Anatomical Diversity of the Adult Corticospinal Tract Revealed by Single-Cell Transcriptional Profiling.

The corticospinal tract (CST) forms a central part of the voluntary motor apparatus in all mammals. Thus, injury, disease, and subsequent degeneration within this pathway result in chronic irreversible functional deficits. Current strategies to repair the damaged CST are suboptimal in part because of underexplored molecular heterogeneity within the adult tract. Here, we combine spinal retrograde CST tracing with single-cell RNA sequencing (scRNAseq) in adult male and female mice to index corticospinal neuron (CSN) subtypes that differentially innervate the forelimb and hindlimb. We exploit publicly available datasets to confer anatomic specialization among CSNs and show that CSNs segregate not only along the forelimb and hindlimb axis but also by supraspinal axon collateralization. These anatomically defined transcriptional data allow us to use machine learning tools to build classifiers that discriminate between CSNs and cortical layer 2/3 and nonspinally terminating layer 5 neurons in M1 and separately identify limb-specific CSNs. Using these tools, CSN subtypes can be differentially identified to study postnatal patterning of the CST in vivo, leveraged to screen for novel limb-specific axon growth survival and growth activators in vitro, and ultimately exploited to repair the damaged CST after injury and disease.SIGNIFICANCE STATEMENT Therapeutic interventions designed to repair the damaged CST after spinal cord injury have remained functionally suboptimal in part because of an incomplete understanding of the molecular heterogeneity among subclasses of CSNs. Here, we combine spinal retrograde labeling with scRNAseq and annotate a CSN index by the termination pattern of their primary axon in the cervical or lumbar spinal cord and supraspinal collateral terminal fields. Using machine learning we have confirmed the veracity of our CSN gene lists to train classifiers to identify CSNs among all classes of neurons in primary motor cortex to study the development, patterning, homeostasis, and response to injury and disease, and ultimately target streamlined repair strategies to this critical motor pathway.

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma.

Failure of CNS neurons to mount a significant growth response after trauma contributes to chronic functional deficits after spinal cord injury. Activator and repressor screening of embryonic cortical neurons and retinal ganglion cells in vitro and transcriptional profiling of developing CNS neurons harvested in vivo have identified several candidates that stimulate robust axon growth in vitro and in vivo Building on these studies, we sought to identify novel axon growth activators induced in the complex adult CNS environment in vivo We transcriptionally profiled intact sprouting adult corticospinal neurons (CSNs) after contralateral pyramidotomy (PyX) in nogo receptor-1 knock-out mice and found that intact CSNs were enriched in genes in the 3-phosphoinositide degradation pathway, including six 5-phosphatases. We explored whether inositol polyphosphate-5-phosphatase K (Inpp5k) could enhance corticospinal tract (CST) axon growth in preclinical models of acute and chronic CNS trauma. Overexpression of Inpp5k in intact adult CSNs in male and female mice enhanced the sprouting of intact CST terminals after PyX and cortical stroke and sprouting of CST axons after acute and chronic severe thoracic spinal contusion. We show that Inpp5k stimulates axon growth in part by elevating the density of active cofilin in labile growth cones, thus stimulating actin polymerization and enhancing microtubule protrusion into distal filopodia. We identify Inpp5k as a novel CST growth activator capable of driving compensatory axon growth in multiple complex CNS injury environments and underscores the veracity of using in vivo transcriptional screening to identify the next generation of cell-autonomous factors capable of repairing the damaged CNS.SIGNIFICANCE STATEMENT Neurologic recovery is limited after spinal cord injury as CNS neurons are incapable of self-repair post-trauma. In vitro screening strategies exploit the intrinsically high growth capacity of embryonic CNS neurons to identify novel axon growth activators. While promising candidates have been shown to stimulate axon growth in vivo, concomitant functional recovery remains incomplete. We identified Inpp5k as a novel axon growth activator using transcriptional profiling of intact adult corticospinal tract (CST) neurons that had initiated a growth response after pyramidotomy in plasticity sensitized nogo receptor-1-null mice. Here, we show that Inpp5k overexpression can stimulate CST axon growth after pyramidotomy, stroke, and acute and chronic contusion injuries. These data support in vivo screening approaches to identify novel axon growth activators.

Nogo receptor decoy promotes recovery and corticospinal growth in non-human primate spinal cord injury.

