Netrin-1 signaling for sensory axons: Involvement in sensory axonal development and regeneration

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a “waiting period” of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.Key words: netrin-1, dorsal root ganglion, axon guidance, chemorepellent, Unc5, spinal cord, axon regenerationDeveloping axons navigate to their targets by responding to attractive and repulsive guidance cues working in a contact-dependent or diffusible fashion in their environment (reviewed in ref. ¹). During early development of the primary sensory system, centrally projecting sensory axons from dorsal root ganglion (DRG) neurons extend toward the dorsolateral region of the spinal cord (Fig. 1A and C), where they enter the spinal cord exclusively through the dorsal root entry zone (DREZ), and never orient themselves toward the notochord or the ventral spinal cord (Fig. 1A; reviewed in ref. ²). We previously showed that the notochord but not the ventral spinal cord secretes semaphorin 3A (Sema3A), which is known to be a chemorepellent for DRG axons at early developmental stages (Fig. 1A).³ This is the reason why DRG axons never project toward the notochord. Along the same line, it is highly possible that the ventral spinal cord may secrete some chemorepulsive cue other than Sema3A for DRG axons.Open in a separate windowFigure 1Netrin-1 plays a critical role in sensory axonal guidance as an axon chemorepellent. (A) A schematic diagram of a thoracic transverse section of an E10 mouse embryo, summarizing the possible mechanism of netrin-1 action in early DRG axonal guidance. When DRG axons project toward the DREZ in the dorsal spinal cord (dSC), ventrally derived netrin-1 chemorepels DRG axons to prevent them from orienting aberrantly toward the ventral spinal cord (vSC) (upper). NC; notochord. In netrin-1-deficient embryos, some DRG axons misorient themselves toward the ventral spinal cord, because of the absence of netrin-1 proteins in the ventral spinal cord (lower). (B) At E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally (arrows) to form the dorsal funiculus (DF), netrin-1 proteins are transiently expressed in a subpopulation of dorsal spinal cord cells adjacent to the dorsal funiculus (upper). In netrin-1-deficient embryos, the dorsal funiculus is disorganized because DRG axons are no longer waiting for invading the dorsal mantle layer (lower). (C) Gain-of-function experiments by electroporation confirm the repulsive activity of netrin-1 toward DRG axons. When netrin-1 is misexpressed in the dorsal spinal cord, the number of DRG axons that enter the DREZ is significantly reduced compared with the control, because some DRG axons fail to project toward the DREZ and turn in the wrong direction.After entering the spinal cord, DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus without projecting to the dorsal mantle layer for a few days (this delay of the axonal projection to the mantle layer is referred to as the ‘waiting period;’ Fig. 1B). A few days later, proprioceptive afferents of DRGs begin to send collaterals into the dorsal layers, and cutaneous afferents project ventrally through the dorsal layers.⁴ This evidence raises the possibility that some repulsive cues transiently prevent the collaterals of DRGs from penetrating the dorsal spinal cord during this waiting period.Netrins are a family of secreted proteins that play a key role in axonal guidance, cell migration, morphogenesis and angiogenesis.⁵ Netrin-1 is a bifunctional axonal guidance cue, attracting some axons including commissural axons via the Deleted in Colorectal Cancer (DCC) receptor and repelling others via Unc5 receptors (reviewed in ref. ⁶). However, it has not been clear whether netrin-1 plays a role in sensory axonal guidance during development.Several observations strongly suggest a role for netrin-1 in DRG axonal guidance as a repulsive guidance cue during development.^7,8 First, in the mouse embryo at embryonic day (E) 10–11.⁵ when many DRG axons orient themselves to reach the DREZ, netrin-1 is strongly expressed in the floor plate of the ventral spinal cord but not in the dorsal spinal cord (Fig. 1A). Second, at E12.5 when DRG neurons extend their axons longitudinally along the dorsolateral margin of the spinal cord, netrin-1 is expressed in the dorsolateral region adjacent to the DREZ (Fig. 1B), but its expression is down-regulated in the dorsal spinal cord at E13.5 when many collaterals have entered the mantle layer. Third, repulsive netrin-1 receptor Unc5c is expressed in the DRG neurons during development.These observations motivated us to explore whether netrin-1/Unc5c signaling contributes to DRG axonal guidance. We used cell and tissue cultures combined with tissues from netrin-1-deficient mice. We clearly showed that netrin-1 exerts a chemorepulsive activity toward developing DRG axons and that the ventral spinal cord-derived repulsive activity depends on netrin-1 in vitro.⁸ Additional evidence for a chemorepulsive role of netrin-1 came from the observation of DRG axonal trajectories in netrin-1-deficient mice.^7,8 In netrin-1-deficient embryos at E10, we showed that some DRG axons became misoriented toward the ventral spinal cord, probably because of the absence of netrin-1 proteins in the ventral spinal cord (Fig. 1A). In addition, at E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus, the dorsal funiculus is disorganized in netrin-1-deficient embryos, because in the absence of netrin-1 DRG axons are not waiting for invading the dorsal mantle layer adjacent to the dorsal funiculus (Fig. 1B). Gain-of-function experiments further confirmed the repulsive activity of netrin-1 toward DRG axons (Fig. 1C). These lines of evidence lead us to the conclusion that dorsally derived netrin-1 plays an important role in providing the ‘waiting period’ for extension of collaterals from sensory afferents and that ventrally derived netrin-1 prevents sensory axons from misorienting themselves toward the ventral spinal cord.At later developmental stages (E13.5), DRG axons still possess a weak responsiveness to the chemorepulsive activity of netrin-1 in vitro.⁸ In addition, both postnatal and adult DRG neurons respond to netrin-1-induced axon inhibition.⁹ Consistent with these results, DRG neurons at not only later developmental stages (E13.5) but also postnatal stages express the repulsion-mediating netrin-1 receptor Unc5c.^8,9Generally, lesioning of the dorsal column projection of sensory axons results in a complete lack of regeneration. The possible explanation for the complete lack of regeneration is that the environment, the lesion site itself and/or oligodendrocytes adjacent to the lesion, may be non-permissive for regenerating axons.¹⁰ Sema3A and chondroitin sulfate proteoglycans (CSPGs) are candidates as major inhibitors of sensory axonal regeneration in the spinal cord, because they are expressed in the lesion site and can inhibit DRG axonal growth in vitro.^3,11–14 Recently, Kaneko et al. showed that a selective inhibitor of Sema3A also enhances axonal regeneration and functional recovery in a subpopulation of sensory neurons after lesioning of the dorsal column.¹² More recently, McMahon''s group clearly demonstrated that enzymatic degradation of CSPGs on the dorsal column lesion of the spinal cord promotes sensory axonal regeneration and functional recovery.^13,14 Although these treatments greatly improved functional recovery, complete sensory axonal growth and functional recovery have not been yet achieved after the spinal cord injury. To promote further recovery of sensory axonal regeneration in the CNS, we should focus on other candidate inhibitors of CNS injury sites.Following spinal cord injury, the expression of the attraction- mediating netrin-1 receptor DCC decreases, while the expression level of the repulsive receptor Unc5c returns to normal.¹⁵ Levels of netrin-1 expression also stay unchanged in neurons and oligodendrocytes adjacent to the lesion site. Together with the in vitro evidence described above, these data strongly suggest a possible role for netrin-1 as a novel inhibitor of CNS myelin for regenerating DRG axons in the dorsal column-lesioned spinal cord. Further studies will be required to show directly the functional recovery of sensory axons in the spinal cord by perturbation of netrin-1 in and around the lesion site after spinal cord injury.     

