord injury. Reactive astrocytes are major contributors to the glial scar that is a physical and chemical barrier to axonal regeneration. Yet, consistent with a supportive role in development, astrocytes secrete neurotrophic factors and protect neurons and glia spared by the injury. In development and after injury, local cues are modulators of astrocyte phenotype and function. When multipotent cells are transplanted into the injured spinal cord, they differentiate into astrocytes and other glial cells as opposed to neurons, which is commonly viewed as a challenge to be overcome in developing stem cell technology. However, several examples show that astrocytes provide support and guidance for axonal growth and aid in improving functional recovery after spinal cord injury. Notably, transplantation of astrocytes of a developmentally immature phenotype promotes tissue sparing and axonal regeneration. Furthermore, interventions that enhance endogenous astrocyte migration or reinvasion of the injury site result in greater axonal growth. These studies demonstrate that astrocytes are dynamic, diverse cells that have the capacity to promote axon growth after injury. The ability of astrocytes to be supportive of recovery should be exploited in devising regenerative strategies.

Loss of spinal motoneurones results in severe functional impairment. The most successful way to replace missing motoneurones is the use of embryonic postmitotic motoneurone grafts. It has been shown that grafted motoneurones survive, differentiate and integrate into the host cord. If grafted motoneurones are provided with a suitable conduit for axonal regeneration (e.g. a reimplanted ventral root) the grafted cells are able to grow their axons along the whole length of the peripheral nerves to reach muscles in the limb and restore function. Grafted motoneurones show excellent survival in motoneurone-depleted adult host cords, but the developing spinal cord appears to be an unfavourable environment for these cells. The long term survival and maturation of the grafted neurones are dependent on the availability of a nerve conduit and one or more target muscles, no matter whether these are ectopic nerve-muscle implants or limb muscles in their original place.

Thus, grafted and host motoneurones induce functional recovery of the denervated limb muscles when their axons regenerate into an avulsed and reimplanted ventral root. On the other hand, motoneurone-enriched embryonic grafts placed into a hemisection cavity in the cervical spinal cord induce axonal regeneration from great numbers of host motoneurones, possibly by the bridging effect of the grafts. In this case the regenerating host motoneurones reinnervate their original target muscles while the graft provides few axons for the reinnervation of muscles.

These results suggest that reconstruction of the injured spinal cord with embryonic motoneurone-enriched spinal cord graft is a feasible method to improve severe functional motor deficits.

Spinal nerve root injuries have a profound effect on the different parts (PNS and CNS) of the root itself as well as the pertinent spinal cord segment. A root avulsion from the spinal cord is a longitudinal spinal cord injury. There is degeneration of sensory and motor axons, loss of synapses, deterioration of local segmental connections, nerve cell death and reactions among non neuronal cells with scar formation, i.e. a cascade of events similar to those known to occur in any injury to the spinal cord. For function to be restored, nerve cells must survive and there must be regrowth of new nerve fibres along a trajectory consisting of CNS growth-inhibitory tissue in the spinal cord as well as PNS growth-promoting tissue in nerves. Problems in PNS regeneration such as non directional growths and unspecific reinnervation of target organs lead to unpredictable sensorimotor activity and conspires against a useful recovery of function. From the results of basic science experiments, a surgical strategy to treat root avulsion with spinal cord injury has been developed. In humans this technique is currently the most promising treatment of any spinal cord injury, with return of useful function together with pain alleviation in cases where all nerves to the extremity have been avulsed from the spinal cord. At present the shortcomings of this technique are proportionate to the delay before surgery, which leads to death of nerve cells and incomplete and unpredictable recovery. In order to improve this situation and achieve further recovery of useful function including sensory perceptions and to fully alleviate pain it is necessary to pursue research and development of both basic and clinical science.