After CNS trauma such as spinal cord injury, the ability of surviving neural elements to sprout axons, reorganize neural networks and support recovery of function is severely restricted, contributing to chronic neurological deficits. Among limitations on neural recovery are myelin-associated inhibitors functioning as ligands for neuronal Nogo receptor 1 (NgR1). A soluble decoy (NgR1-Fc, AXER-204) blocks these ligands and provides a means to promote recovery of function in multiple preclinical rodent models of spinal cord injury. However, the safety and efficacy of this reagent in non-human primate spinal cord injury and its toxicological profile have not been described. Here, we provide evidence that chronic intrathecal and intravenous administration of NgR1-Fc to cynomolgus monkey and to rat are without evident toxicity at doses of 20 mg and greater every other day (≥2.0 mg/kg/day), and far greater than the projected human dose. Adult female African green monkeys underwent right C5/6 lateral hemisection with evidence of persistent disuse of the right forelimb during feeding and right hindlimb during locomotion. At 1 month post-injury, the animals were randomized to treatment with vehicle (n = 6) or 0.10-0.17 mg/kg/day of NgR1-Fc (n = 8) delivered via intrathecal lumbar catheter and osmotic minipump for 4 months. One animal was removed from the study because of surgical complications of the catheter, but no treatment-related adverse events were noted in either group. Animal behaviour was evaluated at 6-7 months post-injury, i.e. 1-2 months after treatment cessation. The use of the impaired forelimb during spontaneous feeding and the impaired hindlimb during locomotion were both significantly greater in the treatment group. Tissue collected at 7-12 months post-injury showed no significant differences in lesion size, fibrotic scar, gliosis or neuroinflammation between groups. Serotoninergic raphespinal fibres below the lesion showed no deficit, with equal density on the lesioned and intact side below the level of the injury in both groups. Corticospinal axons traced from biotin-dextran-amine injections in the left motor cortex were equally labelled across groups and reduced caudal to the injury. The NgR1-Fc group tissue exhibited a significant 2-3-fold increased corticospinal axon density in the cervical cord below the level of the injury relative to the vehicle group. The data show that NgR1-Fc does not have preclinical toxicological issues in healthy animals or safety concerns in spinal cord injury animals. Thus, it presents as a potential therapeutic for spinal cord injury with evidence for behavioural improvement and growth of injured pathways in non-human primate spinal cord injury.

Plexina2 and CRMP2 Signaling Complex Is Activated by Nogo-A-Liganded Ngr1 to Restrict Corticospinal Axon Sprouting after Trauma.

After brain or spinal cord trauma, interaction of Nogo-A with neuronal NgR1 limits regenerative axonal sprouting and functional recovery. Cellular signaling by lipid-anchored NgR1 requires a coreceptor but the relevant partner in vivo is not clear. Here, we examined proteins enriched in NgR1 immunoprecipitates by Nogo-A exposure, identifying CRMP2, a cytosolic protein implicated in axon growth inhibition by Semaphorin/Plexin complexes. The Nogo-A-induced association of NgR1 with CRMP2 requires PlexinA2 as a coreceptor. Non-neuronal cells expressing both NgR1 and PlexinA2, but not either protein alone, contract upon Nogo-A exposure. Inhibition of cortical axon regeneration by Nogo-A depends on a NgR1/PlexinA2 genetic interaction because double-heterozygous NgR1+/-, PlexinA2+/- neurons, but not single-heterozygote neurons, are rescued from Nogo-A inhibition. NgR1 and PlexinA2 also interact genetically in vivo to restrict corticospinal sprouting in mouse cervical spinal cord after unilateral pyramidotomy. Greater post-injury sprouting in NgR1+/-, PlexinA2+/- mice supports enhanced neurological recovery of a mixed female and male double-heterozygous cohort. Thus, a NgR1/PlexinA2/CRMP2 ternary complex limits neural repair after adult mammalian CNS trauma.SIGNIFICANCE STATEMENT Several decades of molecular research have suggested that developmental regulation of axon growth is distinct in most regards from titration of axonal regenerative growth after adult CNS trauma. Among adult CNS pathways, the oligodendrocyte Nogo-A inhibition of growth through NgR1 is thought to have little molecular relationship to axonal guidance mechanisms active embryonically. Here, biochemical analysis of NgR1 function uncovered a physical complex with CRMP cytoplasmic mediators, and this led to appreciation of a role for PlexinA2 in concert with NgR1 after adult trauma. The data extend molecular understanding of neural repair after CNS trauma and link it to developmental processes.

