Disinhibition of neurite growth to repair the injured adult CNS: Focusing on Nogo

Investigations into mechanisms that restrict the recovery of functions after an injury to the brain or the spinal cord have led to the discovery of specific neurite growth inhibitory factors in the adult central nervous system (CNS) of mammals. Blocking their growth-suppressive function resulted in disinhibition of axonal growth, i.e. growth of cultured neurons on inhibitory CNS tissue in vitro and regeneration of injured axons in vivo. The enhanced regenerative and compensatory fibre growth was often accompanied by a substantial improvement in the functional recovery after CNS injury. The first clinical studies to assess the therapeutic potential of compounds that neutralize growth inhibitors or interfere with their downstream signalling are currently in progress. This review discusses recent advances in the understanding of how the ‘founder molecule’ Nogo-A and other glialderived growth inhibitors restrict the regeneration and repair of disrupted neuronal circuits, thus limiting the functional recovery after CNS injuries. Received 5 April 2007; received after revision 28 September 2007; accepted 1 October 2007     

Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors

Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.Received 19 January 2004; received after revision 8 March 2004: accepted 17 March 2004     

Galanin – 25 years with a multitalented neuropeptide

The neuropeptide galanin is widely, but not ubiquitously, expressed in the adult nervous system. Its expression is markedly upregulated in many neuronal tissues after nerve injury or disease. Over the last 10 years we have demonstrated that the peptide plays a developmental survival role to subsets of neurons in the peripheral and central nervous systems with resulting phenotypic changes in neuropathic pain and cognition. Galanin also appears to play a trophic role to adult sensory neurons following injury, via activation of GalR2, by stimulating neurite outgrowth. Furthermore, galanin also plays a neuroprotective role to the hippocampus following excitotoxic injury, again mediated by activation of GalR2. In summary, these studies demonstrate that a GalR2 agonist might have clinical utility in a variety of human diseases that affect the nervous system.     

Involvement of Akt in neurite outgrowth

The regulation of neuronal differentiation and neurite outgrowth is essential during development of the nervous system and is crucial in developing therapies to promote axon regeneration after nerve injury or in neurodegenerative diseases. The serine/threonine kinase Akt has been well documented to promote neuronal survival. More recently Akt has also been revealed as key mediator of several aspects of neurite outgrowth, including elongation, branching and calibre. Downstream of Akt, several substrates have been identified that are likely to play key roles in Akt-mediated neurite outgrowth, such as glycogen synthase kinase 3β, peripherin, mammalian target of rapamycin and δ-catenin. The physical interaction between Akt and Hsp27, another protein that has been linked with neurite outgrowth, may also be significant in the process of neurite outgrowth. This review will unite and discuss the research to date that has examined the functionality of Akt in neuronal differentiation during development and neurite outgrowth.     

Myelination in the hippocampus during development and following lesion

Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate for outgrowing axons. Therefore, one major factor which accounts for the lack of axonal regeneration in the mature brain is myelin. Here we report on the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal lesion with the myelin-specific marker Black Gold. Although entorhinal axons enter the hippocampal formation at embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibers in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long-lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganization pha-ses in the hippocampus. In contrast, electroconvulsive seizures resulted in brief demyelination and a faster recovery time course. In conclusion, we could show that the appearance of mature axons in the hippocampus is temporally regulated during development. In the adult hippocampus, demyelination was found after anterograde degeneration and also following seizures, suggesting that independent types of insult lead to demyelination. Reappearing mature axons were found in the hippocampus following axonal sprouting. Therefore, our quantitative analysis of mature axons and myelination effectively reflects the readjusted axonal density and possible electrophysiological balance following lesion. Received 22 December 2003; received after revision 11 February 2004; accepted 17 February 2004     

Metallothionein expression by NG2 glial cells following CNS injury

Metallothionein (MT) expression is rapidly up-regulated following CNS injury, and there is a strong correlation between the presence or absence of MTand improved or impaired (respectively) recovery from such trauma.We now report that a distinct subset of NG2-positive, GFAP-negative glial cells bordering the injury tract express MT following focal injury to the adult rat neocortex. To confirm the ability of these NG2 glial cells to express MT, we have isolated and cultured them and identified that they can express MT following stimulation with zinc. To investigate the functional importance of MT expression by NG2 glial cells, we plated cortical neurons onto these cells and found that expression of MT enhanced the permissivity of NG2 glial cells to neurite outgrowth. Our data suggest that expression of MT by NG2 glial cells may contribute to the overall permissiveness of these cells to axon regeneration.     