Stimulating regeneration in the damaged spinal cord.

Great progress has been made in recent years in experimental strategies for spinal cord repair. In this review we describe two of these strategies, namely the use of neurotrophic factors to promote functional regeneration across the dorsal root entry zone (DREZ), and the use of synthetic fibronectin conduits to support directed axonal growth. The junction between the peripheral nervous system (PNS) and central nervous system (CNS) is marked by a specialized region, the DREZ, where sensory axons enter the spinal cord from the dorsal roots. After injury to dorsal roots, axons will regenerate as far as the DREZ but no further. However, recent studies have shown that this barrier can be overcome and function restored. In animals treated with neurotrophic factors, regenerating axons cross the DREZ and establish functional connections with dorsal horn cells. For example, intrathecal delivery of neurotrophin 3 (NT3) supports ingrowth of A fibres into the dorsal horn. This ingrowth is revealed using a transganglionic anatomical tracer (cholera toxin subunit B) and analysis at light and electron microscopic level. In addition to promoting axonal growth, spinal cord repair is likely to require strategies for supporting long-distance regeneration. Synthetic fibronectin conduits may be useful for this purpose. Experimental studies indicate that fibronectin mats implanted into the spinal cord will integrate with the host tissue and support extensive and directional axonal growth. Growth of both PNS and CNS axons is supported by the fibronectin, and axons become myelinated by Schwann cells. Ongoing studies are aimed at developing composite conduits and promoting axonal growth from the fibronectin back into the spinal cord.     

Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.

Regeneration is abortive following adult mammalian CNS injury. We have investigated whether increasing the intrinsic growth state of primary sensory neurons by a conditioning peripheral nerve lesion increases regrowth of their central axons. After dorsal column lesions, all fibers stop at the injury site. Animals with a peripheral axotomy concomitant with the central lesion show axonal growth into the lesion but not into the spinal cord above the lesion. A preconditioning lesion 1 or 2 weeks prior to the dorsal column injury results in growth into the spinal cord above the lesion. In vitro, the growth capacity of DRG neurite is also increased following preconditioning lesions. The intrinsic growth state of injured neurons is, therefore, a key determinant for central regeneration.     

Dorsally derived netrin 1 provides an inhibitory cue and elaborates the 'waiting period' for primary sensory axons in the developing spinal cord

Dorsal root ganglion (DRG) neurons extend axons to specific targets in the gray matter of the spinal cord. During development, DRG axons grow into the dorsolateral margin of the spinal cord and projection into the dorsal mantle layer occurs after a ;waiting period' of a few days. Netrin 1 is a long-range diffusible factor expressed in the ventral midline of the developing neural tube, and has chemoattractive and chemorepulsive effects on growing axons. Netrin 1 is also expressed in the dorsal spinal cord. However, the roles of dorsally derived netrin 1 remain totally unknown. Here, we show that dorsal netrin 1 controls the correct guidance of primary sensory axons. During the waiting period, netrin 1 is transiently expressed or upregulated in the dorsal spinal cord, and the absence of netrin 1 results in the aberrant projection of sensory axons, including both cutaneous and proprioceptive afferents, into the dorsal mantle layer. Netrin 1 derived from the dorsal spinal cord, but not the floor plate, is involved in the correct projection of DRG axons. Furthermore, netrin 1 suppresses axon outgrowth from DRG in vitro. Unc5c(rcm) mutant shows abnormal invasion of DRG axons as observed in netrin 1 mutants. These results are the first direct evidence that netrin 1 in the dorsal spinal cord acts as an inhibitory cue for primary sensory axons and is a crucial signal for the formation of sensory afferent neural networks.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

Live imaging of dorsal root axons after rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo(1). These mice have been used extensively for in vivo imaging of muscle(2-4) and brain(5-7), and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy(8,9). Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished(10). Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord(11). In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord

Sensory axonal projections into the spinal cord display a highly stereotyped pattern of T- or Y-shaped axon bifurcation at the dorsal root entry zone (DREZ). Here, we provide evidence that embryonic mice with an inactive receptor guanylyl cyclase Npr2 or deficient for cyclic guanosine monophosphate-dependent protein kinase I (cGKI) lack the bifurcation of sensory axons at the DREZ, i.e., the ingrowing axon either turns rostrally or caudally. This bifurcation error is maintained to mature stages. In contrast, interstitial branching of collaterals from primary stem axons remains unaffected, indicating that bifurcation and interstitial branching are processes regulated by a distinct molecular mechanism. At a functional level, the distorted axonal branching at the DREZ is accompanied by reduced synaptic input, as revealed by patch clamp recordings of neurons in the superficial layers of the spinal cord. Hence, our data demonstrate that Npr2 and cGKI are essential constituents of the signaling pathway underlying axonal bifurcation at the DREZ and neuronal connectivity in the dorsal spinal cord.     

Spinal axon regeneration induced by elevation of cyclic AMP

Myelin inhibitors, including MAG, are major impediments to CNS regeneration. However, CNS axons of DRGs regenerate if the peripheral branch of these neurons is lesioned first. We show that 1 day post-peripheral-lesion, DRG-cAMP levels triple and MAG/myelin no longer inhibit growth, an effect that is PKA dependent. By 1 week post-lesion, DRG-cAMP returns to control, but growth on MAG/myelin improves and is now PKA independent. Inhibiting PKA in vivo blocks the post-lesion growth on MAG/myelin at 1 day and attenuates it at 1 week. Alone, injection of db-cAMP into the DRG mimics completely a conditioning lesion as DRGs grow on MAG/myelin, initially, in a PKA-dependent manner that becomes PKA independent. Importantly, DRG injection of db-cAMP results in extensive regeneration of dorsal column axons lesioned 1 week later. These results may be relevant to developing therapies for spinal cord injury.     