Comprehensive Corticospinal Labeling with mu-crystallin Transgene Reveals Axon Regeneration after Spinal Cord Trauma in ngr1-/- Mice.

Spinal cord injury interrupts descending motor tracts and creates persistent functional deficits due to the absence of spontaneous axon regeneration. Of descending pathways, the corticospinal tract (CST) is thought to be the most critical for voluntary function in primates. Even with multiple tracer injections and genetic tools, the CST is visualized to only a minor degree in experimental studies. Here, we identify and validate the mu-crystallin (crym) gene as a high-fidelity marker of the CST. In transgenic mice expressing green fluorescent protein (GFP) under crym regulatory elements (crym-GFP), comprehensive and near complete CST labeling is achieved throughout the spinal cord. Bilateral pyramidotomy eliminated the 17,000 GFP-positive CST axons that were reproducibly labeled in brainstem from the spinal cord. We show that CST tracing with crym-GFP is 10-fold more efficient than tracing with biotinylated dextran amine (BDA). Using crym-GFP, we reevaluated the CST in mice lacking nogo receptor 1 (NgR1), a protein implicated in limiting neural repair. The number and trajectory of CST axons in ngr1(-/-) mice without injury was indistinguishable from ngr1(+/+) mice. After dorsal hemisection in the midthoracic cord, CST axons did not significantly regenerate in ngr1(+/+) mice, but an average of 162 of the 6000 labeled thoracic CST axons (2.68%) regenerated >100 µm past the lesion site in crym-GFP ngr1(-/-) mice. Although traditional BDA tracing cannot reliably visualize regenerating ngr1(-/-) CST axons, their regenerative course is clear with crym-GFP. Therefore the crym-GFP transgenic mouse is a useful tool for studies of CST anatomy in experimental studies of motor pathways. SIGNIFICANCE STATEMENT: Axon regeneration fails in the adult CNS, resulting in permanent functional deficits. Traditionally, inefficient extrinsic tracers such a biotinylated dextran amine (BDA) are used to label regenerating fibers after therapeutic intervention. We introduce crym-green fluorescent protein (GFP) transgenic mice as a comprehensive and specific tool with which to study the primary descending motor tract, the corticospinal tract (CST). CST labeling with crym-GFP is 10 times more efficient compared with BDA. The enhanced sensitivity afforded by crym-GFP revealed significant CST regeneration in NgR1 knock-out mice. Therefore, crym-GFP can be used as a standardized tool for future CST spinal cord injury studies.

Plasticity of intact rubral projections mediates spontaneous recovery of function after corticospinal tract injury.

Axons in the adult CNS fail to regenerate after injury, and therefore recovery from spinal cord injury (SCI) is limited. Although full recovery is rare, a modest degree of spontaneous recovery is observed consistently in a broad range of clinical and nonclinical situations. To define the mechanisms mediating spontaneous recovery of function after incomplete SCI, we created bilaterally complete medullary corticospinal tract lesions in adult mice, eliminating a crucial pathway for voluntary skilled movement. Anatomic and pharmacogenetic tools were used to identify the pathways driving spontaneous functional recovery in wild-type and plasticity-sensitized mice lacking Nogo receptor 1. We found that plasticity-sensitized mice recovered 50% of normal skilled locomotor function within 5 weeks of lesion. This significant, yet incomplete, spontaneous recovery was accompanied by extensive sprouting of intact rubrofugal and rubrospinal projections with the emergence of a de novo circuit between the red nucleus and the nucleus raphe magnus. Transient silencing of this rubro-raphe circuit in vivo via activation of the inhibitory DREADD (designer receptor exclusively activated by designer drugs) receptor hM4di abrogated spontaneous functional recovery. These data highlight the pivotal role of uninjured motor circuit plasticity in supporting functional recovery after trauma, and support a focus of experimental strategies on enhancing intact circuit rearrangement to promote functional recovery after SCI.

Gene-Silencing Screen for Mammalian Axon Regeneration Identifies Inpp5f (Sac2) as an Endogenous Suppressor of Repair after Spinal Cord Injury.