Molecular analysis of axonal target specificity and synapse formation

The development of neuronal connectivity requires the growth of axons to their target region and the formation of dendritic trees that extend into specific layers. Within the target region growth cones, the tips of extending axons are guided to finer target fields including specific subcellular compartments where they form synapses. In this article we highlight recent progress on molecular aspects of axonal subcellular target selection such as the axon initial segment or specific sublaminae of the vertebrate retina. We then discuss the very recent progress on the molecular analysis of synapse formation in the central nervous system, including the direction of differentiation into an inhibitory or excitatory synapse. Apparently, initial synaptic contacts are structurally and functionally modulated by neuronal activity, raising the question how neuronal activity can modify synaptic circuits. We therefore also focus on neural proteins that are up-regulated, secreted or converted by synaptic activity and, thus, might represent molecular candidates for experience-driven refinement or remodeling of synaptic connections. Received 5 July 2005; received after revision 19 August 2005; accepted 2 September 2005     

Human embryonic stem cell-derived neurons establish region-specific, long-range projections in the adult brain

While the availability of pluripotent stem cells has opened new prospects for generating neural donor cells for nervous system repair, their capability to integrate with adult brain tissue in a structurally relevant way is still largely unresolved. We addressed the potential of human embryonic stem cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) to establish axonal projections after transplantation into the adult rodent brain. Transgenic and species-specific markers were used to trace the innervation pattern established by transplants in the hippocampus and motor cortex. In vitro, lt-NES cells formed a complex axonal network within several weeks after the initiation of differentiation and expressed a composition of surface receptors known to be instrumental in axonal growth and pathfinding. In vivo, these donor cells adopted projection patterns closely mimicking endogenous projections in two different regions of the adult rodent brain. Hippocampal grafts placed in the dentate gyrus projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsule into the cervical spinal cord and via the corpus callosum into the contralateral cortex. Interestingly, acquisition of these region-specific projection profiles was not correlated with the adoption of a regional phenotype. Upon reaching their destination, human axons established ultrastructural correlates of synaptic connections with host neurons. Together, these data indicate that neurons derived from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain.     

Neurotrophins and neuronal differentiation in the central nervous system

The central nervous system requires the proper formation of exquisitely precise circuits to function properly. These neuronal circuits are assembled during development by the formation of synaptic connections between hundreds of thousands of differentiating neurons. For these circuits to form correctly, neurons must elaborate precisely patterned axonal and dendritic arbors. Although the cellular and molecular mechanisms that guide neuronal differentiation and formation of connections remain mostly unknown, the neurotrophins have emerged recently as attractive candidates for regulating neuronal differentiation in the developing brain. The experiments reviewed here provide strong support for a bifunctional role for the neurotrophins in axonal and dendritic growth and are consistent with the exciting possibility that the neurotrophins might mediate activity-dependent synaptic plasticity.     

Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth

Axons of primary olfactory neurons are intimately associated with olfactory ensheathing cells (OECs) from the olfactory epithelium until the final targeting of axons within the olfactory bulb. However, little is understood about the nature and role of interactions between OECs and axons during development of the olfactory nerve pathway. We have used high resolution time-lapse microscopy to examine the growth and interactions of olfactory axons and OECs in vitro. Transgenic mice expressing fluorescent reporters in primary olfactory axons (OMP-ZsGreen) and ensheathing cells (S100ß-DsRed) enabled us to selectively analyse these cell types in explants of olfactory epithelium. We reveal here that rather than providing only a permissive substrate for axon growth, OECs play an active role in modulating the growth of pioneer olfactory axons. We show that the interactions between OECs and axons were dependent on lamellipodial waves on the shaft of OEC processes. The motility of OECs was mediated by GDNF, which stimulated cell migration and increased the apparent motility of the axons, whereas loss of OECs via laser ablation of the cells inhibited olfactory axon outgrowth. These results demonstrate that the migration of OECs strongly regulates the motility of axons and that stimulation of OEC motility enhances axon extension and growth cone activity.     

Collagens in the developing and diseased nervous system

Collagens are extracellular proteins characterized by a structure in triple helices. There are 28 collagen types which differ in size, structure and function. Their architectural and functional roles in connective tissues have been widely assessed. In the nervous system, collagens are rare in the vicinity of the neuronal soma, occupying mostly a “marginal” position, such as the meninges, the basement membranes and the sensory end organs. In neural development, however, where various ECM molecules are known to be determinant, recent studies indicate that collagens are no exception, participating in axonal guidance, synaptogenesis and Schwann cell differentiation. Insights on collagens function in the brain have also been derived from neural pathophysiological conditions. This review summarizes the significant advances which underscore the function and importance of collagens in the nervous system. Received 09 September 2008; received after revision 24 October 2008; accepted 28 October 2008     

Application to the study of connections in the CNS of the retrograde axonal transport of an iron-dextran complex]

The retrograde axonal transport of an iron-dextran complex was observed in neurons of the substantia nigra and of the intralaminar nuclei of the thalamus, after previous injection into the striatum. The histochemical demonstration of iron is simple and rapid, and can be combined with that of horseradish peroxidase, under precise conditions in the sequence of reactions. The iron-dextran complex revealed to be a valuable material for neuronal connectivity studies in the central nervous system.     