Lentiviral-Mediated Netrin-1 Overexpression Improves Motor and Sensory Functions in SCT Rats Associated with SYP and GAP-43 Expressions

Spinal cord injury (SCI), as a major cause of disability, usually causes serious loss of motor and sensory functions. As a bifunctional axonal guidance cue, netrin-1 can attract axons via the deleted in colorectal cancer (DCC) receptors and repelling others via Unc5 receptors, but its exact role in the recovery of motor and sensory function has not well been studied, and the mechanisms remains elusive. The aim of this experiment is to determine whether lentiviral (LV)-mediated overexpression of netrin-1 or RNA interference (RNAi) can regulate the functional recovery in rats subjected to spinal cord transection (SCT). Firstly, two lentiviral vectors including Lv-exNtn-1 (netrin-1 open reading frame (ORF)) and Lv-shNtn-1 (netrin-1 sh) were constructed and injected into spinal cords rostral and caudal to the transected lesion site. Overexpressing netrin-1 enhanced significantly locomotor function, and reduced thermal and mechanical stimuli in vivo, compared with the control, while silencing netrin-1 did not significantly change the situation. Western blot and immunostaining analysis confirmed that netrin-1 ORF treatment not only effectively increased the expression level of netrin-1, also up-regulated the level of synaptophysin (SYP) in spinal cord rostral to the lesion, but also enhanced growth-associated protein-43 (GAP-43) expression in spinal cord caudal to the lesion site. Comparatively, knockdown of netrin-1 did not give rise to positive findings in our experimental condition. These findings therefore pointed that Lv-mediated netrin-1 overexpression could promote motor and sensory functional recoveries following SCT, and the underlying mechanisms were associated with SYP and GAP-43 expressions. The present study therefore provided a novel strategy for the treatment of SCI and explained the possible mechanisms for the functional improvement.     

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate (DiI)¹. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching² (Fig. 1).The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)³. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself⁴ (Fig. 2), however, other options have been discussed as well⁵.This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.     

PlexinA1 signaling directs the segregation of proprioceptive sensory axons in the developing spinal cord

As different classes of sensory neurons project into the CNS, their axons segregate and establish distinct trajectories and target zones. One striking instance of axonal segregation is the projection of sensory neurons into the spinal cord, where proprioceptive axons avoid the superficial dorsal horn-the target zone of many cutaneous afferent fibers. PlexinA1 is a proprioceptive sensory axon-specific receptor for sema6C and sema6D, which are expressed in a dynamic pattern in the dorsal horn. The loss of plexinA1 signaling causes the shafts of proprioceptive axons to invade the superficial dorsal horn, disrupting the organization of cutaneous afferents. This disruptive influence appears to involve the intermediary action of oligodendrocytes, which accompany displaced proprioceptive axon shafts into the dorsal horn. Our findings reveal a dedicated program of axonal shaft positioning in the mammalian CNS and establish a role for plexinA1-mediated axonal exclusion in organizing the projection pattern of spinal sensory afferents.     

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.     

Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury

Studying regeneration in the central nervous system (CNS) is hampered by current histological and imaging techniques because they provide only partial information about axonal and glial reactions. Here we developed a tetrahydrofuran-based clearing procedure that renders fixed and unsectioned adult CNS tissue transparent and fully penetrable for optical imaging. In large spinal cord segments, we imaged fluorescently labeled cells by 'ultramicroscopy' and two-photon microscopy without the need for histological sectioning. We found that more than a year after injury growth-competent axons regenerated abundantly through the injury site. A few growth-incompetent axons could also regenerate when they bypassed the lesion. Moreover, we accurately determined quantitative changes of glial cells after spinal cord injury. Thus, clearing CNS tissue enables an unambiguous evaluation of axon regeneration and glial reactions. Our clearing procedure also renders other organs transparent, which makes this approach useful for a large number of preclinical paradigms.     

The Nogo receptor, its ligands and axonal regeneration in the spinal cord; A review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.