Axonal growth and neuronal rewiring facilitate functional recovery after spinal cord injury. Known interventions that promote neural repair remain limited in their functional efficacy. To understand genetic determinants of mammalian CNS axon regeneration, we completed an unbiased RNAi gene-silencing screen across most phosphatases in the genome. We identified one known and 17 previously unknown phosphatase suppressors of injury-induced CNS axon growth. Silencing Inpp5f (Sac2) leads to robust enhancement of axon regeneration and growth cone reformation. Results from cultured Inpp5f(-/-) neurons confirm lentiviral shRNA results from the screen. Consistent with the nonoverlapping substrate specificity between Inpp5f and PTEN, rapamycin does not block enhanced regeneration in Inpp5f(-/-) neurons, implicating mechanisms independent of the PI3K/AKT/mTOR pathway. Inpp5f(-/-) mice develop normally, but show enhanced anatomical and functional recovery after mid-thoracic dorsal hemisection injury. More serotonergic axons sprout and/or regenerate caudal to the lesion level, and greater numbers of corticospinal tract axons sprout rostral to the lesion. Functionally, Inpp5f-null mice exhibit enhanced recovery of motor functions in both open-field and rotarod tests. This study demonstrates the potential of an unbiased high-throughput functional screen to identify endogenous suppressors of CNS axon growth after injury, and reveals Inpp5f (Sac2) as a novel suppressor of CNS axon repair after spinal cord injury. Significance statement: The extent of axon regeneration is a critical determinant of neurological recovery from injury, and is extremely limited in the adult mammalian CNS. We describe an unbiased gene-silencing screen that uncovered novel molecules suppressing axonal regeneration. Inpp5f (Sac2) gene deletion promoted recovery from spinal cord injury with no side effects. The mechanism of action is distinct from another lipid phosphatase implicated in regeneration, PTEN. This opens new pathways for investigation in spinal cord injury research. Furthermore the screening methodology can be applied on a genome wide scale to discovery the entire set of mammalian genes contributing to axonal regeneration.

Myelin-derived ephrinB3 restricts axonal regeneration and recovery after adult CNS injury.

Recovery of neurological function after traumatic injury of the adult mammalian central nervous system is limited by lack of axonal growth. Myelin-derived inhibitors contribute to axonal growth restriction, with ephrinB3 being a developmentally important axonal guidance cue whose expression in mature oligodendrocytes suggests a role in regeneration. Here we explored the in vivo regeneration role of ephrinB3 using mice lacking a functional ephrinB3 gene. We confirm that ephrinB3 accounts for a substantial portion of detergent-resistant myelin-derived inhibition in vitro. To assess in vivo regeneration, we crushed the optic nerve and examined retinal ganglion fibers extending past the crush site. Significantly increased axonal regeneration is detected in ephrinB3(-/-) mice. Studies of spinal cord injury in ephrinB3(-/-) mice must take into account altered spinal cord development and an abnormal hopping gait before injury. In a near-total thoracic transection model, ephrinB3(-/-) mice show greater spasticity than wild-type mice for 2 mo, with slightly greater hindlimb function at later time points, but no evidence for axonal regeneration. After a dorsal hemisection injury, increased corticospinal and raphespinal growth in the caudal spinal cord are detected by 6 wk. This increased axonal growth is accompanied by improved locomotor performance measured in the open field and by kinematic analysis. Thus, ephrinB3 contributes to myelin-derived axonal growth inhibition and limits recovery from adult CNS trauma.

PlexinA2 limits recovery from corticospinal axotomy by mediating oligodendrocyte-derived Sema6A growth inhibition.

Axonal growth from both intact and severed fibers is limited after adult mammalian CNS injury. Myelin proteins contribute to inhibition of axonal growth. Semaphorin6A protein inhibits the extension of developing axons and is highly expressed in adult oligodendrocytes. This expression pattern suggests that a developmental axon guidance cue contributes to the restriction of adult CNS growth. Here, we assessed the role of a Sema6A receptor, PlexinA2, in recovery from adult trauma. Adult sensory neuron inhibition by Sema6A requires PlexinA2, with complete protection in PlexinA2-/- cultures. Mice lacking another myelin inhibitor receptor, NgR1, are known to exhibit greater axonal sprouting and functional recovery after lesions of the corticospinal tract at the medullary pyramid, so we investigated PlexinA2 in this lesion. Without injury, the corticofugal projection into the cervical spinal cord is normal in adult PlexinA2 null mice. After unilateral pyramidotomy, unlesioned PlexinA2-/- corticospinal fibers sprout across the midline to innervate the contralateral gray matter of the spinal cord to a significantly greater extent than do fibers in wild type mice. Sprouted fibers display frequent synaptophysin-positive synaptic puncta. The increased axonal growth in PlexinA2-/- mice after injury is accompanied by improved behavioral recovery in a pellet retrieval task using the impaired forelimb, and in a tape removal task. Thus, PlexinA2, as a receptor for oligodendrocyte-derived Sema6A and for secreted class 3 Semaphorins, plays a role in limiting adult axon growth and recovery after trauma.