Vagal afferent innervation in regenerated rat liver

Summary The possible presence of neural sprouting in the afferent neurons of regenerated rat liver after hepatectomy was investigated bu retrograde transport of horesradish peroxidase. This experiment was carried out to see if the increase in hepatic parenchyma could provide an adequate stimulus for the sprouting precess. The study was limited to the vagal afferents, particularly the left ones, because they are the principal contributors to hepatic afferent innervation in the rat. The results show that neural sprouting does not occur in regenerated rat liver after 3 weeks In fact, the number of intensely labeled neurons in the left nodose ganglia of hepatectomized rats was significantly smaller than in controls. This could be due to a lessened availability of horseradish peroxidase to nerve terminals in hepatectomized and control animals. This could be a consequence of their possible distribution in hepatic areas not involved in the regenerative process.     

Axon guidance at the central nervous system midline

A key feature of the central nervous system of most higher organisms is their bilateral symmetry about the midline. The specialised cells that lie at the midline have an essential role in regulating the axon guidance decisions of both neurons that project axons across the midline and those that project on one side. The midline cells produce both attractive and repellent short- and long-range signals to guide axonal growth. The axons themselves express specific receptors that can be dynamically regulated in response to midline-derived signals. In this way, axons extend toward or away from the midline and those that do cross change their behaviour to respond to longitudinal signals on the contralateral side.     

Central terminals of capsaicin-sensitive primary afferent make synaptic contacts with neuronal soma in the mouse substantia gelatinosa

Degeneration of primary afferent central terminals (C-terminals) that contact neuronal soma in the substantia gelatinosa of the spinal dorsal horn was examined by electron microscopy 2 h after s.c. injection of capsaicin into newborn and adult mice. The C-terminals were small, dark, sinuous or slender terminals with clear synaptic vesicles in the early postnatal period. They are thought to develop into scalloped CI-terminals, surrounded by dendrites and a few axonal endings, forming synaptic glomeruli. The same type of nonglomerular terminals making presynaptic contacts with neuronal soma showed degeneration in both the newborn and adult animals, and were identified as capsaicin-sensitive CI-terminals. This finding suggests that capsaicin-sensitive C-fibers have a modulatory role on their own nociceptive input besides functioning in nociceptive transmission in the substantia gelatinosa.     

Vagal afferent innervation in regenerated rat liver

The possible presence of neural sprouting in the afferent neurons of regenerated rat liver after hepatectomy was investigated by retrograde transport of horseradish peroxidase. This experiment was carried out to see if the increase in hepatic parenchyma could provide an adequate stimulus for the sprouting process. The study was limited to the vagal afferents, particularly the left ones, because they are the principal contributors to hepatic afferent innervation in the rat. The results show that neural sprouting does not occur in regenerated rat liver after 3 weeks. In fact, the number of intensely labeled neurons in the left nodose ganglia of hepatectomized rats was significantly smaller than in controls. This could be due to a lessened availability of horseradish peroxidase to nerve terminals because of the increased non-innervated hepatic mass. There was no difference between right nodose ganglia neurons in hepatectomized and control animals. This could be a consequence of their possible distribution in hepatic areas not involved in the regenerative process.     

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons

Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent major dementia-associated abnormalities in Alzheimer’s disease (AD). This study examined the role of oxidative stress as a factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated with H₂O₂ (9–90 μM) or desferrioxamine (2–25 μM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function, and pro-apoptosis and sprouting gene expression. H₂O₂ treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The H₂O₂-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin, and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection), and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast, hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD, both oxidative stress and hypoxic injury can contribute to AD neurodegeneration. Received 24 May 2000; received after revision 7 July 2000; accepted 27 July 2000     

Glia as mediators of growth cone guidance: studies from insect nervous systems

Growth cones experience many different cues in their journey to their final target. They can respond to a variety of attractive and repulsive cues that can be secreted or cellular. These cues are generated by a wide range of cell types. One subset of cells that play an important role in growth cone guidance are glial cells. Glia secrete guidance cues and express cellular cues on their surface that guide axonal outgrowth. In doing so, glia can act as intermediate targets in growth cone guidance, a process that is conserved between vertebrate and invertebrate nervous systems. Recent work in grasshopper, Drosophila and moth nervous system development has underscored the importance of the instructive role glia play during axonal outgrowth.