Recovery from chronic spinal cord contusion after Nogo receptor intervention.

OBJECTIVE: Several interventions promote axonal growth and functional recovery when initiated shortly after central nervous system injury, including blockade of myelin-derived inhibitors with soluble Nogo receptor (NgR1, RTN4R) decoy protein. We examined the efficacy of this intervention in the much more prevalent and refractory condition of chronic spinal cord injury. METHODS: We eliminated the NgR1 pathway genetically in mice by conditional gene targeting starting 8 weeks after spinal hemisection injury and monitored locomotion in the open field and by video kinematics over the ensuing 4 months. In a separate pharmacological experiment, intrathecal NgR1 decoy protein administration was initiated 3 months after spinal cord contusion injury. Locomotion and raphespinal axon growth were assessed during 3 months of treatment between 4 and 6 months after contusion injury. RESULTS: Conditional deletion of NgR1 in the chronic state results in gradual improvement of motor function accompanied by increased density of raphespinal axons in the caudal spinal cord. In chronic rat spinal contusion, NgR1 decoy treatment from 4 to 6 months after injury results in 29% (10 of 35) of rats recovering weight-bearing status compared to 0% (0 of 29) of control rats (p < 0.05). Open field Basso, Beattie, and Bresnahan locomotor scores showed a significant improvement in the NgR-treated group relative to the control group (p < 0.005, repeated measures analysis of variance). An increase in raphespinal axon density caudal to the injury is detected in NgR1 decoy-treated animals by immunohistology and by positron emission tomography using a serotonin reuptake ligand. INTERPRETATION: Antagonizing myelin-derived inhibitors signaling with NgR1 decoy augments recovery from chronic spinal cord injury.

MAG and OMgp synergize with Nogo-A to restrict axonal growth and neurological recovery after spinal cord trauma.

Functional recovery after adult CNS damage is limited in part by myelin inhibitors of axonal regrowth. Three molecules, Nogo-A, MAG, and OMgp, are produced by oligodendrocytes and share neuronal receptor mechanisms through NgR1 and PirB. While each has an axon-inhibitory role in vitro, their in vivo interactions and relative potencies have not been defined. Here, we compared mice singly, doubly, or triply mutant for these three myelin inhibitor proteins. The myelin extracted from Nogo-A mutant mice is less inhibitory for axons than is that from wild-type mice, but myelin lacking MAG and OMgp is indistinguishable from control. However, myelin lacking all three inhibitors is less inhibitory than Nogo-A-deficient myelin, uncovering a redundant and synergistic role for all three proteins in axonal growth inhibition. Spinal cord injury studies revealed an identical in vivo hierarchy of these three myelin proteins. Loss of Nogo-A allows corticospinal and raphespinal axon growth above and below the injury, as well as greater behavioral recovery than in wild-type or heterozygous mutant mice. In contrast, deletion of MAG and OMgp stimulates neither axonal growth nor enhanced locomotion. The triple-mutant mice exhibit greater axonal growth and improved locomotion, consistent with a principal role for Nogo-A and synergistic actions for MAG and OMgp, presumably through shared receptors. These data support the hypothesis that targeting all three myelin ligands, as with NgR1 decoy receptor, provides the optimal chance for overcoming myelin inhibition and improving neurological function.

Dissociation of intact adult mouse cortical projection neurons for single-cell RNA-seq.

This protocol provides an improved pipeline for dissociating intact projection neurons from adult mouse cortex for applications including droplet and plate-based single-cell RNA sequencing, qPCR, immunocytochemistry, and long-term in vitro cell culture. This protocol provides a robust and reproducible dissociation pipeline that uses exclusively off-the-shelf reagents, not requiring the use of expensive dissociation kits. The unique incubation steps, in combination with the FACS gating strategy, results in unparalleled enrichment for intact cortical neurons from the adult brain. For complete details on the use and execution of this protocol, please refer to Golan et al. (2021).

Rho-associated kinase II (ROCKII) limits axonal growth after trauma within the adult mouse spinal cord.

Rho GTPases are thought to mediate the action of several axonal growth inhibitors in the adult brain and spinal cord. RhoA has been targeted pharmacologically in both humans and animals to promote neurite outgrowth and functional recovery following CNS trauma. However, rat spinal cord injury studies suggest a complicated and partial benefit of inhibiting Rho or its downstream effector, Rho-associated kinase (ROCKII). This limited benefit may reflect inhibition of other kinases, poor access, or a minimal role of ROCKII in vivo. Therefore, we studied ROCKII mutant mice to probe this pathway genetically. ROCKII(-/-) dorsal root ganglion neurons are less sensitive to inhibition by Nogo protein or by chondroitin sulfate proteoglycan in vitro. We examined adult ROCKII(-/-) mice in two injury paradigms, cervical multilevel dorsal rhizotomy and midthoracic dorsal spinal cord hemisection. After dorsal root crush injury, the ROCKII(-/-) mice recovered use of the affected forepaw more quickly than did controls. Moreover, multiple classes of sensory axons regenerated across the dorsal root entry zone into the spinal cord of mice lacking ROCKII. After the spinal cord injury, ROCKII(-/-) mice showed enhanced local growth of raphespinal axons in the caudal spinal cord and corticospinal axons into the lesion site. Improved functional recovery was not observed by Basso Mouse Scale score following dorsal hemisection, likely due to developmental defects in the nervous system. Together, these findings demonstrate that the ROCKII gene product limits axonal growth after CNS trauma.

Axonal growth therapeutics: regeneration or sprouting or plasticity?

Loss of function after neurological injury frequently occurs through the interruption of axonal connectivity, rather than through cell loss. Functional deficits persist because a multitude of inhibitory factors in degenerating myelin and astroglial scar prevent axonal growth in the adult brain and spinal cord. Given the high clinical significance of achieving functional recovery through axonal growth, substantial research effort has been, and will be, devoted toward this desirable goal. Unfortunately, the labels commonly used in the literature to categorize post-injury axonal anatomy might hinder advancement. In this article, we present an argument for the importance of developing precise terms that describe axonal growth in terms of the inciting event, the distance of axonal extension and the timing of axonal growth. The phenotypes produced by molecular interventions that overcome astroglial scar or myelin-associated inhibitors are reframed and discussed in this context.

Chondroitinase ABC-mediated plasticity of spinal sensory function.

Experimental therapeutics designed to enhance recovery from spinal cord injury (SCI) primarily focus on augmenting the growth of damaged axons by elevating their intrinsic growth potential and/or by nullifying the influence of inhibitory proteins present in the mature CNS. However, these strategies may also influence the wiring of intact pathways. The direct contribution of such effects to functional restoration after injury has been mooted, but as yet not been described. Here, we provide evidence to support the hypothesis that reorganization of intact spinal circuitry enhances function after SCI. Adult rats that underwent unilateral cervical spared-root lesion (rhizotomy of C5, C6, C8, and T1, sparing C7) exhibited profound sensory deficits for 4 weeks after injury. Delivery of a focal intraspinal injection of the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC (ChABC) was sufficient to restore sensory function after lesion. In vivo electrophysiological recordings confirm that behavioral recovery observed in ChABC-treated rats was consequent on reorganization of intact C7 primary afferent terminals and not regeneration of rhizotomized afferents back into the spinal cord within adjacent segments. These data confirm that intact spinal circuits have a profound influence on functional restoration after SCI. Furthermore, comprehensive understanding of these targets may lead to therapeutic interventions that can be spatially tailored to specific circuitry, thereby reducing unwanted maladaptive axon growth of distal pathways.

Functional axonal regeneration through astrocytic scar genetically modified to digest chondroitin sulfate proteoglycans.

Axotomized neurons within the damaged CNS are thought to be prevented from functional regeneration by inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors. Here, we provide a transgenic test of the role of CSPGs in limiting regeneration, using the gfap promotor to express a CSPG-degrading enzyme chondroitinase ABC (ChABC) in astrocytes. Corticospinal axons extend within the lesion site, but not caudal to it, after dorsal hemisection in the transgenic mice. The presence of the gfap-ChABC transgene yields no significant improvement in motor function recovery in this model. In contrast, functionally significant sensory axon regeneration is observed after dorsal rhizotomy in transgenic mice. These transgenic studies confirm a local efficacy for reduced CSPG to enhance CNS axon growth after traumatic injury. CSPGs appear to function in a spatially distinct role from myelin inhibitors, implying that combination-based therapy will be especially advantageous for CNS injuries.

The nociceptin receptor inhibits axonal regeneration and recovery from spinal cord injury.

Axonal growth after traumatic spinal cord injury is limited by endogenous inhibitors, selective blockade of which promotes partial neurological recovery. The partial repair phenotypes suggest that compensatory pathways limit improvement. Gene expression profiles of mice deficient in Ngr1, which encodes a receptor for myelin-associated inhibitors of axonal regeneration such as Nogo, revealed that trauma increased the mRNA expression of ORL1, which encodes the receptor for the opioid-related peptide nociceptin. Endogenous and overexpressed ORL1 coimmunoprecipitated with immature NgR1 protein, and ORL1 enhanced the O-linked glycosylation and surface expression of NgR1 in HEK293T and Neuro2A cells and primary neurons. ORL1 overexpression inhibited cortical neuron axon regeneration independently of NgR1. Furthermore, regeneration was inhibited by an ORL1 agonist and enhanced by the ORL1 antagonist J113397 through a ROCK-dependent mechanism. Mice treated with J113397 after dorsal hemisection of the mid-thoracic spinal cord recovered greater locomotor function and exhibited lumbar raphespinal axon sprouting. These effects were further enhanced by combined Ngr1 deletion and ORL1 inhibition. Thus, ORL1 limits neural repair directly and indirectly by enhancing NgR1 maturation, and ORL1 antagonists enhance recovery from traumatic CNS injuries in wild-type and Ngr1 null mice.

Functional Genome-wide Screen Identifies Pathways Restricting Central Nervous System Axonal Regeneration.

Axonal regrowth is crucial for recovery from CNS injury but is severely restricted in adult mammals. We used a genome-wide loss-of-function screen for factors limiting axonal regeneration from cerebral cortical neurons in vitro. Knockdown of 16,007 individual genes identified 580 significant phenotypes. These molecules share no significant overlap with those suggested by previous expression profiles. There is enrichment for genes in pathways related to transport, receptor binding, and cytokine signaling, including Socs4 and Ship2. Among transport-regulating proteins, Rab GTPases are prominent. In vivo assessment with C. elegans validates a cell-autonomous restriction of regeneration by Rab27. Mice lacking Rab27b show enhanced retinal ganglion cell axon regeneration after optic nerve crush and greater motor function and raphespinal sprouting after spinal cord trauma. Thus, a comprehensive functional screen reveals multiple pathways restricting axonal regeneration and neurological recovery after injury.

Human neuroepithelial stem cell regional specificity enables spinal cord repair through a relay circuit.

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option, but optimal cell type and mechanistic aspects remain poorly defined. Here, we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons, requires the matching of neural identity to the anatomical site of injury, and is accompanied by expression of specific marker proteins. Thus, human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery.

The Nogo-Nogo receptor pathway limits a spectrum of adult CNS axonal growth.

The hypothesis that Nogo-A (Reticulon 4A) and Nogo-66 receptor (NgR1) limit adult CNS axonal growth after injury is supported by both in vitro experiments and in vivo pharmacological studies. However, genetic assessment of the role of Nogo-A in corticospinal tract (CST) axons after spinal cord dorsal hemisection has yielded conflicting results. CST regeneration is detected in homozygous nogo-ab(trap/trap) mice, but not in nogo-ab(atg/atg) mice. CST regeneration is also present after pharmacological NgR blockade, but not in ngr1(-/-) mice. To assess the nogo-ab(atg) and ngr1-null alleles for other axon growth phenotypes, we created unilateral pyramidotomies and monitored the uninjured CST. There is robust pyramidotomy-induced growth of nogo-ab(atg/atg) and ngr1(-/-) CST axons into denervated cervical gray matter. This fiber growth correlates with recovery of fine motor skill in the affected forelimb. Thus nogo-ab and ngr1 play a modulated role in limiting CNS axonal growth across a spectrum of different tracts in various lesion models.